US20110312796A1 - Test module that updates medical databases - Google Patents

Abstract

A test module for analyzing a sample fluid and communicating data to a medical database, the test module having a receptacle for receiving the sample, functional sections for processing and analyzing the sample, a communication interface for communication with the medical database, and, a controller for operative control of the communication interface.

Description

FIELD OF THE INVENTION
• The present invention relates to diagnostic devices that use microsystems technologies (MST). In particular, the invention relates to microfluidic and biochemical processing and analysis for molecular diagnostics.
CO-PENDING APPLICATIONS
• The following applications have been filed by the Applicant which relate to the present application:

• GBS001US	GBS002US	GBS003US	GBS005US	GBS006US
  GSR001US	GSR002US	GAS001US	GAS002US	GAS003US
  GAS004US	GAS006US	GAS007US	GAS008US	GAS009US
  GAS010US	GAS012US	GAS013US	GAS014US	GAS015US
  GAS016US	GAS017US	GAS018US	GAS019US	GAS020US
  GAS021US	GAS022US	GAS023US	GAS024US	GAS025US
  GAS026US	GAS027US	GAS028US	GAS030US	GAS031US
  GAS032US	GAS033US	GAS034US	GAS035US	GAS036US
  GAS037US	GAS038US	GAS039US	GAS040US	GAS041US
  GAS042US	GAS043US	GAS044US	GAS045US	GAS046US
  GAS047US	GAS048US	GAS049US	GAS050US	GAS054US
  GAS055US	GAS056US	GAS057US	GAS058US	GAS059US
  GAS060US	GAS061US	GAS062US	GAS063US	GAS065US
  GAS066US	GAS067US	GAS068US	GAS069US	GAS070US
  GAS080US	GAS081US	GAS082US	GAS083US	GAS084US
  GAS085US	GAS086US	GAS087US	GAS088US	GAS089US
  GAS090US	GAS091US	GAS092US	GAS093US	GAS094US
  GAS095US	GAS096US	GAS097US	GAS098US	GAS099US
  GAS100US	GAS101US	GAS102US	GAS103US	GAS104US
  GAS105US	GAS106US	GAS108US	GAS109US	GAS110US
  GAS111US	GAS112US	GAS113US	GAS114US	GAS115US
  GAS117US	GAS118US	GAS119US	GAS120US	GAS121US
  GAS122US	GAS123US	GAS124US	GAS125US	GAS126US
  GAS127US	GAS128US	GAS129US	GAS130US	GAS131US
  GAS132US	GAS133US	GAS134US	GAS135US	GAS136US
  GAS137US	GAS138US	GAS139US	GAS140US	GAS141US
  GAS142US	GAS143US	GAS144US	GAS146US	GAS147US
  GRR001US	GRR002US	GRR003US	GRR004US	GRR005US
  GRR006US	GRR007US	GRR008US	GRR009US	GRR010US
  GVA001US	GVA002US	GVA004US	GVA005US	GVA006US
  GVA007US	GVA008US	GVA009US	GVA010US	GVA011US
  GVA012US	GVA013US	GVA014US	GVA015US	GVA016US
  GVA017US	GVA018US	GVA019US	GVA020US	GVA021US
  GVA022US	GHU001US	GHU002US	GHU003US	GHU004US
  GHU006US	GHU007US	GHU008US	GWM001US	GWM002US
  GDI001US	GDI002US	GDI003US	GDI004US	GDI005US
  GDI006US	GDI007US	GDI009US	GDI010US	GDI011US
  GDI013US	GDI014US	GDI015US	GDI016US	GDI017US
  GDI019US	GDI023US	GDI028US	GDI030US	GDI039US
  GDI040US	GDI041US	GPC001US	GPC002US	GPC003US
  GPC004US	GPC005US	GPC006US	GPC007US	GPC008US
  GPC009US	GPC010US	GPC011US	GPC012US	GPC014US
  GPC017US	GPC018US	GPC019US	GPC023US	GPC027US
  GPC028US	GPC029US	GPC030US	GPC031US	GPC033US
  GPC034US	GPC035US	GPC036US	GPC037US	GPC038US
  GPC039US	GPC040US	GPC041US	GPC042US	GPC043US
  GLY001US	GLY002US	GLY003US	GLY004US	GLY005US
  GLY006US	GIN001US	GIN002US	GIN003US	GIN004US
  GIN005US	GIN006US	GIN007US	GIN008US	GMI001US
  GMI002US	GMI005US	GMI008US	GLE001US	GLE002US
  GLE003US	GLE004US	GLE005US	GLE006US	GLE007US
  GLE008US	GLE009US	GLE010US	GLE011US	GLE012US
  GLE013US	GLA001US	GGA001US	GGA003US	GRE001US
  GRE002US	GRE003US	GRE004US	GRE005US	GRE006US
  GRE007US	GCF001US	GCF002US	GCF003US	GCF004US
  GCF005US	GCF006US	GCF007US	GCF008US	GCF009US
  GCF010US	GCF011US	GCF012US	GCF013US	GCF014US
  GCF015US	GCF016US	GCF020US	GCF021US	GCF022US
  GCF023US	GCF024US	GCF025US	GCF027US	GCF028US
  GCF029US	GCF030US	GCF031US	GCF032US	GCF033US
  GCF034US	GCF035US	GCF036US	GCF037US	GSA001US
  GSA002US	GSE001US	GSE002US	GSE003US	GSE004US
  GDA001US	GDA002US	GDA003US	GDA004US	GDA005US
  GDA006US	GDA007US	GPK001US	GMO001US	GMV001US
  GMV002US	GMV003US	GMV004US	GRD001US	GRD002US
  GRD003US	GRD004US	GPD001US	GPD003US	GPD004US
  GPD005US	GPD006US	GPD007US	GPD008US	GPD009US
  GPD010US	GPD011US	GPD012US	GPD013US	GPD014US
  GPD015US	GPD016US	GPD017US	GAL001US	GPA001US
  GPA003US	GPA004US	GPA005US	GSS001US	GSL001US
  GCA001US	GCA002US	GCA003US

• The disclosures of these co-pending applications are incorporated herein by reference. The above applications have been identified by their filing docket number, which will be substituted with the corresponding application number, once assigned.
BACKGROUND OF THE INVENTION
• Molecular diagnostics has emerged as a field that offers the promise of early disease detection, potentially before symptoms have manifested. Molecular diagnostic testing is used to detect:
• • Inherited disorders
  • Acquired disorders
  • Infectious diseases
  • Genetic predisposition to health-related conditions.
• With high accuracy and fast turnaround times, molecular diagnostic tests have the potential to reduce the occurrence of ineffective health care services, enhance patient outcomes, improve disease management and individualize patient care. Many of the techniques in molecular diagnostics are based on the detection and identification of specific nucleic acids, both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), extracted and amplified from a biological specimen (such as blood or saliva). The complementary nature of the nucleic acid bases allows short sequences of synthesized DNA (oligonucleotides) to bond (hybridize) to specific nucleic acid sequences for use in nucleic acid tests. If hybridization occurs, then the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease a person will contract in the future, determine the identity and virulence of an infectious pathogen, or determine the response a person will have to a drug.
Nucleic Acid Based Molecular Diagnostic Test
• A nucleic acid based test has four distinct steps:
• 1. Sample preparation
• 2. Nucleic acid extraction
• 3. Nucleic acid amplification (optional)
• 4. Detection
• Many sample types are used for genetic analysis, such as blood, urine, sputum and tissue samples. The diagnostic test determines the type of sample required as not all samples are representative of the disease process. These samples have a variety of constituents, but usually only one of these is of interest. For example, in blood, high concentrations of erythrocytes can inhibit the detection of a pathogenic organism. Therefore a purification and/or concentration step at the beginning of the nucleic acid test is often required.
• Blood is one of the more commonly sought sample types. It has three major constituents: leukocytes (white blood cells), erythrocytes (red blood cells) and thrombocytes (platelets). The thrombocytes facilitate clotting and remain active in vitro. To inhibit coagulation, the specimen is mixed with an agent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Erythrocytes are usually removed from the sample in order to concentrate the target cells. In humans, erythrocytes account for approximately 99% of the cellular material but do not carry DNA as they have no nucleus. Furthermore, erythrocytes contain components such as haemoglobin that can interfere with the downstream nucleic acid amplification process (described below). Removal of erythrocytes can be achieved by differentially lysing the erythrocytes in a lysis solution, leaving remaining cellular material intact which can then be separated from the sample using centrifugation. This provides a concentration of the target cells from which the nucleic acids are extracted.
• The exact protocol used to extract nucleic acids depends on the sample and the diagnostic assay to be performed. For example, the protocol for extracting viral RNA will vary considerably from the protocol to extract genomic DNA. However, extracting nucleic acids from target cells usually involves a cell lysis step followed by nucleic acid purification. The cell lysis step disrupts the cell and nuclear membranes, releasing the genetic material. This is often accomplished using a lysis detergent, such as sodium dodecyl sulfate, which also denatures the large amount of proteins present in the cells.
• The nucleic acids are then purified with an alcohol precipitation step, usually ice-cold ethanol or isopropanol, or via a solid phase purification step, typically on a silica matrix in a column, resin or on paramagnetic beads in the presence of high concentrations of a chaotropic salt, prior to washing and then elution in a low ionic strength buffer. An optional step prior to nucleic acid precipitation is the addition of a protease which digests the proteins in order to further purify the sample.
• Other lysis methods include mechanical lysis via ultrasonic vibration and thermal lysis where the sample is heated to 94° C. to disrupt cell membranes.
• The target DNA or RNA may be present in the extracted material in very small amounts, particularly if the target is of pathogenic origin. Nucleic acid amplification provides the ability to selectively amplify (that is, replicate) specific targets present in low concentrations to detectable levels.
• The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). PCR is well known in this field and comprehensive description of this type of reaction is provided in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008.
• PCR is a powerful technique that amplifies a target DNA sequence against a background of complex DNA. If RNA is to be amplified (by PCR), it must be first transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Afterwards, the resulting cDNA is amplified by PCR.
• PCR is an exponential process that proceeds as long as the conditions for sustaining the reaction are acceptable. The components of the reaction are:
• 1. pair of primers—short single strands of DNA with around 10-30 nucleotides complementary to the regions flanking the target sequence
• 2. DNA polymerase—a thermostable enzyme that synthesizes DNA
• 3. deoxyribonucleoside triphosphates (dNTPs)—provide the nucleotides that are incorporated into the newly synthesized DNA strand
• 4. buffer—to provide the optimal chemical environment for DNA synthesis
• PCR typically involves placing these reactants in a small tube (−10-50 microlitres) containing the extracted nucleic acids. The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocol for each thermal cycle involves a denaturation phase, an annealing phase, and an extension phase. The extension phase is sometimes referred to as the primer extension phase. In addition to such three-step protocols, two-step thermal protocols can be employed, in which the annealing and extension phases are combined. The denaturation phase typically involves raising the temperature of the reaction to 90-95° C. to denature the DNA strands; in the annealing phase, the temperature is lowered to ˜50-60° C. for the primers to anneal; and then in the extension phase the temperature is raised to the optimal DNA polymerase activity temperature of 60-72° C. for primer extension. This process is repeated cyclically around 20-40 times, the end result being the creation of millions of copies of the target sequence between the primers.
• There are a number of variants to the standard PCR protocol such as multiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR and reverse-transcriptase PCR, amongst others, which have been developed for molecular diagnostics.
• Multiplex PCR uses multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several experiments. Optimization of multiplex PCR is more difficult though and requires selecting primers with similar annealing temperatures, and amplicons with similar lengths and base composition to ensure the amplification efficiency of each amplicon is equivalent.
• Linker-primed PCR, also known as ligation adaptor PCR, is a method used to enable nucleic acid amplification of essentially all DNA sequences in a complex DNA mixture without the need for target-specific primers. The method firstly involves digesting the target DNA population with a suitable restriction endonuclease (enzyme). Double-stranded oligonucleotide linkers (also called adaptors) with a suitable overhanging end are then ligated to the ends of target DNA fragments using a ligase enzyme. Nucleic acid amplification is subsequently performed using oligonucleotide primers which are specific for the linker sequences. In this way, all fragments of the DNA source which are flanked by linker oligonucleotides can be amplified.
• Direct PCR describes a system whereby PCR is performed directly on a sample without any, or with minimal, nucleic acid extraction. It has long been accepted that PCR reactions are inhibited by the presence of many components of unpurified biological samples, such as the haem component in blood. Traditionally, PCR has required extensive purification of the target nucleic acid prior to preparation of the reaction mixture. With appropriate changes to the chemistry and sample concentration, however, it is possible to perform PCR with minimal DNA purification, or direct PCR. Adjustments to the PCR chemistry for direct PCR include increased buffer strength, the use of polymerases which have high activity and processivity, and additives which chelate with potential polymerase inhibitors.
• Tandem PCR utilises two distinct rounds of nucleic acid amplification to increase the probability that the correct amplicon is amplified. One form of tandem PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in separate rounds of nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence at regions external to the target nucleic acid sequence. The second pair of primers (nested primers) used in the second round of amplification bind within the first PCR product and produce a second PCR product containing the target nucleic acid, that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake during the first round of nucleic acid amplification, the probability is very low that it would also be amplified a second time by a second pair of primers and thus ensures specificity.
• Real-time PCR, or quantitative PCR, is used to measure the quantity of a PCR product in real time. By using a fluorophore-containing probe or fluorescent dyes along with a set of standards in the reaction, it is possible to quantitate the starting amount of nucleic acid in the sample. This is particularly useful in molecular diagnostics where treatment options may differ depending on the pathogen load in the sample.
• Reverse-transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. Reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus.
• Isothermal amplification is another form of nucleic acid amplification which does not rely on the thermal denaturation of the target DNA during the amplification reaction and hence does not require sophisticated machinery. Isothermal nucleic acid amplification methods can therefore be carried out in primitive sites or operated easily outside of a laboratory environment. A number of isothermal nucleic acid amplification methods have been described, including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification and Loop-Mediated Isothermal Amplification.
• Isothermal nucleic acid amplification methods do not rely on the continuing heat denaturation of the template DNA to produce single stranded molecules to serve as templates for further amplification, but instead rely on alternative methods such as enzymatic nicking of DNA molecules by specific restriction endonucleases, or the use of an enzyme to separate the DNA strands, at a constant temperature.
• Strand Displacement Amplification (SDA) relies on the ability of certain restriction enzymes to nick the unmodified strand of hemi-modified DNA and the ability of a 5′-3′ exonuclease-deficient polymerase to extend and displace the downstream strand. Exponential nucleic acid amplification is then achieved by coupling sense and antisense reactions in which strand displacement from the sense reaction serves as a template for the antisense reaction. The use of nickase enzymes which do not cut DNA in the traditional manner but produce a nick on one of the DNA strands, such as N. Alw1, N. BstNB1 and Mly1, are useful in this reaction. SDA has been improved by the use of a combination of a heat-stable restriction enzyme (Ava1) and heat-stable Exo-polymerase (Bst polymerase). This combination has been shown to increase amplification efficiency of the reaction from 10⁸fold amplification to 10¹⁰fold amplification so that it is possible using this technique to amplify unique single copy molecules.
• Transcription Mediated Amplification (TMA) and Nucleic Acid Sequence Based Amplification (NASBA) use an RNA polymerase to copy RNA sequences but not corresponding genomic DNA. The technology uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase and optionally RNase H (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, this primer hybridizes to the target ribosomal RNA (rRNA) at a defined site. Reverse transcriptase creates a DNA copy of the target rRNA by extension from the 3′ end of the promoter primer. The RNA in the resulting RNA:DNA duplex is degraded by the RNase activity of the reverse transcriptase if present or the additional RNase H. Next, a second primer binds to the DNA copy. A new strand of DNA is synthesized from the end of this primer by reverse transcriptase, creating a double-stranded DNA molecule. RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each of the newly synthesized RNA amplicons re-enters the process and serves as a template for a new round of replication.
• In Recombinase Polymerase Amplification (RPA), the isothermal amplification of specific DNA fragments is achieved by the binding of opposing oligonucleotide primers to template DNA and their extension by a DNA polymerase. Heat is not required to denature the double-stranded DNA (dsDNA) template. Instead, RPA employs recombinase-primer complexes to scan dsDNA and facilitate strand exchange at cognate sites. The resulting structures are stabilised by single-stranded DNA binding proteins interacting with the displaced template strand, thus preventing the ejection of the primer by branch migration. Recombinase disassembly leaves the 3′ end of the oligonucleotide accessible to a strand displacing DNA polymerase, such as the large fragment ofBacillus subtilisPol I (Bsu), and primer extension ensues. Exponential nucleic acid amplification is accomplished by the cyclic repetition of this process.
• Helicase-dependent amplification (HDA) mimics the in vivo system in that it uses a DNA helicase enzyme to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. In the first step of the HDA reaction, the helicase enzyme traverses along the target DNA, disrupting the hydrogen bonds linking the two strands which are then bound by single-stranded binding proteins. Exposure of the single-stranded target region by the helicase allows primers to anneal. The DNA polymerase then extends the 3′ ends of each primer using free deoxyribonucleoside triphosphates (dNTPs) to produce two DNA replicates. The two replicated dsDNA strands independently enter the next cycle of HDA, resulting in exponential nucleic acid amplification of the target sequence.
• Other DNA-based isothermal techniques include Rolling Circle Amplification (RCA) in which a DNA polymerase extends a primer continuously around a circular DNA template, generating a long DNA product that consists of many repeated copies of the circle. By the end of the reaction, the polymerase generates many thousands of copies of the circular template, with the chain of copies tethered to the original target DNA. This allows for spatial resolution of target and rapid nucleic acid amplification of the signal. Up to 10¹²copies of template can be generated in 1 hour. Ramification amplification is a variation of RCA and utilizes a closed circular probe (C-probe) or padlock probe and a DNA polymerase with a high processivity to exponentially amplify the C-probe under isothermal conditions.
• Loop-mediated isothermal amplification (LAMP), offers high selectivity and employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10⁹copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand.
• After completion of the nucleic acid amplification, the amplified product must be analysed to determine whether the anticipated amplicon (the amplified quantity of target nucleic acids) was generated. The methods of analyzing the product range from simply determining the size of the amplicon through gel electrophoresis, to identifying the nucleotide composition of the amplicon using DNA hybridization.
• Gel electrophoresis is one of the simplest ways to check whether the nucleic acid amplification process generated the anticipated amplicon. Gel electrophoresis uses an electric field applied to a gel matrix to separate DNA fragments. The negatively charged DNA fragments will move through the matrix at different rates, determined largely by their size. After the electrophoresis is complete, the fragments in the gel can be stained to make them visible. Ethidium bromide is a commonly used stain which fluoresces under UV light.
• The size of the fragments is determined by comparison with a DNA size marker (a DNA ladder), which contains DNA fragments of known sizes, run on the gel alongside the amplicon. Because the oligonucleotide primers bind to specific sites flanking the target DNA, the size of the amplified product can be anticipated and detected as a band of known size on the gel. To be certain of the identity of the amplicon, or if several amplicons have been generated, DNA probe hybridization to the amplicon is commonly employed.
• DNA hybridization refers to the formation of double-stranded DNA by complementary base pairing. DNA hybridization for positive identification of a specific amplification product requires the use of a DNA probe around 20 nucleotides in length. If the probe has a sequence that is complementary to the amplicon (target) DNA sequence, hybridization will occur under favourable conditions of temperature, pH and ionic concentration. If hybridization occurs, then the gene or DNA sequence of interest was present in the original sample.
• Optical detection is the most common method to detect hybridization. Either the amplicons or the probes are labelled to emit light through fluorescence or electrochemiluminescence. These processes differ in the means of producing excited states of the light-producing moieties, but both enable covalent labelling of nucleotide strands. In electrochemiluminescence (ECL), light is produced by luminophore molecules or complexes upon stimulation with an electric current. In fluorescence, it is illumination with excitation light which leads to emission.
• Fluorescence is detected using an illumination source which provides excitation light at a wavelength absorbed by the fluorescent molecule, and a detection unit. The detection unit comprises a photosensor (such as a photomultiplier tube or charge-coupled device (CCD) array) to detect the emitted signal, and a mechanism (such as a wavelength-selective filter) to prevent the excitation light from being included in the photosensor output. The fluorescent molecules emit Stokes-shifted light in response to the excitation light, and this emitted light is collected by the detection unit. Stokes shift is the frequency difference or wavelength difference between emitted light and absorbed excitation light.
• ECL emission is detected using a photosensor which is sensitive to the emission wavelength of the ECL species being employed. For example, transition metal-ligand complexes emit light at visible wavelengths, so conventional photodiodes and CCDs are employed as photosensors. An advantage of ECL is that, if ambient light is excluded, the ECL emission can be the only light present in the detection system, which improves sensitivity.
• Microarrays allow for hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are powerful tools for molecular diagnostics with the potential to screen for thousands of genetic diseases or detect the presence of numerous infectious pathogens in a single test. A microarray consists of many different DNA probes immobilized as spots on a substrate. The target DNA (amplicon) is first labelled with a fluorescent or luminescent molecule (either during or after nucleic acid amplification) and then applied to the array of probes. The microarray is incubated in a temperature controlled, humid environment for a number of hours or days while hybridization between the probe and amplicon takes place. Following incubation, the microarray must be washed in a series of buffers to remove unbound strands. Once washed, the microarray surface is dried using a stream of air (often nitrogen). The stringency of the hybridization and washes is critical. Insufficient stringency can result in a high degree of nonspecific binding. Excessive stringency can lead to a failure of appropriate binding, which results in diminished sensitivity. Hybridization is recognized by detecting light emission from the labelled amplicons which have formed a hybrid with complementary probes.
• Fluorescence from microarrays is detected using a microarray scanner which is generally a computer controlled inverted scanning fluorescence confocal microscope which typically uses a laser for excitation of the fluorescent dye and a photosensor (such as a photomultiplier tube or CCD) to detect the emitted signal. The fluorescent molecules emit Stokes-shifted light (described above) which is collected by the detection unit.
• The emitted fluorescence must be collected, separated from the unabsorbed excitation wavelength, and transported to the detector. In microarray scanners, a confocal arrangement is commonly used to eliminate out-of-focus information by means of a confocal pinhole situated at an image plane. This allows only the in-focus portion of the light to be detected. Light from above and below the plane of focus of the object is prevented from entering the detector, thereby increasing the signal to noise ratio. The detected fluorescent photons are converted into electrical energy by the detector which is subsequently converted to a digital signal. This digital signal translates to a number representing the intensity of fluorescence from a given pixel. Each feature of the array is made up of one or more such pixels. The final result of a scan is an image of the array surface. The exact sequence and position of every probe on the microarray is known, and so the hybridized target sequences can be identified and analysed simultaneously.
• More information regarding fluorescent probes can be found at: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlight s/Fluorescence-Resonance-Energy-Transfer-FRET.html
Point-of-Care Molecular Diagnostics
• Despite the advantages that molecular diagnostic tests offer, the growth of this type of testing in the clinical laboratory has been slower than expected and remains a minor part of the practice of laboratory medicine. This is primarily due to the complexity and costs associated with nucleic acid testing compared with tests based on methods not involving nucleic acids. The widespread adaptation of molecular diagnostics testing to the clinical setting is intimately tied to the development of instrumentation that significantly reduces the cost, provides a rapid and automated assay from start (specimen processing) to finish (generating a result) and operates without major intervention by personnel.
• A point-of-care technology serving the physician's office, the hospital bedside or even consumer-based, at home, would offer many advantages including:
• • rapid availability of results enabling immediate facilitation of treatment and improved quality of care.
  • ability to obtain laboratory values from testing very small samples.
  • reduced clinical workload.
  • reduced laboratory workload and improved office efficiency by reducing administrative work.
  • improved cost per patient through reduced length of stay of hospitalization, conclusion of outpatient consultation at the first visit, and reduced handling, storing and shipping of specimens.
  • facilitation of clinical management decisions such as infection control and antibiotic use.
Lab-on-a-Chip (LOC) Based Molecular Diagnostics
• Molecular diagnostic systems based on microfluidic technologies provide the means to automate and speed up molecular diagnostic assays. The quicker detection times are primarily due to the extremely low volumes involved, automation, and the low-overhead inbuilt cascading of the diagnostic process steps within a microfluidic device. Volumes in the nanoliter and microliter scale also reduce reagent consumption and cost. Lab-on-a-chip (LOC) devices are a common form of microfluidic device. LOC devices have MST structures within a MST layer for fluid processing integrated onto a single supporting substrate (usually silicon). Fabrication using the VLSI (very large scale integrated) lithographic techniques of the semiconductor industry keeps the unit cost of each LOC device very low. However, controlling fluid flow through the LOC device, adding reagents, controlling reaction conditions and so on necessitate bulky external plumbing and electronics. Connecting a LOC device to these external devices effectively restricts the use of LOC devices for molecular diagnostics to the laboratory setting. The cost of the external equipment and complexity of its operation precludes LOC-based molecular diagnostics as a practical option for point-of-care settings.
• In view of the above, there is a need for a molecular diagnostic system based on a LOC device for use at point-of-care.
SUMMARY OF THE INVENTION
• Accordingly, the present invention provides a test module for analyzing a sample fluid and communicating data to a medical database, the test module comprising:
• a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the medical database; and,
• a controller for operative control of the communication interface.
• Preferably, the test module ofclaim1 further comprising a universal serial bus (USB) plug wherein the communication interface is a USB device driver for operative control of the USB plug to communicate with an external device.
• Preferably, the test module ofclaim2 further comprising digital memory wherein the medical database stores electronic health records (EHR), electronic medical records (EMR) and personal health records (PHR) and, the digital memory is configured for storing data relating to EHR, EMR and PHR.
• Preferably, the test module ofclaim3 further comprising a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory includes the reagent identities.
• Preferably, the data stored in the digital memory includes a unique identifier for test module.
• Preferably, the sample is a biological sample including cells of different sizes, and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample.
• Preferably, the test module ofclaim6 further comprising CMOS circuitry and a temperature sensor wherein the CMOS circuitry incorporates the digital memory and uses the temperature sensor output for feedback control of the PCR section.
• Preferably, one of the functional sections is a dialysis section, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• Preferably, one of the functional sections is a lysis section, the lysis section being configured to release nucleic acid sequences within the cells smaller than the predetermined threshold.
• Preferably, the test module ofclaim9 further comprising an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to excitation, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• Preferably, the test module ofclaim11 further comprising a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• Preferably, the CMOS circuitry is configured to communicate hybridization data to an external device.
• Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• Preferably, the test module ofclaim5 further comprising a LOC device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the CMOS circuitry incorporates the digital memory, the communication interface, the controller, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biochemical sample and processes and analyzes the sample, updating patients' databases based on the diagnostic results derived from the sample.
• The updating of patients' databases with the diagnostics results and the location data provides for improved provision of health care for the patients, automated maintenance of patient's medical records, improved science-base for the functioning of the diagnostic test modules, and optimal higher-level responses to epidemiological situations. The diagnostic test module based automation of updating the databases would provide for massive quality and economic gains for health information systems.
BRIEF DESCRIPTION OF THE DRAWINGS
• Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:
• FIG. 1 shows a test module and test module reader configured for fluorescence detection;
• FIG. 2 is a schematic overview of the electronic components in the test module configured for fluorescence detection;
• FIG. 3 is a schematic overview of the electronic components in the test module reader;
• FIG. 4 is a schematic representation of the architecture of the LOC device;
• FIG. 5 is a perspective of the LOC device;
• FIG. 6 is a plan view of the LOC device with features and structures from all layers superimposed on each other;
• FIG. 7 is a plan view of the LOC device with the structures of the cap shown in isolation;
• FIG. 8 is a top perspective of the cap with internal channels and reservoirs shown in dotted line;
• FIG. 9 is an exploded top perspective of the cap with internal channels and reservoirs shown in dotted line;
• FIG. 10 is a bottom perspective of the cap showing the configuration of the top channels;
• FIG. 11 is a plan view of the LOC device showing the structures of the CMOS+MST device in isolation;
• FIG. 12 is a schematic section view of the LOC device at the sample inlet;
• FIG. 13 is an enlarged view of Inset AA shown inFIG. 6;
• FIG. 14 is an enlarged view of Inset AB shown inFIG. 6;
• FIG. 15 is an enlarged view of Inset AE shown inFIG. 13;
• FIG. 16 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 17 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 18 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 19 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 20 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 21 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 22 is schematic section view of the lysis reagent reservoir shown inFIG. 21;
• FIG. 23 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 24 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 25 is a partial perspective illustrating the laminar structure of the LOC device within Inset AI;
• FIG. 26 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 27 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 28 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 29 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 30 is a schematic section view of the amplification mix reservoir and the polymerase reservoir;
• FIG. 31 show the features of a boiling-initiated valve in isolation;
• FIG. 32 is a schematic section view of the boiling-initiated valve taken through line33-33 shown inFIG. 31;
• FIG. 33 is an enlarged view of Inset AF shown inFIG. 15;
• FIG. 34 is a schematic section view of the upstream end of the dialysis section taken through line35-35 shown inFIG. 33;
• FIG. 35 is an enlarged view of Inset AC shown inFIG. 6;
• FIG. 36 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 37 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 38 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 39 is a further enlarged view within Inset AK shown inFIG. 38;
• FIG. 40 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 41 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 42 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 43 is a further enlarged view within Inset AL shown inFIG. 42;
• FIG. 44 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 45 is a further enlarged view within Inset AM shown inFIG. 44;
• FIG. 46 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 47 is a further enlarged view within Inset AN shown inFIG. 46;
• FIG. 48 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 49 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 50 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 51 is a schematic section view of the amplification section;
• FIG. 52 is an enlarged plan view of the hybridization section;
• FIG. 53 is a further enlarged plan view of two hybridization chambers in isolation;
• FIG. 54 is schematic section view of a single hybridization chamber;
• FIG. 55 is an enlarged view of the humidifier illustrated in Inset AG shown inFIG. 6;
• FIG. 56 is an enlarged view of Inset AD shown inFIG. 52;
• FIG. 57 is an exploded perspective view of the LOC device within Inset AD;
• FIG. 58 is a diagram of a FRET probe in a closed configuration;
• FIG. 59 is a diagram of a FRET probe in an open and hybridized configuration;
• FIG. 60 is a graph of the intensity of an excitation light over time;
• FIG. 61 is a diagram of the excitation illumination geometry of the hybridization chamber array;
• FIG. 62 is a diagram of a Sensor Electronic Technology LED illumination geometry;
• FIG. 63 is a schematic plan view of a reagent dispensing robot;
• FIG. 64 is a perspective of a reagent microvial with inbuilt droplet generator;
• FIG. 65 is a schematic plan view of an oligonucleotide ejector robot for loading selected probes into a probe ejector chip;
• FIG. 66 is a schematic plan view of a probe spotting robot for loading probes into the LOC devices on a partial-depth sawn silicon wafer;
• FIG. 67 is an enlarged plan view of the humidity sensor shown in Inset AH ofFIG. 6;
• FIG. 68 is a schematic showing part of the photodiode array of the photo sensor;
• FIG. 69 is a circuit diagram for a single photodiode;
• FIG. 70 is a timing diagram for the photodiode control signals;
• FIG. 71 shows an oligonucleotide ejector chip (ONEC);
• FIG. 72 shows an array of droplet generators from the ONEC shown in Inset AO ofFIG. 71;
• FIG. 73 is a schematic section of the array of droplet generators taken along line91-91 shown inFIG. 72;
• FIG. 74 is an enlarged view of the evaporator shown in Inset AP ofFIG. 55;
• FIG. 75 is a schematic section view through a hybridization chamber with a detection photodiode and trigger photodiode;
• FIG. 76 is a diagram of linker-primed PCR;
• FIG. 77 is a schematic representation of a test module with a lancet;
• FIG. 78 is a diagrammatic representation of the architecture of LOC variant VII;
• FIG. 79 is a diagrammatic representation of the architecture of LOC variant VIII;
• FIG. 80 is a schematic illustration of the architecture of LOC variant XIV;
• FIG. 81 is a schematic illustration of the architecture of LOC variant XLI;
• FIG. 82 is a schematic illustration of the architecture of LOC variant XLIII;
• FIG. 83 is a schematic illustration of the architecture of LOC variant XLIV;
• FIG. 84 is a schematic illustration of the architecture of LOC variant XLVII;
• FIG. 85 is a diagram of a primer-linked, linear fluorescent probe during the initial round of amplification;
• FIG. 86 is a diagram of a primer-linked, linear fluorescent probe during a subsequent amplification cycle;
• FIGS. 87A to 87F diagrammatically illustrate thermal cycling of a primer-linked fluorescent stem-and-loop probe;
• FIG. 88 is a schematic illustration of the excitation LED relative to the hybridization chamber array and the photodiodes;
• FIG. 89 is a schematic illustration of the excitation LED and optical lens for directing light onto the hybridization chamber array of the LOC device;
• FIG. 90 is a schematic illustration of the excitation LED, optical lens, and optical prisms for directing light onto the hybridization chamber array of the LOC device;
• FIG. 91 is a schematic illustration of the excitation LED, optical lens and mirror arrangement for directing light onto the hybridization chamber array of the LOC device;
• FIG. 92 is a schematic plan view of a probe spotting robot for loading probes into the LOC devices on a separable PCB;
• FIG. 93 is a plan view showing all the features superimposed on each other, and showing the location of Insets DA to DK;
• FIG. 94 is an enlarged view of Inset DG shown inFIG. 93;
• FIG. 95 is an enlarged view of Inset DH shown inFIG. 93;
• FIG. 96 shows one embodiment of the shunt transistor for the photodiodes;
• FIG. 97 shows one embodiment of the shunt transistor for the photodiodes;
• FIG. 98 shows one embodiment of the shunt transistor for the photodiodes;
• FIG. 99 is a circuit diagram of the differential imager;
• FIG. 100 schematically illustrates a negative control fluorescent probe in its stem-and-loop configuration;
• FIG. 101 schematically illustrates the negative control fluorescent probe ofFIG. 100 in its open configuration;
• FIG. 102 schematically illustrates a positive control fluorescent probe in its stem-and-loop configuration;
• FIG. 103 schematically illustrates the positive control fluorescent probe ofFIG. 102 in its open configuration;
• FIG. 104 shows a test module and test module reader configured for use with ECL detection;
• FIG. 105 is a schematic overview of the electronic components in the test module configured for use with ECL detection;
• FIG. 106 shows a test module and alternative test module readers;
• FIG. 107 shows a test module and test module reader along with the hosting system housing various databases;
• FIG. 108 is a schematic side view of a reagent spotting robot;
• FIG. 109 is a schematic representation of an electrochemiluminescence-based test module with multidevice microfluidic device;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTSOverview
• This overview identifies the main components of a molecular diagnostic system that incorporates embodiments of the present invention. Comprehensive details of the system architecture and operation are set out later in the specification.
• Referring toFIGS. 1,2,3,104 and105, the system has the following top level components:
• Test modules10 and11 are the size of a typical USB memory key and very cheap to produce.Test modules10 and11 each contain a microfluidic device, typically in the form of a lab-on-a-chip (LOC)device30 preloaded with reagents and typically more than 1000 probes for the molecular diagnostic assay (seeFIGS. 1 and 104).Test module10 schematically shown inFIG. 1 uses a fluorescence-based detection technique to identify target molecules, whiletest module11 inFIG. 104 uses an electrochemiluminescence-based detection technique. TheLOC device30 has an integratedphotosensor44 for fluorescence or electrochemiluminescence detection (described in detail below). Bothtest modules10 and11 use a standardMicro-USB plug14 for power, data and control, both have a printed circuit board (PCB)57, and both have externalpower supply capacitors32 and aninductor15. Thetest modules10 and11 are both single-use only for mass production and distribution in sterile packaging ready for use.
• Theouter casing13 has amacroreceptacle24 for receiving the biological sample and a removablesterile sealing tape22, preferably with a low tack adhesive, to cover the macroreceptacle prior to use. Amembrane seal408 with amembrane guard410 forms part of theouter casing13 to reduce dehumidification within the test module while providing pressure relief from small air pressure fluctuations. Themembrane guard410 protects themembrane seal408 from damage.
• Test module reader12 powers thetest module10 or11 viaMicro-USB port16. Thetest module reader12 can adopt many different forms and a selection of these are described later. The version of thereader12 shown inFIGS. 1,3 and104 is a smart phone embodiment. A block diagram of thisreader12 is shown inFIG. 3.Processor42 runs application software fromprogram storage43. Theprocessor42 also interfaces with thedisplay screen18 and user interface (UI)touch screen17 andbuttons19, acellular radio21,wireless network connection23, and asatellite navigation system25. Thecellular radio21 andwireless network connection23 are used for communications.Satellite navigation system25 is used for updating epidemiological databases with location data. The location data can, alternatively, be entered manually via thetouch screen17 orbuttons19.Data storage27 holds genetic and diagnostic information, test results, patient information, assay and probe data for identifying each probe and its array position.Data storage27 andprogram storage43 may be shared in a common memory facility. Application software installed on thetest module reader12 provides analysis of results, along with additional test and diagnostic information.
• To conduct a diagnostic test, the test module10 (or test module11) is inserted into theMicro-USB port16 on thetest module reader12. Thesterile sealing tape22 is peeled back and the biological sample (in a liquid form) is loaded into thesample macroreceptacle24. Pressingstart button20 initiates testing via the application software. The sample flows into theLOC device30 and the on-board assay extracts, incubates, amplifies and hybridizes the sample nucleic acids (the target) with presynthesized hybridization-responsive oligonucleotide probes. In the case of test module10 (which uses fluorescence-based detection), the probes are fluorescently labelled and theLED26 housed in thecasing13 provides the necessary excitation light to induce fluorescence emission from the hybridized probes (seeFIGS. 1 and 2). In test module11 (which uses electrochemiluminescence (ECL) detection), theLOC device30 is loaded with ECL probes (discussed above) and theLED26 is not necessary for generating the luminescent emission. Instead,electrodes860 and870 provide the excitation electrical current (seeFIG. 105). The emission (fluorescent or luminescent) is detected using aphotosensor44 integrated into CMOS circuitry of each LOC device. The detected signal is amplified and converted to a digital output which is analyzed by thetest module reader12. The reader then displays the results.
• The data may be saved locally and/or uploaded to a network server containing patient records. Thetest module10 or11 is removed from thetest module reader12 and disposed of appropriately.
• FIGS. 1,3 and104 show thetest module reader12 configured as a mobile phone/smart phone28. In other forms, the test module reader is a laptop/notebook101, adedicated reader103, anebook reader107, atablet computer109 or desktop computer105 for use in hospitals, private practices or laboratories (seeFIG. 106). The reader can interface with a range of additional applications such as patient records, billing, online databases and multi-user environments. It can also be interfaced with a range of local or remote peripherals such as printers and patient smart cards.
• Referring toFIG. 107, the data generated by thetest module10 can be used to update, via thereader12 andnetwork125, the epidemiological databases hosted on the hosting system for epidemiological data111, the genetic databases hosted on the hosting system forgenetic data113, the electronic health records hosted on the hosting system for electronic health records (EHR)115, the electronic medical records hosted on the hosting system for electronic medical records (EMR)121, and the personal health records hosted on the hosting system for personal health records (PHR)123. Conversely, the epidemiological data hosted on the hosting system for epidemiological data111, the genetic data hosted on the hosting system forgenetic data113, the electronic health records hosted on the hosting system for electronic health records (EHR)115, the electronic medical records hosted on the hosting system for electronic medical records (EMR)121, and the personal health records hosted on the hosting system for personal health records (PHR)123, can be used to update, vianetwork125 and thereader12, the digital memory in theLOC30 of thetest module10.
• Referring back toFIGS. 1,2,104 and105 thereader12 uses battery power in the mobile phone configuration. The mobile phone reader contains all test and diagnostic information preloaded. Data can also be loaded or updated via a number of wireless or contact interfaces to enable communications with peripheral devices, computers or online servers. AMicro-USB port16 is provided for connection to a computer or mains power supply for battery recharge.
• FIG. 77 shows an embodiment of thetest module10 used for tests that only require a positive or negative result for a particular target, such as testing whether a person is infected with, for example, H1N1 Influenza A virus. Only a purpose built USB power/indicator-onlymodule47 is adequate. No other reader or application software is necessary. Anindicator45 on the USB power/indicator-onlymodule47 signals positive or negative results. This configuration is well suited to mass screening.
• Additional items supplied with the system may include a test tube containing reagents for pre-treatment of certain samples, along with spatula and lancet for sample collection.FIG. 77 shows an embodiment of the test module incorporating a spring-loaded,retractable lancet390 andlancet release button392 for convenience. A satellite phone can be used in remote areas.
Test Module Electronics
• FIGS. 2 and 105 are block diagrams of the electronic components in thetest modules10 and11, respectively. The CMOS circuitry integrated in theLOC device30 has aUSB device driver36, acontroller34, a USB-compatible LED driver29,clock33,power conditioner31,RAM38 and program anddata flash memory40. These provide the control and memory for theentire test module10 or11 including thephotosensor44, thetemperature sensors170, theliquid sensors174, and thevarious heaters152,154,182,234, together with associateddrivers37 and39 and registers35 and41. Only the LED26 (in the case of test module10), externalpower supply capacitors32 and theMicro-USB plug14 are external to theLOC device30. TheLOC devices30 include bond-pads for making connections to these external components. TheRAM38 and the program anddata flash memory40 have the application software and the diagnostic and test information (Flash/Secure storage, e.g. via encryption) for over 1000 probes. In the case oftest module11 configured for ECL detection, there is no LED26 (seeFIGS. 104 and 105). Data is encrypted by theLOC device30 for secure storage and secure communication with an external device. TheLOC devices30 are loaded with electrochemiluminescent probes and the hybridization chambers each have a pair ofECL excitation electrodes860 and870.
• Many types oftest modules10 are manufactured in a number of test forms, ready for off-the-shelf use. The differences between the test forms lie in the on board assay of reagents and probes.
• Some examples of infectious diseases rapidly identified with this system include:
• • Influenza—Influenza virus A, B, C, Isavirus, Thogotovirus
  • Pneumonia—respiratory syncytial virus (RSV), adenovirus, metapneumovirus,Streptococcus pneumoniae, Staphylococcus aureus
  • Tuberculosis—Mycobacterium tuberculosis, bovis, africanum, canetti, andmicroti
  • Plasmodium falciparum, Toxoplasma gondiiand other protozoan parasites
  • Typhoid—Salmonella entericaserovar typhi
  • Ebola virus
  • Human immunodeficiency virus (HIV)
  • Dengue Fever—Flavivirus
  • Hepatitis (A through E)
  • Hospital acquired infections—for exampleClostridium difficile, Vancomycin resistantEnterococcus, and Methicillin resistantStaphylococcus aureus
  • Herpes simplex virus (HSV)
  • Cytomegalovirus (CMV)
  • Epstein-Ban virus (EBV)
  • Encephalitis—Japanese Encephalitis virus, Chandipura virus
  • Whooping cough—Bordetella pertussis
  • Measles—paramyxovirus
  • Meningitis—Streptococcus pneumoniaeandNeisseria meningitidis
  • Anthrax—Bacillus anthracis
• Some examples of genetic disorders identified with this system include:
• • Cystic fibrosis
  • Haemophilia
  • Sickle cell disease
  • Tay-Sachs disease
  • Haemochromatosis
  • Cerebral arteriopathy
  • Crohn's disease
  • Polycistic kidney disease
  • Congenital heart disease
  • Rett syndrome
• A small selection of cancers identified by the diagnostic system include:
• • Ovarian
  • Colon carcinoma
  • Multiple endocrine neoplasia
  • Retinoblastoma
  • Turcot syndrome
• The above lists are not exhaustive and the diagnostic system can be configured to detect a much greater variety of diseases and conditions using nucleic acid and proteomic analysis.
Detailed Architecture of System ComponentsLOC Device
• TheLOC device30 is central to the diagnostic system. It rapidly performs the four major steps of a nucleic acid based molecular diagnostic assay, i.e. sample preparation, nucleic acid extraction, nucleic acid amplification, and detection, using a microfluidic platform. The LOC device also has alternative uses, and these are detailed later. As discussed above,test modules10 and11 can adopt many different configurations to detect different targets Likewise, theLOC device30 has numerous different embodiments tailored to the target(s) of interest. One form of theLOC device30 isLOC device301 for fluorescent detection of target nucleic acid sequences in the pathogens of a whole blood sample. For the purposes of illustration, the structure and operation ofLOC device301 is now described in detail with reference toFIGS. 4 to 26 and27 to57.
• FIG. 4 is a schematic representation of the architecture of theLOC device301. For convenience, process stages shown inFIG. 4 are indicated with the reference numeral corresponding to the functional sections of theLOC device301 that perform that process stage. The process stages associated with each of the major steps of a nucleic acid based molecular diagnostic assay are also indicated: sample input andpreparation288,extraction290,incubation291,amplification292 anddetection294. The various reservoirs, chambers, valves and other components of theLOC device301 will be described in more detail later.
• FIG. 5 is a perspective view of theLOC device301. It is fabricated using high volume CMOS and MST (microsystems technology) manufacturing techniques. The laminar structure of theLOC device301 is illustrated in the schematic (not to scale) partial section view ofFIG. 12. TheLOC device301 has asilicon substrate84 which supports the CMOS+MST chip48, comprisingCMOS circuitry86 and anMST layer87, with acap46 overlaying theMST layer87. For the purposes of this patent specification, the term ‘MST layer’ is a reference to a collection of structures and layers that process the sample with various reagents. Accordingly, these structures and components are configured to define flow-paths with characteristic dimensions that will support capillary driven flow of liquids with physical characteristics similar to those of the sample during processing. In light of this, the MST layer and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other fabrication methods can also produce structures and components dimensioned for capillary driven flows and processing very small volumes. The specific embodiments described in this specification show the MST layer as the structures and active components supported on theCMOS circuitry86, but excluding the features of thecap46. However, the skilled addressee will appreciate that the MST layer need not have underlying CMOS or indeed an overlying cap in order for it to process the sample.
• The overall dimensions of the LOC device shown in the following figures are 1760 μm×5824 μm. Of course, LOC devices fabricated for different applications may have different dimensions.
• FIG. 6 shows the features of theMST layer87 superimposed with the features of the cap. Insets AA to AD, AG and AH shown inFIG. 6 are enlarged inFIGS. 13,14,35,56,55 and67, respectively, and described in detail below for a comprehensive understanding of each structure within theLOC device301.FIGS. 7 to 10 show the features of thecap46 in isolation whileFIG. 11 shows the CMOS+MST device48 structures in isolation.
Laminar Structure
• FIGS. 12 and 22 are sketches that diagrammatically show the laminar structure of the CMOS+MST device48, thecap46 and the fluidic interaction between the two. The figures are not to scale for the purposes of illustration.FIG. 12 is a schematic section view through thesample inlet68 andFIG. 22 is a schematic section through thereservoir54. As best shown inFIG. 12, the CMOS+MST device48 has asilicon substrate84 which supports theCMOS circuitry86 that operates the active elements within theMST layer87 above. Apassivation layer88 seals and protects theCMOS layer86 from the fluid flows through theMST layer87.
• Fluid flows through both thecap channels94 and the MST channels90 (see for exampleFIGS. 7 and 16) in thecap layer46 andMST channel layer100, respectively. Cell transport occurs in thelarger channels94 fabricated in thecap46, while biochemical processes are carried out in thesmaller MST channels90. Cell transport channels are sized so as to be able to transport cells in the sample to predetermined sites in theMST channels90. Transportation of cells with sizes greater than 20 microns (for example, certain leukocytes) requires channel dimensions greater than 20 microns, and therefore a cross sectional area transverse to the flow of greater than 400 square microns. MST channels, particularly at locations in the LOC where transport of cells is not required, can be significantly smaller.
• It will be appreciated thatcap channel94 andMST channel90 are generic references andparticular MST channels90 may also be referred to as (for example) heated microchannels or dialysis MST channels in light of their particular function.MST channels90 are formed by etching through aMST channel layer100 deposited on thepassivation layer88 and patterned with photoresist. TheMST channels90 are enclosed by aroof layer66 which forms the top (with respect to the orientation shown in the figures) of the CMOS+MST device48.
• Despite sometimes being shown as separate layers, thecap channel layer80 and thereservoir layer78 are formed from a unitary piece of material. Of course, the piece of material may also be non-unitary. This piece of material is etched from both sides in order to form acap channel layer80 in which thecap channels94 are etched and thereservoir layer78 in which thereservoirs54,56,58,60 and62 are etched. Alternatively, the reservoirs and the cap channels are formed by a micromolding process. Both etching and micromolding techniques are used to produce channels with cross sectional areas transverse to the flow as large as 20,000 square microns, and as small as8 square microns.
• At different locations in the LOC device, there can be a range of appropriate choices for the cross sectional area of the channel transverse to the flow. Where large quantities of sample, or samples with large constituents, are contained in the channel, a cross-sectional area of up to 20,000 square microns (for example, a 200 micron wide channel in a 100 micron thick layer) is suitable. Where small quantities of liquid, or mixtures without large cells present, are contained in the channel, a very small cross sectional area transverse to the flow is preferable.
• Alower seal64 encloses thecap channels94 and theupper seal layer82 encloses thereservoirs54,56,58,60 and62.
• The fivereservoirs54,56,58,60 and62 are preloaded with assay-specific reagents. In the embodiment described here, the reservoirs are preloaded with the following reagents, but other reagents can easily be substituted:
• • reservoir54: anticoagulant with option to include erythrocyte lysis buffer
  • reservoir56: lysis reagent
  • reservoir58: restriction enzymes, ligase and linkers (for linker-primed PCR (seeFIG. 76, extracted from T. Stachan et al.,Human Molecular Genetics 2, Garland Science, NY and London, 1999))
  • reservoir60: amplification mix (dNTPs, primers, buffer) and
  • reservoir62: DNA polymerase.
• Thecap46 and the CMOS+MST layers48 are in fluid communication via corresponding openings in thelower seal64 and theroof layer66. These openings are referred to asuptakes96 anddowntakes92 depending on whether fluid is flowing from theMST channels90 to thecap channels94 or vice versa.
LOC Device Operation
• The operation of theLOC device301 is described below in a step-wise fashion with reference to analysing pathogenic DNA in a blood sample. Of course, other types of biological or non-biological fluid are also analysed using an appropriate set, or combination, of reagents, test protocols, LOC variants and detection systems. Referring back toFIG. 4, there are five major steps involved in analysing a biological sample, comprising sample input andpreparation288,nucleic acid extraction290,nucleic acid incubation291,nucleic acid amplification292 and detection andanalysis294.
• The sample input andpreparation step288 involves mixing the blood with an anticoagulant116 and then separating pathogens from the leukocytes and erythrocytes with thepathogen dialysis section70. As best shown inFIGS. 7 and 12, the blood sample enters the device via thesample inlet68. Capillary action draws the blood sample along thecap channel94 to thereservoir54. Anticoagulant is released from thereservoir54 as the sample blood flow opens its surface tension valve118 (seeFIGS. 15 and 22). The anticoagulant prevents the formation of clots which would block the flow.
• As best shown inFIG. 22, the anticoagulant116 is drawn out of thereservoir54 by capillary action and into theMST channel90 via thedowntake92. Thedowntake92 has a capillary initiation feature (CIF)102 to shape the geometry of the meniscus such that it does not anchor to the rim of thedowntake92. Vent holes122 in theupper seal82 allows air to replace the anticoagulant116 as it is drawn out of thereservoir54.
• TheMST channel90 shown inFIG. 22 is part of asurface tension valve118. The anticoagulant116 fills thesurface tension valve118 and pins ameniscus120 to theuptake96 to ameniscus anchor98. Prior to use, themeniscus120 remains pinned at theuptake96 so the anticoagulant does not flow into thecap channel94. When the blood flows through thecap channel94 to theuptake96, themeniscus120 is removed and the anticoagulant is drawn into the flow.
• FIGS. 15 to 21 show Inset AE which is a portion of Inset AA shown inFIG. 13. As shown inFIGS. 15,16 and17, thesurface tension valve118 has threeseparate MST channels90 extending between respective downtakes92 anduptakes96. The number ofMST channels90 in a surface tension valve can be varied to change the flow rate of the reagent into the sample mixture. As the sample mixture and the reagents mix together by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample flow. Hence, the surface tension valve for each of the reservoirs is configured to match the desired reagent concentration.
• The blood passes into a pathogen dialysis section70 (seeFIGS. 4 and 15) where target cells are concentrated from the sample using an array ofapertures164 sized according to a predetermined threshold. Cells smaller than the threshold pass through the apertures while larger cells do not pass through the apertures. Unwanted cells, which may be either the larger cells withheld by the array ofapertures164 or the smaller cells that pass through the apertures, are redirected to awaste unit76 while the target cells continue as part of the assay.
• In thepathogen dialysis section70 described here, the pathogens from the whole blood sample are concentrated for microbial DNA analysis. The array of apertures is formed by a multitude of 3micron diameter holes164 fluidically connecting the input flow in thecap channel94 to atarget channel74. The 3micron diameter apertures164 and the dialysis uptake holes168 for thetarget channel74 are connected by a series of dialysis MST channels204 (best shown inFIGS. 15 and 21). Pathogens are small enough to pass through the 3micron diameter apertures164 and fill thetarget channel74 via thedialysis MST channels204. Cells larger than 3 microns, such as erythrocytes and leukocytes, stay in thewaste channel72 in thecap46 which leads to a waste reservoir76 (seeFIG. 7).
• Other aperture shapes, sizes and aspect ratios can be used to isolate specific pathogens or other target cells such as leukocytes for human DNA analysis. Greater detail on the dialysis section and dialysis variants is provided later.
• Referring again toFIGS. 6 and 7, the flow is drawn through thetarget channel74 to thesurface tension valve128 of thelysis reagent reservoir56. Thesurface tension valve128 has sevenMST channels90 extending between thelysis reagent reservoir56 and thetarget channel74. When the menisci are unpinned by the sample flow, the flow rate from all seven of theMST channels90 will be greater than the flow rate from theanticoagulant reservoir54 where thesurface tension valve118 has three MST channels90 (assuming the physical characteristics of the fluids are roughly equivalent). Hence the proportion of lysis reagent in the sample mixture is greater than that of the anticoagulant.
• The lysis reagent and target cells mix by diffusion in thetarget channel74 within thechemical lysis section130. A boiling-initiatedvalve126 stops the flow until sufficient time has passed for diffusion and lysis to take place, releasing the genetic material from the target cells (seeFIGS. 6 and 7). The structure and operation of the boiling-initiated valves are described in greater detail below with reference toFIGS. 31 and 32. Other active valve types (as opposed to passive valves such as the surface tension valve118) have also been developed by the Applicant which may be used here instead of the boiling-initiated valve. These alternative valve designs are also described later.
• When the boiling-initiatedvalve126 opens, the lysed cells flow into amixing section131 for pre-amplification restriction digestion and linker ligation.
• Referring toFIG. 13, restriction enzymes, linkers and ligase are released from thereservoir58 when the flow unpins the menisci at thesurface tension valve132 at the start of themixing section131. The mixture flows the length of themixing section131 for diffusion mixing. At the end of themixing section131 is downtake134 leading into theincubator inlet channel133 of the incubation section114 (seeFIG. 13). Theincubator inlet channel133 feeds the mixture into a serpentine configuration ofheated microchannels210 which provides an incubation chamber for holding the sample during restriction digestion and ligation of the linkers (seeFIGS. 13 and 14).
• FIGS. 23,24,25,26,27,28 and29 show the layers of theLOC device301 within Inset AB ofFIG. 6. Each figure shows the sequential addition of layers forming the structures of the CMOS+MST layer48 and thecap46. Inset AB shows the end of theincubation section114 and the start of theamplification section112. As best shown inFIGS. 14 and 23, the flow fills themicrochannels210 of theincubation section114 until reaching the boiling-initiatedvalve106 where the flow stops while diffusion takes place. As discussed above, themicrochannel210 upstream of the boiling-initiatedvalve106 becomes an incubation chamber containing the sample, restriction enzymes, ligase and linkers. Theheaters154 are then activated and held at constant temperature for a specified time for restriction digestion and linker ligation to occur.
• The skilled worker will appreciate that this incubation step291 (seeFIG. 4) is optional and only required for some nucleic acid amplification assay types. Furthermore, in some instances, it may be necessary to have a heating step at the end of the incubation period to spike the temperature above the incubation temperature. The temperature spike inactivates the restriction enzymes and ligase prior to entering theamplification section112. Inactivation of the restriction enzymes and ligase has particular relevance when isothermal nucleic acid amplification is being employed.
• Following incubation, the boiling-initiatedvalve106 is activated (opened) and the flow resumes into theamplification section112. Referring toFIGS. 31 and 32, the mixture fills the serpentine configuration ofheated microchannels158, which form one or more amplification chambers, until it reaches the boiling-initiatedvalve108. As best shown in the schematic section view ofFIG. 30, amplification mix (dNTPs, primers, buffer) is released fromreservoir60 and polymerase is subsequently released fromreservoir62 into theintermediate MST channel212 connecting the incubation and amplification sections (114 and112 respectively).
• FIGS. 35 to 51 show the layers of theLOC device301 within Inset AC ofFIG. 6. Each figure shows the sequential addition of layers forming the structures of the CMOS+MST device48 and thecap46. Inset AC is at the end of theamplification section112 and the start of the hybridization anddetection section52. The incubated sample, amplification mix and polymerase flow through themicrochannels158 to the boiling-initiatedvalve108. After sufficient time for diffusion mixing, theheaters154 in themicrochannels158 are activated for thermal cycling or isothermal amplification. The amplification mix goes through a predetermined number of thermal cycles or a preset amplification time to amplify sufficient target DNA. After the nucleic acid amplification process, the boiling-initiatedvalve108 opens and flow resumes into the hybridization anddetection section52. The operation of boiling-initiated valves is described in more detail later.
• As shown inFIG. 52, the hybridization anddetection section52 has an array ofhybridization chambers110.FIGS. 52,53,54 and56 show thehybridization chamber array110 andindividual hybridization chambers180 in detail. At the entrance to thehybridization chamber180 is adiffusion barrier175 which prevents diffusion of the target nucleic acid, probe strands and hybridized probes between thehybridization chambers180 during hybridization so as to prevent erroneous hybridization detection results. Thediffusion barriers175 present a flow-path-length that is long enough to prevent the target sequences and probes diffusing out of one chamber and contaminating another chamber within the time taken for the probes and nucleic acids to hybridize and the signal to be detected, thus avoiding an erroneous result.
• Another mechanism to prevent erroneous readings is to have identical probes in a number of the hybridization chambers. TheCMOS circuitry86 derives a single result from thephotodiodes184 corresponding to thehybridization chambers180 that contain identical probes. Anomalous results can be disregarded or weighted differently in the derivation of the single result.
• The thermal energy required for hybridization is provided by CMOS-controlled heaters182 (described in more detail below). After the heater is activated, hybridization occurs between complementary target-probe sequences. TheLED driver29 in theCMOS circuitry86 signals theLED26 located in thetest module10 to illuminate. These probes only fluoresce when hybridization has occurred thereby avoiding washing and drying steps that are typically required to remove unbound strands. Hybridization forces the stem-and-loop structure of the FRET probes186 to open, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as discussed in greater detail later. Fluorescence is detected by aphotodiode184 in theCMOS circuitry86 underlying each hybridization chamber180 (see hybridization chamber description below). Thephotodiodes184 for all hybridization chambers and associated electronics collectively form the photosensor44 (seeFIG. 68). In other embodiments, the photosensor may be an array of charge coupled devices (CCD array). The detected signal from thephotodiodes184 is amplified and converted to a digital output which is analyzed by thetest module reader12. Further details of the detection method are described later.
Additional Details for the LOC DeviceModularity of the Design
• TheLOC device301 has many functional sections, including thereagent reservoirs54,56,58,60 and62, thedialysis section70,lysis section130,incubation section114, andamplification section112, valve types, the humidifier and humidity sensor. In other embodiments of the LOC device, these functional sections can be omitted, additional functional sections can be added or the functional sections can be used for alternative purposes to those described above.
• For example, theincubation section114 can be used as thefirst amplification section112 of a tandem amplification assay system, with the chemicallysis reagent reservoir56 being used to add the first amplification mix of primers, dNTPs and buffer andreagent reservoir58 being used for adding the reverse transcriptase and/or polymerase. A chemical lysis reagent can also be added to thereservoir56 along with the amplification mix if chemical lysis of the sample is desired or, alternatively, thermal lysis can occur in the incubation section by heating the sample for a predetermined time. In some embodiments, an additional reservoir can be incorporated immediately upstream ofreservoir58 for the mix of primers, dNTPs and buffer if there is a requirement for chemical lysis and a separation of this mix from the chemical lysis reagent is desired.
• In some circumstances it may be desirable to omit a step, such as theincubation step291. In this case, a LOC device can be specifically fabricated to omit thereagent reservoir58 andincubation section114, or the reservoir can simply not be loaded with reagents or the active valves, if present, not activated to dispense the reagents into the sample flow, and the incubation section then simply becomes a channel to transport the sample from thelysis section130 to theamplification section112. The heaters are independently operable and therefore, where reactions are dependent on heat, such as thermal lysis, programming the heaters not to activate during this step ensures thermal lysis does not occur in LOC devices that do not require it. Thedialysis section70 can be located at the beginning of the fluidic system within the microfluidic device as shown inFIG. 4 or can be located anywhere else within the microfluidic device. For example, dialysis after theamplification phase292 to remove cellular debris prior to the hybridization anddetection step294 may be beneficial in some circumstances. Alternatively, two or more dialysis sections can be incorporated at any location throughout the LOC device. Similarly, it is possible to incorporateadditional amplification sections112 to enable multiple targets to be amplified in parallel or in series prior to being detected in thehybridization chamber arrays110 with specific nucleic acid probes. For analysis of samples like whole blood, in which dialysis is not required, thedialysis section70 is simply omitted from the sample input andpreparation section288 of the LOC design. In some cases, it is not necessary to omit thedialysis section70 from the LOC device even if the analysis does not require dialysis. If there is no geometric hindrance to the assay by the existence of a dialysis section, a LOC with thedialysis section70 in the sample input and preparation section can still be used without a loss of the required functionality.
• Furthermore, thedetection section294 may encompass proteomic chamber arrays which are identical to the hybridization chamber arrays but are loaded with probes designed to conjugate or hybridize with sample target proteins present in non-amplified sample instead of nucleic acid probes designed to hybridize to target nucleic acid sequences.
• It will be appreciated that the LOC devices fabricated for use in this diagnostic system are different combinations of functional sections selected in accordance with the particular LOC application. The vast majority of functional sections are common to many of the LOC devices and the design of additional LOC devices for new application is a matter of compiling an appropriate combination of functional sections from the extensive selection of functional sections used in the existing LOC devices.
• Only a small number of the LOC devices are shown in this description and some more are shown schematically to illustrate the design flexibility of the LOC devices fabricated for this system. The person skilled in the art will readily recognise that the LOC devices shown in this description are not an exhaustive list and many additional LOC designs are a matter of compiling the appropriate combination of functional sections.
Sample Types
• LOC variants can accept and analyze the nucleic acid or protein content of a variety of sample types in liquid form including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord blood, breast milk, sweat, pleural effusion, tear, pericardial fluid, peritoneal fluid, environmental water samples and drink samples. Amplicon obtained from macroscopic nucleic acid amplification can also be analysed using the LOC device; in this case, all the reagent reservoirs will be empty or configured not to release their contents, and the dialysis, lysis, incubation and amplification sections will be used solely to transport the sample from thesample inlet68 to thehybridization chambers180 for nucleic acid detection, as described above.
• For some sample types, a pre-processing step is required, for example semen may need to be liquefied and mucus may need to be pre-treated with an enzyme to reduce the viscosity prior to input into the LOC device.
Sample Input
• Referring toFIGS. 1 and 12, the sample is added to themacroreceptacle24 of thetest module10. Themacroreceptacle24 is a truncated cone which feeds into theinlet68 of theLOC device301 by capillary action. Here it flows into the 64 μm wide×60 μmdeep cap channel94 where it is drawn towards theanticoagulant reservoir54, also by capillary action.
Reagent Reservoirs
• The small volumes of reagents required by the assay systems using microfluidic devices, such asLOC device301, permit the reagent reservoirs to contain all reagents necessary for the biochemical processing with each of the reagent reservoirs having a small volume. This volume is easily less than 1,000,000,000 cubic microns, in the vast majority of cases less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns and in the case of theLOC device301 shown in the drawings, less than 20,000,000 cubic microns.
Dialysis Section
• Referring toFIGS. 15 to 21,33 and34, thepathogen dialysis section70 is designed to concentrate pathogenic target cells from the sample. As previously described, a plurality of apertures in the form of 3 micron diameter holes164 in theroof layer66 filter the target cells from the bulk of the sample. As the sample flows past the 3micron diameter apertures164, microbial pathogens pass through the holes into a series of dialysis MSTchannels204 and flow back up into thetarget channel74 via 16 μm dialysis uptake holes168 (seeFIGS. 33 and 34). The remainder of the sample (erythrocytes and so on) stay in thecap channel94. Downstream of thepathogen dialysis section70, thecap channel94 becomes thewaste channel72 leading to thewaste reservoir76. For biological samples of the type that generate a substantial amount of waste, a foam insert or otherporous element49 within theouter casing13 of thetest module10 is configured to be in fluid communication with the waste reservoir76 (seeFIG. 1).
• Thepathogen dialysis section70 functions entirely on capillary action of the fluid sample. The 3micron diameter apertures164 at the upstream end of thepathogen dialysis section70 have capillary initiation features (CIFs)166 (seeFIG. 33) so that the fluid is drawn down into thedialysis MST channel204 beneath. Thefirst uptake hole198 for thetarget channel74 also has a CIF202 (seeFIG. 15) to avoid the flow simply pinning a meniscus across the dialysis uptake holes168.
• The smallconstituents dialysis section682 schematically shown inFIG. 81 can have a similar structure to thepathogen dialysis section70. The small constituents dialysis section separates any small target cells or molecules from a sample by sizing (and, if necessary, shaping) apertures suitable for allowing the small target cells or molecules to pass into the target channel and continue for further analysis. Larger sized cells or molecules are removed to awaste reservoir766. Thus, the LOC device30 (seeFIGS. 1 and 104) is not limited to separating pathogens that are less than 3 μm in size, but can be used to separate cells or molecules of any size desired.
Lysis Section
• Referring back toFIGS. 7,11 and13, the genetic material in the sample is released from the cells by a chemical lysis process. As described above, a lysis reagent from thelysis reservoir56 mixes with the sample flow in thetarget channel74 downstream of thesurface tension valve128 for thelysis reservoir56. However, some diagnostic assays are better suited to a thermal lysis process, or even a combination of chemical and thermal lysis of the target cells. TheLOC device301 accommodates this with theheated microchannels210 of theincubation section114. The sample flow fills theincubation section114 and stops at the boiling-initiatedvalve106. The incubation microchannels210 heat the sample to a temperature at which the cellular membranes are disrupted.
• In some thermal lysis applications, an enzymatic reaction in thechemical lysis section130 is not necessary and the thermal lysis completely replaces the enzymatic reaction in thechemical lysis section130.
Boiling-Initiated Valve
• As discussed above, theLOC device301 has three boiling-initiatedvalves126,106 and108. The location of these valves is shown inFIG. 6.FIG. 31 is an enlarged plan view of the boiling-initiatedvalve108 in isolation at the end of theheated microchannels158 of theamplification section112.
• Thesample flow119 is drawn along theheated microchannels158 by capillary action until it reaches the boiling-initiatedvalve108. The leadingmeniscus120 of the sample flow pins at ameniscus anchor98 at thevalve inlet146. The geometry of themeniscus anchor98 stops the advancing meniscus to arrest the capillary flow. As shown inFIGS. 31 and 32, themeniscus anchor98 is an aperture provided by an uptake opening from theMST channel90 to thecap channel94. Surface tension in themeniscus120 keeps the valve closed. Anannular heater152 is at the periphery of thevalve inlet146. Theannular heater152 is CMOS-controlled via the boiling-initiatedvalve heater contacts153.
• To open the valve, theCMOS circuitry86 sends an electrical pulse to thevalve heater contacts153. Theannular heater152 resistively heats until theliquid sample119 boils. The boiling unpins themeniscus120 from thevalve inlet146 and initiates wetting of thecap channel94. Once wetting thecap channel94 begins, capillary flow resumes. Thefluid sample119 fills thecap channel94 and flows through the valve downtake150 to thevalve outlet148 where capillary driven flow continues along the amplificationsection exit channel160 into the hybridization anddetection section52.Liquid sensors174 are placed before and after the valve for diagnostics.
• It will be appreciated that once the boiling-initiated valves are opened, they cannot be re-closed. However, as theLOC device301 and thetest module10 are single-use devices, re-closing the valves is unnecessary.
Incubation Section and Nucleic Acid Amplification Section
• FIGS. 6,7,13,14,23,24,25,35 to45,50 and51 show theincubation section114 and theamplification section112. Theincubation section114 has a single,heated incubation microchannel210 etched in a serpentine pattern in theMST channel layer100 from the downtake opening134 to the boiling-initiated valve106 (seeFIGS. 13 and 14). Control over the temperature of theincubation section114 enables enzymatic reactions to take place with greater efficiency. Similarly, theamplification section112 has aheated amplification microchannel158 in a serpentine configuration leading from the boiling-initiatedvalve106 to the boiling-initiated valve108 (seeFIGS. 6 and 14). These valves arrest the flow to retain the target cells in the heated incubation oramplification microchannels210 or158 while mixing, incubation and nucleic acid amplification takes place. The serpentine pattern of the microchannels also facilitates (to some extent) mixing of the target cells with reagents.
• In theincubation section114 and theamplification section112, the sample cells and the reagents are heated by theheaters154 controlled by theCMOS circuitry86 using pulse width modulation (PWM). Each meander of the serpentine configuration of theheated incubation microchannel210 andamplification microchannel158 has three separatelyoperable heaters154 extending between their respective heater contacts156 (seeFIG. 14) which provides for the two-dimensional control of input heat flux density. As best shown inFIG. 51, theheaters154 are supported on theroof layer66 and embedded in thelower seal64. The heater material is TiAl but many other conductive metals would be suitable. Theelongate heaters154 are parallel with the longitudinal extent of each channel section that forms the wide meanders of the serpentine shape. In theamplification section112, each of the wide meanders can operate as separate PCR chambers via individual heater control.
• The small volumes of amplicon required by the assay systems using microfluidic devices, such asLOC device301, permit low amplification mixture volumes for amplification inamplification section112. This volume is easily less than 400 nanoliters, in the vast majority of cases less than 170 nanoliters, typically less than 70 nanoliters and in the case of theLOC device301, between 2 nanoliters and 30 nanoliters.
Increased Rates of Heating and Greater Diffusive Mixing
• The small cross section of each channel section increases the heating rate of the amplification fluid mix. All the fluid is kept a relatively short distance from theheater154. Reducing the channel cross section (that is theamplification microchannel158 cross section) to less than 100,000 square microns achieves appreciably higher heating rates than that provided by more ‘macro-scale’ equipment. Lithographic fabrication techniques allow theamplification microchannel158 to have a cross sectional area transverse to the flow-path less than 16,000 square microns which gives substantially higher heating rates. Feature sizes on the order of 1 micron are readily achievable with lithographic techniques. If very little amplicon is needed (as is the case in the LOC device301), the cross sectional area can be reduced to less than 2,500 square microns. For diagnostic assays with 1,000 to 2,000 probes on the LOC device, and a requirement of ‘sample-in, answer out’ in less than 1 minute, a cross sectional area transverse to the flow of between 400 square microns and 1 square micron is adequate.
• The heater element in theamplification microchannel158 heats the nucleic acid sequences at a rate more than 80 Kelvin (K) per second, in the vast majority of cases at a rate greater than 100 K per second. Typically, the heater element heats the nucleic acid sequences at a rate more than 1,000 K per second and in many cases, the heater element heats the nucleic acid sequences at a rate more than 10,000 K per second. Commonly, based on the demands of the assay system, the heater element heats the nucleic acid sequences at a rate more than 100,000 K per second, more than 1,000,000 K per second more than 10,000,000 K per second, more than 20,000,000 K per second, more than 40,000,000 K per second, more than 80,000,000 K per second and more than 160,000,000 K per second.
• A small cross-sectional area channel is also beneficial for diffusive mixing of any reagents with the sample fluid. Before diffusive mixing is complete, diffusion of one liquid into the other is greatest near the interface between the two. Concentration decreases with distance from the interface. Using microchannels with relatively small cross sections transverse to the flow direction, keeps both fluid flows close to the interface for more rapid diffusive mixing. Reducing the channel cross section to less than 100,000 square microns achieves appreciably higher mixing rates than that provided by more ‘macro-scale’ equipment. Lithographic fabrication techniques allows microchannels with a cross sectional area transverse to the flow-path less than 16000 square microns which gives significantly higher mixing rates. If small volumes are needed (as is the case in the LOC device301), the cross sectional area can be reduced to less than 2500 square microns. For diagnostic assays with 1000 to 2000 probes on the LOC device, and a requirement of ‘sample-in, answer out’ in less than 1 minute, a cross sectional area transverse to the flow of between 400 square microns and 1 square micron is adequate.
Short Thermal Cycle Times
• Keeping the sample mixture proximate to the heaters, and using very small fluid volumes allows rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e. denaturing, annealing and primer extension) is completed in less than 30 seconds for target sequences up to 150 base pairs (bp) long. In the vast majority of diagnostic assays, the individual thermal cycle times are less than 11 seconds, and a large proportion are less than 4 seconds.LOC devices30 with some of the most common diagnostic assays have thermal cycles time between 0.45 seconds to 1.5 seconds for target sequences up to 150 bp long. Thermal cycling at this rate allows the test module to complete the nucleic acid amplification process in much less than 10 minutes; often less than 220 seconds. For most assays, the amplification section generates sufficient amplicon in less than 80 seconds from the sample fluid entering the sample inlet. For a great many assays, sufficient amplicon is generated in 30 seconds.
• Upon completion of a preset number of amplification cycles, the amplicon is fed into the hybridization anddetection section52 via the boiling-initiatedvalve108.
Hybridization Chambers
• FIGS. 52,53,54,56 and57 show thehybridization chambers180 in thehybridization chamber array110. The hybridization anddetection section52 has a 24×45array110 ofhybridization chambers180, each with hybridization-responsive FRET probes186,heater element182 and anintegrated photodiode184. Thephotodiode184 is incorporated for detection of fluorescence resulting from the hybridization of a target nucleic acid sequence or protein with the FRET probes186. Eachphotodiode184 is independently controlled by theCMOS circuitry86. Any material between the FRET probes186 and thephotodiode184 must be transparent to the emitted light. Accordingly, thewall section97 between theprobes186 and thephotodiode184 is also optically transparent to the emitted light. In theLOC device301, thewall section97 is a thin (approximately 0.5 micron) layer of silicon dioxide.
• Incorporation of aphotodiode184 directly beneath eachhybridization chamber180 allows the volume of probe-target hybrids to be very small while still generating a detectable fluorescence signal (seeFIG. 54). The small amounts permit small volume hybridization chambers. A detectable amount of probe-target hybrid requires a quantity of probe, prior to hybridization, which is easily less than 270 picograms (corresponding to 900,000 cubic microns), in the vast majority of cases less than 60 picograms (corresponding to 200,000 cubic microns), typically less than 12 picograms (corresponding to 40,000 cubic microns) and in the case of theLOC device301 shown in the accompanying figures, less than 2.7 picograms (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chambers allows a higher density of chambers and therefore more probes on the LOC device. InLOC device301, the hybridization section has more than 1,000 chambers in an area of 1,500 microns by 1,500 microns (i.e. less than 2,250 square microns per chamber). Smaller volumes also reduce the reaction times so that hybridization and detection is faster. An additional advantage of the small amount of probe required in each chamber is that only very small quantities of probe solution need to be spotted into each chamber during production of the LOC device. Embodiments of the LOC device according to the invention can be spotted using a probe solution volume of 1 picoliter or less.
• After nucleic acid amplification, boiling-initiatedvalve108 is activated and the amplicon flows along the flow-path176 and into each of the hybridization chambers180 (seeFIGS. 52 and 56). An end-point liquid sensor178 indicates when thehybridization chambers180 are filled with amplicon and theheaters182 can be activated.
• After sufficient hybridization time, the LED26 (seeFIG. 2) is activated. The opening in each of thehybridization chambers180 provides anoptical window136 for exposing the FRET probes186 to the excitation radiation (seeFIGS. 52,54 and56). TheLED26 is illuminated for a sufficiently long time in order to induce a fluorescence signal from the probes with high intensity. During excitation, thephotodiode184 is shorted. After a pre-programmed delay300 (seeFIG. 2), thephotodiode184 is enabled and fluorescence emission is detected in the absence of the excitation light. The incident light on theactive area185 of the photodiode184 (seeFIG. 54) is converted into a photocurrent which can then be measured usingCMOS circuitry86.
• Thehybridization chambers180 are each loaded with probes for detecting a single target nucleic acid sequence. Eachhybridization chambers180 can be loaded with probes to detect over 1,000 different targets if desired. Alternatively, many or all the hybridization chambers can be loaded with the same probes to detect the same target nucleic acid repeatedly. Replicating the probes in this way throughout thehybridization chamber array110 leads to increased confidence in the results obtained and the results can be combined by the photodiodes adjacent those hybridization chambers to provide a single result if desired. The person skilled in the art will recognise that it is possible to have from one to over 1,000 different probes on thehybridization chamber array110, depending on the assay specification.
Humidifier and Humidity Sensor
• Inset AG ofFIG. 6 indicates the position of thehumidifier196. The humidifier prevents evaporation of the reagents and probes during operation of theLOC device301. As best shown in the enlarged view ofFIG. 55, awater reservoir188 is fluidically connected to threeevaporators190. Thewater reservoir188 is filled with molecular biology-grade water and sealed during manufacturing. As best shown inFIGS. 55 and 74, water is drawn into threedowntakes194 and along respectivewater supply channels192 by capillary action to a set of threeuptakes193 at theevaporators190. A meniscus pins at eachuptake193 to retain the water. The evaporators have annular shaped heaters191 which encircle theuptakes193. The annular heaters191 are connected to theCMOS circuitry86 by theconductive columns376 to the top metal layer195 (seeFIG. 37). Upon activation, the annular heaters191 heat the water causing evaporation and humidifying the device surrounds.
• The position of thehumidity sensor232 is also shown inFIG. 6. However, as best shown in the enlarged view of Inset AH inFIG. 67, the humidity sensor has a capacitive comb structure. A lithographically etchedfirst electrode296 and a lithographically etchedsecond electrode298 face each other such that their teeth are interleaved. The opposed electrodes form a capacitor with a capacitance that can be monitored by theCMOS circuitry86. As the humidity increases, the permittivity of the air gap between the electrodes increases, so that the capacitance also increases. Thehumidity sensor232 is adjacent thehybridization chamber array110 where humidity measurement is most important to slow evaporation from the solution containing the exposed probes.
Feedback Sensors
• Temperature and liquid sensors are incorporated throughout theLOC device301 to provide feedback and diagnostics during device operation. Referring toFIG. 35, ninetemperature sensors170 are distributed throughout theamplification section112. Likewise, theincubation section114 also has ninetemperature sensors170. These sensors each use a 2×2 array of bipolar junction transistors (BJTs) to monitor the fluid temperature and provide feedback to theCMOS circuitry86. TheCMOS circuitry86 uses this to precisely control the thermal cycling during the nucleic acid amplification process and any heating during thermal lysis and incubation.
• In thehybridization chambers180, theCMOS circuitry86 uses thehybridization heaters182 as temperature sensors (seeFIG. 56). The electrical resistance of thehybridization heaters182 is temperature dependent and theCMOS circuitry86 uses this to derive a temperature reading for each of thehybridization chambers180.
• TheLOC device301 also has a number of MSTchannel liquid sensors174 and capchannel liquid sensors208.FIG. 35 shows a line of MSTchannel liquid sensors174 at one end of every other meander in theheated microchannel158. As best shown inFIG. 37, the MSTchannel liquid sensors174 are a pair of electrodes formed by exposed areas of thetop metal layer195 in theCMOS structure86. Liquid closes the circuit between the electrodes to indicate its presence at the sensor's location.
• FIG. 25 shows an enlarged perspective of capchannel liquid sensors208. Opposing pairs ofTiAl electrodes218 and220 are deposited on theroof layer66. Between theelectrodes218 and220 is agap222 to hold the circuit open in the absence of liquid. The presence of liquid closes the circuit and theCMOS circuitry86 uses this feedback to monitor the flow.
Gravitational Independence
• Thetest modules10 are orientation independent. They do not need to be secured to a flat stable surface in order to operate. Capillary driven fluid flows and a lack of external plumbing into ancillary equipment allow the modules to be truly portable and simply plugged into a similarly portable hand held reader such as a mobile telephone. Having a gravitationally independent operation means the test modules are also accelerationally independent to all practical extents. They are resistant to shock and vibration and will operate on moving vehicles or while the mobile telephone is being carried around.
Nucleic Acid Amplification VariantsDirect PCR
• Traditionally, PCR requires extensive purification of the target DNA prior to preparation of the reaction mixture. However, with appropriate changes to the chemistry and sample concentration, it is possible to perform nucleic acid amplification with minimal DNA purification, or direct amplification. When the nucleic acid amplification process is PCR, this approach is called direct PCR. In LOC devices where nucleic acid amplification is performed at a controlled, constant temperature, the approach is direct isothermal amplification. Direct nucleic acid amplification techniques have considerable advantages for use in LOC devices, particularly relating to simplification of the required fluidic design. Adjustments to the amplification chemistry for direct PCR or direct isothermal amplification include increased buffer strength, the use of polymerases which have high activity and processivity, and additives which chelate with potential polymerase inhibitors. Dilution of inhibitors present in the sample is also important.
• To take advantage of direct nucleic acid amplification techniques, the LOC device designs incorporate two additional features. The first feature is reagent reservoirs (forexample reservoir58 inFIG. 8) which are appropriately dimensioned to supply a sufficient quantity of amplification reaction mix, or diluent, so that the final concentrations of sample components which might interfere with amplification chemistry are low enough to permit successful nucleic acid amplification. The desired dilution of non-cellular sample components is in the range of 5× to 20×. Different LOC structures, for example thepathogen dialysis section70 inFIG. 4, are used when appropriate to ensure that the concentration of target nucleic acid sequences is maintained at a high enough level for amplification and detection. In this embodiment, further illustrated inFIG. 6, a dialysis section which effectively concentrates pathogens small enough to be passed into theamplification section292 is employed upstream of thesample extraction section290, and rejects larger cells to awaste receptacle76. In another embodiment, a dialysis section is used to selectively deplete proteins and salts in blood plasma while retaining cells of interest.
• The second LOC structural feature which supports direct nucleic acid amplification is design of channel aspect ratios to adjust the mixing ratio between the sample and the amplification mix components. For example, to ensure dilution of inhibitors associated with the sample in the preferred 5×-20× range through a single mixing step, the length and cross-section of the sample and reagent channels are designed such that the sample channel, upstream of the location where mixing is initiated, constitutes aflow impedance 4×-19× higher than the flow impedance of the channels through which the reagent mixture flows. Control over flow impedances in microchannels is readily achieved through control over the design geometry. The flow impedance of a microchannel increases linearly with the channel length, for a constant cross-section. Importantly for mixing designs, flow impedance in microchannels depends more strongly on the smallest cross-sectional dimension. For example, the flow impedance of a microchannel with rectangular cross-section is inversely proportional to the cube of the smallest perpendicular dimension, when the aspect ratio is far from unity.
Reverse-Transcriptase PCR (RT-PCR)
• Where the sample nucleic acid species being analysed or extracted is RNA, such as from RNA viruses or messenger RNA, it is first necessary to reverse transcribe the RNA into complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction can be performed in the same chamber as the PCR (one-step RT-PCR) or it can be performed as a separate, initial reaction (two-step RT-PCR). In the LOC variants described herein, a one-step RT-PCR can be performed simply by adding the reverse transcriptase toreagent reservoir62 along with the polymerase and programming theheaters154 to cycle firstly for the reverse transcription step and then progress onto the nucleic acid amplification step. A two-step RT-PCR could also be easily achieved by utilizing thereagent reservoir58 to store and dispense the buffers, primers, dNTPs and reverse transcriptase and theincubation section114 for the reverse transcription step followed by amplification in the normal way in theamplification section112.
Isothermal Nucleic Acid Amplification
• For some applications, isothermal nucleic acid amplification is the preferred method of nucleic acid amplification, thus avoiding the need to repetitively cycle the reaction components through various temperature cycles but instead maintaining the amplification section at a constant temperature, typically around 37° C. to 41° C. A number of isothermal nucleic acid amplification methods have been described, including Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), Nucleic Acid Sequence Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), Helicase-Dependent isothermal DNA Amplification (HDA), Rolling Circle Amplification (RCA), Ramification Amplification (RAM) and Loop-mediated Isothermal Amplification (LAMP), and any of these, or other isothermal amplification methods, can be employed in particular embodiments of the LOC device described herein.
• In order to perform isothermal nucleic acid amplification, thereagent reservoirs60 and62 adjoining the amplification section will be loaded with the appropriate reagents for the specified isothermal method instead of PCR amplification mix and polymerase. For example, for SDA,reagent reservoir60 contains amplification buffer, primers and dNTPs andreagent reservoir62 contains an appropriate nickase enzyme and Exo-DNA polymerase. For RPA,reagent reservoir60 contains the amplification buffer, primers, dNTPs and recombinase proteins, withreagent reservoir62 containing a strand displacing DNA polymerase such as Bsu. Similarly, for HDA,reagent reservoir60 contains amplification buffer, primers and dNTPs andreagent reservoir62 contains an appropriate DNA polymerase and a helicase enzyme to unwind the double stranded DNA strand instead of using heat. The skilled person will appreciate that the necessary reagents can be split between the two reagent reservoirs in any manner appropriate for the nucleic acid amplification process.
• For amplification of viral nucleic acids from RNA viruses such as HIV or hepatitis C virus, NASBA or TMA is appropriate as it is unnecessary to first transcribe the RNA to cDNA. In this example,reagent reservoir60 is filled with amplification buffer, primers and dNTPs andreagent reservoir62 is filled with RNA polymerase, reverse transcriptase and, optionally, RNase H.
• For some forms of isothermal nucleic acid amplification it may be necessary to have an initial denaturation cycle to separate the double stranded DNA template, prior to maintaining the temperature for the isothermal nucleic acid amplification to proceed. This is readily achievable in all embodiments of the LOC device described herein, as the temperature of the mix in theamplification section112 can be carefully controlled by theheaters154 in the amplification microchannels158 (seeFIG. 14).
• Isothermal nucleic acid amplification is more tolerant of potential inhibitors in the sample and, as such, is generally suitable for use where direct nucleic acid amplification from the sample is desired. Therefore, isothermal nucleic acid amplification is sometimes useful inLOC variant XLIII673,LOC variant XLIV674 andLOC variant XLVII677, amongst others, shown inFIGS. 82,83 and84, respectively. Direct isothermal amplification may also be combined with one or more pre-amplification dialysis steps70,686 or682 as shown inFIGS. 82 and 84 and/or apre-hybridization dialysis step682 as indicated inFIG. 83 to help partially concentrate the target cells in the sample before nucleic acid amplification or remove unwanted cellular debris prior to the sample entering thehybridization chamber array110, respectively. The person skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybridization dialysis can be used.
• Isothermal nucleic acid amplification can also be performed in parallel amplification sections such as those schematically represented inFIGS. 78,79 and80, multiplexed and some methods of isothermal nucleic acid amplification, such as LAMP, are compatible with an initial reverse transcription step to amplify RNA.
Additional Details on the Fluorescence Detection System
• FIGS. 58 and 59 show the hybridization-responsive FRET probes236. These are often referred to as molecular beacons and are stem-and-loop probes, generated from a single strand of nucleic acid, that fluoresce upon hybridization to complementary nucleic acids.FIG. 58 shows asingle FRET probe236 prior to hybridization with a targetnucleic acid sequence238. The probe has aloop240,stem242, afluorophore246 at the 5′ end, and aquencher248 at the 3′ end. Theloop240 consists of a sequence complementary to the targetnucleic acid sequence238. Complementary sequences on either side of the probe sequence anneal together to form thestem242.
• In the absence of a complementary target sequence, the probe remains closed as shown inFIG. 58. Thestem242 keeps the fluorophore-quencher pair in close proximity to each other, such that significant resonant energy transfer can occur between them, substantially eliminating the ability of the fluorophore to fluoresce when illuminated with theexcitation light244.
• FIG. 59 shows theFRET probe236 in an open or hybridized configuration. Upon hybridization to a complementary targetnucleic acid sequence238, the stem-and-loop structure is disrupted, the fluorophore and quencher are spatially separated, thus restoring the ability of thefluorophore246 to fluoresce. Thefluorescence emission250 is optically detected as an indication that the probe has hybridized.
• The probes hybridize with very high specificity with complementary targets, since the stem helix of the probe is designed to be more stable than a probe-target helix with a single nucleotide that is not complementary. Since double-stranded DNA is relatively rigid, it is sterically impossible for the probe-target helix and the stem helix to coexist.
Primer-Linked Probes
• Primer-linked, stem-and-loop probes and primer-linked, linear probes, otherwise known as scorpion probes, are an alternative to molecular beacons and can be used for real-time and quantitative nucleic acid amplification in the LOC device. Real-time amplification could be performed directly in the hybridization chambers of the LOC device. The benefit of using primer-linked probes is that the probe element is physically linked to the primer, thus only requiring a single hybridization event to occur during the nucleic acid amplification rather than separate hybridizations of the primers and probes being required. This ensures that the reaction is effectively instantaneous and results in stronger signals, shorter reaction times and better discrimination than when using separate primers and probes. The probes (along with polymerase and the amplification mix) would be deposited into thehybridization chambers180 during fabrication and there would be no need for a separate amplification section on the LOC device. Alternatively, the amplification section is left unused or used for other reactions.
Primer-Linked Linear Probe
• FIGS. 85 and 86 show a primer-linkedlinear probe692 during the initial round of nucleic acid amplification and in its hybridized configuration during subsequent rounds of nucleic acid amplification, respectively. Referring toFIG. 85, the primer-linkedlinear probe692 has a double-strandedstem segment242. One of the strands incorporates the primer linkedprobe sequence696 which is homologous to a region on the targetnucleic acid696 and is labelled on its 5′ end withfluorophore246, and linked on its 3′ end to anoligonucleotide primer700 via anamplification blocker694. The other strand of thestem242 is labelled at its 3 end with aquencher moiety248. After an initial round of nucleic acid amplification has completed, the probe can loop around and hybridize to the extended strand with the, now complementary,sequence698. During the initial round of nucleic acid amplification, theoligonucleotide primer700 anneals to the target DNA238 (FIG. 85) and is then extended, forming a DNA strand containing both the probe sequence and the amplification product. Theamplification blocker694 prevents the polymerase from reading through and copying theprobe region696. Upon subsequent denaturation, theextended oligonucleotide primer700/template hybrid is dissociated and so is the double strandedstem242 of the primer-linked linear probe, thus releasing thequencher248. Once the temperature decreases for the annealing and extension steps, the primer linkedprobe sequence696 of the primer-linked linear probe curls around and hybridizes to the amplifiedcomplementary sequence698 on the extended strand and fluorescence is detected indicating the presence of the target DNA. Non-extended primer-linked linear probes retain their double-stranded stem and fluorescence remains quenched. This detection method is particularly well suited for fast detection systems as it relies on a single-molecule process.
Primer-Linked Stem-and-Loop Probes
• FIGS. 87A to 87F show the operation of a primer-linked stem-and-loop probe704. Referring toFIG. 87A, the primer-linked stem-and-loop probe704 has astem242 of complementary double-stranded DNA and aloop240 which incorporates the probe sequence. One of thestem strands708 is labelled at its 5′ end withfluorophore246. Theother strand710 is labelled with a 3′-end quencher248 and carries both theamplification blocker694 andoligonucleotide primer700. During the initial denaturation phase (seeFIG. 87B), the strands of the targetnucleic acid238 separate, as does the stem242 of the primer-linked, stem-and-loop probe704. When the temperature cools for the annealing phase (seeFIG. 87C), theoligonucleotide primer700 on the primer-linked stem-and-loop probe704 hybridizes to the targetnucleic acid sequence238. During extension (seeFIG. 87D) thecomplement706 to the targetnucleic acid sequence238 is synthesized forming a DNA strand containing both theprobe sequence704 and the amplified product. Theamplification blocker694 prevents the polymerase from reading through and copying theprobe region704. When the probe next anneals, following denaturation, the probe sequence of theloop segment240 of the primer-linked stem-and-loop probe (seeFIG. 87F) anneals to thecomplementary sequence706 on the extended strand. This configuration leaves thefluorophore246 relatively remote from thequencher248, resulting in a significant increase in fluorescence emission.
Control Probes
• Thehybridization chamber array110 includes somehybridization chambers180 with positive and negative control probes used for assay quality control.FIGS. 100 and 101 schematically illustrate negative control probes without afluorophore796, andFIGS. 102 and 103 are sketches of positive control probes without aquencher798. The positive and negative control probes have a stem-and-loop structure like the FRET probes described above. However, afluorescence signal250 will always be emitted frompositive control probes798 and nofluorescence signal250 is ever emitted fromnegative control probes796, regardless of whether the probes hybridize into an open configuration or remain closed.
• Referring toFIGS. 100 and 101, thenegative control probe796 has no fluorophore (and may or may not have a quencher248). Hence, whether the targetnucleic acid sequence238 hybridizes with the probe (seeFIG. 101), or the probe remains in its stem-and-loop configuration (seeFIG. 100), the response to theexcitation light244 is negligible. Alternatively, thenegative control probe796 could be designed so that it always remains quenched. For example, by synthesizing theloop240 to have a probe sequence that will not hybridize to any nucleic acid sequence within the sample under investigation, thestem242 of the probe molecule will re-hybridize to itself and the fluorophore and quencher will remain in close proximity and no appreciable fluorescence signal will be emitted. This negative control signal would correspond to low level emissions fromhybridization chambers180 in which the probes has not hybridized but the quencher does not quench all emissions from the reporter.
• Conversely, thepositive control probe798 is constructed without a quencher as illustrated inFIGS. 102 and 103. Nothing quenches thefluorescence emission250 from thefluorophore246 in response to theexcitation light244 regardless of whether thepositive control probe798 hybridizes with the targetnucleic acid sequence238.
• FIG. 52 shows a possible distribution of the positive and negative control probes (378 and380 respectively) throughout thehybridization chamber array110. The control probes378 and380 are placed inhybridization chambers180 positioned in a line across thehybridization chamber array110. However, the arrangement of the control probes within the array is arbitrary (as is the configuration of the hybridization chamber array110).
Fluorophore Design
• Fluorophores with long fluorescence lifetimes are required in order to allow enough time for the excitation light to decay to an intensity below that of the fluorescence emission at which time thephotosensor44 is enabled, thereby providing a sufficient signal to noise ratio. Also, longer fluorescence lifetime translates into larger integrated fluorescence photon count.
• The fluorophores246 (seeFIG. 59) have a fluorescence lifetime greater than 100 nanoseconds, often greater than 200 nanoseconds, more commonly greater than 300 nanoseconds and in most cases greater than 400 nanoseconds.
• The metal-ligand complexes based on the transition metals or lanthanides have long lifetimes (from hundreds of nanoseconds to milliseconds), adequate quantum yields, and high thermal, chemical and photochemical stability, which are all favourable properties with respect to the fluorescence detection system requirements.
• A particularly well-studied metal-ligand complex based on the transition metal ion Ruthenium (Ru (II)) is tris(2,2′-bipyridine) ruthenium (II) ([Ru(bpy)₃]²⁺) which has a lifetime of approximately 1 μs. This complex is available commercially from Biosearch Technologies under the brand name Pulsar 650.

• TABLE 1
  Photophysical properties of Pulsar 650 (Ruthenium chelate)

  Parameter	Symbol	Value	Unit

  Absorption Wavelength	λabs	460	nm
  Emission Wavelength	λem	650	nm
  Extinction Coefficient	E	14800	M−1cm−1
  Fluorescence Lifetime	τf	1.0	μs
  Quantum Yield	H	1 (deoxygenated)	N/A

• Terbium chelate, a lanthanide metal-ligand complex has been successfully demonstrated as a fluorescent reporter in a FRET probe system, and also has a long lifetime of 1600 μs.

• TABLE 2
  Photophysical properties of terbium chelate

  Parameter	Symbol	Value	Unit
  Absorption Wavelength	λabs	330-350	nm
  Emission Wavelength	λem	548	nm
  Extinction Coefficient	E	13800	M−1cm−1
  		(λabsand ligand depen-
  		dent, can be up to
  		30000 @ λe = 340 nm)
  Fluorescence Lifetime	τf	1600	μs
  		(hybridized probe)
  Quantum Yield	H	1	N/A
  		(ligand dependent)

• The fluorescence detection system used by theLOC device301 does not utilize filters to remove unwanted background fluorescence. It is therefore advantageous if thequencher248 has no native emission in order to increase the signal-to-noise ratio. With no native emission, there is no contribution to background fluorescence from thequencher248. High quenching efficiency is also important so that fluorescence is prevented until a hybridization event occurs. The Black Hole Quenchers (BHQ), available from Biosearch Technologies, Inc. of Novato Calif., have no native emission and high quenching efficiency, and are suitable quenchers for the system. BHQ-1 has an absorption maximum at 534 nm, and a quenching range of 480-580 nm, making it a suitable quencher for the Tb-chelate fluorophore. BHQ-2 has an absorption maximum at 579 nm, and a quenching range of 560-670 nm, making it a suitable quencher for Pulsar 650.
• Iowa Black Quenchers (Iowa Black FQ and RQ), available from Integrated DNA Technologies of Coralville, Iowa, are suitable alternative quenchers with little or no background emission. Iowa Black FQ has a quenching range from 420-620 nm, with an absorption maximum at 531 nm and would therefore be a suitable quencher for the Tb-chelate fluorophore. Iowa Black RQ has an absorption maximum at 656 nm, and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 650.
• In the embodiments described here, thequencher248 is a functional moiety which is initially attached to the probe, but other embodiments are possible in which the quencher is a separate molecule free in solution.
Excitation Source
• In the fluorescence detection based embodiments described herein, a LED is chosen as the excitation source instead of a laser diode, high power lamp or laser due to the low power consumption, low cost and small size. Referring toFIG. 88, theLED26 is positioned directly above thehybridization chamber array110 on an external surface of theLOC device301. On the opposing side of thehybridization chamber array110, is the photosensor44, made up of an array of photodiodes184 (seeFIGS. 53,54 and68) for detection of fluorescence signals from each of the chambers.
• FIGS. 89,90 and91 schematically illustrate other embodiments for exposing the probes to excitation light. In theLOC device30 shown inFIG. 89, theexcitation light244 generated by theexcitation LED26 is directed onto thehybridization chamber array110 by thelens254. Theexcitation LED26 is pulsed and the fluorescence emissions are detected by thephotosensor44.
• In theLOC device30 shown inFIG. 90, theexcitation light244 generated by theexcitation LED26 is directed onto thehybridization chamber array110 by thelens254, a firstoptical prism712 and secondoptical prism714. Theexcitation LED26 is pulsed and the fluorescence emissions are detected by thephotosensor44.
• Similarly, theLOC device30 shown inFIG. 91, theexcitation light244 generated by theexcitation LED26 is directed onto thehybridization chamber array110 by thelens254, afirst minor716 andsecond minor718. Again, theexcitation LED26 is pulsed and the fluorescence emissions are detected by thephotosensor44.
• The excitation wavelength of theLED26 is dependent on the choice of fluorescent dye. The Philips LXK2-PR14-R00 is a suitable excitation source for the Pulsar 650 dye. The SET UVTOP335TO39BL LED is a suitable excitation source for the Tb-chelate label.

• TABLE 3
  Philips LXK2-PR14-R00 LED specifications

  	Parameter	Symbol	Value	Unit
  	Wavelength	λex	460	nm
  	Emission Frequency	νem	6.52(10)14	Hz
  	Output Power	pl	0.515 (min) @ 1 A	W
  	Radiation pattern		Lambertian profile	N/A

• TABLE 4
  SET UVTOP334TO39BL LED Specifications

  Parameter	Symbol	Value	Unit
  Wavelength	λe	340	nm
  Emission Frequency	νe	8.82(10)14	Hz
  Power	pl	0.000240 (min) @ 20 mA	W
  Pulse Forward Current	I	200	mA
  Radiation pattern		Lambertian	N/A

Ultra Violet Excitation Light
• Silicon absorbs little light in the UV spectrum. Accordingly, it is advantageous to use UV excitation light. A UV LED excitation source can be used but the broad spectrum of theLED26 reduces the effectiveness of this method. To address this, a filtered UV LED can be used. Optionally, a UV laser can be the excitation source unless the relatively high cost of the laser is impractical for the particular test module market.
LED Driver
• TheLED driver29 drives theLED26 at a constant current for the required duration. A lower power USB 2.0-certifiable device can draw at most 1 unit load (100 mA), with a minimum operating voltage of 4.4 V. A standard power conditioning circuit is used for this purpose.
Photodiode
• FIG. 54 shows thephotodiode184 integrated into theCMOS circuitry86 of theLOC device301. Thephotodiode184 is fabricated as part of theCMOS circuitry86 without additional masks or steps. This is one significant advantage of a CMOS photodiode over a CCD, an alternate sensing technology which could be integrated on the same chip using non-standard processing steps, or fabricated on an adjacent chip. On-chip detection is low cost and reduces the size of the assay system. The shorter optical path length reduces noise from the surrounding environment for efficient collection of the fluorescence signal and eliminates the need for a conventional optical assembly of lenses and filters.
• Quantum efficiency of thephotodiode184 is the fraction of photons impinging on itsactive area185 that are effectively converted to photo-electrons. For standard silicon processes, the quantum efficiency is in the range of 0.3 to 0.5 for visible light, depending on process parameters such as the amount and absorption properties of the cover layers.
• The detection threshold of thephotodiode184 determines the smallest intensity of the fluorescence signal that can be detected. The detection threshold also determines the size of thephotodiode184 and hence the number ofhybridization chambers180 in the hybridization and detection section52 (seeFIG. 52). The size and number of chambers are technical parameters that are limited by the dimensions of the LOC device (in the case of theLOC device301, the dimensions are 1760 μm×5824 μm) and the real estate available after other functional modules such as thepathogen dialysis section70 and amplification section(s)112 are incorporated.
• For standard silicon processes, thephotodiode184 detects a minimum of 5 photons. However, to ensure reliable detection, the minimum can be set to 10 photons. Therefore with the quantum efficiency range being 0.3 to 0.5 (as discussed above), the fluorescence emission from the probes should be a minimum of 17 photons but 30 photons would incorporate a suitable margin of error for reliable detection.
Calibration Chambers
• The non-uniformity of the electrical characteristic of thephotodiode184, autofluorescence, and residual excitation photon flux that has not yet completely decayed, introduce background noise and offset into the output signal. This background is removed from each output signal using one or more calibration signals. Calibration signals are generated by exposing one ormore calibration photodiodes184 in the array to respective calibration sources. A low calibration source is used for determining a negative result in which a target has not reacted with a probe. A high calibration source is indicative of a positive result from a probe-target complex. In the embodiment described here, the low calibration light source is provided bycalibration chambers382 in thehybridization chamber array110 which:
• do not contain any probes;
• contain probes that have no fluorescent reporter; or,
• contain probes with a reporter and quencher configured such that quenching is always expected to occur.
• The output signal fromsuch calibration chambers382 closely approximates the noise and offset in the output signal from all the hybridization chambers in the LOC device. Subtracting the calibration signal from the output signals generated by the other hybridization chambers substantially removes the background and leaves the signal generated by the fluorescence emission (if any). Signals arising from ambient light in the region of the chamber array are also subtracted.
• It will be appreciated that the negative control probes described above with reference toFIGS. 100 to 103 can be used in calibration chambers. However, as shown inFIGS. 94 and 95, which are enlarged views of insets DG and DH of LOC variant X728 shown inFIG. 93, another option is to fluidically isolate thecalibration chambers382 from the amplicon. The background noise and offset can be determined by leaving the fluidically isolated chambers empty, or containing reporterless probes, or indeed any of the ‘normal’ probes with both reporter and quencher as hybridization is precluded by fluidic isolation.
• Thecalibration chambers382 can provide a high calibration source to generate a high signal in the corresponding photodiodes. The high signal corresponds to all probes in a chamber having hybridized. Spotting probes with reporters and no quenchers, or just reporters will consistently provide a signal approximating that of a hybridization chamber in which a predominant number of the probes have hybridized. It will also be appreciated thatcalibration chambers382 can be used instead of control probes, or in addition to control probes.
• The number and arrangement of thecalibration chambers382 throughout the hybridization chamber array is arbitrary. However, the calibration is more accurate ifphotodiodes184 are calibrated by acalibration chamber382 that is relatively proximate. Referring toFIG. 56, thehybridization chamber array110 has onecalibration chamber382 for every eighthybridization chambers180. That is, acalibration chamber382 is positioned in the middle of every three by three square ofhybridization chambers180. In this configuration, thehybridization chambers180 are calibrated by acalibration chamber382 that is immediately adjacent.
• FIG. 99 shows adifferential imager circuit788 used to substract the signal from thephotodiode184 corresponding to thecalibration chamber382 as a result of excitation light, from the fluorescence signal from the surroundinghybridization chambers180. Thedifferential imager circuit788 samples the signal from thepixel790 and a “dummy”pixel792. In one embodiment, the “dummy”pixel792 is shielded from light, so its output signal provides a dark reference. Alternatively, the “dummy”pixel792 can be exposed to the excitation light along with the rest of the array. In the embodiment where the “dummy”pixel792 is open to light, signals arising from ambient light in the region of the chamber array are also subtracted. The signals from thepixel790 are small (i.e. close to dark signal), and without a reference to a dark level it is hard to differentiate between the background and a very small signal.
• During use, the “read_row”794 and “read_row_d”795 are activated andM4797 andMD4801 transistors are turned on.Switches807 and809 are closed such that the outputs from thepixel790 and “dummy”pixel792 are stored onpixel capacitor803 anddummy pixel capacitor805 respectively. After the pixel signals have been stored, switches807 and809 are deactivated. Then the “read_col”switch811 and dummy “read_col”switch813 are closed, and the switchedcapacitor amplifier815 at the output amplifies thedifferential signal817.
Suppression and Enablement of the Photodiode
• Thephotodiode184 needs to be suppressed during excitation by theLED26 and enabled during fluorescence.FIG. 69 is a circuit diagram for asingle photodiode184 andFIG. 70 is a timing diagram for the photodiode control signals. The circuit hasphotodiode184 and six
• MOS transistors, M_shunt394, M_tx396, M_reset398, M_sf400, M_read402 and M_bias404. At the beginning of the excitation cycle, t1, the transistors M_shunt394, and M_reset398 are turned on by pulling the M_shuntgate384 and the reset gate388 high. During this period, the excitation photons generate carriers in thephotodiode184. These carriers have to be removed, as the amount of generated carriers can be sufficient to saturate thephotodiode184. During this cycle, M_shunt394 directly removes the carriers generated inphotodiode184, while M_reset398 resets any carriers that have accumulated on node ‘NS’406 due to leakage in transistors or due to diffusion of excitation-produced carriers in the substrate. After excitation, a capture cycle commences at t4. During this cycle, the emitted response from the fluorophore is captured and integrated in the circuit on node ‘NS’406. This is achieved by pulling tx gate386 high, which turns on the transistor M_tx396 and transfers any accumulated carriers on thephotodiode184 to node ‘NS’406. The duration of the capture cycle can be as long as the fluorophore emits. The outputs from allphotodiodes184 in thehybridization chamber array110 are captured simultaneously.
• There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. This delay is due to the requirement to read eachphotodiode184 in the hybridization chamber array110 (seeFIG. 52) separately following the capture cycle. Thefirst photodiode184 to be read will have the shortest delay before the read cycle, while thelast photodiode184 will have the longest delay before the read cycle. During the read cycle, transistor M_read402 is turned on by pulling the read gate393 high. The ‘NS’ node406 voltage is buffered and read out using the source-follower transistor M_sf400.
• There are additional, optional methods of enabling or suppressing the photodiode as discussed below:
1. Suppression Methods
• FIGS. 96,97 and98 show threepossible configurations778,780,782 for the M_shunttransistor394. The M_shunttransistor394 has a very high off ratio at maximum |V_GS|=5 V which is enabled during excitation. As shown inFIG. 96, the M_shuntgate384 is configured to be on the edge of thephotodiode184. Optionally, as shown inFIG. 97, the M_shuntgate384 may be configured to surround thephotodiode184. A third option is to configure the M_shuntgate384 inside thephotodiode184, as shown inFIG. 98. Under this third option there would be less photodiodeactive area185.
• These threeconfigurations778,780 and782 reduce the average path length from all locations in thephotodiode184 to the M_shuntgate384. InFIG. 96, the M_shuntgate384 is on one side of thephotodiode184. This configuration is simplest to fabricate and impinges the least on the photodiodeactive area185. However, any carriers lingering on the remote side of thephotodiode184 would take longer to propagate through to the M_shuntgate384.
• InFIG. 97, the M_shuntgate384 surrounds thephotodiode184. This further reduces the average path length for carriers in thephotodiode184 to the M_shuntgate384. However, extending the M_shuntgate384 about the periphery of thephotodiode184 imposes a greater reduction of the photodiodeactive area185. Theconfiguration782 inFIG. 98 positions the M_shuntgate384 within theactive area185. This provides the shortest average path length to the M_shuntgate384 and hence the shortest transition time. However, the impingement on theactive area185 is greatest. It also poses a wider leakage path.
2. Enabling Methods
• a. A trigger photodiode drives the shunt transistor with a fixed delay.
  b. A trigger photodiode drives the shunt transistor with programmable delay.
  c. The shunt transistor is driven from the LED drive pulse with a fixed delay.
  d. The shunt transistor is driven as in 2c but with programmable delay.
• FIG. 75 is a schematic section view through ahybridization chamber180 showing aphotodiode184 andtrigger photodiode187 embedded in theCMOS circuitry86. A small area in the corner of thephotodiode184 is replaced with thetrigger photodiode187. Atrigger photodiode187 with a small area is sufficient as the intensity of the excitation light will be high in comparison with the fluorescence emission. Thetrigger photodiode187 is sensitive to theexcitation light244. Thetrigger photodiode187 registers that theexcitation light244 has extinguished and activates thephotodiode184 after a short time delay Δt300 (seeFIG. 2). This delay allows thefluorescence photodiode184 to detect the fluorescence emission from the FRET probes186 in the absence of theexcitation light244. This enables detection and improves the signal to noise ratio.
• Bothphotodiodes184 and triggerphotodiodes187 are located in theCMOS circuitry86 under eachhybridization chamber180. The array of photodiodes combines, along with appropriate electronics, to form the photosensor44 (seeFIG. 68). Thephotodiodes184 are pn-junction fabricated during CMOS structure manufacturing without additional masks or steps. During MST fabrication, the dielectric layer (not shown) above thephotodiodes184 is optionally thinned using the standard MST photolithography techniques to allow more fluorescent light to illuminate theactive area185 of thephotodiode184. Thephotodiode184 has a field of view such that the fluorescence signal from the probe-target hybrids within thehybridization chamber180 is incident on the sensor face. The fluorescent light is converted into a photocurrent which can then be measured usingCMOS circuitry86.
• Alternatively, one ormore hybridization chambers180 can be dedicated to atrigger photodiode187 only. These options can be used in these in combination with 2a and 2b above.
Delayed Detection of Fluorescence
• The following derivations elucidate the delayed detection of fluorescence using a long-lifetime fluorophore for the LED/fluorophore combinations described above. The fluorescence intensity is derived as a function of time after excitation by an ideal pulse of constant intensity I, between time t₁and t₂as shown inFIG. 60.
• Let [S1](t) equal the density of excited states at time t, then during and after excitation, the number of excited states per unit time per unit volume is described by the following differential equation:
• $\begin{array}{cc}\frac{\left[S1\right]}{t}\left(t\right)+\frac{\left[S1\right]\left(t\right)}{{\tau }_F}=\frac{I_e\varepsilon c}{{\mathrm{hv}}_e}& \left(1\right)\end{array}$
• where c is the molar concentration of fluorophores, ∈ is the molar extinction coefficient, ν_eis the excitation frequency, and h=6.62606896(10)⁻³⁴Js is the Planck constant.
  This differential equation has the general form:
• $\frac{y}{x}+p\left(x\right)y=q\left(x\right)$
• which has the solution:
• $\begin{array}{cc}y\left(x\right)=\frac{\int {}^{\int p\left(x\right)x}q\left(x\right)x+k}{{}^{\int p\left(x\right)x}}& \left(2\right)\end{array}$
• Using this now to solve equation (1),
• $\begin{array}{cc}\left[S1\right]\left(t\right)=\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}+k{}^{-t/{\tau }_f}& \left(3\right)\end{array}$
• Now at time t₁, [S1](t₁)=0, and from (3):
• $\begin{array}{cc}k=-\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}{}^{{\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00013.png,US20110312796A1-20111222-D00033.png"\; state="\{\{state\}\}">t</figure-callout>}}_1/{\tau }_f}& \left(4\right)\end{array}$
• Substituting (4) into (3):
• $\left[S1\right]\left(t\right)=\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}-\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}{}^{-\left(t-t_1\right)/{\tau }_f}$
• At time t₂:
• $\begin{array}{cc}\left[S1\right]\left(t_2\right)=\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}-\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}{}^{-\left(t_2-t_1\right)/{\tau }_f}& \left(5\right)\end{array}$
• For t≧t₂, the excited states decay exponentially and this is described by:
• 
  [S1](t)=[S1](t₂)e^{−(t−t2)/τf}  (6)
• Substituting (5) into (6):
• $\begin{array}{cc}\left[S1\right]\left(t\right)=\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}\left[1-{}^{-\left(t_2-t_1\right)/{\tau }_f}\right]{}^{-\left(t-t_2\right)/{\tau }_f}& \left(7\right)\end{array}$
• The fluorescence intensity is given by the following equation:
• $\begin{array}{cc}{I}_f\left(t\right)=-\frac{\left[S1\right]\left(t\right)}{x}{\mathrm{hv}}_f\eta l& \left(8\right)\end{array}$
• where ν_fis the fluorescence frequency, η is the quantum yield and l is the optical path length.
• Now from (7):
• $\begin{array}{cc}\frac{\left[S1\right]\left(t\right)}{t}=-\frac{I_e\varepsilon c}{{\mathrm{hv}}_e}\left[1-{}^{-\left(t_2-t_1\right)/{\tau }_f}\right]{}^{-\left(t-t_2\right)/{\tau }_f}& \left(9\right)\end{array}$
• Substituting (9) into (8):
• $\begin{array}{cc}{I}_f\left(t\right)=I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}\left[1-{}^{-\left(t_2-t_1\right)/{\tau }_f}\right]{}^{-\left(t_2-t_1\right)/{\tau }_f}\mathrm{For}\frac{t_2-{\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00013.png,US20110312796A1-20111222-D00033.png"\; state="\{\{state\}\}">t</figure-callout>}}_1}{{\tau }_f}\to \infty ,I_f\left(t\right)\to I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}{}^{-\left(t-t_2\right)/{\tau }_f}& \left(10\right)\end{array}$
• Therefore, we can write the following approximate equation which describes the fluorescence intensity decay after a sufficiently long excitation pulse (t₂−t₁>>τ_f:
• $\begin{array}{cc}{I}_f\left(t\right)=I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}{}^{-\left(t-t_2\right)/{\tau }_f}\mathrm{for}t\ge t_2& \left(11\right)\end{array}$
• In the previous section, we concluded that for t₂−t₁>>τ_f,
• $I_f\left(t\right)=I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}{}^{-\left(t-t_2\right)/{\tau }_f}\mathrm{for}t\ge {\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00012.png,US20110312796A1-20111222-D00013.png"\; state="\{\{state\}\}">t</figure-callout>}}_2.$
• From the above equation, we can derive the following:
• $\begin{array}{cc}{\stackrel{⃛}{n}}_f\left(t\right)={\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}{\mathrm{\eta }}^{-\left(t-t_2\right)/{\tau }_f}\mathrm{where}{\stackrel{⃛}{n}}_f\left(t\right)=\frac{I_f\left(t\right)}{{\mathrm{hv}}_f}& \left(12\right)\end{array}$
• is the number of fluorescent photons per unit time per unit area and
• ${\stackrel{⃛}{n}}_e=\frac{I_e}{{\mathrm{hv}}_e}$
• is the number of excitation photons per unit time per unit area.
• Consequently,
• $\begin{array}{cc}{\ddot{n}}_f\left(t\right)={\int }_{{\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00013.png,US20110312796A1-20111222-D00030.png"\; state="\{\{state\}\}">t</figure-callout>}}_3}^{\infty }{\stackrel{⃛}{n}}_f\left(t\right)t& \left(13\right)\end{array}$
• where {umlaut over (n)}_fis the number of fluorescent photons per unit area and t₃is the instant of time at which the photodiode is turned on. Substituting (12) into (13):
• $\begin{array}{cc}{\ddot{n}}_f={\int }_{{\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00013.png,US20110312796A1-20111222-D00030.png"\; state="\{\{state\}\}">t</figure-callout>}}_3}^{\infty }{\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}\eta {}^{-\left(t-t_2\right)/{\tau }_f}t& \left(14\right)\end{array}$
• Now, the number of fluorescent photons that reach the photodiode per unit time per unit area,

  

  (t), is given by the following:
• $\begin{array}{cc}{\stackrel{⃛}{n}}_s\left(t\right)={\stackrel{⃛}{n}}_f\left(t\right){\phi }_0& \left(15\right)\end{array}$
• where φ₀is the light gathering efficiency of the optical system.
• Substituting (12) into (15) we find
• $\begin{array}{cc}{\stackrel{⃛}{n}}_s\left(t\right)={\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0{\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}\eta {}^{-\left(t-t_2\right)/{\tau }_f}& \left(16\right)\end{array}$
• Similarly, the number of fluorescence photons that reach the photodiode per unit fluorescent area {umlaut over (n)}_s, will be as follows:
• ${\ddot{n}}_s={\int }_{{\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00013.png,US20110312796A1-20111222-D00030.png"\; state="\{\{state\}\}">t</figure-callout>}}_3}^{\infty }{\stackrel{⃛}{n}}_s\left(t\right)t$
• and substituting in (16) and integrating:
• $\begin{array}{cc}{\stackrel{⃛}{n}}_s={\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0{\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}\eta {\tau }_f{}^{-\left(t_3-t_2\right)/{\tau }_f}& \left(17\right)\end{array}$
• Therefore,
• 
  n_s=φ₀{dot over (n)}_e∈clητ_fe^−Δt/τf  (17)
• The optimal value of t₃is when the rate of electrons generated in thephotodiode184 due to fluorescence photons becomes equal to the rate of electrons generated in thephotodiode184 by the excitation photons, as the flux of the excitation photons decays much faster than that of the fluorescence photons.
• The rate of sensor output electrons per unit fluorescent area due to fluorescence is:
• ${\stackrel{⃛}{e}}_f\left(t\right)={\phi }_f{\stackrel{⃛}{n}}_s\left(t\right)$
• where φ_fis the quantum efficiency of the sensor at the fluorescence wavelength.
• Substituting in (17) we have:
• $\begin{array}{cc}{\stackrel{⃛}{e}}_f\left(t\right)={\phi }_f{\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0{\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}\eta {}^{-\left(t-t_2\right)/{\tau }_f}& \left(18\right)\end{array}$
• Similarly, the rate of sensor output electrons per unit fluorescent area due to the excitation photons is:
• $\begin{array}{cc}{\stackrel{⃛}{e}}_e\left(t\right)={\phi }_e{\stackrel{⃛}{n}}_e{}^{-\left(t-t_2\right)/{\tau }_e}& \left(19\right)\end{array}$
• where φ_eis the quantum efficiency of the sensor at the excitation wavelength, and τ_eis the time-constant corresponding to the “off” characteristics of the excitation LED. After time t₂, the LED's decaying photon flux would increase the intensity of the fluorescence signal and extend its decay time, but we are assuming that this has a negligible effect on I_f(t), thus we are taking a conservative approach.
• Now, as mentioned earlier, the optimal value of t₃is when:
• ${\stackrel{⃛}{e}}_f\left(t_3\right)={\stackrel{⃛}{e}}_e\left(t_3\right)$
• Therefore, from (18) and (19) we have:
• ${\phi }_f{\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0{\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}\eta {}^{-\left(t_3-t_2\right)/{\tau }_f}={\phi }_e{\stackrel{⃛}{n}}_e{}^{-\left(t_3-t_2\right)/{\tau }_e}$
• and rearranging we find:
• $\begin{array}{cc}{t}_3-{\mathrm{<figure-callout\; label="t"\; filenames="US20110312796A1-20111222-D00012.png,US20110312796A1-20111222-D00013.png"\; state="\{\{state\}\}">t</figure-callout>}}_2=\frac{\ln \left(\varepsilon \mathrm{cl}\eta \frac{{\phi }_f{\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0}{{\phi }_e}\right)}{\frac{1}{{\tau }_f}-\frac{1}{{\tau }_e}}& \left(20\right)\end{array}$
• From the previous two sections, we have the following two working equations:
• $\begin{array}{cc}{n}_s={\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0{\stackrel{.}{n}}_eF{\tau }_{f}{}^{-\Delta t/{\tau }_f}& \left(21\right)\\ \Delta t=\frac{\ln \left(F\frac{{\phi }_f{\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0}{{\phi }_e}\right)}{\frac{1}{{\tau }_f}-\frac{1}{{\tau }_e}}& \left(22\right)\end{array}$
• where F=∈clη and Δt=t₃−t₂. We also know that, in practice, t₂−t₁>>τ_f.
• The optimal time for fluorescence detection and the number of fluorescence photons detected using the Philips LXK2-PR14-R00 LED and Pulsar 650 dye are determined as follows. The optimum detection time is determined using equation (22):
• Recalling the concentration of amplicon, and assuming that all amplicons hybridize, then the concentration of fluorescent fluorophores is: c=2.89(10)⁻⁶mol/L
• The height of the chamber is the optical path length l=8(10)⁻⁶m.
• We have taken the fluorescence area to be equal to our photodiode area, yet our actual fluorescence area is substantially larger than our photodiode area; consequently we can approximately assume φ₀=0.5 for the light gathering efficiency of our optical system. From the photodiode characteristics,
• $\frac{{\phi }_f}{{\phi }_e}=10$
• is a very conservative value for the ratio of the photodiode quantum efficiency at the fluorescence wavelength to its quantum efficiency at the excitation wavelength.
• With a typical LED decay lifetime of τ_e=0.5 ns and using Pulsar 650 specifications, Δt can be determined:
• $F=\left[1.48{\left(10\right)}^6\right]\left[2.89{\left(10\right)}^{-6}\right]\left[8{\left(10\right)}^{-6}\right]\left(1\right)=3.42{\left(10\right)}^{-5}$$\Delta t=\frac{\ln \left(\left[3.42{\left(10\right)}^{-5}\right]\left(10\right)\left(0.5\right)\right)}{\frac{1}{1{\left(10\right)}^{-6}}-\frac{1}{0.5{\left(10\right)}^{-9}}}=4.34{\left(10\right)}^{-9}s$
• The number of photons detected is determined using equation (21). First, the number of excitation photons emitted per unit time {dot over (n)}_eis determined by examining the illumination geometry.
• The Philips LXK2-PR14-R00 LED has a Lambertian radiation pattern, therefore:
• $\begin{array}{cc}{\stackrel{⃛}{n}}_l={\stackrel{⃛}{n}}_{l0}\cos \left(\theta \right)& \left(23\right)\end{array}$
• where

  

  the number of photons emitted per unit time per unit solid angle at an angle of θ off the LED's forward axial direction, and

  

  is the valve of

  

  in the forward axial direction.
• The total number of photons emitted by the LED per unit time is:
• $\begin{array}{cc}{\stackrel{.}{n}}_l={\int }_{\Omega }^{}{\stackrel{⃛}{n}}_l\Omega ={\int }_{\Omega }^{}{\stackrel{⃛}{n}}_{l0}\cos \left(\theta \right)\Omega \mathrm{Now},\mathrm{\Delta \Omega }=2\pi \left[1-\cos \left(\theta +\mathrm{\Delta \theta }\right)\right]-2\pi \left[1-\cos \left(\theta \right)\right]\begin{array}{c}\mathrm{\Delta \Omega }=2\pi \left[\cos \left(\theta \right)-\cos \left(\theta +\mathrm{\Delta \theta }\right)\right]\\ =4\pi \sin \left(\theta \right)\cos \left(\frac{\mathrm{\Delta \theta }}{2}\right)\sin \left(\frac{\mathrm{\Delta \theta }}{2}\right)+4\pi \cos \left(\theta \right){\mathrm{<figure-callout\; label="sin"\; filenames="US20110312796A1-20111222-D00012.png,US20110312796A1-20111222-D00013.png"\; state="\{\{state\}\}">sin</figure-callout>}}^2\left(\frac{\mathrm{\Delta \theta }}{2}\right)\end{array}\Omega =2\pi \sin \left(\theta \right)\theta & \left(24\right)\end{array}$
• Substituting this into (24):
• ${\stackrel{.}{n}}_l={\int }_0^{\frac{\mathrm{<figure-callout\; label="\pi "\; filenames="US20110312796A1-20111222-D00012.png,US20110312796A1-20111222-D00013.png"\; state="\{\{state\}\}">\pi </figure-callout>}}{2}}2\pi {\stackrel{⃛}{n}}_{l0}\cos \left(\theta \right)\sin \left(\theta \right)\theta =\pi {\stackrel{⃛}{n}}_{l0}$
• Rearranging, we have:
• $\begin{array}{cc}{\stackrel{⃛}{n}}_{l0}=\frac{{\stackrel{.}{n}}_l}{\pi }& \left(26\right)\end{array}$
• The LED's output power is 0.515 W and ν_e=6.52(10)¹⁴Hz, therefore:
• $\begin{array}{cc}\begin{array}{c}\stackrel{.}{n}=\frac{p_l}{{\mathrm{hv}}_e}\\ =\frac{0.515}{\left[6.63{\left(10\right)}^{-34}\right]\left[6.52{\left(10\right)}^{14}\right]}\\ =1.19{\left(10\right)}^{18}\mathrm{photons}\text{/}s\end{array}& \left(27\right)\end{array}$
• Substituting this value into (26) we have:
• ${\stackrel{⃛}{n}}_{l0}=\frac{1.19{\left(10\right)}^{18}}{\pi }=3.79{\left(10\right)}^{17}\mathrm{photons}\text{/}s\text{/}\mathrm{sr}$
• Referring toFIG. 61, theoptical centre252 and thelens254 of theLED26 are schematically shown. The photodiodes are 16 μm×16 μm, and for the photodiode in the middle of the array, the solid angle (Ω) of the cone of light emitted from theLED26 to thephotodiode184 is approximately:
• $\begin{array}{c}\Omega =\mathrm{area}\mathrm{of}\mathrm{sensor}\text{/}<figure-callout\; label="\; r"\; filenames="US20110312796A1-20111222-D00012.png,US20110312796A1-20111222-D00013.png"\; state="\{\{state\}\}"></figure-callout>r^{}{}^2\\ =\frac{\left[16{\left(10\right)}^{-6}\right]\left[16{\left(10\right)}^{-6}\right]}{{\left[2.85{\left(10\right)}^{-3}\right]}^2}\\ =3.21{\left(10\right)}^{-5}\mathrm{sr}\end{array}$
• It will be appreciated that thecentral photodiode184 of thephotodiode array44 is used for the purpose of these calculations. A sensor located at the edge of the array would only receive 2% less photons upon a hybridization event for a Lambertian excitation source intensity distribution.
• The number of excitation photons emitted per unit time is:
• $\begin{array}{cc}{\stackrel{.}{n}}_e={\stackrel{⃛}{n}}_l\Omega =\left[3.79{\left(10\right)}^{17}\right]\left[3.21{\left(10\right)}^{-5}\right]=1.22{\left(10\right)}^{13}\mathrm{photons}\text{/}s& \left(28\right)\end{array}$
• Now referring to equation (29):
• $n_s={\mathrm{<figure-callout\; label="\phi "\; filenames="US20110312796A1-20111222-D00008.png,US20110312796A1-20111222-D00012.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0{\stackrel{.}{n}}_eF{\tau }_f{}^{-\Delta t/{\tau }_f}$$\begin{array}{c}{n}_s=\left(0.5\right)\left[1.22{\left(10\right)}^{13}\right]\left[3.42{\left(10\right)}^{-5}\right]\left[1/{\left(10\right)}^{-6}\right]{}^{-4.34{\left(10\right)}^{-9}/1{\left(10\right)}^{-6}}\\ =208\mathrm{photons}\mathrm{per}\mathrm{sensor}.\end{array}$
• Therefore, using the Philips LXK2-PR14-R00 LED and Pulsar 650 fluorophore, we can easily detect any hybridization events which results in this number of photons being emitted.
• The SET LED illumination geometry is shown inFIG. 62. At I_D=20 mA, the LED has a minimum optical power output of p₁=240 μW centred at λ_e=340 nm (the absorption wavelength of the terbium chelate). Driving the LED at I_D=200 mA would increase the output power linearly to p₁=2.4 mW. By placing the LED'soptical centre252, 17.5 mm away from thehybridization chamber array110, we would approximately concentrate this output flux in a circular spot size which has a maximum diameter of 2 mm.
• The photon flux in the 2 mm-diameter spot at the hybridization away plane is given byequation 27.
• $\begin{array}{c}{\stackrel{.}{n}}_l=\frac{p_l}{{\mathrm{hv}}_e}\\ =\frac{2.4{\left(10\right)}^{-3}}{\left[6.63{\left(10\right)}^{-34}\right]\left[8.82{\left(10\right)}^{14}\right]}\\ =4.10{\left(10\right)}^{15}\mathrm{photons}\text{/}s\end{array}$
• Usingequation 28, we have:
• $\begin{array}{c}{\stackrel{.}{n}}_e={\stackrel{⃛}{n}}_l\Omega \\ =4.10\left(10\right)15\frac{{\left[16{\left(10\right)}^{-6}\right]}^2}{{\pi \left[1{\left(10\right)}^{-3}\right]}^2}\\ =3.34{\left(10\right)}^{11}\mathrm{photons}\text{/}s\end{array}$
• Now, recallingequation 22 and using the Tb chelate properties listed previously,
• $\Delta t=\frac{\ln \left[\left(6.94{\left(10\right)}^{-5}\right)\left(10\right)\left(0.5\right)\right]}{\frac{1}{1{\left(10\right)}^{-3}}-\frac{1}{0.5{\left(10\right)}^{-9}}}=3.98{\left(10\right)}^{-9}s$
• Now from equation 21:
• $\begin{array}{c}{n}_s=\left(0.5\right)\left[3.34{\left(10\right)}^{11}\right]\left[6.94{\left(10\right)}^{-5}\right]\left[1{\left(10\right)}^{-3}\right]{}^{-3.98{\left(10\right)}^{-9}/1{\left(10\right)}^{-3}}\\ =11,600\mathrm{photons}\mathrm{per}\mathrm{sensor}.\end{array}$
• The theoretical number of photons emitted by hybridization events using the SET LED and terbium chelate system are easily detectable and well over the minimum of 30 photons required for reliable detection by the photosensor as indicated above.
Maximum Spacing Between Probes and Photodiode
• The on-chip detection of hybridization avoids the needs for detection via confocal microscopy (see Background of the Invention). This departure from traditional detection techniques is a significant factor in the time and cost savings associated with this system. Traditional detection requires imaging optics which necessarily uses lenses or curved mirrors. By adopting non-imaging optics, the diagnostic system avoids the need for a complex and bulky optical train. Positioning the photodiode very close to the probes has the advantage of extremely high collection efficiency: when the thickness of the material between the probes and the photodiode is of the order of 1 micron, the angle of collection of emission light is up to 173°. This angle is calculated by considering light emitted from a probe at the centroid of the face of the hybridization chamber closest to the photodiode, which has a planar active surface area parallel to that chamber face. The cone of emission angles within which light is able to be absorbed by the photodiode is defined as having the emitting probe at its vertex and the corner of the sensor on the perimeter of its planar face. For a 16 micron×16 micron sensor, the vertex angle of this cone is 170°; in the limiting case where the photodiode is expanded so that its area matches that of the 29 micron×19.75 micron hybridization chamber, the vertex angle is 173°. A separation between the chamber face and the photodiode active surface of 1 micron or less is readily achievable.
• Employing a non-imaging optics scheme does require thephotodiode184 to be very close to the hybridization chamber in order to collect sufficient photons of fluorescence emission. The maximum spacing between the photodiode and probes is determined as follows with reference toFIG. 54.
• Utilizing a terbium chelate fluorophore and a SET UVTOP335TO39BL LED, we calculated11600 photons reaching our 16 micron×16micron photodiode184 from therespective hybridization chamber180. In performing this calculation we assumed that the light-collecting region of ourhybridization chamber180 has a base area which is the same as our photodiodeactive area185, and half of the total number of the hybridization photons reaches thephotodiode184. That is, the light gathering efficiency of the optical system is φ₀=0.5.
• More accurately we can write φ₀=[(base area of the light-collecting region of the hybridization chamber)/(photodiode area)][Ω/4π], where Ω=solid angle subtended by the photodiode at a representative point on the base of the hybridization chamber. For a right square pyramid geometry:
• 
  Ω=4 arcsin(a²/(4d₀²+a²)),
• where d₀=distance between the chamber and the photodiode, and a is the photodiode dimension.
• Each hybridization chamber releases 23200 photons. The selected photodiode has a detection threshold of 17 photons; therefore, the minimum optical efficiency required is:
• 
  φ₀=17/23200=7.33×10⁻⁴
• The base area of the light-collecting region of thehybridization chamber180 is 29 micron×19.75 micron.
• Solving for d₀, we will get the maximum limiting distance between the bottom of our hybridization chamber and ourphotodiode184 to be d₀=249 microns. In this limit, the collection cone angle as defined above is only 0.8°. It should be noted this analysis ignores the negligible effect of refraction.
• Test Module with Microfluidic Device Having Dialysis Device, LOC and Interconnecting Cap
• Atest module11 for analysing a sample fluid containing target molecules is shown inFIG. 109. Thetest module11 comprises anouter casing13 with areceptacle24 for receiving the sample fluid, a removablesterile sealing tape22 to cover thereceptacle24 prior to use, amembrane seal408 with amembrane guard410 forming part of theouter casing13 to reduce dehumidification within the test module while providing pressure relief from small air pressure fluctuations with themembrane guard410 protecting themembrane seal408 from damage, a printed circuit board (PCB)57, amicrofluidic device783, aporous element49, a standardMicro-USB plug14 for power, data and control, externalpower supply capacitors32, andinductor15.
• Themicrofluidic device783 has adialysis device784 in fluid communication with thereceptacle24 and configured to separate the target molecules from other constituents of the sample, aLOC device785 for analysing the target molecules and acap51 overlaying theLOC device785 and thedialysis device784 for establishing fluid communication between theLOC device785 and thedialysis device784.
Reagent Loading and Probe Spotting System
• Reagent reservoirs54,56,58,60 and62 (seeFIG. 6) are filled with reagents and water from a robotic, droplet ejection system shown inFIGS. 63 to 66. The robotic system also spots the oligonucleotide FRET probes186 or ECL probes237 into thehybridization chambers180. Droplet dispensing technology is an inexpensive spotting technique, delivers small droplets with reproducible volumes and many droplets of different solutions can be dispensed simultaneously. This allows the LOC devices to be mass produced at extremely high throughput and low cost.
• The reagent and probe spotting system includes three robotic subsystems:
• 1. Reagent dispensing robot256 (see FIG.63)—microvials258 (seeFIG. 64), each with adroplet dispenser262, dispense reagents into thereservoirs54,56,58,60 and62 and water into the water reservoir188 (seeFIG. 6). It then applies the patterned upper seal82 (if necessary) to thecap46.
• 2. ONEC refill robot274 (see FIG.65)—microvials258 with adroplet dispenser262 dispense probes into thereservoirs278 of an oligonucleotide ejector chip (ONEC)272 (seeFIGS. 71 and 72). TheONEC reservoirs278 feed an array ofthermal droplet generators271. The ONEC is then used in the third robotic subsystem, the LOC spotting robot.
• 3. LOC spotting robot289 (schematically shown in FIG.66)—ONEC272 spots eachhybridization chamber180 of theLOC device30 with probes using a thermal droplet generator271 (seeFIG. 72).
Microvials
• Thereagent dispensing robot256 and theONEC refill robot274 both use microvials258 as shown schematically inFIG. 64. Probes and reagents are ordered directly from the suppliers in macrovials (not shown). Liquids are micropipetted from the macrovials into acontainer259 on each of themicrovials258 to form small aliquots (typically between 282 microliters and 400 microliters) that can be refrigerated along with the macrovials until required. Eachmicrovial258 has apiezoelectric droplet dispenser262 and an enclosed quality assurance chip (i.e. integrated circuit)266 with flash memory andelectrical contacts264 for power and data transmission. Thedroplet dispenser262 has a piezo-electric actuator261 configured to eject drops with a volume between 50 picoliters and 150 picoliters for reasonably quick reagent loading while maintaining accurate drop placement.
Probe and Reagent Identification Scheme
• The quality assurance chip266 (seeFIG. 64) has digital memory used to store, identify and track the specification data characterizing the reagent or oligonucleotide probe solution within themicrovial258. At the end of the spot and load process, the data from each microvial258, along with other loading and spotting data, is downloaded and stored in the program anddata flash memory40 of theLOC device30 via thecontrol microprocessor263 controlling the reagent dispensing robot or probe dispensing robot. This data is used for diagnostic information and processing tasks, quality control and auditing.
• Referring toFIG. 73,ONEC272 also has digital memory such asflash memory281 in theONEC CMOS structure285 to store oligonucleotide specification data such as probe identities, batch numbers and so on. As with the LOC device, theONEC refill robot274 downloads the specification data to theONEC flash memory281 from the quality assurance chips266 on themicrovials258.
• Automated information transfer minimizes the possibility of errors occurring and in the event an incorrect microvial is used, thetest module reader12 or other system component identifies this error when processing the diagnostic information.
Reagent Dispensing Robot
• A simplified top and side view of thereagent dispensing robot256 are shown inFIGS. 63 and 108. It includes:
• • microvials258 containing reagents and molecular biology grade water (only some of the microvials are shown)
  • mechanical/electrical rack286 (shown only in outline) which holds and provides electrical connectivity to microvials258
  • XY stage268 providing a surface for detachably mounting a partial-depthsawn silicon wafer260 or other fixed array such asseparable PCB wafer720
  • Registration camera270 providing feedback to thecontrol microprocessor263 for mapping the exact location of thepiezoelectric droplet dispensers262
• Thepiezoelectric droplet dispensers262 on themicrovials258 are used to dispense the reagents and water directly into theLOC device reservoirs54,56,58,60 and62 and thehumidifier water reservoir188 respectively.
ONEC Refill Robot
• TheONEC refill robot274 is shown inFIG. 65. It is similar to thereagent dispensing robot256 and includes:
• • 1080 microvials258 containing solutions of oligonucleotide probes (for the purposes of illustration, not all microvials are shown)
  • mechanical/electrical rack286 (shown only in outline)—holds and provides electrical connectivity to microvials258
  • oligonucleotide ejector chip (ONEC)272—with 1080ONEC reservoirs278 supplyingrespective ejectors287 with four ONECthermal droplet generators271 each (seeFIGS. 71 and 72)
  • XY stage268: holds the oligonucleotide ejector chip/s (ONEC/s)272
  • Registration camera270 providing feedback to thecontrol microprocessor263 for mapping the exact location of thethermal droplet generators271
• TheONEC272 is moved under the mechanical/electrical rack286. A unique probe solution is dispensed from each microvial258 into eachONEC reservoir278. TheONEC272 is then used in the probe spotting robot273 to spot the LOCdevice hybridization chambers180 with a single droplet of probe solution.
ONEC
• FIGS. 71,72 and73 show theONEC272 in detail. TheONEC272 is an oligonucleotide spotting device for contactless spotting of probes onto a surface such as the hybridization chamber array in any of the LOC devices. It has overall dimensions of 23,296 μm×1,760 μm and is fabricated using well-established high volume photolithography fabrication techniques. Each ONEC has 1080reservoirs278 etched into thereservoir side277 of a monolithic silicon substrate275 (seeFIG. 73). With more than 1000reservoirs278, each ONEC has the complete assay of probes needed to spot the LOC devices described herein. This allows the spotting process of each LOC to be one-step in the sense that there is no need to use more than one ONEC to spot LOCs configured for each particular analysis. TheONEC reservoirs278 have a rectangular base (96 μm×208 μm) with a depth of 200 μm. EachONEC reservoir278 feeds a probe suspension to arespective ejector287. The liquid suspension of probes fill acommon chamber282 via a pair of chamber inlets284 (seeFIG. 72). The chamber inlets284 are two 21 μm diameter holes from thereservoir278 to thecommon chamber282. One of fourthermal droplet generators271 ejects probe droplets throughnozzles283 in theejector side279 into thehybridization chambers180 by heating theactuator280 to generate a vapor bubble. Having fourthermal droplet generators271 allows for redundancy if there is a droplet generator failure.
LOC Probe Spotting Robot
• The LOCprobe spotting robot289 is shown inFIGS. 66 and 92. For clarity, components other than theLOC device30 on thePCB wafer720 are not shown. It includes the following:
• • ONEC272 —oligonucleotide ejector chip with 1080reservoirs278, each filled with a probe solution (seeFIGS. 71 and 72)
  • XY stage268: holds the partial-depth sawn silicon LOC wafer260 (seeFIG. 66) or alternatively the separable PCB wafer720 (seeFIG. 92)
  • Registration camera270 providing feedback to thecontrol processor263 for mapping the exact location of the ONECthermal droplet generators271
• TheLOC silicon wafer260 or theseparable PCB wafer720 is detachably mounted to a stage that can translate along two orthogonal axes. TheONEC272 is detachably held in achuck265 that is closely adjacent the stage with theejectors287 facing the stage (seeFIG. 66). TheLOC silicon wafer260 or theseparable PCB wafer720 is moved relative to theONEC272 by thecontrol processor263. Each LOCdevice hybridization chamber180 is spotted by the ejectors under the operative control of thecontrol processor263. Using volumes less than 100 picoliters reduces the reaction times and allows the density of the hybridization chamber array to increase. Spotting low-volume probe droplets has not been previously adopted because of the difficulty associated with ejecting very small droplets precisely and reliably. Misdirected drops can fail to spot the correct chamber and may contaminate an adjacent chamber.
• TheONEC272 can be driven to generate a range of droplet volumes. For accurate dispensing, the droplets generated by theONEC272 would be less than 100 picoliters. To improve the accuracy of the probes and reagents dispensed (in terms of volume and position on the LOC device), the droplets generated by the ONEC can be reduced to less than 25 picoliters, and preferably less than 6 picoliters. TheONEC272 dispenses probe solution into the 1080hybridization chambers180 in droplets with volumes between 0.1 picoliters and 1.6 picoliters and a high degree of positional accuracy.
• Thehybridization chamber array110 is configured as 24 rows with 45 adjacent chambers in each row (seeFIG. 52). The sample flow-path176 extends between every second row such that the overall array has a substantially square shape for approximately uniform illumination by theLED26. As thehybridization chamber array110 is confined to an area less than 1500 microns by 1500 microns, the spotting accuracy of theONEC272 is necessarily high. Aregistration camera270 is used by thecontrol processor263 to determine the exact position of the ONECthermal droplet generators271 and the droplet generator drive pulses are synchronized with theXY stage268 via the ONEC bond-pads276.
• The LOC probe spotting robot273 using theONEC272 andcamera270 can easily spot probes onto a surface (such as the hybridization chamber array110) at a rate greater than 100 probes per second; in the vast majority of cases at a rate greater than 1,400 probes per second. Typically, the array of droplet generators spot the probes onto the surface at a rate greater than 20,000 probes per second and in many cases, the array of droplet generators spot the probes onto the surface at a rate between 300,000 probes per second and 1,000,000 probes per second.
• The array of droplet generators lithographically fabricated on a silicon substrate allows theONEC272 to spot oligonucleotides onto a surface at a density far greater than existing probe spotters.ONEC272 easily spots at a density of more than 1 probe per square millimetre. In the vast majority of cases, the spotting density is greater than 8 probes per square millimetre. In most cases, the spotting density is more than 60 probes per square millimetre, and typically the density is between 500 probes per square millimetre and 1,500 probes per square millimetre.
• The LOC probe spotting robot273, using theONEC272 as a biochemical deposition device, can easily deposit biochemicals onto a surface at a rate greater than 100 droplets per second, in the vast majority of cases at a rate greater than 1,400 droplets per second. Typically, the array of droplet generators spot the droplets onto the surface at a rate greater than 20,000 droplets per second, and in many cases, the array of droplet generators spot the droplets onto the surface at a rate between 300,000 droplets per second and 1,000,000 droplets per second.
• The LOC probe spotting robot273, using theONEC272 as a biochemical deposition device, can easily deposit biochemicals onto a surface at a density of more than 1 droplet per square millimetre. In the vast majority of cases, the spotting density is greater than 8 droplets per square millimetre. In most cases, the spotting density is more than 60 droplets per square millimetre, and typically the density is between 500 droplets per square millimetre and 1,500 droplets per square millimetre.
CONCLUSION
• The devices, systems and methods described here facilitate molecular diagnostic tests at low cost with high speed and at the point-of-care.
• The system and its components described above are purely illustrative and the skilled worker in this field will readily recognize many variations and modifications which do not depart from the spirit and scope of the broad inventive concept.

Claims (20)

1. A test module for analyzing a sample fluid and communicating data to a medical database, the test module comprising:

a receptacle for receiving the sample;

functional sections for processing and analyzing the sample;

a communication interface for communication with the medical database; and,

a controller for operative control of the communication interface.

2. The test module ofclaim 1 further comprising a universal serial bus (USB) plug wherein the communication interface is a USB device driver for operative control of the USB plug to communicate with an external device.

3. The test module ofclaim 2 further comprising digital memory wherein the medical database stores electronic health records (EHR), electronic medical records (EMR) and personal health records (PHR) and, the digital memory is configured for storing data relating to EHR, EMR and PHR.

4. The test module ofclaim 3 further comprising a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory includes the reagent identities.

5. The test module ofclaim 4 wherein the data stored in the digital memory includes a unique identifier for test module.

6. The test module ofclaim 5 wherein the sample is a biological sample including cells of different sizes, and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample.

7. The test module ofclaim 6 further comprising CMOS circuitry and a temperature sensor wherein the CMOS circuitry incorporates the digital memory and uses the temperature sensor output for feedback control of the PCR section.

8. The test module ofclaim 7 wherein one of the functional sections is a dialysis section, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.

9. The test module ofclaim 8 wherein one of the functional sections is a lysis section, the lysis section being configured to release nucleic acid sequences within the cells smaller than the predetermined threshold.

10. The test module ofclaim 9 further comprising an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.

11. The test module ofclaim 10 wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to excitation, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.

12. The test module ofclaim 11 further comprising a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.

13. The test module ofclaim 12 wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.

14. The test module ofclaim 13 wherein the CMOS circuitry is configured to communicate hybridization data to an external device.

15. The test module ofclaim 14 wherein the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.

16. The test module ofclaim 15 wherein the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.

17. The test module ofclaim 16 wherein the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.

18. The test module ofclaim 5 further comprising a LOC device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the CMOS circuitry incorporates the digital memory, the communication interface, the controller, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.

19. The test module ofclaim 18 wherein the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.

20. The test module ofclaim 6 wherein the PCR section has a thermal cycle time of less than 4 seconds.

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