US20160066828A1 - Personal diagnostic device having an air sample collection unit and a fluidic circuit - Google Patents

Abstract

Personal diagnostic devices including diagnostic patches (bio-patches) and interactive medical bracelets (bio-bracelets) are provided with a skin/patch interface, at least one analysis layer, a signal processing layer, and a user output interface. Embodiments of the interactive diagnostic devices may include micro-fluidic circuits with reaction chambers, analysis chambers, mixing cambers, and various pre-disposed chemistries or reagents for performing a wide verity of tests by transdermal transport of blood or perspiration. Sample collection chambers for the fluidic circuit may include minimally invasive tubules that penetrate the skin surface to acquire blood samples from capillaries near the epidermis. Alternate implementations of the personal diagnostic device may be equipped with logic processing, input/output devices, acoustic microphones, cryogenic circuits, embedded processors, electrical control circuitry, and battery current sources or photovoltaic sources of electrical power.

Description

RELATED APPLICATIONS
• Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet, or any correction thereto, are hereby incorporated by reference into this application under 37 CFR 1.57.
STATEMENT REGARDING COPYRIGHTED MATERIAL
• Portions of the disclosure of this patent document contain material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office file or records, but otherwise reserves all U.S. and foreign rights whatsoever relating to the copyright material contained herein.
BACKGROUND OF THE INVENTION
• 1. Field of the Invention
• This invention relates in general to personal diagnostic devices such as dermal patches or diagnostic bracelets and, in particular, to interactive adhesive dermal patches and medical diagnostic bracelets. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to interactive adhesive dermal patches and personal diagnostic bracelets including a skin/patch interface, at least one analysis or processing layer, and a user output and/or input interface.
• 2. Description of the Related Art
• Prior hereto, typical dermal patches have been employed essentially as passive drug release devices for use on the human body. Examples of such devices include the nicotine patch used for cigarette smoking cessation and the sea-sickness patch employed to administer predetermined amounts of Dramamine, for example, to relieve the effects of sea-sickness.
• Medical monitoring devices for diagnostic purposes are numerous. Such well known devices include, for example, x-ray machines, ultra sound devices, computerized axial tomography scanners (CAT scanners) which produce a tomograph that constructs a 3-D model of an object by combining parallel planes, electrocardiograms (ECG/EKG) monitoring systems for the electrical recording of the heart and use in the investigation of heart disease, electroencephalogram (EEG) systems for brain mapping and neuro-feedback, and Positron Emission Tomography (PET) scanning for brain imaging and related analysis.
• More particularly, the arts related hereto have been contributed to by several artisans including, for example, Sarussi et al., Conn et al., and S. Berlin. As an illustration of such contributions, there is the device disclosed in U.S. Patent Application Publication No. 2003/0229276 in the name of Sarussi et al. This publication discloses a device for monitoring blood constituent levels having a microprocessing unit. In one embodiment, the device contains an alarm which is activated when a particular blood constituent falls below a predetermined level. In other embodiments, the device may include a display unit for displaying output signals or indicating the well being of the wearer. The Sarussi device may be contained within a housing which adheres to the surface of the skin. The device may also contain a light source such as a light emitting diode (LED).
• U.S. Patent Application Publication No. 2002/0004640 (Conn et al.) discloses a device for measuring the concentration of target chemical analytes present in a biological system which involves application of a sensor device to the skin. In one embodiment, the analyte is extracted by means of iontrophoresis. The analyte is detected by including analyte-specific enzymes in the collection reservoir which react to produce a detectable product. The invention also involves use of a microprocessor.
• In the disclosure of U.S. Pat. No. 6,585,646 issued to Stuart Berlin there is presented and discussed a skin patch for use in collecting and detecting specific markers in apocrine sweat which may indicate the existence of various diseases. In one embodiment, the Berlin patch contains monoclonal antibodies or other chemical compounds which produce a visible signal upon reaction with specific markers. In another embodiment, an electronic sensor is utilized.
• U.S. Pat. No. 6,251,083 (Yum et al.) discloses a disposable patch for use in body fluid analysis. The patch includes a skin interface layer having an attachment means for attaching to the skin, a plurality of test zones, and an indicating reagent system for detecting the presence or concentration of an analyte by means of a spectrophotometric change. The reagents employed therein may include indicating dyes.
• With reference next to U.S. Pat. No. 5,443,080 (D'Angelo et al.), there is disclosed a device having a skin interface membrane layer, a chemical reactant layer, an indicator means which displays a color response, and an electro-optical interpretation means.
• U.S. Pat. No. 4,821,733 as issued to Peck discloses a transdermal detection systems for detecting a target substance which migrates to the surface of the skin. An adhesive means is used to attach the system to the skin surface. A detectable signal is produced which may be in the form of a visible color change. In one embodiment, the system is used to detect ethanol on the surface of the skin.
• In U.S. Patent Application Publication No. 2002/0091312 in the name of Berner et al., there is disclosed a device and method for measuring the concentration of an analyte in a biological system wherein the device is in operative contact with a skin or mucosal surface. The analyte is extracted transdermally using an iontophoretic sampling means. A sensing means is used to obtain a detectable analyte-specific signal and a microprocessor is used to provide for one or more measurement cycles.
• U.S. Pat. No. 4,732,153 issued to Phillips discloses a dosimeter which is attached to the skin for the collection of substances from the surface of the skin. The device produces an observable color change upon reaction of the collected substances with stored chemical compounds in the patch. The assay may be performed in situ in the transdermal dosimeter.
• And as a further examples of contributions to the arts related hereto, there is disclosedzzz in U.S. Patent Application Publication No. 2002/0099308 (Bojan et al.) a multiple-layer device for collecting and detecting an analyte in interstitial fluid. This device includes a skin contacting layer and a detecting layer for detecting or measuring an analyte. A light source is utilized to transdermally extract the interstitial fluid. In U.S. Pat. No. 6,479,015 awarded to Long et al., there is disclosed a skin patch having an indicator layer which turns color in the presence of a lactate solution, while EP 1262559 (Deigner et al.) also discloses a dermal patch having an adhering means and a reagent layer.
• Notwithstanding the advances in the arts related to the present invention, there has not been proposed a personal diagnostic device in the nature of an interactive dermal patch or bracelet including a skin/patch interface, at least one analysis or processing layer, an integrated micro-processor or computer, and a user output and/or input interface that incorporates the advantages of MEMS, bio-MEMS, or nano-based technologies to provide a wide variety of real-time patient diagnostic information or results directly to the patient user or patient caretaker without necessarily requiring involvement or intervention from medical professionals.
• Thus with the advent of MEMS and Bio-MEMS in conjunction with related micro-systems and nano-systems, there is a need for an integrated interactive personal diagnostic device in the nature of a patch or bracelet that may be easily employed by a user to perform a wide variety of real-time clinical diagnostic tests that otherwise require complicated lab-based medical equipment or a visit to the doctor's office and the subsequent delay associated with the processing of blood or urine samples by established laboratories.
• As a significant advancement over the prior art and related apparatus or methods, the present invention provides various embodiments of such personal diagnostic devices as embodied in an interactive patch or “bid-patch” that includes a skin/patch interface, at least one analysis or processing layer, and a user output interface integrated with MEMS, Bio-MEMS, and/or related micro-systems or nano-systems to perform a wide variety of desired tests in real-time while dermatologically employed by a patient or user. The bio-patch is implemented in a number of integrated flexible layers to form a light weight adhesively applied interactive dermal patch. The present invention and its various embodiments may also be implemented in a hard or semi-hard case or housing bracelet-type device which is conveniently referred to herein as a “bio-bracelet”. Furthermore, the present invention is not limited to use on humans. Certain embodiments hereof are directed to use on animals relative to particular and certain needs of the livestock, breading, or dairy industries for example.
SUMMARY OF THE INVENTION
• The present invention is directed to an interactive diagnostic patch or bracelet having a skin interface layer, one or more analysis or processing layers, and a user output interface. According to the invention, the patch or bracelet may perform a variety of in-situ diagnostic tests utilizing Micro Electrical Machine Systems (MEMS) or related micro- and nano-technologies. In one or more embodiments, the patch contains an adhesive layer for attachment to the skin. It may also contain means for collecting a transdermal biological sample such as blood or sweat. The collected sample may undergo one or more real-time qualitative or quantitative analyses to detect or monitor physiological changes in the body. The processing layer of the patch may contain various chemical reagents for reacting with the biological sample in order to produce a detectable signal. Results may be detected by CCD detector, laser emitting diodes (LED), or nano-wires. The signal may be visible via the user output interface layer in the form of a bar code, color change, numeric output, or alphanumeric output which may be color or monochromatic presented on a LED/LCD display, or it may be transmitted to a remote computer. The patch may contain other features including micro-fluidics, audio capabilities, minimally invasive tubules, fiber optic cables, light emitting or detecting nano-wires, cryogenic liquids, and photo emitters.
• In another embodiment, the patch or bracelet device may be utilized to detect airborne biological agents that collect on the top surface thereof.
• More particularly there is provided a dermal adhesive and/or non-adhesive patch or diagnostic bracelet incorporating any or all of a multitude of features enabling testing, analysis, diagnosis, treatment, input interfaces and output interfaces. The dermal patch or bracelet includes any or all options involving fluidic, micro-fluidic, micro-fluidic valves, reagent testing chambers, sensing equipment, logic processing in fluidic circuits, logic processing in electrical circuits, and connections from processing circuits to input and or output interfaces. The logic processing, and/or the input interfaces, and/or the output interfaces may reside on a reusable application which attaches to the surface of the testing patch or bracelet.
• The testing patch and/or the reusable processing/interface may be any of a multitude of shapes including but not limited to rectangular, circular, or any other shape. It may also be of many sizes including but not limited to small over all measurements on the order of 1 millimeter, as large as 30 centimeters in outside dimensions, or any desired combination of lengths, widths, and thicknesses within the range of about 1 millimeter to 30 centimeters.
• The testing patch or bracelet is capable of performing qualitative and/or quantitative tests and providing a user output and/or user input and/or processing capabilities in order to generate a logical result of the test.
• The interface abilities of optional input and optional output include user viewable quantitative and/or qualitative data presented by any of a number of means including but not limited to LED/LCD flexible or rigid display within or on the device and or the reusable surface. User selectable buttons' include, but not limited to, buttons, sliders, pressure switches, and remote input devices such as wireless interfaces and/or connected (wired) interfaces.
• The personal diagnostic device of the present invention and its various embodiments may include one or more qualitative or quantitative tests that may be a number of the same test to be performed at intervals determined by logic and/or human input. The testing device may alternatively include a number of different tests each providing a qualitative and or quantitative result.
• The present personal diagnostic device may also include cavities which may be employed for pressure testing and/or for providing energy in the form of compressed gasses such as air or vacuum.
• More specifically, the present invention is directed to a personal diagnostic device that includes a sample acquisition layer for obtaining a fluid sample from a user; a fluid sample layer for processing the fluid sample obtained from the user, the fluid sample layer being in fluid communication with the sample acquisition layer; means for detecting diagnostic results derived from the processing of the fluid sample; and means for displaying the diagnostic results. The device may include at least one cavity containing a pressurized gas, or at least one cavity containing a vacuum. The device may further include hydrophobic surfaces for retarding fluid flow or hydrophilic surfaces for promoting fluid flow. The device may be equipped with a sound emitter for providing the user with feedback. The device may also include a logic processing system and an Internet Protocol address within the logic processing.
• According to another aspect of this invention there is provided a personal diagnostic device including an air sample acquisition member for obtaining an air sample from the ambient environment of a user; means for testing the air sample for at least one specific air born contaminant; a fluid sample acquisition layer for obtaining a fluid sample from a user; a fluid sample layer for processing the fluid sample obtained from the user, the fluid sample layer being in fluid communication with the sample acquisition layer; a detector implemented to detect diagnostic results derived from the processing of the fluid sample; and a display unit that displays the diagnostic result. This device may further include a logic processing system having TCP/IP, and may include separately or in combination therewith a receiver and a transmitter to provide telemetry information regarding location of the user. The device is advantageously provided with a processor for processing the diagnostic results detected by the detector wherein the processor generates output information regarding a medical state of the user. A memory for storing the output information may also be provided in the device or external to the device.
• In accordance with another aspect of this invention, the is further provided a method of using a personal diagnostic device. This method may include the steps of applying a respective personal diagnostic device to a user; allowing the personal diagnostic device to obtain a biological sample from the user; processing the biological sample in the personal diagnostic device to obtain medical or health information regarding the user; transmitting the information to a receiving device; and displaying output results associated with the medical information. This may include the further step of storing the information in the personal diagnostic device before the transmitting step is performed. The receiving device may be in the personal diagnostic device or it may be a personal computer, or linked to a communications network.
• Various aspects of the present invention, described in detail below in conjunction with the drawing figures, include methods of qualitative and quantitative analysis of biological and chemical test samples carried out in-part or fully within the personal diagnostic device of the present invention. These test samples may include, but are not limited to, whole blood, serum, plasma, saliva, urine, sputum, stool, sweat, tissue samples, tumor or biopsy samples, water and other samples that may contain a chemical or biochemical target of interest. These targets of interest may include for example, specific nucleic acid sequences, proteins, antibodies, toxic chemicals, pollutants, stress indicators, cardiovascular health indicators, tumor markers, cells, bacteria, viruses, biochemical weapons, and other targets of a biological or chemical nature.
• It is, therefore, an object of the present invention to improve upon limitations in the prior art. These and other objects are attained in accordance with the present invention wherein there is provided several embodiments of an interactive bio-patch, bio-bracelet, and various methods and processes relating thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
• Further objects of the present invention together with additional features contributing thereto and advantages accruing therefrom will be apparent from the following description of preferred embodiments of the invention which are shown in the accompanying drawing figures with like reference numerals indicating like components throughout, wherein:
• FIG. 1 is a perspective view of a user of the personal diagnostic device of the present invention sitting at a PC which is illustrated down-loading test results wirelessly from the device to the PC for the display of test results on the PC monitor;
• FIG. 2 is a perspective view of one embodiment of the analytical personal diagnostic device shown inFIG. 1 including cut away sections to reveal the components of each of the principal layers associated therewith;
• FIG. 3A is an exploded perspective view of an alternate embodiment of the analytical diagnostic device shown inFIG. 1 including removable and interchangeable portions illustrated with cut away sections to reveal the components of each of the principal layers and interchangeable portions;
• FIG. 3B is a perspective view of a reader device used in one embodiment to down-load results stored in the removal top portion of the device illustrated inFIG. 3A;
• FIG. 4 is a perspective view of an alternate embodiment of the personal diagnostic devices shown inFIGS. 1-3 including the aspect of a sample collection inlet port on the top surface thereof;
• FIG. 5 is an exploded perspective view of another alternate embodiment of the personal diagnostic device shown inFIG. 1 including sound chambers and acoustic sensing microphones implemented for audio input/output applications;
• FIG. 6 is an exploded perspective view of yet a further alternative embodiment of the personal diagnostic device shown inFIG. 1 including a cryogenic tumor treatment assembly with a CCD for imaging the progress of treatment;
• FIG. 7A is an exploded perspective view of the principal sample acquisition, fluid processing, and assay results detection layers of the personal diagnostic device according to the present invention;
• FIG. 7B is an exploded perspective view which illustrates the principal assay results detection layer, electronic processing layer, logic control layer, and input/output layer of the personal diagnostic device according to the present invention;
• FIG. 8A is a perspective view of the personal diagnostic device of the present invention as implemented in a bio-bracelet configuration;
• FIG. 8B is a perspective view of the personal diagnostic device according to the present invention as implemented in a bio-patch configuration;
• FIG. 9 is an enlarged detailed plan view of a single micro-fluidic circuit which may be employed in conjunction with various embodiments of the interactive analytical personal diagnostic device of the present invention;
• FIG. 10 is a view similar toFIG. 9 including an emitter and detector assembly implemented in conjunction with the illustrated micro-fluidic circuit;
• FIG. 11A is an exploded perspective view of a sample acquisition layer, fluidic circuit layer, heat and wash layer, and results detection layer as employed in one specific embodiment of the present personal diagnostic device implemented to perform a DNA assay;
• FIG. 11B is a perspective view with cut-away sections showing a fully assembled personal diagnostic device including the layers illustrated inFIG. 11A for performing a DNA assay;
• FIGS. 12A to 12G present a series of related cross-sectional side views showing the progression of a test sample through the fluidic processing circuit of the device illustrated inFIGS. 11A and 11B as implemented to perform a DNA assay;
• FIG. 13A is a diagrammatic representation of a pressure valve utilized in the fluidic circuits of the present invention to control the flow of fluids;
• FIG. 13B is a schematic presentation of a normally closed melt valve employed in the fluidic circuits of the present invention to control and direct the flow of fluids therein;
• FIG. 13C is a schematic representation of a normally open melt valve used in the fluidic circuits of the present invention to direct and control fluid flow;
• FIG. 13D is a schematic presentation of another normally open melt valve used in the fluidic circuits of this invention to control fluid flow;
• FIG. 13E is a diagrammatic representation of a dissolvable plug used to form a timed fluid valve shown closed, partially dissolved, and then open to allow flow;
• FIGS. 14A to 14K are detailed schematic and diagrammatic isolation views of the semiconductor nano-wire and micro-wire assemblies employed in this invention as light emitters and detectors for illuminating, detecting, and imaging DNA, small molecule, and cellular assay results derived from use of the personal diagnostic devices disclosed herein;
• FIG. 15A is an exploded perspective view of a sample acquisition layer, fluid processing layer, results detection layer, and wash buffer reservoir layer as employed in another specific embodiment of the present personal diagnostic device implemented to perform a cellular assay;
• FIG. 15B is an exploded perspective isolation view of the wash buffer reservoir, micro-wire detector, and capture zone layer of the persona) diagnostic device ofFIG. 15A;
• FIG. 15C is a perspective view with cut-away sections showing a fully assembled personal diagnostic device including the layers illustrated inFIG. 15A for performing a cellular assay;
• FIGS. 16A to 16F present a series of related cross-sectional schematic side views showing the progression of a test sample through the fluidic processing circuit of the device ofFIGS. 15A,15B, and15C as implemented to perform a cellular assay;
• FIGS. 17A and 17B are schematic plan views of the semiconductor micro-wire assemblies employed in this invention as light emitters and detectors for illuminating, detecting, and imaging cellular assay results derived from use of the present personal diagnostic devices;
• FIG. 18A is an exploded perspective view of a sample acquisition layer, fluid processing layer, and alternative results detection layers as employed in yet another specific embodiment of the present personal diagnostic device implemented to perform small molecule assays;
• FIG. 18B is a perspective view with cut-away sections illustrating a fully assembled personal diagnostic device including the layers shown inFIG. 18A for performing a small molecule assay;
• FIG. 18C is an exploded perspective isolation view of the insulin reservoir and related supply lancets of the personal diagnostic device ofFIGS. 18A and 18B;
• FIGS. 19A and 19B are two related cross-sectional side views showing the progression of a sample through the fluidic processing circuit of the device ofFIGS. 18A and 18B as implemented to perform small molecule assays;
• FIG. 20A is an exploded perspective view of a sample acquisition layer, results illumination layer, fluid processing layer, results detection layer, and wash buffer reservoir layer as employed in still another alternative specific embodiment of the present personal diagnostic device implemented to perform stress analysis;
• FIG. 20B is a perspective view with cut-away sections showing a fully assembled personal diagnostic device including the layers illustrated inFIG. 20A for performing stress analysis according to certain aspects of the present invention;
• FIGS. 21A to 21D are a series of related cross-sectional side views illustrating the progression of a test sample through the fluidic processing circuit of the device shown inFIGS. 20A and 20B as implemented to perform stress analysis;
• FIG. 22A is an exploded perspective view of a sample acquisition layer, repeated fluid processing layers, and a results detection layer as employed in yet still another alternative specific embodiment of the personal diagnostic device according to the present invention implemented to perform sports performance analysis during real-time use;
• FIG. 22B is a perspective view with cut-away sections illustrating a fully assembled personal diagnostic device including the layers shown inFIG. 22A for conducting sports performance analysis according to other certain aspects of the present invention;
• FIGS. 23A to 23E present a series of a cross-sectional side views showing the progression of a sample through the fluidic processing circuit of the device ofFIGS. 22A and 22B as implemented to perform sports performance analysis;
• FIG. 24A is an exploded perspective view of a sample acquisition layer, fluid processing layer, and results detection layer as employed in another alternate specific embodiment of the personal diagnostic device hereof implemented to perform elderly care monitoring in older patients;
• FIG. 24B is a perspective view with cut-away sections showing a fully assembled personal diagnostic device including the layers illustrated inFIG. 24A as utilized to perform elderly care monitoring in older patients; and
• FIGS. 25A to 25D present a series cross-sectional side views showing the progression of a sample through the fluidic processing circuit of the device illustrated inFIGS. 24A and 24B as employed for elderly care monitoring in older patients.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
• The following description is first directed to a discussion of the basic structural and functional components of the personal diagnostic device (PDD) according to the present invention as embodied in bio-patches and bio-bracelets. Next presented is a detailed description of DNA assays and related methods as implemented on the PDD bio-patches and bio-bracelets according to specific embodiments of this invention. Then a detailed discussion of PDD cellular assay implementations and methods is presented. Thereafter, small molecule assay PDD implementations and related methods are discussed in detail. Then stress monitoring implementations and various related methods are presented. Next discussed are certain sports performance monitoring implementations and related methods illustrating various real-time use aspects of the present invention. And lastly, a detailed discussion of elderly care monitoring PDD implementations, applications, and related methods is presented to illustrate in detail certain further aspects of the present invention.
Structural and Functional Components
• With reference now toFIG. 1 there is shown auser102 wearing a personaldiagnostic device104 that may be implemented in either a bio-bracelet configuration (FIG. 8A) or a bio-patch configuration (FIG. 8B). Theuser102 ofFIG. 1 is seated atworkstation106 which includes apersonal computer108, akeyboard110, and adisplay monitor112. According to one principal embodiment of the personaldiagnostic device104, the device includes a radio frequency transmitter223 (FIGS. 8A and 8B) which is employed to an transmit electronic signal from the personaldiagnostic device104 to thepersonal computer108 which is outfitted with asuitable RF receiver114. In this manner, diagnostic test results processed by and stored in thedevice104 may be wirelessly transmitted to thepersonal computer108 and then conveniently displayed on thedisplay monitor112.
• Referring next toFIG. 2 there is shown a prospective view of one embodiment of the analytical personaldiagnostic device104 including cutaway sections to reveal the various components in each of the principal layers associated herewith. This embodiment of thedevice104 includes asample acquisition layer116, afluid processing layer118, a results processing andcontrol layer120, and anoutput layer122. Theoutput layer122 is illustrated with avideo display monitor124 and individual fixed-display results windows126. Thevideo display monitor124 may display real time video information in the nature of a PC monitor or a television monitor. The fixeddisplay result windows126 are one time use result display windows that display in a fixed permanent manner the various results of a particular assay or assays performed in thedevice104. The results displayed on themonitor124 and thewindows126 may be qualitative, quantitative, or semi-quantitative.
• FIG. 3A is an exploded perspective view of an alternate embodiment of the analyticaldiagnostic device104 including a removable and interchangeabletop portion128 as illustrated.FIG. 3A includes cutaway sections to reveal the components of each of the principal layers and interchangeable portions. More specifically, thedevice104 shown inFIG. 3A includes theremovable cap portion128 which is provided with the processing andcontrolling layer120, thevideo display monitor124, and the fixed results displaywindows126. Thedevice104 illustrated inFIG. 3A further includes a bracelet orbase portion130 which is provided with thesample acquisition layer116 and thefluid processing layer118. In the bio-bracelet embodiment hereof, the bracelet orbase portion130 may be made of a rigid material such as aluminum while in the bio-patch embodiment hereof, the bracelet orbase portion130 is made up of a flexible plastic layers. Thebase portion130 further includesinterface connectors132 so that theremovable cap portion128 may be readily mounted and disconnected from thebase portion130. Thus in this implementation the present invention, a user may apply the base orbracelet portion130 with a firstremovable cap portion128 to receive and process results within a first time period. Thereafter, the user may then remove the firstremovable cap portion128 and replace it with secondremovable cap portion128 to continue to collect and process diagnostic information from the user's acquired biological samples. In an alternate embodiment, the individualremovable cap portions128 may be inserted into areceptacle131 of areader device129 that down-loads the collected diagnostic information from the removabletop portion128 in a secured manner as illustrated inFIG. 3B. In this embodiment, the patient may not be allowed access to the collected information. Thereader device129, for example, would be in the custody of an attending doctor or caretaker in the doctor's office or hospital and the results then analyzed by the attending physician or medical professional rather than directly by the patient by interactive use of thePC108 and monitor112 as represented inFIG. 3B. Thus in various applications of this embodiment, theremovable cap portion128 may not include the devicevideo display monitor224 and/or thedisplay windows126. Alternatively, the devicevideo display monitor224 and/or thedisplay windows126 may be only partially operative so that certain test results are made available to the patient user while other test results are restricted to only doctor analysis via the down-loading aspect hereof.
• With reference now toFIG. 4, there is shown a perspective view of alternate embodiment of the personaldiagnostic device104 of the present invention including air sample collection inlet ports or detectors on the top surface thereof. As illustrated inFIG. 4, thedevice104 includes thesample collection layer116, thefluid processing layer118, the results processing andcontrol layer120, and theoutput layer122 further alternatively including thevideo display monitor124 and the fixed results displays126. In addition thereto, the embodiment of thedevice104 illustrated inFIG. 4 is further provided with a first air sample collection member, detector, orunit134 and a second air sample collection member, detector, orunit136. In this embodiment the airsample collection units134 and136 are intended to receive, capture, and detect various airborne particles that may be in the ambient environment in which a user is working or otherwise present. Such applications would include firemen fighting house fires, building fires, or forest fires; health care workers working in hospitals or research laboratories; employees and inspectors working in various chemical processing plants, oil refineries, or similar types of processing and power generating plants wherein exposure to airborne chemicals or other particular matter is a health and safety issue. In another implementation hereof, military personal and police officers may utilize such an airborne PDD system to detect potentially harmful biological, viral, or chemical particulate matter that has been accidentally or intentionally released into the environment. Thus the user of this embodiment of the personaldiagnostic device104 will be informed in the event there are any harmful levels of airborne particulate matter present in the surrounding environment. In addition thereto, the various sample acquisition, processing, and assaying aspects of thedevice104 are then employed to monitor the user's biological and physiological responses to any such airborne particular matter that has been detected and identified by the first and/or second airsample collection units134 and136.
• FIG. 5 shows an exploded perspective view of yet another alternate embodiment of the personaldiagnostic device104 according to the present invention. Thedevice104 illustrated inFIG. 5 is provided with sound chambers138,microphones140, and asignal processing unit142. The system elements including the sound chamber138,microphones140, andsignal processing unit142 are provided operating power by asmall battery144 preferably implemented in an integral manner in the bottom layer as illustrated. As an alternative to thebattery144 as the internal power source, thedevice104 may be equipped with a solar cell type power source that converts incident light to electrical power for internal device use. As another alternative thereto, thedevice104 may be provided with internal reactive chemicals that upon a controlled command react to thereby generate needed electrical power internally.
• The top layer of thedevice104 illustrated inFIG. 5 may include thevideo display monitor124 and the fixed results displaywindows126. In addition thereto, the top layer of thedevice104 illustrated inFIG. 5 may further include aninput selection switch146 and an on/offswitch148. In this manner, the personaldiagnostic device104 illustrated inFIG. 5 includes both output capabilities as well as input capabilities. The one or combination of the principal elements of the device illustrated schematically inFIG. 5, may be combined with any other embodiments or subsystems of the various PDDs disclosed herein for specific application implementations.
• FIG. 6 schematically illustrates an example of an implementation of thePDD104 for use in the treatment of skin lesions. More particularly,FIG. 6 is a perspective view a personaldiagnostic device104 including a cryogenic tumor treatment assembly with imaging capability to visually monitor progress of the treatment. Thedevice104 illustrated inFIG. 6 similarly includes thevideo display monitor124, the fixed results displaywindows126, theinput selection member146, and the on/offswitch148. The device ofFIG. 6 is then further provided with acryogenic chamber150, a charge couple device (CCD)152 for imaging, and cryogenicmicro-fluidic channels154. The flow of a cryogenic fluid such as liquid nitrogen is controlled in thechannels154 by employment of a series ofvalves156 which may be electronically controlled by thesignal processing unit142 to direct flow in a pre-determined and timed manner. Cryogenic fluid such as liquid nitrogen may be supplied to thedevice104 by means ofinput supply lines158. It is contemplated that use of this implementation of the present invention would be in a doctor's office or other professionally supervised setting. The device is applied to an appropriately diagnose location on the skin lesion, cryogenic fluid such as liquid nitrogen is applied, and various steps in the lesion treatment are monitored in real time and displayed on thevideo monitor124 as treatment progresses. In this implementation, the fixeddisplay windows126 may be utilized to take various diagnostic readings at desired intervals throughout the entire time duration of the skin lesion treatment process.
• With reference now toFIG. 7A, there is shown an exploded perspective view four of the principal layers of one preferred embodiment of the personaldiagnostic device104 according to the present invention. More specifically,FIG. 7A shows thesample acquisition layer116 which is provided with minimally invasive tubules, lancets, ormicro-probes159 that penetrate the skin surface to acquire blood samples from capillaries near the epidermis. Alternatively, the tubules, lancets, or micro-probes159 may be non-invasive and then implemented to acquire sweat or perspiration form the skin surface of a user. In this specific embodiment illustrated inFIG. 7A, the lancets or micro-probes159 are implemented to transdermally draw blood or interstitial cell fluid (ICF) from the user. The tubules, lancets, ormicro-probes159 include a piercingend160 and adischarge end161. The piercingend160 is implemented to penetrate the epidermis of a user and draw blood by capillary action up through the micro-probe orlancet159 and into the device as further described in detail below.
• Next provided is aspacing layer162 as shown inFIG. 7A. Thespacing layer162 may be formed from any suitable flexible plastic material such as polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the like. Thespacing layer162 may be of a thickness on the order of 25 microns to 500 microns depending on the sample acquisition volume requirements and other assay specific requirements. As illustrated inFIG. 7A, thespacing layer162 is provided withreservoir openings164. Thereservoir openings164 are cutouts in thelayer162 which are positioned to be in register withcorresponding inlet formations166 integrally formed in thesample acquisition layer116. Theinlet formations166 may be achieved by recessed areas, hollows, or dimples formed in the top surface of thesample acquisition layer116 around the lancet ormicro-probe discharge end161 but which do not penetrate through to the bottom surface thereof. Thus in this manner when themicroprobes159 penetrate the user's skin, blood begins to flow by capillary action and then fills the sample collection chamber224 (FIGS. 9 and 10) formed by arespective reservoir opening164 and itscorresponding inlet formation166. Thus the sample collection chamber may be formed to collect a metered amount of blood of a specific or desired volume. As further illustrated inFIG. 7A, thefluidic processing layer118 similarly includes thereservoir openings164 which are formed and positioned in register with the correspondingreservoir openings164 formed in thespacing layer162. In this manner thereservoir openings164 in thespacing layer162 combined with the corresponding reservoir opening164 in thefluidic processing layer118 in further combination with theinlet formations166 to create thesample collection chamber224 illustrated schematically inFIGS. 9 and 10.
• Thefluid processing layer118 may be similarly formed from any suitable flexible plastic material such as polymethylmethacrylate (PMMA), polycarbonate, polytetra-fluoroethylene (TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the like. Thefluid processing layer118 may also be on the order of anywhere between 25 microns and 500 microns depending on the assay application and related volumetric and flow requirements within the fluidic processing circuits formed therein. More specifically in the embodiment of thefluidic processing layer118 illustrated inFIG. 7A, there is shown a firstfluidic channel168 which is in fluid communication with thesample collection chamber224,FIGS. 9 and 10, formed by thereservoir openings164 and theinlet formations166 as described above. The firstfluidic channel168 is in fluid communication with afluidic chamber170. To continue flow through the fluidic circuit, thefluidic chamber170 is in fluid communication with a secondfluid channel172 which in turn is in fluid communication with areaction zone174. Thereaction zone174 may be utilized in differing embodiments in a wide variety of assay applications described below in conjunction withFIGS. 11A to 25D including DNA, cellular, small molecule, or other similar types of assays which are performed in conjunction with blood samples, DNA samples, or sweat samples obtained from or through the skin surface of the user. Thereaction zone174 may be in fluid communication with at least one waste chamber orcollection chamber176 to promote fluid flow and/or to collect wash buffer after the capture agents, for example, have captured, bound, or otherwise identified targets in thereaction zone174.
• The last principal layer illustrated inFIG. 7A is an assay resultsdetection layer178 which includesresults detectors180 each having aninput182 andoutput184. Thedetector180 may be embodied in any number of suitable types of detection formats including by way of example and not limitation, CCD imaging devices, electrical sensing or detecting electrodes, optical fibers, silicon-based sensors and bio-sensors, and individual or bundles of semi-conductor nano-wires or semi-conductor micro-wires which are implemented herein in various combinations as illuminating sources, detector devices, and/or imaging devices as discussed in further detail herein below. Thedetectors180 are each respectively positioned over a correspondingreaction zone174 as illustrated. In this manner, after assay results are manifest in thereaction zone174, thedetector180 may be activated to detect, identify, or otherwise determine the existence, presence, or absence of expected, unexpected, desired, undesired, anticipated, or unanticipated assay results. One implementation of theresults detector180 according to the teachings hereof, includes the use of individual or bundles of semi-conductor nano-wires or micro-wires of the type currently being developed by Nanosys Inc. of Palo Alto, Calif. Further details, aspects, and implementations of the types, uses, and functions of the various detection and illumination systems employed herein according to various embodiments of the present invention are discussed with further specificity herein below.
• Returning now to the principal component layers illustrated inFIG. 7A, it should be understood that when thesample acquisition layer116,spacing layer162, thefluid processing layer118, and the assayresults detection layer178 are assembled and theinlet formations166 thereby positioned in register with thereservoir openings164; the sample collection chamber224 (FIGS. 9 and 10) is closed at the top by the bottom surface of the results detection layer covering and sealing around thereservoir openings164 formed in thespacing layer162 and thefluid processing layer118. In one embodiment hereof, the surfaces of the various layers include an adhesive to thereby obtain adhesion and sealing of the layers to each other. In other embodiments, the layers may be assembled together in a fluid tight manner by employing suitable plastic melting or micro-welding techniques. Similarly, when thesample acquisition layer116,spacing layer162, thefluid processing layer118, and the assayresults detection layer178 are assembled, theresults detectors180 are each thereby respectively positioned in register with a correspondingreaction zone174.
• The thickness of thefluid processing layer118 relative to the thickness of thespacing layer162 may be varied to achieve any desired volumetric relationship between thesample collection chamber224,FIGS. 9 and 10, and the various components of the fluidic circuit represented in thefluid processing layer118. For example, in a specific assay implementation it may be desired to have a relatively large sample volume collected in thechamber224 while having the fluidic circuit of relatively smaller dimensions. In this case, for example, the spacing layer may be 500 microns thick while the fluid processing layer is, for example, 25 microns thick. Furthermore, thesample collection layer116 may be, for example, 100 microns thick while the inlet formations formed therein may be, for example, 25 microns deep. Thus when thesample acquisition layer116,spacing layer162, thefluid processing layer118, and the assayresults detection layer178 are assembled and theinlet formations166 thereby positioned in register with thereservoir openings164, thesample collection chamber224 will have a height of 550 microns (500+25+25) while the fluidic circuit elements will have a height of 25 microns. Alternatively, for example, thefluid processing layer118 may be 150 microns thick, thespacing layer162 not utilized, thesample acquisition layer116 also with a thickness of 150 microns with the inlet formations made 50 microns deep. In this case, thesample collection chamber224 will have a height of 200 microns (150+50) while the fluidic circuit elements will have a height of 150 microns. In yet still alternate combinations hereof, both thespacing layer162 and the inlet formations may not be utilized. In this case, the sample collection chamber224 (FIGS. 9 and 10) will be formed only by thereservoir opening164 in thefluid processing layer118 and thus the fluidic circuit elements formed therein will have the same height as thesample collection chamber224. Thus in view hereof, it should be understood that any desired combination of relative volumes and/or heights between and among thesample collection chamber224, the first and secondfluid channels168 and172, thefluid chamber170, and thewaste chambers176 may be achieved by utilizing desired thicknesses of thespacing layer162 and thefluid processing layer118 with corresponding cutouts in thespacing layer162 to add depth to any, all, or any combination for the fluid circuit elements in thefluid processing layer118. Similarly, theinlet formations166 as achieved by recessed areas, hollows, or dimples formed in the top surface of thesample acquisition layer116 around the lancet or micro-probe discharge ends161 may be utilized at any desired depth, or alternately not so employed to add volume to thesample collection chamber224. Furthermore, additional volume may be selectively added to discrete circuit elements in thefluid processing layer118 by utilizing recessed areas, hollows, or dimples formed in the bottom surface of the assayresults detection layer178.
• FIG. 7B is a view similar toFIG. 7A illustrating further principal layers of the personaldiagnostic device104 according to the present invention. More particularly,FIG. 7B shows again the assayresults detection layer178 ofFIG. 7A, next a separatesignal processing layer186, a separate logic and input/output controller layer188, and then theoutput layer122 as described above in conjunction with FIGS.2 and4-6. The assay resultsdetection layer178 includes theresults detectors180 each with theirrespective input182 andoutput184. As illustrated, thesignal processing layer186 includes theprocessor142 and the logic and input/output controller layer188 includes a logic and I/O controller190 hereinafter simply referred to for convenience as “controller190”. As shown, theprocessor142 has a variety of electrical connection lines which each connect either to theinputs182 andoutputs184 of theresults detector180 in theresults detection layer178 or to circuit connections associated with thecontroller190. Theprocessor142 includes correspondingconnections192 and194 as illustrated. More specifically as shown, each of theinput connectors182 andoutput connectors184 of theresults detector180 are in register and connect respectively to acorresponding connection192 or194 associated with theprocessor142. Similarly, theprocessor142 includesoutput connectors196 andinput connectors198 which upon assembly of the various layers illustrated inFIG. 7B correspond to and connect respectively withoutput connectors198 andinput connectors196 associated with thecontroller190 in the controller layer. In the embodiment of the device illustrated inFIG. 7B, thecontroller190 further includes several input connectors which are referenced200,202,204,206,208,210,212,213,216,218,220, and222. As indicated inFIG. 7B, theinput connector202 of thecontroller190 is situated for connection with the input key number “7” in theoutput layer122. Similarly, theinput connector202 is positioned to make a connection with the number “8” key, the input connector204 is situated to make a connection to the number “9” key, theinput connector206 is placed to make a connection with the number “4” key, theinput connector208 is positioned to make a connection to the number “5” key, theinput connector210 is located to make a connection with the number “6” key, theinput connector212 is situated to make a connection to the number “1” key, theinput connector214 is placed to make a connection with the number “3” key, theinput connector218 is situated to make a connection to the “start” key, theinput connector220 is positioned to make a connection with the “stop” key, and the input connector222 is located to make a connection to the “pause” key. As further illustrated, other connections from thecontroller190 are provide to facilitate desired communication between the input/output layer122, theprocessor142, and thecontroller190. These connections and communication networks will be described in further detail below in connection with the discussion of specific assay implementations. Thus in this manner, the personaldiagnostic device104 is provided with both input capability as well as output capability for interactive use by a patient wearing the device as intended.
• As represented in the input/output layer122 ofFIG. 7B, assay results may be displayed in either thevideo display monitor124 or the fixedresults windows126. These results may be qualitative and in the form of a “Yes” or “No” result display, or a “Positive” or “Negative” result display; quantitative and in the form of calibrated bar graphs as shown; or semi-quantitative giving an indication such as “High” or “Low” depending on the particular assay implementation and desired results.
• With reference now toFIG. 8A, there is illustrated a perspective view of the personal diagnostic device of the present invention as implemented in a bio-bracelet configuration. In this configuration, the is provided with theradio frequency transmitter223 as discussed above. Thedevice104 as shown also includes connection indicator lights225 which may be enabled to blink or otherwise emit light to indicate a proper wireless connection between thedevice104 and thePC108 via theRF receiver114,FIG. 1. The bio-bracelet104 ofFIG. 8A is provided with aband219 and aclasp221 so that a user may ware the bans around the wrist or ankle, for example. Theband219 may be made of a flexible, stretch material to provide good holding force between the device and the user, or may be made of a hard material such as stainless steel or aluminum.FIG. 8B is a perspective view of the personaldiagnostic device104 according to the present invention as implemented in a bio-patch configuration. In this particular embodiment, thedevice104 includes thevideo display monitor124, the individual fixed-display results windows126, a pair of RF transmitters, and the connection indicator lights225. As with any of the embodiments of thedevice104 discussed herein, theRF transmitters223 may include both transmission capabilities as well as receiving capabilities to thereby establish two-way communication between thedevice104 and thePC108, and to further establish interactive two-way communication with any networks to which thePC108 may be connected or otherwise linked.
• Turning now toFIGS. 9 and 10, there is shown an enlarged detail schematic representation of a single micro-fluidic circuit123 which may be employed in conjunction with the various elements and layers of the personaldiagnostic patch104 of the present invention. The micro-fluidic circuit123 shown inFIGS. 9 and 10 includes thesample collection chamber224 which may be formed by thereservoir openings164 andinlet formations166 as discussed above in conjunction withFIGS. 7A and 7B. As represented inFIGS. 9 and 10, thesample collection chamber224 is in fluid communication with the firstfluid channel168 which in turn is fluidly connected to thefluidic chamber170 which is in fluidic communication with the secondfluid channel172 which is next in fluid communication with thereaction zone174. As discussed in further detail for the various assay implantations discussed below, a wide variety of assays may be conducted within thereaction zone174. Thereaction zone174 may be in fluid communication with at least one waste orcollection chamber176. To promote flow control within the fluidic circuit123 illustrated inFIGS. 9 and 10, the circuit may includemicro-valves226 which may be implemented in a wide variety of different configurations. Additional details regarding the valving and fluid control elements and methods utilized in the various embodiments hereof are presented and discussed herein below in connection withFIGS. 13A-13E and in further connection with specific assay implementations thereafter described.
• FIG. 10 is a view similar toFIG. 9 further illustrating anoptical emitter228 and an optical detector230. In order to detect and image manifest results of a number of different types of assays, theresults detector180 is implemented as the optical detector230 which may be employed in combination with theoptical emitter228. The various optical and electrochemical phenomena that may be detected with theoptical detector228 or generally theresults detector180 are presented below in Table 1. Further detailed explanation relating thereto may be found in “Point of Care Testing”, 2nd edition, edited, by Christopher Price, Andrew St John, and Jocelyn Hicks, 2004.

• TABLE 1
  Signal Generation	Types
  Optical Detection	Absorbance, Reflectance, Transmission,
  	Fluorescence, Luminescence,
  	Turbidimetry
  	and Nephelometry
  Electrochemical Signals	Amperometric, Impedimetric,
  	Potentiometric
  Optical Motion	Light Scattering, Paramagnetic Particles,
  	Interference Pattern, Image Analysis
  Surface Interrogation	Optical Interference, Pattern Recognition,
  	Surface Enhancement, Diffraction,
  	Ellipsometry, Surface Plasmon Resonance

• Theoptical emitter228 and optical detector230 may be embodied in a variety of different optical devices or formats including for example, but not limited to, charged coupled devices (CCD), fiber optics, nano-wires, micro-wires, semi-conductor light emitting and/or detecting materials, or other suitable light emitting and detecting materials or devices.
DNA Assay Implementations and Methods
• The ability to detect target nucleic acid analytes of specific nucleic acid sequence using nucleic acid probe hybridization methods has many applications. Among these applications are diagnoses of infectious or genetic diseases or determination of susceptibility to cancer in humans or other animals; identification of viral or microbial contamination of cosmetics, food or water; and identification or characterization of, or discrimination among, individuals at the genetic level, for forensic or paternity testing in humans and breeding analysis and stock improvement in plants and animals. The basis for applications of nucleic acid probe hybridization methods is the ability of an oligonucleotide or nucleic-acid-fragment probe to hybridize, i.e., form a stable, double-stranded hybrid through complementary base-pairing, specifically with nucleic acid segments which have a particular sequence and occur only in particular species, strains, individual organisms or cells taken from an organism.
• One of the basic limitations in nucleic acid probe hybridization assays has been the sensitivity of the assays, which depends on the ability of a probe to bind to a target molecule and on the magnitude of signal that is generated from each probe that binds to a target molecule and that can be detected in a time period available for detection. Known detection methods in the assays include methods dependent on signal generated from a probe, as from fluorescent moieties or radioactive isotopes included in the probe, or an enzyme, such as an alkaline phosphatase or a peroxidase, linked to the probe and, after probe hybridization and separation of hybridized from unhybridized probe, incubated with a specific substrate to produce a characteristic colored product. However, the practical detection limit of these assays is about 200,000 target molecules (3 femtomolar concentration in 100 μl), which is not sufficiently sensitive for many applications. Much effort is therefore being expended in increasing the sensitivity of detection systems for nucleic acid probe hybridization assays.
• Achieving adequate limits of detection has been one of the difficulties facing the clinical application of nucleic hybridization methods. Several amplification methods have been developed to alleviate this difficulty by increasing either the sample or detection product. The Polymerase Chain Reaction (PCR) is the best known and most widely applied of these methods. With respect to PCR amplification, reference is made to Current Protocols in Molecular Biology, Suppl. 4,Section 5, Unit 3.17, which is incorporated herein by reference, for a basic description of PCR. Other references which describe PCR include Erlich, H. A., (Ed.) 1989, PCR Technology, Stockton Press; Erlich, H. A., et al. (1988), Nature 331:461-462; Mullis, K. B. and Faloona, F. A. (1987), Methods in Enzymology, 155:335-350; Saiki, R. K., et al. (1986), Nature 324:163-166; Saiki, R. K., et al. (1988), Science 239:487-491; Saiki, R. K., et al. (1985), Science 230:1350-1354; U.S. Pat. No. 4,683,195 to Mullis, et al.; and U.S. Pat. No. 4,683,202 to Mullis. All of which are incorporated by reference as if fully repeated herein.
• First invented by Kary Mullis, PCR has been widely adopted and adapted for many research purposes, and commercial kits for clinical diagnosis are now available. However, these amplification methods are time-consuming, labor intensive, costly, and susceptible to contamination, resulting in false positives.
• Therefore, the development of a quick, easy to use, inexpensive medical devices that allow for the detection of very low level of target nucleic acids without target amplification and probe labeling are of particular interest.
• The nucleic acid analysis device implementation of the personaldiagnostic device104 is herein referred to as the DNA patch or bracelet which provides a quick, effortless, and automatic method for nucleic acid detection using only a simple patch or bracelet that interfaces with a personal computer or a data display unit as described above.
• With reference now toFIG. 11A, there are shown layers of an exemplary embodiment of the present invention for nucleic acid analysis including the base orsample acquisition layer116, thefluidic processing layer118, a fluid chamber andsample processing layer300, and a specific assayresults detection layer302. These layers are similar to or a combination of the layers described above in conjunction withFIGS. 2 to 7B. As mentioned earlier, the layers are placed in register with each other such that a fluidic circuit is formed containing different chambers for sample processing, detection, and analysis. The fluidic circuit of the DNA patch may include the lancet or micro-probe159 operatively connected to ablood metering chamber304, a red blood cell (RBC)capture chamber306, a cell lysis andDNA extraction chamber308, a DNA shearing or DNA nicking anddenaturing chamber310, a capture or analysis orreaction chamber312, awaste chamber314, and washbuffer reservoir316. All of which are operatively attached to each other to form the fluidic circuit.Chamber308 may alternatively be two separate chambers for cell lysis and DNA extraction. As illustrated, washbuffer reservoir316 is located inlayer300 which also includesheating elements317 which are used to heat the sample to aid in cell lysis and denaturation of the DNA in the sample for analysis.Heating elements317 are accordingly placed in register with lysis andDNA extraction chambers308 anddenaturation chamber310.FIG. 11A further shows alight source320 inlayer116 and adetector322 inlayer302.Light source320,analysis chamber312, anddetector322 are in register with each other such that light emitted fromsource320 passes throughchamber312 and is detected by thedetector322. Thelight source320 anddetector322 are implemented in a nano-wire format described in further detail below
• FIG. 11B is a perspective view with cut-away sections showing a fully assembled personaldiagnostic device104 including the layers illustrated inFIG. 11A combines with the signal processing lay186, thecontroller layer188, and the input/output layer122 for performing and reporting the results of the DNA assay described below in further detail.
• FIGS. 12A to 12G next illustrate the steps of a method for detection of a nucleic acid sequence of interest using the DNA patch.FIGS. 12A to 12G show a simplified diagrammatic cross-sectional view of the fluidic circuit described above.
• More particularly now, the first step is sample collection depicted inFIG. 12A. Blood sample is drawn by the microprobe159 (FIG. 11A). A wide range of sample sizes, from 1 μl to 200 μl, and preferably 5- to 10 μl can be collected. The blood sample follows the fluidic circuit to the metering reservoir orchamber304 of pre-determined volume. When the blood level in themetering chamber304 reaches a pre-determined volume, the connection between themicroprobe159 and themetering chamber304 is closed using a pinch or melt valve as shown and described below. A liquid sensor may be situated in themetering reservoir304 such that when blood reaches that sensor, a signal is sent to theprocessor142 which in turn closes the pinch or melt valve as described below in conjunction withFIGS. 13A and 13B. Blood from themetering chamber304 is prevented from entering theRBC capture chamber306 by a closed valve. This valve may be a dissolvable plug,FIG. 13E, that dissolves when it comes in contact with a liquid or a melt plug324 (as shown).Melt plug324 is made of a material that melts when heated such as wax.Plug324 is preferably formed from inert biocompatible materials that melt when heated. Since heat is needed to openplug324, aheating element326 is embedded therein. When the reservoir is filled up to its pre-determined volume, the signal from the above-mentioned liquid also causes the processor to activate theheating element326 which then meltsplug324. This opens the connection between the metering reservoir theRBC capture chamber306 as shown inFIG. 12B and in further detail inFIG. 13B.
• In order to effectively detect a target nucleic acid, it is usually necessary to isolate that nucleic acid from cellular and other specimen debris. It is also known that many target nucleic acids in whole blood are found in specific cell populations, such as in white blood cells (WBCs or leucocytes) as opposed to the red blood cells (RBCs or erythrocytes). RBC capture may be carried out using a solid support matrix, such as a membrane capable of lateral flow that contains a capture reagent for red blood cells. An example of membrane material which could be used is the high density or ultra high molecular weight polyethylene sheet material manufactured by Porex Technologies Corp. of Fairburn, Ga., U.S.A. This membrane has an open pore structure with a typical density, at 40% void volume, of 0.57 gm/cc and an average pore diameter of 1 to 250 micrometers, the average generally being from 3 to 100 micrometers. The membranes are from about 0.1 mm to 5.0 mm in thickness. While membranes made of polyethylene have been found to be highly satisfactory, membranes made from thermoplastic materials, e.g., polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride polyamide, polycarbonate, polystyrene, and similar materials can also be used. A sponge-like matrix material, a three dimensional grid or micro-posts that span the height or width of the RBC capture chamber having attached thereto RBC capture agents such as lectin can also be used to remove RBCs from the blood sample. The RBC-binding reagent is immobilized on the solid support matrix using standard techniques, which are well known to those skilled in the art. The RBC capture agent used to immobilize the RBCs that is contained in the solid support matrix, such as those described above, is typically and most preferably an antibody, polyclonal or monoclonal, which is specific for red blood cells. Alternatively, other reagents which are known to bind RBCs, such as lectins or polymeric amino acids, e.g., polylysine and polyarginine, may also be used.
• With continuing reference toFIG. 12B, the metered blood then enters theRBC capture chamber306, the blood sample is prevented from flowing back into the metering reservoir by a detent pressure valve330 (further described below in conjunction withFIG. 13A). Whenchamber306 is filled, it may be sealed off by amelt valve332, as demonstrated inFIG. 12C showing the melt valve in aclosed position334.Melt valve332 is similar tovalve324 but is used to close fluidic circuits instead of open them. Melt valves can be controlled as discussed above by the processor using theheating element326 wherein the valve is melted into the fluidic circuit effectively blocking portions of the circuit at desired time points during the assay process. Each of the chambers in the fluidic circuit may include avent port340 that allows air within the chamber to escape thereby preventing air blockages within the fluidic circuit.Vent port340 may include afilter342 that prevents contamination of the sample while allowing air to pass through.
• RBCs are separated from the sample withinchamber306. In the illustrated example,RBCs334 are captured onmicro-posts328 coated with a RBC capture agent. After allowing sufficient time for RBC capture, asecond melt plug344 is opened (FIG. 12D) allowing theWBCs338 inchamber306 to flow into the cell lysis andDNA extraction chamber308. The sample containing WBCs is prevented from flowing back intochamber306 by adetent pressure valve330 situated betweenchamber306 and308. The white blood cells which contain genetic material may be moved by capillary action to thelysis chamber308 which contains pre-immobilized lysing reagent. Various lysing procedures are well known to those skilled in the art, including freezing, treatment with digesting enzymes such as proteases (for example, Proteinase K), boiling, and use of detergents (see for example U.S. Ser. No. 178,202, filed Apr. 6, 1988 by Higuchi, and EP-A-0 428 197, published May 22, 1991). By way of non-limiting example, a lysis reagent containing guanidine isothiocyanate and Triton X-100 detergent, and Proteinase K, which facilitates the release of nucleic acid fromlyzed WBCs346 can be used. Double strandedDNA348 is released from the lysedWBCs346 and is extracted from the solution. The extraction of DNA from white cells can be carried out using commercially available magnetic beads that allow extraction of nucleic acids without organic solvents. By way of non-limiting example, Dynabeads DNA Directs that allows for the rapid isolation of DNA from small volumes of blood can be used. In one embodiment hereof, these beads are preloaded into the lysis chamber and upon application or removal of the magnetic field; the DNA is isolated from other cellular materials and eluted (step not shown). After the DNA is extracted or isolated, the solution containing the isolated DNA is then moved into the DNA nicking anddenaturation chamber310 by opening athird melt plug350 as represented inFIGS. 12D and 12E.FIG. 12E also depicts remainingWBC cell membranes352 inchamber308 after the cell lysis and DNA isolation step described above. The double strandedDNA348 is sheared or nicked inchamber310 into short double stranded DNA fragments354 which are then denatured into single stranded DNA fragments356.
• The genomic DNA extracted can be sheared by using restriction enzymes which are preloaded in thenicking chamber310. The methodologies for genomic DNA digestions are well known to those skilled in the art. The temperature in this chamber can be controlled viaheating elements316. Alternatively, magnetic beads may be used to promote the shearing of the genomic DNA, thus facilitating target capture. The solution in the nicking chamber containing the double stranded DNA fragments354 may be heated up to between about 65° C. and 75° C. to thereby denature the double strandedDNA354. After the DNA denaturation step, a fourth melt plug358 (FIG. 12E) is opened to allow the sample containing the single strandedDNA fragments356 to enter theanalysis chamber312. Theanalysis chamber312 has nucleic acid capture probes360 which are attached to a capture zone. Each of theprobes360 may be attached to a single capture zone. Anytarget DNA362 in the sample is then allowed to hybridize with thecapture probe360 to thereby “capture” the target nucleic acid in the target zone for detection as illustrated inFIG. 12F and later described in further detail in conjunction withFIGS. 14A to 14G. Capture DNA may be single stranded or partially double stranded near its attachment point to the capture zone. The double strand is located at the reactive end, such as the amino end, of the probe where it is attached to a solid support like the capture zone because a double strand has been found to more effectively project the capture probe erectly or upwardly from the active layer as compared to ssDNA in some instances. An extension or spacer including, for example, ssDNA and PEG (polyethylene glycol), may be used to extend the probe further from the surface so as to increase the hybridization efficiency of the capture probe with the target. Furthermore, to prevent the capture probes from collapsing onto the solid phase and to prevent the target and non-target nucleic acid sequences from binding non-specifically onto the capture zone, the capture zone surface can be treated with standard blocking buffers well known to those skilled in this particular art. The sequence of the capture DNA is selected so as to hybridize directly with target DNA or RNA, thereby forming a partially double stranded complex364 comprising capture DNA, target DNA or RNA (FIG. 12F).
• The length of the capture probes can be from 15 to 70 bases long, preferably 25 to 40 bases long. The attachment of DNA probes to the solid support in the capture zone can be achieved by covalent or non-covalent attachment strategies, which are well known to those skilled in this art.
• Probe density is a critical factor for the efficiency of target capture as well as the kinetics of the target and probe hybridization. According to studies reported on the effect of surface probe density on DNA hybridization (Nucleic Acid Research, 2001, Vol. 29, number 24, pages 5163-5168), the probe density strongly affects the target hybridization efficiency. The hybridization efficiency is optimal when the probe density is below 2×10¹²probes/cm², preferably 1×10¹¹probes/cm²to 1×10¹²probes/cm².
• The capture probes can be immobilized on the nano-wires directly in the format of acapture unit371,FIG. 14B. Eachcapture unit371 may consist, for example, of one hundred 5 nm diameter nano-wires for an area of 50 nm×50 nm, as illustrated inFIG. 14B. The probe density on each capture unit may vary from 1 to 100 probes per capture unit, and preferably about 10 capture probes/capture unit of area 50 nm×50 nm. The total number of capture units per reaction zone in the assay area may vary from 10⁶to 5×10⁶, and preferably about 10⁶. The capture units may be distributed over an area from about 10⁻⁴cm²(for example a square of dimensions 0.1 mm by 0.1 mm) to about 10⁻²cm²(1 mm by 1 mm), and preferably about 10⁻⁴cm². The spacing between the capture units on the capture area can vary from none to 10 as represented inFIG. 14B. The total number of capture probes in theentire reaction zone312,FIG. 11A, may range from about 10⁶to about 5×10⁷, and preferably on the order of about 10⁷.
• When target RNA or DNA of a specific sequence is present in the test sample, the target RNA or DNA hybridizes with the capture DNA. In this manner, the target DNA is retained within thereaction zone312, as discussed above. The hybridization step can be performed at room temperature or higher in any standard buffer well known to those skilled in the art. By way of non-limiting example, a standard buffer containing 5×SSC, 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS, 1% Blocking Reagent can be used for this purpose. Hybridization may be further facilitated by heating the analysis orreaction chamber312. The hybridization procedure can be performed from a few minutes up to several hours.
• With reference now toFIG. 12G, there is shown the last steps of the analysis method. After hybridization, thereaction zone312 may be washed to get rid of any unattached single stranded nucleic acid sequences. The wash process may be initiated by a time-controlled opening of afifth melt plug367 connecting thewash reservoir316 to the analysis chamber312 (FIG. 12F). Wash buffer366 stored in thewash reservoir316 is released into the analysis chamber, as shown, to wash the capture zone of unbound DNA or RNA. The wash can be performed at high temperature to minimize or eliminate any non-specific binding. A fifth melt plug368 (FIG. 12F) is opened allowing the wash buffer and sample to be emptied into the waste chamber314 (FIG. 12G) removing unbound nucleic acid sequences from theanalysis chamber312.
• With continuing reference toFIG. 12G, an alternate embodiment of the fluidic circuit described in conjunction withFIGS. 12A to 12F is shown. In the embodiment ofFIG. 12G, the vents from each chamber are interconnected as shown. Quantification of the complex364 can be carried out by image analysis, pattern recognition, or optical interference as presented in Table 1 above. The required resolution to distinguish the double stranded DNA resulted from target capture from the single stranded capture probe can be achieved using the nano-wire technology of the present invention described below in more detail connection withFIGS. 14A to 14G.FIG. 12G also illustrates nano-wire bundles associated with each capture probe used as a light source and a detector. As shown, the nano-wire bundle on the bottom emits a focused light through the capture or reaction zone containing a single capture probe or complex364, since complex364 is significantly larger than the single stranded probe, the optical motion or the surface interrogation pattern of the double strandedDNA364 as detected by the nano-wire detectors will be distinct from those of a single probe. This difference can thus be detected and the number ofcomplexes364 quantitated.
• Referring now toFIGS. 13A to 13E, there are shown several flow control elements and related methods as employed herein to direct and control fluid flow in a timed and pre-determined manner as may be centrally controlled by thesignal processing unit142 and thelogic controller190 illustrated, for example, above inFIGS. 7B and 11B. More specifically,FIG. 13A is an enlarged detailed diagrammatic representation of thedetent pressure valve330 utilized in the fluidic circuits of the present invention to control the flow of fluids. As illustrated thedetent pressure valve330 includes a pair ofdetent lips331 which are each provided with a pre-stressed to hold them together in a normally closed position as shown inFIG. 13A-I. When the down stream pressure in the flow channel is less that the pressure upstream of the pair ofdetent lips331, and the pressure difference therebetween is greater than the pre-stressed force holding thedetents331 in a normally closed condition, thedetents331 will open and allow flow as illustrated inFIG. 13A-II. After the pressure normalized up stream and down stream of thedetents331, the pre-stress force will close the valve as illustrated inFIG. 13A-III. As further illustrated, thedetents331 are shaped to prevent back flow in the channel.
• FIG. 13B is a schematic presentation of a normally closedmelt valve332 employed in the fluidic circuits of the present invention to control and direct the flow of fluids therein. The normally closedmelt valve332 includes theheating element326 positioned in themelt plug324. Upon receiving a signal from thesignal processing unit142 and/or thelogic controller190 illustrated, for example above inFIGS. 7B and 11B, theheating element326 receives a charge of current, heats to a predetermined temperature, and then melts themelt plug324 to thereby open the channel and allow flow therein.
• FIG. 13C is a schematic representation of a normallyopen melt valve332 used in the fluidic circuits of the present invention to direct and control fluid flow. The normallyopen melt valve332 includes theheating element326 positioned in themelt plug324. As illustrated, themelt plug324 is positioned above the flow channel to normally allow flow. Upon receiving a signal from thesignal processing unit142 and/or thelogic controller190, theheating element326 receives a charge of current, heats to a predetermined temperature, and then melts themelt plug324 to thereby close the channel and prevent further flow therein.
• FIG. 13D is a schematic presentation of another normally open melt valve orpinch valve332 used in the fluidic circuits of this invention to control fluid flow. In this embodiment, theheating element326 is wrapped around the flow channel as illustrated. The flow channel is made of a material that contracts when heated. Thus when theheating element326 receives a signal from thesignal processing unit142 and/or thelogic controller190, theheating element326 receives a charge of current, heats to a predetermined temperature, and then causes the channel to collapse and contract to thereby close the channel and prevent further flow therein.
• FIG. 13E is a diagrammatic representation of a dissolvable plug used to form a timed fluid valve shown closed, partially dissolved, and then open to allow flow. In this embodiment, a biocompatible material that dissolves upon contact with a fluid is pre-loaded in the channel which is dry until use of thedevice104 in an intended manner. Upon use of the device, a fluid such as blood, sweat, or interstitial cell fluid (ICF) from the user is introduced into the channel. Once the dissolvable plug is in contact with the fluid in the channel, the plug begins to dissolve. With proper selection of the composition of the plug material, the dissolvable plug may be designed to dissolve after a pre-determined time duration starting from the point of first contact by fluid in the channel. In this embodiment, fluid control is passively achieve, rather than actively achieved by intervention from thesignal processing unit142 and/or thelogic controller190 as in the case of the flow elements discussed above in connection withFIGS. 13B,13C, and13D.
• Turning now toFIGS. 14A to 14K, there are shown several detailed isolation views of semi-conductor nano-wire and micro-wire assemblies utilized in various embodiment of the present invention as light emitters and detectors for illuminating, detecting, and imaging assay results obtained in the reaction zones or capture zones of the personal diagnostic devices hereof.
• Generally, the inventors hereof propose that semi-conductor nano-wires and micro-wires represent a unique material system for investigating low dimensional biochemistry. Such nano-wire elements, components, and systems are anticipated to play increasingly important roles as both interconnects and functional device elements in nano-scale electronics, optoelectronics, MENS, bio-MENS, and nano-scale bio-chemical systems. Such biological and bio-chemical applications are utilized herein. These small wires are made from semi-conductor material. They include silicone (Si) that is doped with all known, traditional, or newly created or discovered dopants to produce different various desired semi-conductor materials. The nano-wire may be used as light tubes or optical fibers with a diameter as small of 5 nm. The wires can conduct current, produce laser light, or detect incident light. The inorganic semi-conductor nano-structures may be custom fabricated with virtually any desired composition, size, shape, crystal structure, doping, and surface chemistry characteristics. The semi-conductor nano-structures currently being developed commercially include three major categories: nano-dots, nano-rods, and nano-wires. In addition, it has been reported that more complex shapes, such as cones, teardrops, and tetra-pods are also possible.
• As utilized herein for certain applications, the nano-wires may be employed as nano-piercing probes for intracellular illumination and/or detection. Additionally, the nano-wires and micro-wires may be prepared by chemically treating and functionalizing the surfaces thereof. In this manner, the need for performing the assays in a separate reagent layer or capture layer is eliminated. The nano-wires can be made of a semi-conductor material that is electrochemically responsive. That is, if a chemical reaction takes place, be it exothermic or endothermic, the nano-wire will detect a change in local temperature and measure the heat absorbed or produced. Also, a change in electrical conductivity in an electro-chemical results assay may be detected by a nano-wire or micro-wire implemented as an electrode and then converted to an electrical signal which is processed by theprocessor142 and/or thelogic controller190 illustrated inFIGS. 7B and 11B to thereby produce a readable results display on thevideo display monitor124 or the fixed results displaywindows126 illustrated inFIGS. 2-6 andFIG. 7B.
• FIG. 14A shows 5 by 5 a bundle of nano-wires365 comprised of individualsemi-conductor wires369 on the nano-scale or micro-scale in diameter. For convenience, the individualsemi-conductor wires369 hereinafter are alternatively referred to as either “nano-wires369” or “micro-wires369” depending on the specific application.
• As discussed above, the 5 by 5bundle365 may be employed as acapture unit371,FIG. 14B. The DNA capture probes360 in the assay discussed above are immobilized on the nano-wires directly to form thecapture units371 as illustratedFIG. 14B. Thecapture units371 may include any number of individual nano-wires369 in any preferred geometric arrangement and any desired number and placement of DNA capture probes360 as illustrated inFIG. 14C. Eachcapture unit371 may consist of, for example, one hundred 5 nm diameter nano-wires for an area of 50 nm×50 nm, as illustrated inFIG. 14B and preferably utilized in the above DNA assay. The spacing between thecapture units371 as arranged in the assay reaction zone may be of any desired or preferred density or distribution. As illustrated inFIG. 14D the individual nano-wires369 may be individually addressable so that a signal from each nano-wire may be processed by thesignal processor142 and/or thelogic controller190.FIG. 14D shows an emitter unit and a detector unit each formed by a bundle of nano-wires. In this configuration, the individual nano-wires369 in the emitter unit are enabled to emit an incident beam of light or electro-magnetic energy373 of a pre-determined wave length. As further illustrated, individual nano-wires369 in the detector unit are enabled to detect theincident beam373 and generate an output signal associated therewith. When there is an absence of any matter positioned between the emitter unit and the detector unit, theincident energy373 on the detector unit may be converted to a baseline output signal in the form of a steady state flat output as illustrated in the graph ofFIG. 14E.FIG. 14F shows the nano-wire emitted unit with aDNA capture probe360 secured in the center thereof. Thus according to one aspect of this invention, whenincident energy373 is applied to the emitter unit by a directed input signal there will be a light/matter interaction between some of the incident electro-magnetic energy373 and theDNA capture probe360 to thereby produce electro-magnetic energy that has been modified by this light/matter interaction. Thus a modified electro-magnetic beam375 will be detected by some of the individual nano-wires369 in the detector unit as illustrated inFIG. 14F. Thus the output signal generated by the nano-wire detector unit will include a perturbation in the baseline signal as illustrated in the graph ofFIG. 14F. After hybridization between arespective capture probe360 and acomplementary target probe360, a double stranded segment of DNA is formed in the area of thecapture unit371 as illustrated inFIG. 14G. The scientifically determined and reported dimensions of a segment of double stranded DNA are shown inFIG. 14H. Since the nano-wires may be made as small as at least 5 nm and the dimensions of a segment of double stranded DNA are on the same order,FIG. 14H, there will be a detectable difference between the output signal generated by the light/matter interaction associated with ssDNA and that associated with double stranded DNA as represented by a comparison for the graphs ofFIGS. 14F and 14G.
• FIGS. 14I and 14J are views respectively similar toFIGS. 14F and 14G. In this embodiment, the individualsemi-conductor wires369 are of a diameter on the order of microns, preferably 1 to 5 microns for the intended cellular detection and imaging applications illustrated inFIGS. 14F and 14G, and in further detail herein below. The μ-wire emitter unit shown inFIG. 14I similarly produces incident electro-magnetic energy373 which may be detected by the μ-wire detector unit to generate a baseline output signal as illustrated. InFIG. 14J, a capturedcell406 is shown bound specifically to the μ-wire emitter unit. As illustrated, the μ-wire emitter unit emitsincident energy373 some of which interacts with the capturedcell406 to produce modified electro-magnetic energy375. Both theincident energy373 and the modified electro-magnetic energy375 are detected by the μ-wire detector unit to produce an output signal indicative of the capturedcell406. Since blood cells, for example, are of a size on the order of microns, (RBCs about 3×5 μm and WBCs about 8 to 15 μm) the sizing of the individualsemi-conductor wires369 on the order of microns is suitable for the purposes intended in the present cellular assays discussed below in further detail.
• InFIG. 14K, there is shown a capturedcell406 with anucleus407. As a further application of the micro-wires described herein, it is proposed that micro-wires may be designed as piercing μ-probe emitters and piercing μ-probe detectors that penetrate the cell wall and are thus employed to obtain signal information derived by intracellular illumination and detection. In this manner, the piercing μ-probe emitter producesincident energy373 which is modified by interaction with, for example, thenucleus407 to produced modified electro-magnetic energy375 which in turn is detected by a corresponding piercing μ-probe detector to produce an output signal that may be converted to an image as illustrated. In this manner, useful intracellular information such as the morphology of thenucleus407 may be obtained by the methods and apparatus so described.
Cellular Assay Implementations and Methods
• The immune system is made up of cells and organs that protect the human body from outside invaders such as bacteria, viruses, fungi, and parasites (germs) that can cause infection, disease, and even death. The immune system also eliminates abnormal cancerous cells that are growing out of control. When functioning properly, the immune system fights off infection and keeps a person healthy. But when it malfunctions, germs that enter the body can more easily cause disease or death. Two very important cell types that play a significant role in the immune system are Helper T cells (also known as CD4 cells) and suppressor T cells, known as CD8 cells. The human acquired immune deficiency syndrome (AIDS) is characterized by a depletion of Helper T cells (CD4 bearing cells or CD4+ cells). Upon infection with HIV, CD4+ T-Helper cells are rendered non-functional and become depleted. The depletion of these CD4+ lymphocytes leads to immunosuppression, with the patient becoming susceptible to a wide range of opportunistic infections and malignancies.
• The ratio of the CD4+ and CD8+ cells, and the absolute counts of CD4+ T Helper cells are the current standard used to monitor the progression of the HIV infection to AIDS. During the course of an infection, the number of CD8+ cells remains constant, while the number of CD4+ cells present in the blood fall precipitously. The ratios of CD4+/CD8+ and the absolute CD4+ count in patients who are treated with pharmacological agents for the virus can be an important indicator of whether their system has developed drug-resistant viral strains.
• Currently, a measurement of the CD4+/CD8+ ratio and the absolute CD4+ count requires the use of a high-priced cell sorting machine, or cytometer. Furthermore, specialized technical personnel with a high degree of expertise are required to operate the cytometer. Therefore, the development of portable, easy to use, and inexpensive diagnostic devices, which are capable of quantifying the CD4+/CD8+ ratio and absolute CD4+ counts in real-time, is of particular interest.
• The personaldiagnostic device104 of the present invention may be used for cellular assays such as CD4+ and CD8+ cellular analyses. The cellular analysis device is also herein referred to as a cellular patch or bracelet which could provide a quick, easy-to-use, and inexpensive method for diagnosing and monitoring the progression of an HIV infection using only a simple patch or bracelet that interfaces with a personal computer.
• Referring now toFIG. 15A, there is depicted an exploded perspective view of asample acquisition layer116,fluid processing layer118,results detection layer178, and washbuffer reservoir layer370 as employed in the cellular patch embodiment of the present invention. The fluidic circuit of the cellular patch may include asample metering chamber304, andRBC capture chamber306, ananalysis chamber312, awaste chamber314, and washbuffer reservoir316. Each of these reservoirs are operatively connected to each other and may include one ormore vents340 to prevent air blockages with the fluidic circuit as described above. The connections and flow between each chamber may be controlled by melt plugs, detent pressure valves, melt valves, and pinch valves in any desired combination of the valves described above in conjunction withFIGS. 13A-13E that allows controlled movement of the samples and buffers between the different chambers. In one particular embodiment, thecapture zone312 may be provided withseveral waste cambers314 disposed around the periphery thereof to aid in the collection of waste fluids.
• FIG. 15B is an exploded perspective isolation view of thewash buffer reservoir316,micro-wire detector322, and capturezone area312. As illustrated, thewash buffer reservoir316 is equipped with dispensingnozzles377 withheating elements326 that may be activated upon a specific command to open thenozzles377 and thereby release wash buffer into thecapture chamber312. In this particular embodiment, the is provided with pass through holes so that thenozzles377 may pass there through and direct released wash buffer directly into thecapture camber312.FIGS. 17A and 17B show in greater schematic detail the arrangement of the micro-wires369, the pass throughholes379, and the capturedcells406 in the micro-wire emitter anddetector assemblies320 and322 utilized according to this embodiment of the present invention. As represented inFIG. 17B, the micro-wires369 may be of different diameters depending on the application and desired wave length.
• FIG. 15C is a perspective view with cut-away sections showing a fully assembled personal diagnostic device including the layers illustrated inFIG. 15A for performing a cellular assay. In this view, there is thus shown the minimally invasive tubules, lancets, or micro-probes159,reservoir openings164,blood metering chamber304, theRBC capture chamber306, themicro-wire detector320, thecapture zone312, analternative waste chamber314, themicro-wire detector322, thewash buffer reservoir316, thesignal processor142, thecontroller190, the outputvideo display monitor124, and the individual fixed-display results windows126.
• Referring next toFIGS. 16A-16F, there is shown a series cross-sectional side views of the fluidic circuit ofFIG. 15 illustrating a method for detection and quantitation of CD4+ and CD8+ cells using the cellular patch. More specifically,FIG. 16A depicts acquisition of a blood sample containing RBCs and WBCs into themetering chamber304. Blood sample is withdrawn viamicroprobes159. A wide range of sample size, from 1 μl to 200 μl, to preferably about 5-10 μl can be collected. Oncechamber304 if filled to a pre-determined volume,pinch valves372 are closed (FIG. 16B). The melt plug between themetering chamber304 and theRBC capture chamber306 is opened (FIGS. 16B and 16C) allowing the blood sample to move into theRBC capture chamber306 where theRBCs336 are captured. In this illustration, a mesh that allows cells to pass through is coated with a RBC capture agent. RBCs are thus captured on the mesh as illustrated.
• Separation of White Blood Cells from Red Blood Cells:
• RBCs are captured onto a solid support matrix, such as a mesh or a membrane capable of lateral flow that contains a capture reagent for red blood cells. An example of membrane material which could be used is the high density or ultra high molecular weight polyethylene sheet material manufactured by Porex Technologies Corp. of Fairburn, Ga., U.S.A. This membrane has an open pore structure with a typical density, at 40% void volume, of 0.57 gm/cc and an average pore diameter of 1 to 250 micrometers, the average generally being from 3 to 100 micrometers. The optimum pore diameter for the membrane for use in this invention is about 5 to about 20 μM. The membranes are from about 0.1 mm to 5 mm in thickness. While membranes made of polyethylene have been found to be highly satisfactory, membranes made from thermoplastic materials, e.g., polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride polyamide, polycarbonate, or polystyrene can also be used.
• The RBC-binding reagent is immobilized on the solid support matrix using standard techniques well known to those skilled in the art. The reagent which is capable of binding red blood cells that is contained in the solid support matrix, such as those described above, is typically and most preferably an antibody, polyclonal or monoclonal, which is specific for red blood cells. Alternatively, other reagents which are known to bind red blood cells, such as lectins or polymeric amino acids, e.g., polylysine and polyarginine, may also be used.
• After the RBCs are removed from the sample, amelt plug374 between the connection ofchamber306 andanalysis chamber312 is melted allowing thesample containing WBCs338 to enter analysis chamber312 (FIG. 16D).Chamber312 contains immobilized capture agents against CD4+ and CD8+ cells which are preferably antibodies (as illustrated) but may include antibody fragments and other substances that specifically bind to CD4 or CD8 markers. Antibodies may be immobilized on a solid phase by a variety of methods known in the art, including covalent coupling, direct adsorption, physical entrapment and attachment to a protein-coated surface. For references describing this methodology, see Silman, I. H. and Katchalski, E. in Annual Review of Biochemistry, Vol. 35, p. 873 (1966); Melrose, G. J. H., in Review of Pure and Applied Chemistry, Vol. 21, p. 83, (1971); and Cuatrecasas, P. and Anfinsen, C. B., in Methods in Enzymology, Vol. 22, (1971). The method of attachment to a protein-coated surface is disclosed by Lai et al. (German OS No. 2,539,657; U.S. Pat. No. 4,066,512).
• Specific antibodies against CD4, CD8 may be immobilized on discreet areas within theanalysis chamber312 to capture CD4+ T cells and CD8+ T cells. Positive and negative control zones may also be included withinchamber312. Cell binding in the negative control detects non specific binding. The capture antibodies can be produced in vivo or in vitro. Methods for the production of antibodies are well known to those skilled in this particular art. For example, see Antibody Production: Essential Techniques, Peter Delves (Ed.), John Wiley & Son Ltd, ISBN: 0471970107 (1997). Alternatively, antibodies may be obtained from commercial sources, e.g., Research Diagnostics Inc., Pleasant Hill Road, Flanders, N.J. 07836 and Ortho Diagnostic Systems).
• After allowing sufficient time for cells to bind with their respective capture agents, theanalysis chamber312 is then washed to remove unbound cells. Washing is performed by flushingchamber312 with a wash buffer. As depicted inFIGS. 16D and 16E, melt plugs274 betweenreservoir316,capture chamber312, andwaste chamber314 are opened and wash buffer fromreservoir316 is moved intochamber312 which carries unbound cells intowaste chamber314. The wash process can be initiated by a time-controlled opening of the melt plug connecting thereservoir316 tochamber312, as well as the melt plugs connectingchamber312 to thewaste chamber314.
• Once the unbound cells are removed (FIG. 16F), the number of CD4+ and CD8+ cells captured can be quantified by image analysis. The program algorithm counts the number of cells in the CD4+ and CD8+ capture zone, calculates the ratio of CD4+/CD8+ and the absolute counts using the volume of whole blood metered prior to red blood cell capture.
Small Molecule Assay Implementations and Methods
• The quantification of chemical and biochemical components in colored aqueous fluids, in particular colored biological fluids such as whole blood and urine and biological fluid derivatives such as blood serum and blood plasma, is of ever-increasing importance. Important applications exist in medical diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, hazardous chemicals, and the like. In some instances, the amounts of materials being determined are either so miniscule (in the range of a microgram or less per deciliter) or so difficult to precisely determine that the apparatus employed is complicated and useful only to skilled laboratory personnel. In this case the results are generally not available for some hours or days after sampling. In other instances, there is often an emphasis on the ability of lay operators to perform the test routinely, quickly and reproducibly outside a laboratory setting with rapid or immediate information display.
• One common medical test is the measurement of blood glucose levels by diabetics. Diabetes is a major health concern, and treatment of the more severe form of the condition, Type I (insulin-dependent) diabetes, requires one or more insulin injections per day. Insulin controls utilization of glucose or sugar in the blood and prevents hyperglycemia which, if left uncorrected, can lead to ketosis. On the other hand, improper administration of insulin therapy can result in hypoglycemic episodes, which can cause coma and death. Hyperglycemia in diabetics has been correlated with several long-term effects of diabetes, such as heart disease, atherosclerosis, blindness, stroke, hypertension, and kidney failure.
• Current teaching counsels diabetic patients to measure their blood glucose level from two to seven times a day depending on the nature and severity of their individual cases. Based on the observed pattern in the measured glucose levels, the patient and physician together make adjustments in diet, exercise and insulin intake to better manage the disease. Clearly, this information should be available to the patient immediately.
• Conventional blood glucose monitoring methods generally require the drawing of a blood sample (e.g., by finger prick) for each test, and a determination of the glucose level using an instrument that reads glucose concentrations by electrochemical or colorimetric methods. Type I diabetics must obtain several finger prick blood glucose measurements each day in order to maintain tight glycemic control. However, the discomfort and inconvenience associated with this method of measurement, along with the fear of hypoglycemia, has lead to poor patient compliance, despite strong evidence that tight control dramatically reduces long-term diabetic complications. Accordingly, there remains a need in the art for medical devices which provide effortless analyte measurements in field or home-testing applications, particularly where continual and/or automatic monitoring is desired.
• Now with reference toFIGS. 18A and 18B, there is shown exploded and cutaway views of the personaldiagnostic device104 of the present invention for detection of small molecules herein referred to as the small molecules patch. This bio-patch provides a quick, effortless, and automatic device and method for analyte quantification using only a simple patch or bracelet that interfaces with a personal computer. The small molecules patch may include asample acquisition layer116, asample processing layer380, and anelectrochemical detector layer382 or alternatively, (or in combination therewith), aphoto detector layer384. Sample processing is carried out in a biocompatible lateral flow matrix preloaded with analysis reagents as described in further detail blow with reference toFIGS. 19A and 19B.
• Thesample processing layer380 includes a particularmicro-fluidic circuit227 with individual fluid components that will be described in detail below. Thesample processing layer380 may include onemicro-fluidic circuit227 or may alternatively include severalmicro-fluidic circuits227 formed therein as represented in broken-line fashion by the four referencedreservoir openings164 and their respective firstfluid channels168. Alternatively, thedevice104 may include several sample processing layers380 each with a single or a multiple number ofmicro-fluidic circuits227. In this manner, several blood samples may be taken over time by using flow control at the firstfluid channel168 as described with reference toFIGS. 13A to 13E. Each of these separate blood samples may then be processed in real time in a separatemicro-fluidic circuit227. For example, thedevice104 may include 12micro-fluidic circuits227 either all formed and situated on onesample processing layer380, or each formed and situated on separate individual sample processing layers380 for a total for 12 such sample processing layers. As understood, the 12 circuits would be preferably fluidly isolated from each other. Alternatively, the 12micro-fluidic circuits227 may be formed and situated in pairs, groups of three, or groups of four, for example, with each pair or group then being formed on onesuch layer380. The various layers are then assembled accordingly to function as intended. In this specific embodiment, each of the 12micro-fluidic circuits227 would be fluidly connected to arespective lancet159 as discussed above. With blood sampling and fluid control directed and timed by thecontroller142, a melt plug associated with eachlancet159 may be activated at a specific time to put arespective metering chamber304 in fluid communication with itsrespective lancet159 to thereby draw a fresh sample of blood by capillary action at a specific predetermined time. With 12circuits227 and 12corresponding lancets159, for example, a user may wear thebio-patch device104 for a twenty-four hour period with a blood sample taken every two hours.
• Themicro-fluidic circuit227 includes theblood metering chamber304, a separateblood separator zone385 and acapture zone312. In some implementations of this embodiment, the separateblood separator zone385 and thecapture zone312 may be combined into a single zone or chamber with serial processing of the sample fluid through a biocompatible lateral flow matrix or membrane which may be comprised of separate pads or segments, or alternatively comprised of a single lateral flow matrix provided with various gradations for different fluid processing results along the length thereof. InFIGS. 19A and 19B, theblood separator zone385 and thecapture zone312 are combined into a single membrane ormatrix386 including three lateral flow pads.
• In the glucose assay implementation hereof, the bio-patch or bio-bracelet may advantageously include a pre-loaded supply reservoir ofinsulin395,FIGS. 18A and 18C. Here software stored in thelogic controller190 orsignal processor142 is interactive to direct a control system to release a determined dose of insulin. To release the insulin, a supply duct may be provided with a one-way valve of the flexible detent lip type described above. Also a plug will be used to hold the insulin in the supply reservoir. This plug is not dissolvable upon contact with liquid because it needs to hold the insulin in the supply reservoir until directed by the control system to release the dosage in the reservoir. Here we use an electric signal generated by the control system to send a current to the plug which is pre-wired with a resistive element and designed to melt when current is supplied to the resistive element embedded in the plastic type plug material. In this manner, the insulin form the supply is released in a controlled, timed fashion. Metering the release flow and closing the channel or supply duct may also be achieved. The dosage is preferably controlled by the size of the supply reservoir wherein a simple discharge of the entire volume from the reservoir is initiated when needed. The reservoirs can be of different volumes and the desired volume selected by the control system by sending the melt current to the reservoir with the correct volume.
• With continuing reference toFIG. 18A, thephoto detector layer384 includes theinsulin reservoir395 which may be pre-loaded with a predetermined amount of insulin according to the time-release aspects of this invention. Theinsulin reservoir395 may have one pre-loaded reservoir or alternatively it may be subdivided into several smaller reservoirs each having a pre-determined amount of insulin stored therein for time-release application. According to this aspect of the present invention, a prescribed dosage of insulin, for example, is released from theinsulin reservoir395, directed to a respective supply lancet, tubule, or micro-probe397 through suitable fluid circuits, and then thereby caused to enter the blood stream of the user.FIG. 18C shows in greater schematic detail this aspect of the present invention. As shown, theinsulin reservoir395 is provided with the dispensingnozzles377 that are opened by a command from thecontroller142 by use of theheating elements326 as described above in regard toFIG. 15B. Through suitable supply fluid circuits, a pre-determined dose of insulin is thereby administered to the user of the device. Thelogic controller190 may include expert software that determines the insulin needs of the patient and then administers a proper dosage of insulin as based on the detected results of the glucose assay. As would be readily apparent to one of skill in these arts, the device so described herein is not specifically limited to conducting glucose tests and administering a determined amount of insulin as based on those tests, but may be readily applied and adapted to a wide verity of different assays and hormone or drug release therapies or treatments.
• FIG. 18B is a perspective view with cut-away sections showing a fully assembled personaldiagnostic device104 including the layers illustrated inFIG. 18A for performing a small molecule assay. In this view, there is thus shown the minimally invasive tubules, lancets, or micro-probes159, thereservoir openings164, theblood metering chamber304, theinsulin reservoir395, thesupply lancets397, thesignal processor142, thecontroller190, the outputvideo display monitor124, and the individual fixed-display results windows126.
• Referring now toFIG. 19A, there is illustrated a cross-sectional side view showing theblood metering chamber304 and the biocompatible lateral flow matrix ormembrane386 which is divided into three sections including acell separation pad388, areaction pad390, and anadsorbent pad392. Blood sample is withdrawn via themicroprobe159 at specified time intervals throughout the life time of the small molecules patch. There may be multiple collection sites and/or multiple inlet ducts as described above. Also as described above, there may be several microprobes to extract blood into the micro-fluidic circuit in the patch. For purposes of clear explanation here in regard toFIGS. 19A and 19B, the present assay will be described with reference to one such fluid circuit.
• Preferably about 5-10 μl of blood is withdrawn at specified time intervals. Themicroprobes159 are preferably coated with anticoagulant to prevent blood clotting. The micro-fluidic circuit may include a reservoir with programmable valves to flush out any old blood accumulated at the tip of the microprobes as described in further detail below in conjunction withFIGS. 20A and 20B. With continuing reference toFIG. 19A, blood enters themetering chamber304 which is closed when the amount of blood reaches a desired or pre-determined level. The circuit may be closed using thepinch valve372. The metered blood is then allowed to enter thecell separation pad388 by openingmelt plug374.
• For electrochemical detection method, glucose can be measured directly from whole blood in themetering reservoir304 where appropriate electrodes, shown schematically inlayer382 ofFIG. 18A, are placed in contact with the blood sample to measure glucose levels. In this case, glucose oxidase is preloaded into themetering chamber304 which produces hydrogen peroxide (H₂O₂) in the presence of glucose which is detectable electrochemically.
• However, should optical detection (absorbance, reflectance, or transmittance, for example) be the method of choice for the detection of results, a cell separation step can be included as shown. In this embodiment, thecell separation pad388 separates RBC and WBC from the plasma or serum. In a typical operation, a 5-10 μl blood sample is collected and directed into thecell separation pad388. As the sample migrates throughpad388, the fibrous network material making uppad388 retards the movement of particulate matter, including blood cells, acting to partially remove blood cells before the sample reaches thereaction pad390,FIGS. 19A and 19B.
• All enzymes necessary for the glucose reaction are immobilized or preloaded in thereaction pad390. For the glucose assay, the reaction pad(s) contains glucose oxidase, a peroxidase, and a substrate reagent which is converted by the peroxidase in the presence of H₂O₂to adetectable reaction product391 as illustrated inFIG. 19B. This reaction is presented below. Preferably, the reaction pads are porous, fused polymer substrate membranes having a thickness (after complete penetration of the fluid) of about 125 μm and side dimensions of about 1 mm. The absorption volume of each pad is preferably between about 1 and 2 μl.
Glucose Assay:
• 
• When the method of detection is optical, themicro-wire emitter320 is activated to emitincident illumination373 toward thedetectable reaction product391 as shown inFIG. 19B. After light/matter interaction between theincident energy373 and thedetectable reaction product391, modified electro-magnetic energy is transmitted toward themicro-wire detector322. Optical and/or image analysis is then performed to quantify the results as described above. In the alternate embodiment hereof where the method of detection is electrochemical, the enzyme peroxidase and chromagen substrate are omitted. In this case, the H₂O₂produced is oxidized at a platinum electrode to produce an electric current which is proportional to the sample glucose concentration.
• The amount of hydrogen peroxide produced is quantified either by absorbance (via a chromagen that changes color upon being oxidized as depicted inFIG. 19B or by the change in electrochemical force via the electrodes, for example, as alternatively illustrated inFIG. 18A. The small molecule patch may have an algorithm installed in thelogic controller190 that will result in a beep or beeping type sound should the analyte concentration drop below a prescribed threshold or become higher than a certain level so that the patient can take appropriate action.
• The device hereof may be readily adopted for use in conjunction with other small molecules such as cholesterol, low density lipoproteins or high density lipoproteins, triglycerides, and others. For example, in an assay for total serum cholesterol, the reaction pad(s) will contain cholesterol esterase, cholesterol oxidase, a peroxidase, and a substrate reagent which is converted by the peroxidase in the presence of H₂O₂to thedetectable reaction product391. If the method of detection is electrochemical, the enzyme peroxidase and chromagen substrate are omitted as discussed above.
• As another example, the small molecule patch may be used in determining triglycerides in a blood-fluid sample, in this case the reaction pad will contain lipase, a source of ATP, glycerol kinase, and glycerol-phosphate oxidase, for generating H₂O₂, and the above components for generating thedetectable reaction product391 in the presence of H₂O₂.
Stress Monitoring Implementations and Methods
• In daily life, individuals encounter many sources of stress. The detrimental effects of stress on general health are well documented. Health problems such as heart disease, depression, lack of energy, insomnia, and hypertension have all be related to stress. Thus the inventors hereof propose a personal diagnostic device that records a user's stress level throughout the day. At the end of the day, the user downloads the day's collection of analytical stress information to see at what time of the day the user encountered the highest stress levels.
• In this implementation, a user of the personal stress monitoring device would keep a written or voice recorded journal of the day's activities. For example, simple entries such as “wake up, 6:45 am”; “fix breakfast, 7:30 am”; “drive kids to school, 8:00”; “meeting with boss, 9:45 am”; “argue with co-worker, 10:30 am”; “lunch, noon to 1:30 pm”; “afternoon coffee, 3:15 pm”; “pick kids up from day care, 5:15 pm”; “work out at gym, 5:00 to 7:00 pm”; “fix dinner, 7:30 pm”; “put kids to sleep, 9:00 pm”; “fight with spouse, 9:30 pm”; “do family finances, 10:00 to 11:15 pm”; “watch late night television, 11:30 pm”; and “go to sleep, 12:15 am” would be maintained on a daily basis while under the care of a medical doctor or other health care professional.
• In this manner, a patient and the patient's doctor could determine patterns of daily living that cause the highest stress levels. With counseling and professional intervention, the patient's life style could be revised or adjusted to avoid the daily activities which cause unhealthy levels of stress. If stress levels are abnormally high when interacting with a spouse, for example, marriage counseling may be recommended. If stress levels are particularly high with a certain co-worker or the boss, then the employee's company may be liable for exposure to a hostile environment and then be required to take remedial action.
• Many individuals now have private personal physicians who are available 24 hours a day by phone and email. The stress patch described below may be further provided with a fluid release reservoir that is pre-loaded with some type of prescription drug such as theinsulin reservoir395 described above with reference toFIGS. 18A to 18C. According to one method of use of the present invention, if the user of the stress patch desires medication, the user goes to his desk top computer as illustrated inFIG. 1 and clicks on the Life Patch icon. The PC and related software then download the day's collection of stress data stored in thePDD104 via theRF receiver114. The stress data is then relayed to the private personal physician who then determines that a dosage of the drug is needed by the patient. The doctor then inputs a command to his computer that is transmitted to the patient's computer. The patient's computer in turn sends a command to the user's patch to release a pre-loaded dosage of prescription medication. In this manner, over medication of dangerous or addictive drugs is avoided.
• According to another method of use of the present invention, the above method is modified to remotely control use and dosage of experimental drugs during clinical trials. The use and dosage is thus doctor-controlled remotely by patch interaction, after the doctor analyzes downloaded bio-feedback information. Also, the doctor may monitor remotely the patient's biological and physiological response to the released drug via real-time patch/PC interaction.
• In a similar manner, the inventors hereof propose that implementations of the present invention may be used to monitor and treat various psychiatric disorders such as depression, for example. In this use application, the bio-patch monitors in real time a patient's mental health and reports to the attending physician or psychiatrist when mental health is outside of normal parameters. In this case, the bio-patch or bio-bracelet then reports remotely to the attending physician or psychiatrist who in turn may remotely authorize the patch to release a dosage of anti-depression drugs. This use method according to the present invention may also be advantageously applied to other mental disorders, such as schizophrenia or paranoia, which are treated with strong drugs. Similarly, the doctor controlled drug release aspects of the present invention may be employed to wean drug addicts off of cocaine or heroine. In an alternate implementation of this embodiment, user/PC interaction is not required. Alternatively, the home or hospital environment to which a patient may be confined is equipped with remote RF receivers. The personal diagnostic device according to this aspect of the invention is then is implemented to transmit a continuous or intermittent signal. As the device transmits this reporting signal, collected diagnostic information is thereby wirelessly communicated to the attending physician without the need of any user/PC interaction.
• Returning now to the stress patch implementation of the present invention, it is understood by artisans in the field that the hormone cortisol is produced by the adrenal gland and is released in the body during stressed or agitated states. Thus cortisol has gained widespread attention as the so-called “stress hormone.” This hormone, however, is more than a simple marker of stress levels, it is necessary for the functioning of almost every part of the body. Excesses or deficiencies of this crucial hormone also lead to various physical symptoms and disease states. Cortisol has been studied as a biomarker for many diseases such as Cushing's disease, chronic fatigue syndrome, and fibromyalgia—an autoimmune disorder. Cortisol has also been correlated with stress levels. It has been found that the level of salivary cortisol serves as a biochemical marker for post-traumatic psychological distress disorders and other conduct disorders. Thus, measurement of cortisol levels in the body can be an important diagnostic tool—both in clinical settings and during certain stress-intensive activities. Cortisol levels in the body, however, fluctuate throughout the day with levels being highest in the morning and lowest in the evening. Dietary intake and metabolic imbalances cause further fluctuations. To identify and allow for these different fluctuations, a real-time determination of cortisol levels is needed, without the use of costly and cumbersome laboratory equipment. Because the clinically significant concentration range of cortisol is very small (e.g. about 12.5 to 800 ng/ml), cortisol concentrations are commonly determined via immunoassay techniques.
• For many cortisol detection applications, the development of portable, semi-invasive sensor devices, which are relatively highly-selective and sensitive to the detection of cortisol, and are capable of continuous monitoring cortisol levels in real-time, is of particular interest.
• In view of the above, the inventors hereof propose a personal diagnostic device for monitoring stress markers or indicators such as cortisol. This specific PDD is herein referred to as a stress patch and as implemented provides a quick, effortless, and automatic device and method for cortisol quantification using only a simple patch or bracelet that can interface with a personal computer or RF wired computer network.
• Turning now toFIG. 20A, there is shown an exploded perspective view of the principal fluid sample processing layers of a stress patch according to another preferred embodiment of the present invention. These include thesample acquisition layer116, a separateresults illumination layer117 as used in this particular embodiment, thefluid processing layer118, theresults detection layer178, andreservoir layer379 as utilized to perform stress monitoring. As illustrated, thesample acquisition layer116 includes the minimally invasive tubules, lancets, ormicro-probes159 that penetrate the skin surface to thereby acquire blood samples from capillaries near the epidermis. In this particular embodiment of the personal diagnostic device, themicro-wire emitter320 is employed as the light source for illuminating the assay results and herein is preferably provided in the separateresults illumination layer117. Alternatively, themicro-wire emitter320 employed herein may be preferably integrated with thesample acquisition layer116 as in the embodiments discussed above. Theresults illumination layer117 is provided with thereservoir openings164 with are respectively positioned in register with theinlet formations166 formed around thedischarge end161 of the lancet ormicro-probe159. In this manner, the blood sample is allowed to pass through theresults illumination layer117 and enter the corresponding reservoir opening164 in thefluid processing layer118. Thereservoir layer379 includes andoptional wash buffer316 which is of the type described with reference toFIGS. 15A and 15B, and a drug-release reservoir389. Theresults detection layer178 is provided with themicro-wire detector322 with is of the type described in detail in conjunction withFIGS. 14A-14K and as employed in the device embodiments shown inFIGS. 15A and 18A.
• With continuing reference toFIG. 20A, thefluid processing layer118 includes themicro-fluidic circuit227. This particular embodiment of themicro-fluidic circuit227 is provided with thereservoir opening164, a draw-off reservoir or an initialsample collection chamber394, theblood metering chamber304, theblood separator zone385, aprotein binding zone399, anantibody binding zone401, and thecapture zone312.
• Thesample processing layer118 may include onemicro-fluidic circuit227 or may alternatively preferably include severalmicro-fluidic circuits227 formed therein as represented by the two referencedreservoir openings164 and their respectivefluid channels168. Alternatively, thedevice104 may include several sample processing layers118 each with a single or a multiple number ofmicro-fluidic circuits227. In this manner, several blood samples may be taken over time by using flow control at the firstfluid channel168 as described with reference toFIGS. 13A to 13E. Each of these separate blood samples may then be processed in real time in a separatemicro-fluidic circuit227. For example, thedevice104 may include 24micro-fluidic circuits227 either all formed and situated on onesample processing layer118, or each formed and situated on separate individual sample processing layers118 for a total for 24 such sample processing layers. As understood, the 24 circuits would be preferably fluidly isolated from each other. Alternatively, the 24micro-fluidic circuits227 may be formed and situated in pairs, groups of three, or groups of four, for example, with each pair or group then being formed on onesuch layer118. The various layers are then assembled accordingly to function as intended. In this particular embodiment of the stress patch, each of the 24micro-fluidic circuits227 would be fluidly connected to a respective lancet or micro-probe159 as discussed above. With blood sampling and fluid control directed and timed by thecontroller142, a melt plug associated with eachlancet159 may be activated at a specific time to put a respective initialsample collection chamber394 in fluid communication with itsrespective lancet159 to thereby draw a fresh sample of blood by capillary action at a pre-determined specific time. With 24circuits227 and 24corresponding lancets159, for example, a user may wear thebio-patch device104 for a twenty-four hour period with a blood sample taken every hour.
• Themicro-fluidic circuit227 illustrated inFIG. 20A includes thereservoir opening164, the draw-off reservoir or initialsample collection chamber394, theblood metering chamber304, theblood separator zone385, theprotein binding zone399, theantibody binding zone401, and thecapture zone312. In some implementations of this embodiment, theblood separator zone385, theprotein binding zone399, theantibody binding zone401, and thecapture zone312 may be combined into a single zone or chamber with serial processing of the sample fluid through a biocompatible lateral flow matrix or membrane which may be comprised of separate pads or segments, or alternatively comprised of a single lateral flow matrix provided with various gradations of density, porosity, or material composition for different fluid processing results along the length thereof. InFIGS. 21A-21D, theblood separator zone385, theprotein binding zone399, theantibody binding zone401, and thecapture zone312 are combined into a series of lateral flow pads as described below.
• As illustrated inFIG. 20A, themicro-fluidic circuit227 is provided with the draw-off reservoir orinitial sample chamber394. In this embodiment, the draw-offreservoir394 is associated with a melt plug and vent. The melt plug is controlled by theprocessor142 and/or thecontroller190. At a pre-determined time, the melt plug associated with the draw-offreservoir394 is first activated to place its associatedlancet159 in fluid communication to thereby draw off any blood that may have resided in the lancet from the time of application of the patch to the time theparticular circuit227 is required by the controller. After the initial draw-off of blood, the draw-off reservoir is then fluidly isolated from the circuit by any one of the means described above. In this manner, when the initialsample collection chamber394 is activated by thecontroller190, a draw of fresh blood having real-time or then current biological characteristics of the user is then directed by capillary action into themetering chamber304 as intended. As would be readily understood by those skilled in these arts, given the present disclosure, the draw-off reservoir aspects of the present invention may be advantageously employed in any of the embodiments hereof, and is preferably used in anyfluidic circuit227 that is employed in connection with the time-delayed sample acquisition aspects of this invention.
• Thereservoir layer379 includes the drug-release reservoir389 as shown. The drug-release reservoir389 may be pre-loaded with a predetermined amount of a prescribed drug according to the time-release aspects of this invention. The drug-release reservoir389 may have one pre-loaded reservoir or alternatively it may be subdivided into several smaller reservoirs each having a pre-determined amount of liquid drug stored therein for time-release application. According to this aspect of the present invention, a prescribed dosage of drug, for example, is released from the drug-release reservoir389, directed to the respective supply lancet, tubule, or micro-probe397 through suitable fluid circuits, and then thereby caused to enter the blood stream of the user. As in the embodiment of theinsulin reservoir395 shown inFIG. 18C, the drug-release reservoir398 may be provided with dispensingnozzles377 that are opened by a command from thecontroller142 by use of theheating elements326 as described above. Through suitable supply fluid circuits, a pre-determined dose of liquid drug is thereby administered to the user of the device. Thelogic controller190 may include expert software that determines the drug needs of the patient and then administers a proper dosage of drug as based on the detected results of the stress assay. As would be readily apparent to one of skill in these arts, the device so described herein is not specifically limited to conducting stress tests and administering a determined amount of liquid drug as based on those tests, but may be readily applied and adapted to a wide verity of different assays and drug or hormone release therapies or treatments. As described above, the drug release command may be doctor administered by patch/PC/network/doctor communications, rather than by patient or patch administration.
• FIG. 20B is a perspective view with cut-away sections showing a fully assembled stress patch including the layers illustrated inFIG. 20A for performing stress analysis according to certain aspects of the present invention. More particularly,FIG. 20B shows the minimally invasive tubules, lancets, or micro-probes159, the supply tubule ormicro-probe397, thereservoir openings164, the draw-off reservoir or an initialsample collection chamber394, theblood metering chamber304, themicro-wire detector322, the optional washbuffer wash buffer316, thesignal processor142 in thesignal processing layer186, thecontroller190 in the logic and input/output controller layer188, the outputvideo display monitor124, and the individual fixed-display results windows126.
• Referring next toFIGS. 21A,21B, and21C, there is depicted a series of related cross-sectional side views showing the progression of a sample through thefluidic processing circuit227 of the device illustrated inFIGS. 20A and 20B as implemented to perform stress analysis. In this implementation of the stress patch, blood sample is withdrawn via themicroprobes159 at specified time intervals throughout the life time of the stress patch. There may be multiple collection sites, and/or multiple inlet ducts, and/or multiple reservoirs as described above. Additionally, there may beseveral microprobes159 that extract blood and direct the sample into a singlemicro-fluidic circuit227 in the patch.
• With reference now specifically toFIG. 21A, there is shown a fluidic circuit for analysis of cortisol. The circuit includes melt plugs324,244, and350,detent pressure valves330, meltvalves332, the initialsample collection chamber394, thesample metering chamber304, ventports340 havingfilter material342, and a biocompatible membrane subdivided into different sections including acell separation pad388, a protein capture orbinding pad396, areagent pad400, a capture andsample analysis pad402, and anadsorbent pad392. Reagents required for the assay are preloaded into thereagent pad400. These reagents may include asignal agent398 made from a micro-particle and a target molecule binding agent such as an antibody that binds specifically to cortisol.Capture agents378 having affinity to cortisol are located in theanalysis pad402. Thecapture agents378 are preferably bound to theanalysis pad402 such that when the target molecule cortisol moves through theanalysis pad402 and binds to thecapture agent378, it is held in place by the capture agent for analysis.
• Preferably about 10 to 30 μl of blood are withdrawn at specified time intervals. The microprobes are preferably coated with anticoagulant to prevent blood clotting. When thefirst melt plug324 is opened, blood in themicroprobe159 enters the initial sample collection chamber394 (FIG. 21B). This is performed, as described above, to flush out any old blood accumulated at the tip of the microprobes so as to provide a fresh blood sample for analysis. The next step is opening thesecond melt plug344 to allow fresh blood to enter the metering chamber304 (FIGS. 21B and 21C). Once the sample reaches a desired volume,chamber304 is fluidly isolated by closing melt plugs332. After metering the sample, thethird melt plug350 is opened and the metered sample is allowed to move into the biocompatible membrane (FIG. 21D) where cells are separated from the sample inpad388 and proteins, specifically cortisol binding proteins, are then removed form the sample inpad396.Pad396 is preferably coated with protein capture agents that remove specific proteins from the sample. The sample then continues to move through the biocompatible membrane through thereagent pad400 wherecortisol410 binds to thesignal agent398. This complex then moves into theanalysis pad402 where the capture agents bind to thecortisol410 having attached thereto asignal agent398. An incident beam ofelectromagnetic radiation373 is then directed throughpad402 into adetector322 which measures the modified light transmitted throughpad402. Thelight source320 and thedetector322 utilized in this embodiment of the stress patch may be preferably of the micro-wire emitter and detector types described above. The presence and amount of cortisol is then analyzed by determining the difference between the intensity of the transmitted light before and after the sample is introduced into theanalysis pad402 and comparing the difference with a sample having a know amount of cortisol.
• As described above, the blood or cell separation is mediated via thecell separation pad388 that will separate RBC and WBC from the plasma or serum. In a typical operation, a blood fluid sample (10-30 μl) is withdrawn from the collection tubule and from here is drawn by capillary action through the cell separation pad. As the sample migrates through the filter, the fibrous network material making up the cell separation pad retards the movement of particulate matter, including blood cells, acting to partially remove blood cells before the sample reaches theprotein binding pad396.
• In blood serum, there exist several cortisol binding proteins which can interfere with a cortisol immunoassay. The most commonly cited interfering protein is the protein transcortin (TC). The second step in the cortisol assay is its displacement from the binding proteins. As the plasma or serum sample migrates into theprotein binding pad396 by capillary action. The proteinbinding pad396 consists of a conjugate pad or membrane, where a protein binding agent such as 8-anilino-1-naphthalene-sulfonic acid (ANS) is impregnated; which at low pH will free the serum cortisol from its endogeneous binding protein.
• Because the clinically significant concentration range of cortisol is very small (e.g. about 12.5 to 800 ng/ml), cortisol concentrations are commonly determined via immunoassay techniques. In an alternative embodiment to the sandwich immunoassay and related method described above, the assay may be implemented as a competitive assay between pre-loaded labeled cortisol and the cortisol in the sample for a limited number of binding sites on the antibody coatedanalysis pad412. A variety of labels or signal agents is known and can be implemented in the present invention. Micro-particles may be used as described above, and fluorogenic materials useful in a fluoroimmunoassay (FIA) described in U.S. Pat. No. 3,940,475 to Gross may also be used as an alternate signal agent. Another alternative signal agent includes enzyme labels coupled to antibodies or antigens used to perform an enzyme immunoassay (EIA) as illustrated in U.S. Pat. No. 3,654,090 to Schuurs et al. As used herein, the expression labeled substance, label, marker, tracer, or the equivalent, includes any of those known labels. By way of non-limiting example, enzyme labels such as Horse radish peroxidase or alkaline phosphatase which produce a detectable signal can be used in this assay.
• The biocompatible membrane is preferably formed from conjugate pads or membrane strips that are commercially available. Membrane strips with good release efficiency are preferred which facilitate the migration of labeled cortisol into the analysis pad. By way of non-limiting example, hydrophilic polyethersulfone membranes can be used for this purpose. The movement of serum through this layer will allow the migration of labeled cortisol to the analysis pad.
• As illustrated inFIGS. 21A-21D, impregnated or immobilized on the analysis pad402 (nitrocellulose or borosilicate paper matrix) areantibody capture agents378 againstcortisol410. By way of non-limiting example, antisera raised against cortisol21 hemisuccinate and cortisol3 carboxy methyloxime conjugates have proved suitable and are widely used for direct immunoassays without sample extraction. Antibodies may be immobilized on a solid phase by a variety of methods known in the art, including covalent coupling, direct adsorption, physical entrapment and attachment to a protein-coated surface. For references describing this methodology, see Silman, I. H. and Katchalski, E. in Annual Review of Biochemistry, Vol. 35, p. 873 (1966); Melrose, G. J. H., in Review of Pure and Applied Chemistry, Vol. 21, p. 83, (1971); and Cuatrecasas, P. and Anfinsen, C. B., in Methods in Enzymology, Vol. 22, (1971).
• After capture, the analysis pad may be washed to remove unbound signal agents and cortisol. The wash process can be initiated by a time-controlled opening of a plug connecting awash reservoir316,FIG. 20A, to the analysis pad. In an enzyme assay implementation wherein the signal agent is an enzyme, a chromagen substrate that reacts with the enzyme to produce a detectable reaction may be mixed with the wash buffer. The wash buffer will elute unbound Enzyme-labeled cortisol while concomitantly allowing the bound enzyme to catalyze substrate cleavage. The wash buffer will be drained into the adsorbent pad.
• The amount of enzyme-labeled cortisol can be quantified by optical detection (absorbance, reflectance, transmittance or fluorescence) and from there the concentration of sample cortisol will be derived. The concentration of cortisol will be computed using a pre-established standard curve.
Sports Performance Monitoring Implementations and Methods
• Timely diagnosis of dehydration, heat stroke, or hypothermia is critical for athletes participating in sporting activities, as these conditions may lead to exercise-associated-collapse, which is linked with morbidity and mortality due to excessive physical activity. The two main causes for exercise-associated-collapse are exercise-induced-hyponatremia and cardiac disorders presenting as arrhythmias, cardiac arrest and myocardial ischemia.
• Exercise-induced-hyponatramia is caused by abnormal levels of electrolytes, more specifically a decreased plasma sodium concentration (<136 mmol/L). Some cardiac disorders are attributed to abnormal lactate levels. Indeed, blood lactate levels have been demonstrated to be useful in establishing a diagnosis of acute myocardial ischemia within 3 hours of symptom development (Schmiechen, N.; Han, C. and Milzman, D. Ann Emerg Med 1997; 30:571-577). Furthermore, elevated lactate values in critical care medicine generally mean that tissues are not getting adequate oxygen. Oxygen deprivation of many tissues (e.g. brain, heart, kidney) correlates strongly with morbidity and mortality. Consequently, elevated lactate in many critical settings demands its rapid discovery, an explanation for the oxygen deprivation and rapid therapy to correct it. Therefore, monitoring lactate and oxygen levels may be useful in ruling out acute myocardial infarctions in athletes presenting with exercise-associated collapse.
• Conventional blood analyte monitoring methods generally require the drawing of a blood sample (e.g., by finger prick) for each test, and a determination of the analyte level using specific diagnostic devices by specialized technical personnel. This may be problematic in sporting activities in areas remote from traditional medical facilities. Accordingly, there remains a need in the art for medical devices which provide effortless analyte measurements in field or home-testing applications, particularly where continual and/or automatic monitoring is desired.
• With reference now toFIG. 22A, there is shown an exploded perspective view of the principal fluid sample processing detection layers of a sports patch according to yet another preferred embodiment of the present invention. These include thesample acquisition layer116, thefluid processing layer118, theresults detection layer178 includingelectrodes414, and anotherfluid processing layer118 as utilized to perform sports performance monitoring. As illustrated, thesample acquisition layer116 includes the minimally invasive tubules, lancets, ormicro-probes159 that penetrate the skin surface to thereby acquire blood samples from capillaries near the epidermis. Each pair ofelectrodes414 is assigned to aparticular analysis chamber412 and situated in register therewith as illustrated.
• Themicro-fluidic circuit227 illustrated inFIG. 22A includes thereservoir opening164, the draw-off reservoir or initialsample collection chamber394, theblood metering chamber304, and a series ofanalysis chambers412. Thesample processing layer118 may include onemicro-fluidic circuit227 or may alternatively preferably include severalmicro-fluidic circuits227 formed therein. Alternatively, thedevice104 may include several sample processing layers118 (as represented by the second layer118) each with a single or a multiple number ofmicro-fluidic circuits227. In this manner, several blood samples may be taken over time by using flow control at the firstfluid channel168 as described with reference toFIGS. 13A to 13E. Each of these separate blood samples may then be processed in real time in a separatemicro-fluidic circuit227.
• FIG. 22B is a perspective view with cut-away sections showing a fully assembled sports patch including the layers illustrated inFIG. 22A for performing sports performance analysis according to certain additional aspects of the present invention. More particularly,FIG. 22B shows the minimally invasive tubules, lancets, or micro-probes159 in thesample acquisition layer116, thereservoir openings164 inlayer118, the draw-off reservoir or an initialsample collection chamber394 also inlayer118, theelectrodes414 in theresults detection layer178, the secondfluid processing layer118, thesignal processor142 in thesignal processing layer186, thecontroller190 in the logic and input/output controller layer188, the outputvideo display monitor124, and the individual fixed-display results windows126.
• FIGS. 23A-23E, illustrate a cross-sectional side views showing the progression of a sample through the fluidic processing circuit of the present sports performance monitoring patch, herein after referred to for convenience as the sport patch.FIG. 23A shows typical components of a fluidic circuit including themicroprobe159, afirst melt valve324, the initialsample collection chamber394, asecond melt valve344, ametering chamber304, avent port340 having afilter342, athird melt plug350, adetent pressure valve330,analysis chambers412, andelectrodes414.
• The first step in the analysis is removal of old blood accumulated in themicroprobe159 so as to provide a fresh blood sample for analysis. This step is illustrated inFIG. 23B where thefirst melt plug324 is opened allowing blood to enter the initialsample collection chamber394.Chamber394 is then sealed by closing amelt valve332 in the channel leading into chamber394 (FIG. 23C). Thesecond melt plug344 is then opened to allow blood to enter themetering chamber304. Once the blood in themetering chamber304 reaches a pre-determined amount, the blood flow intochamber304 is cut off by closing the melt valve in the channel leading into chamber304 (FIG. 23D). Thethird melt valve350 is then opened to allow the sample to enter into theanalysis chambers412 as illustrated inFIG. 23D. The amount of analytes in the sample are then determined electrochemically usingappropriate electrodes414 specific for each analyte as described below. These analytes may include hydrogen peroxide (H₂O₂), oxygen (O₂), chloride (Cl⁻), sodium (Na⁺) and potassium (K⁺) as shown inFIG. 23E.
• More specifically now regarding the methods hereof, blood sample is withdrawn via microprobes at specified time intervals throughout the life time of the sport patch. There may be multiple collection sites and/or multiple inlet ducts and/or reservoirs as described above. There may be several microprobes to extract blood into the micro-fluidic circuit in the sports patch. Preferably about 15-30 μl of blood is withdrawn at specified time intervals. The microprobes are preferably coated with anticoagulant to prevent blood clotting. The micro-fluidic circuit includes the initial collection chamber or draw-offreservoir394 with programmable valves to flush out any old blood accumulated at the tip of the microprobes. Fresh blood sample is then channeled into themetering chamber304 then distributed in toanalysis chambers412.
• As discussed above, the detection of the analytes of interest is electrochemical, hence no blood separation is required prior to analysis. However, should optical detection (absorbance, reflectance, or transmittance for example) be the method of choice, a blood separation step may be included. In this alternate embodiment, the first layer on the sport patch will be a blood or cell separation pad that will separate RBC and WBC from the plasma or serum similar to that described above in conjunction withFIGS. 21A to 21D. In a typical operation, a blood fluid sample (15-30 μl) is withdrawn from the collection tubule and from here is drawn by capillary action through the cell separation pad. As the sample migrates through the cell separation pad, the fibrous network material making up the cell separation pad retards the movement of particulate matter, including blood cells, acting to partially remove blood cells before the sample moves into the subsequent pads.
• As described above in connection withFIG. 23E, the whole blood sample migrates into 5 analyses chambers (situated in parallel configurations). The analysis chamber used to quantify lactate is preloaded with all enzymes necessary for the lactate reaction diagrammed below. For the lactate assay, the analysis chamber may contain lactate oxidase, a peroxidase and a substrate reagent which is converted by the peroxidase in the presence of H₂O₂to the detectable reaction product. Given the method of detection of choice in this patch which is electrochemical, the enzyme peroxidase and chromogen substrate are omitted. In this case, the H₂O₂produced is oxidized at a platinum electrode to produce an electric current which is proportional to the sample lactate concentration. Presented below is the lactate assay reaction related to the present sports patch.
Lactate Assay:
• 
• Oxygen is measured amperometrically. An oxygen permeable membrane allows oxygen to permeate into an internal electrolyte solution where it is reduced at the cathode. The oxygen reduction current is proportional to the dissolved oxygen concentration.
• Oxygen electrodes are used to measure the oxygen concentration. The composition of oxygen electrodes is well known to those skilled in the art. A typical oxygen electrode contains a working electrode (cathode), a counter electrode (anode), an electrolyte, and a gas permeable membrane. The oxygen dissolved in the electrolyte through the gas permeable membrane and water react with electrons on the working electrode (cathode) to form hydroxide ions OH⁻(i.e., O₂+2H₂O+4e⁻=4OH⁻) and cause an electric current in relation to the amount of the dissolved oxygen so that the oxygen concentration can be estimated from the electric current detected.
• A tetrafluoroethylene/hexafluoroethylene copolymer (FEP) film is preferably used as the gas-permeable film, and the thickness of this film is preferably smaller than 20 μm. A gold electrode, a platinum electrode, a carbon electrode and the like are preferably used as the working electrode (cathode), and similarly, a gold electrode, a platinum electrode, carbon electrode and the like are preferably used as the counter electrode (anode). Furthermore, a silver/silver chloride electrode is preferably used as the reference electrode.
• An aqueous solution of potassium chloride, an aqueous solution of potassium hydroxide and the like are preferably used as the electrolyte.
• Sodium, potassium, and chloride are measured by ion-selective electrode potentiometry. Concentrations are calculated from the measured potential through the Nerst Equation. Different ions are measured by specific ion-selective electrode. As known to those skilled in the art, ion-selected electrodes, which are commercially available, are made of membranes which are made of varying composition of the glass electrodes with directed selectivity for the ion of interest.
• An algorithm may be included in firmware or software that will result in a beep should the analyte concentration drop below or is higher than a certain level so that the patient can take appropriate action. Use of the sports patch includes real-time use during sports activity. Such sports may include daily work-outs in the gym or health club by ordinary individuals or more disciplined work-outs by professional athletes such as marathon runners, swimmers, or hockey, baseball, football, and soccer players, for example.
• In the case of daily works-outs in the health club, an individual may monitor the body's response to certain exercise routines. With the aid of a trainer, results from the sports patch may be used to modify the work-out routine and/or the diet of the individual. In this manner, health and nutrition aspects for good health maintenance are integrated with use of the sports patch.
• In the case of professional sports the sports patch may be similarly used. In addition thereto, the sports patch may be equipped with RF detectors and transmitters as discussed above. In this manner, the coaching staff may use wirelessly transmitted diagnostic information to make coaching decision during an event or game. For example, in the game of professional hockey, line changes are of critical importance tactically and strategically. Thus the inventors hereof propose a sports patch that is used to monitor the performance level of a professional during a game or other competitive match. The retail time information is transmitted from the sports field or ice rink to the coaching staff. The coaching staff then utilizes this information in making coaching decisions like line changes in professional ice hockey, substitutions in basketball, football, or soccer, for example. The sports patch may be similarly utilized during training sessions.
Elderly Care Monitoring Implementations and Methods
• One of the major challenges facing the world today is providing health care to our ever-growing aging population. Not only will there be more individuals reaching the retirement age of 65, but they will be living longer. Along with longevity come chronic ailments such as heart diseases, kidney diseases, and diabetes, all of which infringe on one's independence. The need to provide economical healthcare to the elderly is therefore becoming more and more pressing.
• Real time measurements of cardiac biochemical markers are critical in the diagnosis of cardiovascular diseases. Furthermore, patients with kidney diseases are also at high risk for developing cardiovascular diseases. Therefore, early detection of deteriorating kidney function and intervention can improve outcomes. Measurement of serum creatinine can be used to detect a reduction in renal function. Anemia is known to affect up to 80% of patients with renal impairment, with a reduction of life and early death. Measurement of total hemoglobin can be used to monitor anemia status.
• Conventionally, the quantification of the above blood analytes generally require the patient to have their blood drawn at a medical facility and the determination of the analytes of interest is carried out by specialized technical personnel using specific diagnostic devices. However, the discomfort and inconvenience associated with this process may lead to poor patient compliance, resulting in failure of timely diagnosis and treatment.
• Accordingly, there remains a need in the art for medical devices which provide effortless analyte measurements in field or home-testing applications, particularly where continual and/or automatic monitoring is desired.
• In view of the above, the inventors hereof propose a personal diagnostic device for monitoring cardiac, renal, and anemia markers, for example, herein after referred to as an elderly care patch or elder care patch. The proposed elder care patch allows measurements of cardiac markers (CK-MB, TnI, and myoglobin), the renal marker (creatinine), and anemia marker (total hemoglobin). The elder care patch thus provides a quick, effortless device and method for analyte determination using only a simple patch or bracelet that interfaces with a personal computer or alternatively interfaces wirelessly with a smart home environment that may be advantageously networked with a professional health care service.
• With reference now toFIG. 24A, there is shown an exploded perspective view of the principal fluid sample processing and detection layers of an elder care patch according to still yet another preferred embodiment of the present invention. These include thesample acquisition layer116 including thelight sources320, thefluid processing layer118, and theresults detection layer178 including thedetectors322 as utilized to perform elder care monitoring. As illustrated, thesample acquisition layer116 includes the minimally invasive tubules, lancets, ormicro-probes159 that penetrate the skin surface to thereby acquire blood samples from capillaries near the epidermis. The fluid circuit illustrated inFIG. 24A includes thereservoir opening164, theblood metering chamber304 in a rectangular format, and threeanalysis chambers412. In this particular embodiment, thelight sources320 are implemented as micro-wire emitters and thedetectors322 are implemented as corresponding micro-wire detectors as illustrated.
• FIG. 24B is a perspective view with cut-away sections showing a fully assembled elder care patch including the layers illustrated inFIG. 24A for performing elder care monitoring according to certain further and additional aspects of this present invention. More particularly,FIG. 24B shows the minimally invasive tubules, lancets, or micro-probes159 in thesample acquisition layer116, thereservoir openings164 andanalysis chambers412 in thefluid processing layer118, themicro-wire detector322 in theresults detection layer178, thesignal processor142 in thesignal processing layer186, thecontroller190 in the logic and input/output controller layer188, the outputvideo display monitor124, and the individual fixed-display results windows126.
• FIGS. 25A-25D present cross-sectional side views showing the progression of a sample through the fluidic processing circuit of the elder care patch ofFIGS. 24A and 24B as implemented to perform monitoring in older patients. Referring now specifically toFIG. 25A, there is shown a fluidic circuit for analysis of CK-MB, Troponin-I (Tn-I), myoglobin, creatinine, and hemoglobin. The fluidic circuit illustrated includes amicroprobe159, afirst melt plug324, amelt valve332, ametering chamber304, avent340 having afilter342, asecond melt plug344, afirst analysis membrane416, asecond analysis membrane418, and athird analysis membrane420.First analysis membrane416 is used for analysis of hemoglobin while thesecond analysis membrane418 is made for analysis of creatinine and the third analysis membrane is for analysis of the cardiac markers. The various assays and methods for detection and quantitation are described in detail below.
• Moving next toFIG. 25B, plug324 is opened allowing blood to enter themetering chamber304. Once the blood level in the chamber reaches apre-determined volume valve332 is closed (FIG. 25C). Thesecond melt plug344 is then opened allowing the blood sample to flow into theanalysis membranes416,418, and420.FIG. 25D next shows the steps of the sample processing and analyses of the various analytes mentioned above.
• The hemoglobin analysis is carried out inmembrane316 where RBCs are lysed in the RBC lysis pad which releases the hemoglobin. The sample containing hemoglobin then enters the adjacent analysis or reaction pad where preloaded reagents that react with hemoglobin produce a detectable signal422. The incident beam ofelectromagnetic radiation373 is then directed through the analysis pad and into thephoto detector322. The amount of light detected by thephoto detector322 is then analyzed to determine the hemoglobin concentration in the sample.
• Creatinine analysis is carried inmembrane418 where cells are removed from the blood sample in the blood separation pad, serum then moves into the reaction or analysis pad where pre-loaded reagents react with creatinine through a series of reactions to produce a detectable product422. Details of these reactions are described and discussed below. The amount of creatinine is then calculated based on the signal detected by the photo detector.
• With continuing reference toFIG. 25D, the cardiac marker assay is performed inmembrane420 where the plasma is separated from the cells in the sample while the sample passes through the blood separation pad. The blood separation pad is preferably a sieve or filter having a desired pore size that prevents cells from passing through. The serum containing the various cardiac marker analytes then enter a tagging zone containing different types of signal agents specific for each analyte. The different signal agents may include a binding moiety that has specific affinity to the respective analytes. For example, a first signal agent may be a fluorescent micro-particle having attached thereto an antibody that binds to CK-MB, a second signal agent may be an opaque micro-particle having attached thereto an antibody that binds to TnI, and a third signal agent may be an IR (infrared) absorbing micro-particle having attached thereto an antibody that binds to myoglobin. As the sample flows through the tagging zone, each respective analyte is tagged with its respective signal agent.Membrane420 includes a series of analysis pads each containing a binding agent specific for one of the analytes. For example, the first analysis pad may contain capture antibodies against CK-MB, the second capture antibodies against TnI and the third capture antibodies against myoglobin. So as the tagged sample moves through each of the analysis or reaction pads each respective tagged analyte will be bound to its respective capture agent in each respective analysis pad. In this manner, each of the analyte concentrations may then be determined by analysis of the amount of signal agent present in each respective analysis zone. As one of skill in the art would surmise in view of this disclosure, different types of light sources and detectors may be required for interrogation of the analysis pads. The fluorescent micro-particles will require a light source of a specific wavelength to excite the fluorescent label in the micro-particle and a specific photo detector will be need to detect the emitted light of a particular wavelength coming from the fluorescing micro-particle. The IR micro-particle will thus need an IR light source and the opaque micro-particle yet another appropriate light source. As illustrated above inFIG. 17B, the micro-wire detector may be implemented with different diameter individual semi-conductor micro-wires that each emits a specific wave length of light. Any desired packing density and variation of sizes, diameters, and resulting wavelengths may be provided. Themicro-wire emitter320 may be similarly constructed to achieve these desired results in combination with a corresponding micro-wire detector as described.
• The following are details of reactions and detection systems that may be used for the above-described elder care patch of the present invention.
• A. Quantification of Cardiac Markers CK-MB, TnI, and Myoglobin
• Impregnated in the tagging zone or conjugate release pad is a first antibody to a cardiac marker of interest which is labeled with a particular signal label (colored latex particle or fluorescent tags or enzyme tags). The capture antibodies in the analysis pads can be produced in vivo or in vitro. Methods for the production of antibodies are well known to those skilled in the art. For example, see Antibody Production: Essential Techniques, Peter Delves (Ed.), John Wiley & Son Ltd, ISBN: 0471970107 (1997). Alternatively, antibodies may be obtained from commercial sources. Antibodies may be immobilized on a solid phase by a variety of methods known in the art, including covalent coupling, direct adsorption, physical entrapment, and attachment to a protein-coated surface.
• The cardiac markers will bind to the first antibody in the sample layer. The movement of serum through this layer will allow the migration of the analyte-antibody complex to the analysis pad.
• Impregnated on the analysis pad (nitrocellulose or borosilicate paper matrix) is a capture antibody, typically coupled to the analysis pad in a band like format to form a capture zone. The capture antibody captures the labeled analyte. The presence of the analyte creates a detectable signal line or band in the capture zone and a signal is produced (fluorescent or color). Excess labeled antibody can also continue to migrate along the solid phase and be captured by an immobilized antispecie antibody, creating a second signal band. This is used as an internal quality control check to indicate that the device has worked.
• B. Quantification of Creatinine
• The following section describes the quantification of creatinine and the creatinine assay as implemented and utilized in the elder care patch of the present invention.
Creatinine Assay:
• 
• Reaction Zone:
• Impregnated on a membrane are all enzymes necessary for the creatinine reaction shown above. For the creatinine assay, the reaction pad(s) contains creatininase, creatinase, sarcosine oxidase, a peroxidase, and a substrate reagent which is converted by the peroxidase in the presence of H₂O₂to a detectable reaction product. Desirably, the reaction pads are porous, fused polymer substrate membranes having a thickness, after complete penetration of the fluid, of about 125 μm and side dimensions of about 1 mm. The absorption volume of each pad is preferably between about 5-10 μl.
• Creatinine Quantification:
• At the end of the reaction zone is the analysis zone, where the amount of hydrogen peroxide produced is quantified by absorbance via a chromagen that changes color upon being oxidized.
• An algorithm may be included in firmware or software that will result in a beep should the analyte concentration drop below or is higher than a certain level so that the patient can take appropriate action. In addition thereto, various drugs, hormones, or other liquid therapies may be administered from the elder care patch according to the controlled time-release aspects of this invention.
• C. Quantification of Total Hemoglobin
• Hemoglobin assay is used to detect anemia. Anemia is known to affect 60-80% of patients with renal impairment, with a reduction in the quality of life and risk of early death.
• Measurement of hemoglobin concentration has been conducted by means of a cyanmethemoglobin method. With this method, an erythrocytolytic or red blood cell lysing agent is used to lyse the RBCs in the RBC lysis pad. Several red blood cell lysing reagents and methods have been described by previous scientific publications and patents including, for example, U.S. Pat. Nos. 3,874,852; 4,286,963; 4,346,018; 4,485,175; 4,528,274; 4,751,179; and 5,731,206. The cell lysis reagents are available commercially. The RBC lysis pad may also contain a nonionic surfactant to reduce turbidity caused by the erythrocyte cell membranes. The released hemoglobin is oxidized by the action of an oxidizing agent in the reaction pad, such as potassium ferricyanate, to produce methemoglobin. Subsequently, the cyanide ions bind to methemoglobin to form cyanmethemoglobin (HiCN) which produces a stable hemoglobin measurement sample. The absorbance of the cyanmethemoglobin sample is measured at a predetermined wavelength. This method is accepted worldwide as the standard method to determine hemoglobin concentration.
• For the hemoglobin assay, the reaction pad(s) contains potassium ferricyanide (0.6 mM), potassium cyanide 0.7 mM. In an alkaline medium, potassium ferricyanide oxidizes hemoglobin and its derivatives to methoglobin. Subsequent reactions with potassium cyanide produces the more stable cyanmethemoglobin which has a maximum absorbance at 540 nm.
• At the end of the reaction zone is the analysis zone, where the amount of colored product is quantified. The detector measures the change in reflected or transmitted light before and after blood application to determine total hemoglobin.
• An algorithm may be included in firmware or software that will result in a beep should the analyte concentration drop below or is higher than a certain level so that the patient can take appropriate action.
• In this particular embodiment of the present invention, the elderly care patch or bracelet may be advantageously equipped with a voice activated or voice recognition patch sub-system wirelessly connected to and integrated with the patient's smart home environment. Thus according to further use methods of the present invention, if an elderly person is wearing a patch in a retirement community with remote sensing of real time output for all users in the community, as contemplated by the inventors hereof, and the patient has some type of abnormal health condition come on suddenly—the user may simply say aloud, for example, “call 911”. The elder care patch then receives this voice command and dials 911 via remote wireless interfaces implemented in the elder care smart home environment according to these aspects of this invention. According to further steps of this use method, the attending doctor is promptly notified, and vital signs of the patient are then transmitted in real time via the patch to the hospital, the doctor's office, and/or to the paramedics in the ambulance that has been dispatch to the patient's location. To add further functionality to the elder care patch, the device may be advantageously equipped with a GPS locator so that in the event the users is unable to communicate his location to health officials, his location can be determined by the GPS locator and then communicated by wireless transmission within the smart home environment and any networks associated therewith.
CONCLUDING ASPECTS AND APPLICATIONS
• It is presently contemplated by the inventors hereof that certain aspects of the present invention may be implemented in a personal diagnostic device that is employed in the related fields of mental health and brain and cognitive sciences. In this application of the present invention, the inventors contemplate a bio-patch or bio-bracelet that can monitor the brain activity and function of a user and/or related signals in the user's nervous system.
• In the case when a patient feels fear, sadness, or depression; detecting purely physiological parameters like blood pressure and heart rate that are associated with such mental states may be performed by the patch technologies disclosed and discussed herein. In addition thereto, the inventors hereof propose that patterns of mental processes and nervous reactions may be detected by the bio-patches and bio-bracelets and utilized in combination with detected biological and physiological parameters to thereby further treat patients in a more comprehensive manner.
• The Brain Engineering Laboratory at the University of California at Irvine (UCI) has as its goal the attainment of a fundamental understanding of the brain, its mechanisms, operation, and behaviors. There has been explosive growth of information about the brain from a broad range of fields including neuro-anatomy, physiology, biochemistry and behavior, and tools from mathematics, computer science, and engineering are brought to bear to make sense of the voluminous data. The UCI lab and others investigate detailed designs of real brain areas, taking advantage of the explosion of new data and insights from the growing fields of neurobiology.
• The UCI researchers have found that previously ignored details of the anatomical wiring diagrams and physiological operating mechanisms of brain circuits suggest powerful algorithms that differ substantially from those in neural networks, and were unexpected from psychological or neuroscience studies. For example, the UCI models of superficial cortical layers perform the unexpectedly complex task of hierarchical clustering. See for example, Ambros-Ingerson, J., Granger, R., and Lynch, G. (1990); Simulation of Paleocortex Performs Hierarchical Clustering, Science vol. 247, pgs 1344-1348.
• The UCI algorithms derived from various brain areas have turned out to be so unexpectedly effective and efficient that they have found use in a variety of real-world applications. A hardware and software system derived from the UCI cortical models has been used to analyze EEG data in normal and early Alzheimer's subjects, as a potential device for aiding clinicians in the early detection of Alzheimer's Disease. See, Benvenuto, J., Jin, Y., Casale, M., Lynch, G., Granger, R. (2002), Identification of Diagnostic Evoked Response Potential Segments in Alzheimer's Disease, Exper. Neurology, 176: 269-276; and Granger, R. (2001), Method and Computer Program Product for Assessing Neurological Conditions and Treatments using Evoked Response Potentials, U.S. Pat. No. 6,223,074.
• At the Massachusetts Institute of Technology (MIT), researchers in the Brain and Cognitive Sciences Department are exploring how cellular and molecular neuroscience may be employed to understand the brain at its most fundamental level by examining the basic elements of the nervous system. They have determined that the interplay of the complex molecular machinery of the neuronal membrane with the dynamics of electrical potentials is critical to understanding the synaptic contacts where neurons communicate with each other. Such understanding may advantageously be applied to the inventions disclosed herein.
• Thus in view of the basic research and experimental work currently being performed in the fields of cognitive and brain sciences at several leading academic institutions, such as UCI and MIT for example, the inventors hereof contemplate useful applications for the present bio-patch and bio-bracelet in the realm of mental health and brain sciences which heretofore have not been contemplated by any artisan endeavoring in the various arts related hereto. For example, the inventors hereof contemplate that once the signals of the brain are well understood, an implementation of the present inventions may include detection of such signals which may then be converted to useful results in wireless networks such as those described above in connection with the stress patch, controlled drug release patches, and elderly cares patches.
• If not so specifically incorporated herein, all patents, patent applications, and other publications mentioned, cited, or otherwise referred to in this specification are hereby expressly incorporated herein by reference in their entireties.
• And while this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. For example, the bio-patch implementation may be made to conform to any particular surface area of the body and is not intended to necessarily be limited to a rectangular shape for use on the wrist areas. Any number of different size and shape configurations would be applicable. The bio-patch may also have pre-defined contours to promote adhesion with the body surface for use, for example, on the lower back, lower neck, shoulder, chest area, or skull. Similarly, many of the individual layers and layer components such as the fluidic circuits, the processors and controllers, wash buffer reservoirs, drug or hormone release reservoirs, valves, fluid channels, detectors, emitters, and electrodes, for example, are not necessarily restricted to the placements or locations shown but rather may alternatively be located on any suitable layer or otherwise combined on single layers to achieve the intended functions and results hereof.
• The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.

Claims (21)

What is claimed is:

1. A personal diagnostic device, comprising:

at least one air sample collection unit for obtaining an air sample from an ambient environment of a user, said at least one air sample collection unit configured to detect at least one airborne contaminant;

a user sample acquisition layer for obtaining a fluid sample from said user;

a fluidic processing layer in fluid communication with said user sample acquisition layer and configured to process said fluid sample, said fluidic processing layer including a fluidic circuit comprising a plurality of analysis chambers, said fluidic circuit configured to direct said fluid sample to said one or more of said plurality of analysis chambers for sample processing, detection, and analysis;

an assay results detection layer having a detector for detecting diagnostic results derived from said processing of said fluid sample;

a signal processing unit configured to display detection of said at least one air born contaminant and display at least some of said diagnostic results of said fluid sample; said user sample acquisition layer, said fluidic processing layer, and said assay results detection layer being in communication with each other; and said one or more of said plurality of analysis chambers and said detector being in communication with each other such that said one or more of said plurality of analysis chambers is detectable by said detector.

2. The device according toclaim 1 wherein said at least one air sample collection unit is located on a top surface of said personal diagnostic device.

3. The device according toclaim 1 wherein said diagnostic results monitor a response by said user to said at least one airborne contaminant.

4. The device according toclaim 1 wherein said at least one air sample collection unit identifies harmful levels of said at least one airborne contaminant.

5. The device according toclaim 1 further comprising hydrophobic surfaces for retarding fluid flow.

6. The device according toclaim 1 further comprising hydrophilic surfaces for promoting fluid flow.

7. The device according toclaim 1 further comprising a logic processing system with an Internet Protocol address.

8. The device according toclaim 1 further comprising a receiver and a transmitter to provide telemetry information regarding location of said user.

9. The device according toclaim 1 wherein said signal processing unit comprises a receiver, and a controller wherein a return command from a first remote location is received by said receiver, and processed by said controller to thereby activate a desired functionality within said personal diagnostic device.

10. The device according toclaim 1 further configured to perform at least one of the group consisting of: a cellular assay, a small molecule assay, a glucose assay, a stress assay, a cortisol assay, an enzyme assay, a lactate assay, a cardiac marker assay, a creatinine assay, and a hemoglobin assay.

11. The device according toclaim 1 further comprising tubules, lancets, or microprobes configured to draw up blood or fluid samples for testing.

12. The device according toclaim 11 further comprising an anticoagulant in said tubules, lancets, or microprobes.

13. The device according toclaim 11 further comprising programmable valves to flush out any old blood accumulated at tips of said tubules, lancets, or microprobes.

14. The device according toclaim 1 wherein said signal processing unit wirelessly transmits an electronic signal including at least some information about said diagnostic results to a remote location.

15. The device according toclaim 1 further configured to interface with a computer device remotely maintained from said personal diagnostic device, said computer device comprising at least one of the group consisting of: a desk top computer, a lap top computer, a hand-held computer, a receiver that receives at least some information about said diagnostic results, a receiver that receives at least some of said information about said diagnostic results wirelessly, a monitor that displays at least some of said information about said diagnostic results.

16. The device according toclaim 1 wherein said signal processing unit comprises an address so that said personal diagnostic device is addressable remotely over a network.

17. The device according toclaim 1 wherein said detector comprises at least one of the group consisting of: a charged coupled device (CCD), at least one light receiving nano-wire, and a semi-conductor light detecting material.

18. The device according toclaim 1 further comprising at least one drug-release reservoir having supply probes that penetrate into said user and said signal processing unit includes logic and control functionality to release a pre-determined amount of liquid medication from said at least one drug-release reservoir into said user through supply probes in response to a respective control command sent by said medical provider to said personal diagnostic device over said network.

19. The device according toclaim 1 further comprising at least one drug-release reservoir that is subdivided into a pre-selected number of sub-reservoirs each having a pre-determined amount of liquid medication stored therein so that said amount of liquid medication stored in each of said sub-reservoirs is releasable at different times for administration of a time-released application of said liquid medication over an extended period of time.

20. The device according toclaim 19 wherein said liquid medication includes a hormone.

21. The device according toclaim 20 wherein said hormone is insulin.

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