POROUS NAN0 STRUCTURED FILM SENSOR
Introduction
The invention relates to a porous nanostructured film having a sensing element and/or a signal detection element immobilised thereon having a signal transduction capability, sensors comprising such films and methods for preparing such films.
Many sensors work on the principle of recognition of an analyte by a complementary receptor molecule. Such a pair of molecules might comprise two complementary strands of nucleic acids, an enzyme substrate pair or an antigen and its specific antibody. In order to detect the sensor's measurement, a signal must be generated and transduced by passing it to a circuit where it can be digitised. The digital information can then be stored in memory, displayed visually or made accessible via a digital communications port. Signal generation may be achieved electrochemically, (electrochemical sensors), optically by measuring changes in absorbance or luminescence (optical sensors) or by plasmon resonance.
Luminescence methods such as fluorescence and chemiluminescence are currently popular. Luminescent detection relies on a marker in the form of fluorescent dye markers, chemiluminescent systems or fluorescent semiconductor nanoparticles.
The marker is associated with the molecular recognition pair or targeted to the pair, which either directly gives off light, gives off light when an external stimulus is applied or catalyses a chemical reaction that produces light. The amount of light given out from a sensor is proportional to the amount of analyte present in a given sample. The detection means rely on light emission for assay quantification and are capable of extremely high sensitivity assays. In order to detect and quantify this light output a photodiode, photomultiplier tube, charge coupled device (CCD) camera or complementary metal oxide semiconductor (CMOS) image sensor may be used. Most commonly, quantification of the light emission is carried out using a
CCD camera or photomultiplier tube. For applications such as biochip applications, where a number of light signals are detected simultaneously, a CCD device is typically used. While the CCD camera can be quite small, it can require cooling and this adds to the bulk of the sensor apparatus. There is currently an intense effort towards the miniaturisation and simplification of the reader element of sensors. Notably Motorola have developed an electrochemical method of detecting hybridisation between complementary DNA fragments which can be miniaturised to palm-top dimensions and requires no moving parts. This electrochemical method works through self assembled molecular wires on a gold electrode that are capable of detecting hybridisation events (1).
Others have also described the use of photovoltaic cells as elements for the quantification of luminescent signals. Biosite describe a near patient immunosensor biochip which incorporates solid-state photovoltaic cells for the quantification of a fluorescent read out. However, in this case the photovoltaic cell and the sensing element are separate entities. McCaffrey describes a hand held assay device capable of measuring chemiluminescence (2). In this case the photodiode detects luminescence from an assay but the diode is a separate entity to the assay sample. US6,060,327 mentions the use of a photodiode for assay applications (3). A silicon photodiode is used to immobilise conducting polymer strands with associated molecular recognition moieties and redox active molecules. When molecular recognition occurs, the redox active molecules send a signal through the conducting polymer strand. The photoactive properties of the diode are not used to detect luminescence in this application, the diode being used to simplify the electronic configuration of the sensor.
Dye sensitised solar cells are currently a research area of great interest (4,5). These cells work by sensitising a nanostructured, wide band-gap semiconductor with a molecular dye. When light hits the dye and a photon is absorbed, the dye is elevated to an excited state. When this excited state decays an electron is injected into the semiconductor creating an electrical current. The absorbtion characteristics of the dye determine which wavelengths of light the film will be most sensitive, i.e. if the absorbtion maximum of the dye is at 500 nm it will give a higher current efficiency here than at other wavelengths.
The type of nanostructured film employed in such solar cells is usually titanium dioxide (TiO2). This material can be made into a nanostructured film by formulating
TiO2 nanoparticles in a suitable solvent with a polymer binder and depositing the TiO2 nanoparticles using either a simple screen-printing or doctor-blading method. Sintering at elevated temperatures leads to the particles fusing and the binder being burned out to leave a porous network. In addition to their extensive use in solar cells, this type of nanostructured film has also been used to immobilise proteins for biosensor applications (6,7). It has been demonstrated that the mesoporous nature of these films allows for immobilisation of proteins in quantities significantly greater than on a flat surface. Biochemical devices which utilise nanostructured titania films, comprising fused titania particles are described. The technical advantages of biosensors based on these nanostructured films are stated to be: high biomolecule loading, optical transparency, stability and electrical conductivity as well as the possibility of immobilising proteins under mild conditions, so preserving the functionality of the protein. The high capacity for biomolecules is due to the high surface area to geometric area of the nanostructured film. However, due to small pore sizes (ca 15nm) this high surface area is not available to large proteins such as antibodies.
The introduction of large pores into films has previously been shown using templating methodologies. Templating titania around latex spheres forming hollow titania shells followed by removal of the latex has been described (8). The titania is formed by hydrolysis of a titanium alkoxide precursor in situ forming a smooth and well defined layer of titania on the polymer surface.
Polystyrene microspheres have also been used to engineer porosity into thin (thickness of nm), dense silica films (9). The microspheres are removed using an organic solvent. Similarly others have prepared photonic materials by forming titania around a densely packed crystalline structure comprised of latex spheres (10). The ordered packing of the resultant pores imparted useful optical properties on the film, such as inhibition of spontaneous emission or photon localisation.
There is a need however for an improved and more efficient method which allows for very sensitive detection of an analyte on a miniaturised scale.
Statements of invention
According to the invention there is provided a porous nanostructured film having a sensing element covalently immobilised thereon. Preferably the pore size is between 1 and 3000nm in size.
In one embodiment of the invention the film has a bimodal pore distribution.
Preferably the film comprises a combination of mesopores and macropores.
Preferably the mesopores are between 1 and 50nm in size. Preferably the macropores are greater than 50nm in size. Most preferably the macropores are greater than lOOnm in size.
In one embodiment of the invention the macropores are between 500nm and lOOOnm in size.
In another embodiment of the invention the macropores are between lOOOnm and 3000nm in size.
Preferably the film has a loading capacity greater than 5μg/cm2, preferably greater than 10μg/cm2, most preferably greater than 20μg/cm2.
Preferably the film is of a metal oxide material. The metal oxide may be selected from any one or more of titanium, zirconium, hafnium, chromium, molybdenum, tungsten, vanadium, niobium, tantalum, silver, zinc, strontium, iron (Fe2+ and Fe3+), nickel and perovskites thereof, preferably WO3, M0O3, ZnO or SNO2. Most preferably the metal oxide is TiO2.
In one embodiment of the invention the sensing element is selected from any one or more of an antibody; IgG. IgM, IgA, IgD, IgE antibody fragment; Fab, F(ab)'2, Fv, Fc receptors, drug receptors, ligands, enzymes, substrates, aptamers, affibodies™ , affinity binding agents, proteins, cell or tissue samples, cells, antigen or DNA. Preferably any one or more of an oligonucleotide probe, bacteria, fibrin binding protein, EGF receptor, collagenase, plasminogen, vitamin K, somatostatin receptors,
Monoamine oxidase, dopamine receptors, prostaglandin synthase or Hydroxymethyl glutaryl (HMG)Co-A.In one embodiment of the invention the porous nanostructured film has a sensing element and a signal detection element immobilised thereon. Preferably the sensing element and signal detection element are distributed throughout the film.
In one embodiment of the invention at least one portion of the film has a sensing element and at least one portion of the film has a signal detection element immobilised thereon.
Preferably the signal detection element is immobilised by covalent binding, chemisorption or physisorption, most pre erably by covalent binding.
In one embodiment of the invention the signal detection element comprises a dye material. Preferably the dye material has the generic structure
C(R)n wherein C is the chromophore, chemical entity capable of absorbing electromagnetic radiation in the desired region of the spectrum; R is selected from any one or more of NH2, NH-NH2, CHO, C=C, C≡C, SH, COOH, maleimide, PO32", sugar residues, COR2, COOR2 wherein R2 is CH3, (CH2)nCH3; and
n is 1 or more ; and
wherein when n >1 R may be the same or different.
Preferably the dye material has an absorbance range of 300 to 700nm, most preferably an absorbance range of 350 to 550nm.
The dye material may be selected from any one or more of coumarins, porphyrins, ruthenium complexes, Ru tris (bpy) derivatives, or any molecule with a chromophore attached. Preferably the dye is a porphyrin dye. The dye may be a porphyrin dye such as hematin. Most preferably the dye is Protoporphyrin IX.
The invention also provides a method of preparing a film of the invention having a sensing element attached comprising the steps of:-
treating the surface of a film with a functional silane to form a surface with a high density of reactive attachment groups;
reacting the attachment groups with a bifunctional or a polyfunctional cross- linking agent; and
adding a sensing element which attaches to the free end of the cross-linking reagent.
In one embodiment of the invention the sensing element reacts directly with the functional silane. The invention also provides a method of preparing a film comprising attaching a sensing element to the film and subsequently attaching a signal detection element to the film in the spaces between the sensing element on the film.
In another aspect the invention provides a method of preparing a film comprising attaching a signal detection element to the film and subsequently attaching a sensing element to the signal detection element.
In these methods the signal detection element may be a dye and the sensing element may be an antibody.
The invention also provides a method of preparing a film of the invention having a sensing element and a signal detecting element attached comprising the steps of:-
treating the surface of a film with a functional silane to form a surface with a high density of reactive attachment groups;
reacting the attachment groups with a bifunctional or a polyfunctional cross- linking agent; and
adding a sensing element and a signal detecting element which attach to the free end of the cross-linking agent.
Preferably the functional silane has the general formula
Si(0R)3-X-R1
wherein R is CH3, CH2CH3 ;
R1 is selected from any one or more of NH2, NH-NH2, CHO, SH, COOH, C=C, C≡C, maleimide epoxide, PO32", sugar residues, COR2, COOR2 wherein R2 is CH3, (CH2)nCH3; X is -(CH2)n, aromatic, and n is 1 or more
Preferably the bifunctional or a polyfunctional cross linking reagent has the general formula
R-X-R1,
wherein R and R1 are selected from any one or more of NH2, CHO, SH, COOH, C=C, epoxide, COR2, COOR2 wherein R2 is CH3, (CH2)nCH3; wherein R may be the same or different as R ,
X is -(CH2)n or an aromatic, and wherein n is 1 or more.
The invention provides use of a porous nanostructured film of the invention in a bioassay, immunoassay, biocatalysis or any sensor that comprises a chemiluminescent reaction.
The invention further provides a sensor for detecting an analyte comprising a substrate and a porous nanostructured film of the invention immobilised thereon. Preferably the substrate is selected from any one or more glass or metal or plastic having a conducting layer of indium tin oxide (ITO) or fluorine doped tin oxide (FTO) coated thereon.
In one embodiment of the invention the sensor has dual spectrophotometric and electrochemical measurement means.
The invention also provides use of a sensor of the invention in a bioassay or immunoassay. The invention further provides a method of manufacturing a sensor of the invention comprising the steps of:-
formulating nanoparticles comprising a metal oxide with solvent and polymeric binder to form a printable paste;
depositing the paste onto a conducting substrate;
heating the substrate and paste to remove excess solvent and binder, sinter the nanoparticles together to form a porous nanostructured film and adhere the film to the substrate surface; and
covalently attaching a signal detecting element and/or a sensing element to the porous nanostructured film.
Preferably the nanoparticles comprise single nanocrystallites and/or agglomerates of nanocrystallites.
Preferably the nanoparticles are templated around spheres during deposition of the printable paste onto a conducting substrate, most preferably the nanoparticles are templated around polymer fibres, surfactants, starches, or other small molecules used in templating.
In one embodiment of the invention the polymer spheres are removed during sintering.
Preferably the printable paste is deposited onto a conducting substrate by screen printing, doctor-blading or ink-jetting.
According to the invention there is provided a porous nanostructured film having a signal detection element covalently immobilised thereon. The invention further provides a porous nanostructured film having a sensing element and a signal detection element immobilised thereon. Preferably the sensing element and signal detection element are distributed throughout the film. Most preferably at least one portion of the film has a sensing element and at least one portion of the film has a signal detection element immobilised thereon.
In one embodiment of the invention the sensing element is immobilised by covalent binding, chemisorption or physisorption, preferably by covalent binding.
In one embodiment of the invention the signal detection element is immobilised by covalent binding, chemisorption or physisorption, preferably by covalent binding.
Preferably the signal detection element comprises a dye material. Most preferably the dye material has the generic structure
C(R)n
wherein C is the chromophore, chemical entity capable of absorbing electromagnetic radiation in the desired region of the spectrum;
R is selected from any one or more of NH2, NH-NH2, CHO, C=C, C≡C, SH, COOH, maleimide, PO32", sugar residues, COR2, COOR2 wherein R2 is CH3, (CH2)nCH3;
n is 1 or more ; and
wherein when n >1 R may be the same or different. Preferably the dye has an absorbance range of 300 to 700nm. Most preferably the dye has an absorbance range of 350 to 550nm. The dye may be selected from any one or more of coumarins, porphyrins, ruthenium complexes, Ru tris (bpy) derivatives, or any molecule with a chromophore attached. Preferably the dye is a porphyrin dye, most preferably Protoporphyrin IX
The invention further provides a porous nanostructured film having a sensing element covalently immobilised thereon.
Preferably the pore size is between 1 and 3000nm in size. In one embodiment of the invention the film has a bimodal pore distribution.
Preferably the film comprises a combination of mesopores and macropores.
Preferably the mesopores are between 1 and 50nm in size and the macropores are greater than 50nm in size. Most preferably the macropores are greater than lOOnm in size.
In one embodiment of the invention the macropores are between 500nm and lOOOnm in size.
In another embodiment of the invention the macropores are between lOOOnm and 3000nm in size.
Preferably the film is of a metal oxide material. The metal oxide may be selected from any one or more of titanium, zirconium, hafnium, chromium, molybdenum, tungsten, vanadium, niobium, tantalum, silver, zinc, strontium, iron (Fe2+ and Fe3+), nickel and perovskites thereof, preferably WO3, MoO3, ZnO or SNO2. Most preferably the metal oxide is TiO2,
The sensing element may be selected from any one or more of an antibody; IgG, IgM, IgA, IgD, IgE, antibody fragment; Fab, F(ab)'2, Fv, Fc, receptors, drug receptors, ligands, enzymes, substrates, aptamers, affibodies™, affinity binding agents, proteins, cell or tissue samples, cells, antigen or DNA, including, oligonucleotide probe, bacteria, fibrin binding protein, EGF receptor, collagenase, plasminogen, vitamin K, somatostatin receptors, Monoamine oxidase, dopamine receptors, prostaglandin synthase or Hydroxy-methyl glutaryl (HMG)Co-A.The invention provides use of a porous nanostructured film of the invention in a bioassay, immunoassay, biocatalysis or any sensor that comprises a chemiluminescent reaction.The invention further provides a method of preparing a film of the invention having a signal detection element attached comprising the steps of:-
treating the surface of a film with a functional silane to form a surface with a high density of reactive attachment groups; and
adding a signal detecting element which attaches to the free end of the cross- linking agent.
The invention also provides a method of preparing a film having a sensing element and a signal detecting element attached comprising the steps of:-
treating the surface of a film with a functional silane to form a surface with a high density of reactive attachment groups;
reacting the attachment groups with a bifunctional or a polyfunctional cross- linking agent; and
adding a sensing element and a signal detecting element which attach to the free end of the cross-linking agent. The invention also provides a method of preparing a film having a sensing element attached comprising the steps of:-
treating the surface of a film with a functional silane to form a surface with a high density of reactive attachment groups; reacting the attachment groups with a bifunctional or a polyfunctional cross -linking agent; and
adding a sensing element which attaches to the free end of the cross- linking reagent.
In one embodiment of the invention the sensing element reacts directly with the functional silane.
Preferably the functional silane has the general formula
Si(0R)3-X-R1
wherein R is CH3, CH2CH3 ;
R1 is selected from any one or more of NH2, NH-NH2, CHO, SH, COOH,
C=C, C≡C, maleimide epoxide, PO32~ , sugar residues, COR2, COOR2 wherein R2 is CH3, (CH2)nCH3;
X is -(CH2)n or an aromatic, and wherein n is 1 or more.
Pre erably the bifunctional cross linking reagent has the general formula
R-X-R1, wherein R and R1 are selected from any one or more of NH2, CHO, SH,
COOH, C=C, epoxide, COR2, COOR2 wherein R2 is CH3, (CH2)nCH3;
R may be the same or different to R1;
X is -(CH2)n, aromatic, and wherein n is 1 or more. The invention provides a sensor for detecting an analyte comprising a substrate and a porous nanostructured film having a sensing element and signal detection element immobilised thereon. Preferably the substrate is selected from any one or more glass or metal or plastic having a conducting layer of indium tin oxide (ITO) or fluorine doped tin oxide (FTO) coated thereon. Most preferably the porous nanostructured film comprises a pore size between 1 and 3000nm in size.
In one embodiment of the invention the porous nanostructured film has a bimodal pore distribution. Preferably the porous nanostructured film comprises a combination of mesopores and macropores.
The invention provides an integrated sensor comprising a conducting film as a solid phase support.
The invention also provides an integrated sensor comprising a semi-conducting film as a solid phase support.
The integrated sensor comprises a sensing element and signal detecting element immobilised on the solid phase support.
In one embodiment of the invention the sensor has dual spectrophotometric and electrochemical methods.
The invention provides a method of manufacturing a sensor comprising the steps of :-
formulating nanoparticles comprising a metal oxide with solvent and polymeric binder to form a printable paste;
depositing the paste onto a conducting substrate; heating the substrate and paste to remove excess solvent and binder, sinter the nanoparticles together to form a porous nanostructured film and adhere the film to the substrate surface; and
covalently attaching a signal detecting element and/or a sensing element to the porous nanostructured film.
Preferably the nanoparticles comprise single nanocrystallites and/or agglomerates of nanocrystallites. Preferably the nanoparticles are templated around spheres during deposition of the printable paste onto a conducting substrate. The nanoparticles may be templated around polymer fibres, surfactants, starches, or other small molecules used in templating. Preferably the polymer spheres are removed during sintering.
In one embodiment of the invention the printable paste is deposited onto a conducting substrate by ink-jetting, screen printing or doctor blading.
Preferably the porous nanostructured film of the invention comprises mesopores between 1 and 50nm in size and macropores between 50 and lOOOnm in size. The invention also provides a method for detecting an analyte in a test sample comprising the steps of:-
immobilising a sensing element specific for the analyte onto a porous nanostructured film on a support;
incubating the immobilised sensing element with a test sample containing the analyte;
washing to remove any non-specific binding;
incubating the film with a second labelled sensing element specific for the analyte; creating an electrochemical cell with the porous nanostructured film; and
adding a chemiluminescent substrate in solution to the electrochemical cell,
wherein the light released on reaction of the chemiluminescent substrate in solution with the labelled sensing element is absorbed by a signal detecting element immobilised on the porous nanostructured film, elevating molecules in the signal detecting element to an excited state thereby injecting electrons into the porous nanostructured film indicative of the concentration of analyte present.
Fluorescence may alternatively be used as a detection method.
Brief description of the drawings
The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only with reference to the accompanying drawings in which:-
Fig. 1 is a schematic representation of the functioning of the sensor of the invention;
Fig. 2 are surface and cross sectional scanning electron micrograph (SEM) showing a dense screen printed titania film with pore size of approximately 20 nm;
Fig. 3 are surface and cross sectional SEM showing a templated, doctorbladed titania films incorporating 100 nm pores;
Fig. 4 are surface and cross sectional SEM showing a templated, doctorbladed titania films incorporating 400 nm pores; Fig. 5 are surface and cross sectional SEM showing a templated, doctorbladed titania films incorporating 1000 nm pores;
Fig. 6 is a schematic representation of three nanoparticles having a pore size of less than 50nm in a closely packed structure;
Fig. 7 is a schematic representation of nanoparticles having a pore size of from 1 to 50nm and larger macropores approximately lOOnm in size;
Fig. 8 is a schematic representation of nanoparticles having a pore size of from 1 to 50nm and larger macropores approximately 400nm in size;
Fig. 9 is a schematic representation of nanoparticles having a pore size of from 1 to 50nm and larger macropores approximately lOOOnm in size;
Fig. 10 is a schematic representation of nanoparticles having a pore size of from 1 to 50nm and larger macropores approximately 400nm and lOOnm in size;
Fig. 11 is a bar graph comparing the IgG loading capacity of standard screen printed films to films incorporating 100 nm pores;
Fig. 12 is a schematic representation showing the covalent binding of proteins and/or dyes to the titania surface;
Fig. 13 is a schematic representation of the electrochemical cell used with the sensor of the invention;
Fig. 14 shows the structure of Protoporphyrin IX; Fig. 15 is a graph showing the dose response to horse radish peroxidase (HRP) (in solution);
Fig. 16 is a graph showing three traces for potential transients for IgG-HRP covalently bound to the film and three traces for potential transients obtained in the absence of HRP conjugated to the IgG;
Fig 17 is a graph showing the limits of sensitivity of the hCG assay achieved on the porous nanostructured films of the invention.
Fig. 18 is a schematic representation of a titania film with dye and antibody attached;
Fig. 19(a) is a graph showing the sensitivity of the sandwich assay to different concentrations of hCG, 0, 25 and 50mIU; and
Fig. 19(b) is a graph showing the potential transients obtained for concentration of hCG at 0, 25 and 50mIU. The tests were carried out in triplicate.
Detailed description
The invention describes a porous nanostructured film with integrated signal transduction capability where detection of an analyte is via luminescent signal generation. This is achieved by integration of a sensing element and/or a signal detection element into a single porous nanostructured semiconductor metal oxide film surface.
The invention combines two complementary applications of nanostructured titania films to make an integrated sensing device. The invention provides a nanostructured film which comprises not only the molecules necessary to detect an analyte and produce a light signal but also molecules that can harvest the light and turn it into an electrical output thus eliminating the need for external photon measuring instrumentation. The invention significantly simplifies and reduces the cost of transducing a luminescent signal. The invention also provides a sensor comprising the nanostructured film.
Referring in particular to Fig. 1 there is a sensor (1) of the invention, for example an immunosensor, comprising a nanostructured titania film (2) with probe molecules (3) (such as antibodies; IgG, IgM, IgA, IgD, IgE or antibody fragments; Fab, F(ab)'2,
Fv, Fc receptors, drug receptors, ligands, enzymes, substrates, aptamers, affibodies, affinity binding agents, proteins, cell or tissue samples, cells, antigen and DNA, an oligonucleotide probe, bacteria, fibrin binding protein, EGF receptor, collagenase, plasminogen, vitamin K, somatostatin receptors, monoamine oxidase, dopamine receptors, prostaglandin synthase or Hydroxy-methyl glutaryl (HMG) Co-
A), immobilised on the film. The titania film (2) is positioned on a support (4). The bound molecules (3) are capable of recognising and binding the analyte (5) being detected. A reporter molecule (6) (for example a secondary antibody), conjugated to an enzyme is used to indicate the presence of the analyte (5) in a sample that has been brought into contact with the sensor (1). If the analyte (5) is present the reporter molecule (6) binds to it and is retained. If the analyte (5) is not present then the reporter molecule (6) will not bind. A chemiluminescent substrate (7) is then added to the sensor (1) and light is emitted when the enzyme conjugated to the reporter molecule (6) reacts with it. The light produced is absorbed by a dye (8) which is also bound to the titania surface (2). The dye (8) is thus excited to an electronically excited state and injects an electron into the titania film (2). The presence of electrons in the titania film are then measured. The sensor (1) therefore senses the analyte present in a sample and simultaneously converts the light signal indicating the presence of the analyte to an electrical signal which can be measured.
The nanostructured films of the invention are meso and macroporous metal oxide films comprising fused nanoscale particles. The films provide a recipient surface for the immobilisation of probe molecules employed in the sensor. These films typically possess the following characteristics: high surface area, optical transparency, semi- conductivity and are highly receptive surfaces for biomolecules, for biosensor applications.
The term mesoporous is taken to include nanoparticles of between 2 to 50nm in size. The term macroporous is taken to include any particle greater than 50nm in size. These are the accepted international definitions for pores of this size. (IUPAC Recommendation of 2001 (Pure Appl. Chem. 73, 381-394, 2001.)
Nanostructured titania films are deposited by conventional methods either by screen printing, doctor blading or ink-jetting onto a fluorine doped tin oxide (FTO) coated or indium doped tin oxide (ITO) coated substrate. FTO and ITO are typically used for glass and plastic substrates. ITO is the industry standard however, its properties change at high sintering temperatures (400°C), therefore FTO is more suitable for glass substrates and ITO for plastic substrates.
The nanoparticles are formulated with solvent and a polymeric binder to make a printable paste. After printing, the films are heated to remove both the solvent and binder and to sinter the particles together and adhere them to the surface below. Full details on the preparation of dense metal oxide films such as TiO2 films are described in detail in WO9835267 and WOO 127690 both of which are herein incoφorated in full. Such dense films typically comprise mesopores of approximately 2-50nm in size only.
The properties of the films of the invention may be tailored to the sensor application. For example, for immunosensors, which employ large proteins, such as antibodies, it is important that the pores are sufficiently large to allow the probe access to as much of the available surface area as possible (internal as well as external). Pores should also accommodate all of the recognition molecules involved in the sensing event and allow efficient washing where necessary. Bimodal pore distributions are possible, the smaller pores being accessible to small molecules such as dyes while the larger pores can accommodate antibodies or other large proteins. Bimodal pore distribution is also useful for use in multianalyte assays such as microarrays which may require different pore sizes for loading of different sensing agents. Trimodal or polymodal pore distributions are also possible. In this case mesopores are present in addition to two or more sizes of macropores. In this way the pore distribution of the films of the present invention provides a higher loading capacity in comparison to films having a mesopore pore distribution only.
Film porosity is determined by constituent particle size and by the binders used during formation. The particles making up the film can either be single nanocrystallites of the metal oxide in question or can be agglomerates of nanocrystallites, also known as secondary particles. Variation of the above parameters allows control over the porosity of the resulting film. Both unimodal, bimodal, trimodal or polymodal pore distributions may be achieved.
Another method of preparing films with bimodal pore distributions is to template the nanoparticles around polymer spheres of a defined diameter during the film deposition process. The polymer is then removed during sintering, leaving films with high surface area and pore size corresponding to the diameter of the templating spheres. In this case a bimodal pore distribution is obtained with the size of the larger pores being fully controllable as shown in Figs. 3 to 5 and Figs 11 to 15.
The loading of large proteins, such as antibodies; ; IgG, IgM, IgA, IgD, IgE or antibody fragments; Fab, F(ab)'2, Fv, Fc, receptors, drug receptors, ligands, enzymes, substrates, aptamers, affibodies™, affinity binding agents, proteins, cell or tissue samples, cells, antigen and DNA, an oligonucleotide probe, bacteria, fibrin binding protein, EGF receptor, collagenase, plasminogen, vitamin K, somatostatin receptors, Monoamine oxidase, dopamine receptors, prostaglandin synthase or Hydroxy -methyl glutaryl (HMG) Co- A, on nanostructured films with large pores as shown in Fig. 5, is significantly higher than on less porous nanostructured titania films such as those shown in Fig. 2. A comparison of IgG loading on dense films and IgG loading on templated films with 100 nm pores is shown in Fig. 6.
The films may also be templated around polymer fibres, surfactants, starches, or other small molecules to produce films with the required properties for a specific application.
The titania films of the invention are thicker, and are sintered which fuses the nanocrystallites and makes the films conductive. The polymer spheres are burnt out during the sintering process and an organic solvent is not needed. Where the film is deposited on a plastic substrate and high temperature sintering is not used, the polymer spheres may be removed using organic solvents.
In order to covalently attach antibodies and dye molecules to the film, the surface is treated with a functional silane such as an amino silane. The surface may also be treated with polyions, bifunctional polymers or DNA. This creates a surface with a high density of reactive attachment groups. These groups are then reacted with a bifunctional cross-linking reagent such as glutaric dialdehyde, the free end of which then attaches to functional groups on the antibodies or the dye molecules. For example this may be either the amine moieties on the antibody or the allylic double bonds on the dye molecule. Thus, a film with either covalently bound antibody and/or dye may be prepared (Fig. 12). Alternatively a polyfunctional cross-linking agent could be used.
The loading of proteins and/or dye molecules to the film may also be carried out in the absence of a bifunctional or a polyfunctional cross-linking reagent wherein the surface is treated with a functional silane, polyion, bifunctional polymer and the protein is attached directly.
Preferably the functional silanes have the general formula
Si(0R)3-X-R1
wherein R is CH3, CH2CH3 ;
R1 includes but is not limited to any of NH2, NH-NH2, CHO, SH, COOH,
C=C, C≡ CC, maleimide epoxide, PO32\ sugar residues, COR2 or COOR2 wherein R2 is CH3, (CH2)nCH3; and
X is -(CH2)n, aromatic, and n is 1 or more.
Preferably the bi-functional cross linking reagents are compounds having the general formula
R-X-R1,
wherein R and R1 are selected from any one or more of NH2, CHO, SH, COOH, C=C, epoxide, COR2 or COOR2 wherein R2 is CH3, (CH2)nCH3; R may be the same or different to R1, X is (CH2)n, aromatic, and wherein n is 1 or more. In order to have both dye and antibody covalently attached to the film there are two possibilities. Either the antibody is attached first and the film is then placed in a solution of the dye so that dye molecules assemble and are covalently attached in the spaces between the antibody. Alternatively the dye is covalently attached first and then the pendant carboxylate groups of the dye are activated with a compound such as carbodiimide which then attach to amine groups on the antibody. The electrodes are then incubated in a solution of blocking agent such as Bovine Serum Albumin (BSA) or casein to block non-specific binding by analyte or secondary antibodies.
To carry out an assay the films are incubated with an analyte for which the immobilised antibody is specific, washed to eliminate any non-specific binding and then incubated with a labelled secondary antibody, using a label such as horse radish peroxidase (HRP). The secondary antibody is also specific for the analyte and forms a sandwich complex of the two antibodies with the analyte in the middle. In the case of HRP, the label is an enzyme capable of carrying out organic transformations on small molecules to produce a luminous signal.
Other assay types such as a competitive assay may also be carried out using the films of the invention.
To create an electrochemical cell (20), the conducting glass substrate (21) with modified TiO2 film (22) is used as one electrode and a piece of platinised conducting glass (23) is used as the other electrode as shown in Fig. 13. To separate these, a piece of rubber gasket (24) is cut to size, with a hole in the middle to contain solution. A contact is made between each of these electrodes and a potentiostat in order to measure the open circuit potential.
The open circuit potential measurement is started and a chemiluminescent substrate in solution such as luminol is injected into the cell, ensuring that no air bubbles are injected. When the chemiluminescent substrate in solution is in contact with HRP, light is released and this light is absorbed by the dye molecules on the surface, elevating them to an excited electronic state which then inject electrons into the titania electrode. These electrons can be measured as a current, potential or a charge build up. The signal produced is proportional to the amount of light produced and the concentration of analyte captured by the sensor.
The integrated sensor of the invention may form part of an array of such sensors for highly parallel analysis. In this case individual sensors may be addressed by direct drive, passive matrix or active matrix electronic circuitry.
The integrated sensor of the invention has many advantages over currently known sensors.
It provides for the integration of sensing elements (e.g. antibodies) and photovoltaic elements (molecules that turn light into electrons) on the same film.
The sensor provides a simple alternative to conventional instrumentation and software required to collect and analyse a luminescent signal. It allows for the miniaturisation of instrumentation required to collect and process a luminescent signal and adds the capability to the sensor of transportability providing on-site or in the field analysis. The sensor of the invention also presents a significant reduction in the cost of collecting and processing a luminescent signal.
There are many advantages to using nanostructured films as solid phase supports for sensors such as high probe loading due to the high surface area of these materials, scope for miniaturisation, high signal to noise ratios for optical sensors due to optical properties of the materials and the opportunity for integrated readouts due to semiconductive properties of the films etc. For typical mesoporous titania films these advantages can only be realised in practice for sensors which use small molecules, i.e. molecules with dimensions smaller than the pore size in the film. Nanostructured metal oxide films with variable porosity allow all the advantages of using nanostructured films for biosensor applications to be extended to sensors that involve the use of large and bulky molecules for recognition and detection such as immunosensors.
The above features of the invention in combination provide the scope to move diagnostic assays with the sensitivity of luminescent detection, to the point of need.
The integrated sensor of the invention is applicable to any sensor that emits a luminescent signal such as chemiluminescent gas sensors for the detection of ozone, reactive oxygen species and nitrogen, chemiluminescent sensors for the analysis of solutions such as ethanol or glucose. The invention is particularly applicable to biochips where trends are towards highly parallel testing and miniaturisation.
The biochip market is an important and emerging market and has potential in the areas of pharmaceutical research, medical diagnostics for example autoimmune disease and cancer typing, identification of infecting microorganisms, forensics, transplantation, identity testing and environmental testing in the future.
Biochips require high sensitivity and a capacity for high protein or nucleotide loading making the integrated film and sensor of the invention ideally suited to this format. There are two main types of biochip, depending on the probes attached.
Nucleic acid biochips which have DNA/RNA attached and protein biochips that have proteins such as antibodies attached. In addition there are Mini-lab chips which incorporate microfluidics in the housing.
Many of the biochip products on the market use fluorescence readout methods.
However, the substrates commonly used such as glass, PNDF, nitrocellulose or polycarbonate are prone to autofluorescence giving a poor signal to noise ratio. In addition, fluorescent probes can be susceptible to photo-bleaching, and the ambient buffer must be carefully chosen so that there are no quenching species present, the solvent polarity must also be considered and fluorescent probes are pH sensitive.
The integrated system of the invention overcomes these problems. In biochip applications, recent developments in microfluidic technologies have enabled on chip fluid handling and integration of all the steps necessary in an analytical procedure to be performed on the chip. Advantageously the present invention enables standard luminescent detection and signal processing to be also integrated on the chip.
The integrated sensor of the invention is applicable to a wide range of assay types and formats on the biochip. The system has been established in an immunoassay but is applicable to all protein assays including, antibody specificity, receptor-ligand binding assay and enzyme-substrate assay, protein-protein interactions, protein- RNA, protein-DNA, protein-drug interactions. In addition, the system is compatible with DNA assays encompassing gene expression analysis and DNA analysis such as SNP detection.
The invention will be more clearly understood from the following examples.
Example 1 - Preparation of a porous nanostructured film
The water in aliphatic amine white polystyrene latex microspheres of required diameter in surfactant-free de-ionised water at 4% w/v is removed by sublimation using a freeze-dryer. The resultant latex is ground to a fine powder with a pestle and mortar. This is added to a standard "in-house" TiO2 paste at a ratio of 60:100, latex beads: TiO2 particles. For example for a 10% TiO2 paste; for every 10 grams of paste 0.6g of latex is added. The mixture is stirred overnight and deposited onto
FTO coated glass.
Example 2 - Determination of the potential transients for different concentrations of IgG-HRP in solution. 04/011672
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A standard "in-house" TiO2 doctor bladed film as illustrated in Fig. 2 was prepared. The film was silanised, treated with glutaraldehyde and IgG immobilised on it. The film was then dyed in Protoporphyrin IX (Fig. 14), and blocked with BSA. An electrochemical cell as shown in Fig. 13 was assembled and different concentrations of IgG HRP in luminol solution injected. For each injection the open circuit potential was monitored for at least 1500 sees. Between injections the cell was washed and dried thoroughly before reassembly. The highest concentration HRP shows the biggest potential change as shown in Fig. 15.
Example 3 - Determination of the potential transients for films of IgG-HRP covalently bound-
Porous titania films having 400 and 100 nm pores were heated to 110°C, cleaned with UN-ozone and silanised with aminopropyl silane. The films were heated to
110°C, allowed to cool and then reacted with glutaraldehyde. IgG was then spotted onto three of the six films and IgG-HRP conjugate spotted onto the other three. The films were left overnight. The films were then dyed by immersion in a Protoporphyrin IX solution (lOmM in phosphate buffer) for 6 hours, rinsed in PBS- tween and blocked overnight in BSA (3% in PBS-tween).
Each film was assembled into a 2 electrode cell as shown in Fig. 13, an open circuit potential measurement taken and luminol solution injected into the cell. The open circuit potential measurement was continued for 1500 seconds. Fig. 16 shows the potential transients obtained. The three thicker lines indicate films with IgG attached while the three thinner lines indicate film with IgG-HRP attached.
Example 4 - Comparison of detection limits achieved for hCG assay on porous titanium oxide films Latex-templated titanium dioxide films were silanated with amino silane, and reacted with gluteraldehyde. The capture antibody, 0.5ug affinity purified polyclonal anti- hCG IgG (in phosphate buffered saline pH 7.2) was covalently immobilised onto the film at room temperature over 16 to 20 hours. The films were blocked with 3% bovine serum albumen in phosphate buffered with 0.05% Tween-20 (PBS-tween) for 2 hours on an orbital shaker at 50rev/min followed by two 15min washes in PBS- tween. The films were incubated with different concentrations of hCG (diluted in PBS-tween) for one hour and washed twice in PBS-tween for 15 min. Subsequently, the films were incubated with the secondary antibody, monoclonal anti-hCG IgG conjugated to Horse Radish Peroxidase and washed two times in PBS-tween for 15 min. Finally, the films were incubated with precipitating 3,3'5,5-
Tetramethylbenzidine (TMB) for 10 minutes. The reaction was stopped by decanting the TMB and washing the films with dH2O. The films were air-dried and the resultant colour measured by reflectance on a UV-NIS spectrophotometer.
Fig. 17 shows the limits of sensitivity of the hCG assay achieved on the porous nanostructured films.
Example 5 - Sandwich assay
Titania films were prepared by doctor blading, using a single layer of Scotch-tape as a spacer. They were allowed to dry and then heated at 450°C for 45 minutes. The titania films were coated in amino silane by immersing in a solution (1% aminopropyltriethoxysilane in a 95:5 mixture of ethanol/water) for 1 hour. The films were removed from the silane solution, rinsed twice in pure ethanol and then heated to 110°C for 20 minutes. The films were then placed in a solution of glutaraldehyde (1% in PBS (phosphate buffered saline)) for 1 hour, rinsed twice in water and allowed to dry. The films were then placed in a solution of protoporphyrin IX [5mM in phosphate buffer (50mM, pH 10.6)] for three hours and washed thoroughly in phosphate buffer (50mM, pH 10.6) and then water. The porphyrin molecule attaches to the silane film due to the reaction between one of its pendant double bonds and an /011672
amine group in the silane film. The carboxylate groups on the porphyrin were activated for binding by immersing the dyed films in a solution of l-(-3 Dimethylaminopropyl)-3-ethyI-carbodiimide hydrochloride (25mM in phosphate buffer (50mM, pH 8.3)) for 10 minutes. This solution was then drained from the films, IgG (0.5 g) spotted onto each sensor area and left for at least 1 hour during which primary amine groups on the antibody react with the activated carboxy groups on the porphyrin. A schematic representation of the preparation of the titania film is shown in Fig. 18.
The films were then blocked by placing in a solution of BSA (3% in PBS/Tween) overnight. A standard hCG assay was then performed as in example 4, using concentrations of 0, 25 and 50 mlU of hCG. On completion of the incubation with secondary antibody and associated washing, the film was assembled into the 2- electrode cell shown in Fig. 13 and an open circuit potential measurement started when luminol was injected into the cell.
Fig. 19(a) shows the results of an integrated sensor and its sensitivity over a concentration range which is industrially applicable. Fig. 19(b) shows the potential transients obtained for the different concentrations of hCG. Each assay was carried out in triplicate.
The invention is not limited to the embodiments hereinbefore described which may be varied in detail.
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