Patent Application
20090317845
The present invention relates broadly to the area of chemical and biochemical sensors, in particular the area of immunosensors and biosensors, devices comprising such sensors and methods of forming such sensors.
A known sensing technology is based on sandwich type assays for sensing an analyte in a sample, wherein a first receptor is bound to a substrate, the analyte binds to the receptor and a second receptor carrying a signal enhancing group is then allowed to bind to the analyte (see for instance Taussig et al Targets, (2003), 2(4), 169, Seong et al, Proteomics (2003), 3, 2176; Wu et al, Clinica Chimica Acta, 369, (2006), 119 and references therein). These sandwich assays may also be used in a competitive assay format wherein an analyte-mimic containing the signal enhancing group is already bound to the first receptor but is displaced by analyte contained within the test sample.
Generally, optical methods are used to as the readout mechanism for these sensors. This has the drawback that optical systems are relatively expensive as they require precision instruments. This is especially true for assays requiring high sensitivity. Thus methods are required to enhance the sensitivity of sandwich assays without requiring elaborate or costly sensing equipment.
The present inventors have invented a novel sensor transduction method to detect the binding of a second receptor or the displacement of the analyte-mimic using the generation of an impulsive noise due to the interaction of a trigger element, that is attached to the second receptor or analyte-mimic, with a substrate on exposure to a triggering impulse such as bursts of electromagnetic radiation. This has the advantage of being detectable by simple, low-cost microphonic noise detection systems with enhanced sensitivity compared to some optical or other detection means.
The present inventors have further found that the use of trigger elements which generate an impulsive noise when triggered are capable of more general application in the field of analyte sensing than the sandwich assays described above.
Accordingly, in a first aspect, the present invention provides a method for measuring the presence of an analyte in a sample, wherein the presence of the analyte is indicated by the presence or absence of one or more trigger elements that generate an impulsive noise when triggered, comprising the steps of:
(a) exposing said one or more trigger elements to a trigger impulse; and
(b) detecting the impulsive noise generated by said one or more trigger elements.
In a second aspect, the present invention provides a kit for performing the method of the first aspect, said kit comprising one or more of the components required for performing the method of the first aspect.
In a third aspect, the present invention provides a receptor capable of binding to an analyte wherein the receptor is labelled with a trigger element wherein said trigger element generates an impulsive noise when triggered.
In a fourth aspect, the present invention provides an analyte-mimic capable of binding to a receptor for an analyte, wherein said analyte-mimic is labelled with a trigger element wherein said trigger element generates an impulsive noise when triggered.
In a first aspect, the present invention provides a method for measuring the presence of an analyte in a sample, wherein the presence of the analyte is indicated by the presence or absence of one or more trigger elements that generate an impulsive noise when triggered, comprising the steps of:
(a) exposing the one or more trigger elements to a triggering impulse; and
(b) detecting the impulsive noise generated by said one or more trigger elements.
As used herein, the term “analyte” means any substance for which there exists a naturally occurring specific binding member (receptor) or for which a specific binding member (receptor) can be prepared. The analyte may be for example proteins, peptides, antigens, bacterial antigens, glycolipids, viruses or viral fragments (hepatitis A, hepatitis B, hepatitis C, hepatitis delta, hepatitis E, malaria, tuberculosis, human immunodeficiency virus), haptens, antibodies (monoclonal or polyclonal), nucleic acid sequences, enzymes, hormones, vitamins, steroids, drugs (therapeutic or drugs of abuse), carbohydrates, explosive substances, toxins (ricin, nerve gas agents), pollutants (pesticides, endocrine hormone disrupting substances), cancer markers and so on.
The impulsive noise provides an indication of the presence of the analyte in the sample. Preferably, the impulsive noise provides an indication of the amount of analyte present in the sample. In a more preferred form, the intensity of the impulsive noise is directly related to the amount of analyte in the sample. The relationship between the intensity of the noise and the amount of analyte may be proportional or inversely proportional. For instance, in a competitive binding assay, if a greater the amount of analyte is present in the sample, then a lesser amount of trigger element is present and, accordingly, the intensity of the impulsive noise will be lower.
Preferably, the triggering impulse is radiation. Preferably, the radiation is selected from ultra-violet, visible, infra-red and microwave radiation and combinations thereof.
Preferably, the radiation is in the form of a pulse of less than 100 milliseconds duration but greater than one nanosecond duration. More preferably, the duration is less than 5 milliseconds.
The source of the radiation may be derived from a pulsed laser, a photoflash, a high power light emitting diode (LED), a pulsed xenon or a pulsed mercury lamp. More preferably, the source of the radiation is derived from a photoflash. The method for producing the pulse of radiation may be either through turning the radiation source on and off rapidly or through another mechanism such as a mechanical shutter device.
The triggering impulse need not be radiation. It can include other suitable means such as the use of an electrical current.
The trigger element can be any molecule or group that when exposed to specific conditions (ie the particular triggering impulse) is capable of generating an impulsive noise, either by itself or by interaction with a nearby reactive substrate.
Accordingly, in a preferred form, the trigger element interacts with a reactive substrate to generate the impulsive noise. The reactive substrate is preferably made from a material that in the absence of the trigger element is stable towards the triggering impulse but at least partly decomposes rapidly when the trigger element is exposed to the triggering impulse.
In a preferred embodiment, at least a proportion of the reactive substrate is a nitrated cellulose, nitrated starch, nitrated carbohydrate such as nitro-mannitol, 3-nitro-1,2,4-triazol-5-one, 2,4,6-trinitrobenzaldehyde, 2,4,6-trinitrotoluene, 2,4,6-trinitroanisole, 2,4,6-trinitro-m-cresol, N-methyl-N,2,4,6-tetranitroaniline, 2,4-dinitrotoluene, 2,4,6-trinitro-m-xylene, 1,3,6,8-tetranitronapthalene, dipentaerythritol hexanitrate, 2,2′,4,4′6,6′-hexanitrobiphenyl, tetranitropentaerythritol, nonanitroterphenyl, octanitroterphenyl, ammonium-2,4,5-trinitroimidazole, hexahydro-1,3,5-trinitro-1,3,5-triazine, octahydro-1,3,5,7-tetranitro-1,2,5,7-tetrazine, 1,2,4-trinitrobenzene, 1,3,5-trinitrobenzene, 1,3-diamino-2,4,6-trinitrobenzene.
It is preferred that the reactive substrate comprises an oxidising polymer produced from a polymer substance that is partially nitrated, that is, an organic polymer functionalised with organo-nitro groups.
It is further preferred that the reactive substrate contains nitrated cellulose, nitrated starch, nitrated carbohydrate such as nitro-mannitol.
In a preferred embodiment, at least a proportion of the reactive substrate is an inert material. An inert material, in the context of the present invention, is a material that does not generate an impulsive noise during or after exposure to a triggering impulse in either the presence or absence of the trigger element.
It is further preferred that the reactive substrate comprises an inert material that is at least partially coated with a thin film of a substance that does produce an impulsive noise on exposure to a triggering impulse in the presence of the trigger group.
It is further preferred that the reactive substrate comprises a nitrated cellulose material such as nitrocellulose used for filtration membranes.
It is further preferred that the reactive substrate comprises a nitrocellulose material containing a proportion of cellulose acetate.
The proportion of cellulose acetate is preferably between 5 to 20% w/w relative to the nitrocellulose.
It is further preferred that the reactive substrate is in the form of a porous substrate.
The reactive substrate is preferably located in close proximity to the trigger element. For instance, it may form a shell encapsulating the trigger element. Conversely, the substrate may be in the form of a particle with a diameter of between 1 to 10,000 nm wherein said particle is at least partly functionalised with the trigger element. In another preferred form, the substrate forms part of the region in which the sample is analysed. For instance, the surface of the region may comprise the substrate.
In a preferred form, the trigger element is a coloured, insoluble substance or precipitate. The present inventors have found that certain coloured substances or precipitates, such as those produced by enzymes interacting with the enzyme substrates can function as trigger elements. Enzymes capable of producing such coloured insoluble substances are well known to those skilled in the art and include peroxidases, phosphatases, esterases, oxidases such as horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, choline oxidase. These enzymes catalyse the precipitation of a chromogenic substrate in the immediate vicinity of the enzyme. Chromogens may include nitro blue tetrazolium (NBT/BCIP), 3,3′-diaminobenzidine tetrahydrochloride (DAB), and 3-amino-9-ethylcarbazole (AEC).
Preferably the coloured, insoluble substance is produced by a peroxidase reacting with 3,3′diaminobenzidine tetrahydrochloride to produce a red-brown precipitate.
Preferably the coloured, insoluble substance is produced by a peroxidase reacting with 3,3′diaminobenzidine tetrahydrochloride in the presence of nickel ions to produce a blue-grey precipitate.
In a further preferred embodiment an enzyme can be used to produce a metallic deposit on the reactive substrate, said metallic deposit acting as the trigger element. Such enzyme-catalysed metal deposition is known to those skilled in the art and is described in US2005/0100976 A1, which is incorporated by reference in its entirety.
In a preferred form, the trigger element is a nanoparticle. The present inventors have found that nanoparticles when exposed to specific conditions (ie particular triggering impulses) are capable of generating an impulsive noise from nearby reactive substrates.
Preferably, the nanoparticle is between 1 and 100 nm in diameter.
Preferably, the nanoparticle is made from metals such as gold, silver, palladium, copper, platinum, nickel, cobalt, chromium, iron, ruthenium, rhodium, or alloys of such metals; metal oxides such as titanium oxide, iron oxides, zinc oxides, nickel oxides, copper oxides, cobalt oxides and indium oxide; metal sulfides; metal selenides; carbon nanoparticles, carbon black, carbon nanotubes, carbon fullerenes; magnetic nanoparticles; clusters of dye molecules; or core-shell nanoparticles wherein a nanoparticle core is coated with another material to form a shell.
In the case where a nano-particle is a core-shell nanoparticle, the core-shell nanoparticle may be particles with a reactive metal core and an inert shell such as aluminium core-aluminium oxide shell nanoparticles.
The nanoparticles may be spherical, oblate spheroids, platelets, cubic, rod-like, hemi-spherical or other shape, depending on the desired absorption characteristics. It is known in the art that depending on the shape of the nanoparticle it is possible to change its absorption characteristics, thus rod-like gold nanoparticles absorb at longer wavelengths than spherical nanoparticles and hollow hemispherical gold nanoparticles may have significant absorption characteristics in the near infra-red spectrum.
In one embodiment, the trigger element comprises a gold nanoparticle of 5 to 60 nm diameter.
In another embodiment, the trigger element comprises a carbon black nanoparticle.
Nanoparticles are able to absorb part of the burst of radiation, giving rise to a high energy state where the nanoparticles specifically interacts with a reactive substrate such that it causes the substrate to produce an impulsive noise through rapid chemical degradation reactions.
Although not wishing to be bound by scientific theory, it is believed that the nanoparticle when in its high energy state after absorption of radiant energy, is able to catalyse the localised chemical breakdown of the reactive substrate, giving rise to a number of gaseous products. The rapid nature of this chemical reaction gives rise to pressure waves in the surrounding medium that manifest themselves as impulsive noise. This impulsive noise may be detected and analysed using microphone systems or other methods that are sensitive towards changes in pressure in gas, liquid or solid media.
The sensitivity of the method may be enhanced by techniques known in the art. For instance, in circumstances where the trigger element is a gold nanoparticle, the amount of nanoparticles present may be increased by the addition of an LI silver enhancer solution. The LI silver enhancer solution consists of a two-part electroless plating solution that autocatalytically plates silver onto gold nanoparticles, thereby increasing the size and optical density of the nanoparticles.
Accordingly, there is further provided a method according to the first aspect, said method comprising, before steps (a) and (b), the step of:
exposing said one or more trigger elements to a solution of electroless metal plating solution, wherein said electroless metal plating solution deposits metal onto the triggering elements.
The means for detecting impulsive noise is preferably a microphone, a piezoelectric system or other means of detecting pressure changes in gas, solid or liquid media.
In one embodiment, the present invention provides a method for measuring the presence of an analyte in a sample, said method comprising, before steps (a) and (b), the steps of:
(i) immobilising first receptors in a region wherein the first receptors are capable of binding to the analyte;
(ii) adding the sample to the region whereby, when analyte is present in the sample, the analyte binds to said first receptors;
(iii) adding second receptors to the region wherein the second receptors bind to the analyte and wherein the second receptors comprise said one or more trigger elements; and
(iv) removing unbound second receptors from said region.
Steps (ii) and (iv) may be carried out in either order or simultaneously.
For example, the second receptors may be present to the region, perhaps in a dehydrated form, before the addition of the sample. Alternatively, the second receptors may be added to the region simultaneously with or after the addition of the sample.
In another embodiment, the present invention provides a method for measuring the presence of an analyte in a sample, said method comprising, before steps (a) and (b), the steps of:
(i) immobilising first receptors in a region wherein the first receptors are capable of binding to the analyte;
(ii) adding analyte-mimics to the region wherein the analyte-mimics bind to the first receptors and wherein the analyte-mimics comprise a trigger element that generates an impulsive noise when triggered;
(iii) adding the sample to the region whereby, when analyte is present in the sample, the analyte binds to said first receptors and displaces said analyte-mimics; and
(iv) removing unbound analyte-mimics from said region.
Steps (ii) and (iv) may be carried out in either order or simultaneously.
The analyte-mimics may be present in the region and bound to the first receptors before addition of the sample. Alternatively, the analyte-mimics may be added to the region simultaneously with or after the addition of the sample.
In the context of the present invention it is preferred that the receptors are antibodies such as monoclonal or polyclonal antibodies or their fragments capable of binding to an analyte, such as the Fab or Fv fragments, streptavidin, avidin, biotin, protein A, protein A and its complex with antibodies, proteins (natural or recombinant), protein scaffolds, protein G, protein G and its complex with antibodies, lectins, DNA, RNA, nucleic acid aptamers, enzymes, G-protein coupled receptors, G-proteins, anticalins, trinectins, affibody molecules, receptors based on molecularly imprinted polymers.
It is particularly preferred that the receptors are antibodies such as monoclonal or polyclonal antibodies or their fragments capable of binding to an analyte, such as the Fab or Fv fragments.
The analyte-mimics of the present invention are preferably functionalized analyte or hapten molecules that have been modified in order to attach them to the trigger group in such a way that they are capable of binding to the first receptors. A requirement of the analyte-mimic is that the first receptor/analyte-mimic binding properties are such that the analyte-mimic is able to replaced by the analyte of interest.
It is preferred that the first receptors are immobilised either by physical adsorption or via specific chemical interaction. Similarly, it is preferred that the second receptors and analyte-mimics are attached to the trigger element either by physical adsorption or via specific chemical interaction.
In the case of the trigger element being a metal nanoparticle such as a gold, platinum, palladium or silver nanoparticle, it is preferred that the receptor/analyte-mimic is adsorbed onto the metal surfaces via non-specific binding, or that specific chemical coupling strategies such as those known to those skilled in this art are used. Such specific coupling chemistries include, but are not limited to: using metal-sulfur interactions where the receptor/analyte-mimic contains a surface accessible thiol, disulfide, sulphide, or other sulfur moiety known to bind to gold, palladium, platinum or silver.
In a further preferred embodiment, the trigger element that is attached to the receptor/analyte-mimic is further functionalised such that non-specific binding of other moieties in the sample to be analysed is minimised. This may be achieved through adsorbing proteins such as bovine serum albumin, or casein to the trigger group after functionalising with the receptor. It is also possible to minimise non-specific binding to the trigger element by functionalising the trigger element with compounds containing oligoethylene glycol or polyethylene glycol groups.
In a yet further embodiment, the present invention provides a method of measuring the presence of an analyte in a sample, said method comprising, before steps (a) and (b), the step of:
(i) exposing said sample to an analyte-specific substrate, wherein said analyte-specific substrate generate said one or more trigger elements in the presence of analyte.
Preferably the analyte-specific substrate is an enzyme capable of producing one or more trigger elements in the presence of analyte and include peroxidases, phosphatases, esterases, oxidases such as horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, choline oxidase, glucose oxidase.
In a yet further embodiment, the present invention provides a method of measuring the presence of an analyte in a sample, said method comprising, before steps (a) and (b), the steps of:
(i) exposing said sample to an analyte-specific substrate, wherein said analyte-specific substrate adsorbs or binds to said analyte and wherein said analyte-specific substrate comprises one or more trigger elements; and
(ii) removing unadsorbed or unbound analyte-specific substrate.
Preferably, the analyte is a protein and the analyte-specific substrate is a nanoparticle, such as a gold nanoparticle. The present inventors have found that a sample of protein will adsorb gold nanoparticles in proportion to the amount of protein present.
It is further preferred that the analyte is a protein or mixture of proteins that have been subjected to a Western blot analysis wherein a sample containing one or more proteins is subjected to gel electrophoresis, followed by transfer of the separated proteins onto a nitrocellulose membrane. Protein blotting procedures, such as Western blotting techniques, are well known to those of ordinary skill in the art. A review of protein blotting is given in “Protein blotting: a review” by B. J. Kurien, R. H Scoffield; J. Immunol. Methods, 274 (2003) pg 1-15, which is incorporated by reference in its entirety.
It is further preferred that the present embodiment is a Southern (DNA) or Northern (RNA) blotting analysis.
In a second aspect, the present invention provides a kit for performing the method of the first aspect, said kit comprising one or more of the components required for performing the method of the first aspect.
The present invention further provides reagents, such as receptors and analyte-mimics, that can be used in the method of the present invention.
Accordingly, in a third aspect, the present invention provides a receptor capable of binding to an analyte wherein the receptor is labelled with a trigger element wherein said trigger element generates an impulsive noise when triggered.
Further, in a fourth aspect, the present invention provides an analyte-mimic capable of binding to a receptor for an analyte, wherein said analyte-mimic is labelled with a trigger element wherein said trigger element generates an impulsive noise when triggered.
In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples.
Standard synthetic methods were used for the synthesis of nanoparticles. Typical methods may be found in publications such as: Clusters and Colloids, from Theory to Applications, G. Schmid (Ed.), 1994, VCH Publishers New York, USA; Nanoparticles and Nanostructured Films, J. H. Fendler (Ed.), 1998, Wiely-VCH. Unless otherwise specified water-soluble, sodium citrate coated gold nanoparticles of approximately 16 nm in diameter (hereafter known as Au16 gold nanoparticles) were synthesised according to the method of Schmitt, J. et. al. Adv. Mater., 1997, 9, 61. The final concentration of Au16 nanoparticles was 2 nM with respect to the number of particles.
A sample solution of Au16 gold nanoparticles (20 ml) was treated with N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (1 ul of 10 mM, in dimethylsulfoxide). The Au16 gold nanoparticles were left for 30 minutes, resulting in functionalization of the Au16 nanoparticles with biotin (solution Au16-Biotin). The Au16-Biotin solution was stable for several days at 4° C. and was used as a test analyte solution wherein the biotin represent the analyte to be determined conjugated to a Au16 gold nanoparticle as the trigger group.
A solution of Au16 gold nanoparticles (10 ml) was treated with cystamine hydrochloride (100 μl, 0.1M aqueous solution). Rapid cross-linking of the nanoparticles occurred as evidenced by the rapid colour change from burgundy-red to blue-black. Aliquots of this solution were diluted to 1 ml with water and were then filtered through Millipore™ GSWP04700 membranes. These membranes are derived from nitrocellulose and have a nominal pore size of 0.22 um. The filter area used was approximately 0.28 cm2. After filtration the cross-linked nanoparticles formed a thin film on and in the pores of the membrane. The membrane was dried and placed adjacent to a photoflash. The photoflash used was a Canon 550 unit operating at between one-eighth to half power. Other photoflash units from different manufacturers were also trialled and could be readily used. A microphone was placed adjacent to the membrane and connected to a digitally controlled potentiostat and data acquisition system (AdInstruments, Powerlab Data Acquisition System and Potentiostat). A voltage was applied to the microphone (0.5V) and changes in current was monitored as the microphone responded to different noise levels. The photoflash was then triggered, producing a brief burst of irradiation and the impulsive noise generated by the substrate with the absorbed Au16 nanoparticles was monitored. The resulting signal was normalised to a pre-flash initial current value of zero microamps, squared and integrated in order to obtain a value (hereafter called the integrated noise level) for the impulsive noise level generated.
A Millipore™ GSWP04700 nitrocellulose membrane (0.28 cm2) was functionalised with a protein receptor, streptavidin (10 ul of 1 mg/ml) for 3 min. During this time the streptavidin non-specifically bound to the nitrocellulose membrane surface. The membrane was then placed onto a porous adsorbent membrane pad that acts as a reservoir for adsorbing the liquid from the applied sample. The membrane composite was then placed in a holder, forming a well above the membrane. A solution of Au16-Biotin (1 ml) was then placed in the well and allowed to flow into the adsorbent pad below the membrane by capillary forces. The Au16-Biotin nanoparticle analyte was bound to the streptavidin on the surface during this phase. The membrane was subsequently removed, dried and placed onto the photoflash and a microphone connected as in example 3. The integrated noise level was 0.16. A control experiment wherein the Millipore™ GSWP04700 nitrocellulose membrane was not firstly functionalised with streptavidin was also carried out in analogous manner. The integrated noise level was 0.01 clearly indicating that detection of the biotin analyte requires the streptavidin receptor bound onto the substrate.
A solution of Au16 gold nanoparticles (10 ml) was treated with cystamine hydrochloride (100 ul, 0.1M aqueous solution). Rapid cross-linking of the nanoparticles occurred as evidenced by the rapid colour change from burgundy-red to blue-black. Aliquots of this solution were diluted with water as required and were then filtered through Millipore™ GSWP04700 membranes. These membranes are derived from nitrocellulose and have a nominal pore size of 0.22 um. Two samples of each nanoparticle aliquot were prepared and were filtered through membranes with two different areas. For sample A, the filter area used was approximately 0.28 cm2, for sample B the filter area was approximately 0.027 cm2. After filtration the cross-linked nanoparticles formed a thin film on and in the pores of the membrane. The membrane was dried and placed adjacent to a photoflash. The photoflash used was a Canon EX-550 unit operating at full power. A microphone system (Optima N26) was placed adjacent to the membrane and connected to a digitally controlled potentiostat and data acquisition system (AdInstruments, Powerlab Data Acquisition System and Potentiostat) using the preamplifier of the microphone system. The microphone system produced a current that was measured by the Powerlab Data Acquisition System and Potentiostat system. The absolute integral of the output signal was determined over the first four milliseconds after the photoflash was activated and for convenience was multiplied by a factor of 106. This value of the absolute integral will hereafter be known as the integrated noise signal (INS). The results are shown in table 1 below.
As can be seen the size and shape of the nanoparticle film influences the amount of the INS. For instance, by decreasing the area of the filter an effective increase in the sensitivity of the detection limit of the number of nanoparticles may be achieved (i.e. an effective lower detection limit of 1.2×109 nanoparticles for the 0.027 cm2 area film compared to 6×109 nanoparticles for the 0.28 cm2 film). Alternatively by increasing the area it is possible to distinguish higher concentrations of nanoparticles effectively.
A solution of Au16 gold nanoparticles (10 ml) was treated with cystamine hydrochloride (100 ul, 0.1M aqueous solution). Rapid cross-linking of the nanoparticles occurred as evidenced by the rapid colour change from burgundy-red to blue-black. Aliquots of this solution were diluted with water as required and were then filtered through Millipore™ GSWP04700 membranes or Millipore™ Durapore GVWP04700 membranes. These membranes are derived from nitrocellulose and a polyvinylidine fluoride (PVDF) polymer respectively and have a nominal pore size of 0.22 um. The filter area used was approximately 0.28 cm2. After filtration the cross-linked nanoparticles formed a thin film on and in the pores of the membrane. The membrane was dried and placed adjacent to a photoflash. The INS was then obtained as for example 5. Results of the INS for the two types of membrane are shown in table 2 below.
As can be seen the nitrocellulose containing membranes produce a larger INS than the inert PVDF membranes.
A Millipore™ GSWP04700 nitrocellulose membrane was placed on a 3 mm thick absorbent paper pad. A six mm diameter Teflon well was then placed on top of the nitrocellulose membrane. Aliquots of bovine serum albumin (BSA) solution (50 microliters) were allowed to wick through the nitrocellulose membrane into the lower adsorbent pad over a period of approximately 10 seconds. The aliquots of BSA contained 10 mg/ml, 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml and 0.001 mg/ml of BSA in a phosphate buffer (PB) at pH7.4. The membranes were then washed with fifty microliters of deionized water. This procedure effectively adsorbed different quantities of a protein (BSA) onto the nitrocellulose membrane. The amount of protein adsorbed was then detected by allowing two aliquots of Au16 nanoparticles (50 microliters) to pass through the nitrocellulose membrane, followed by deionized water (50 microliters). The Au16 nanoparticles adsorb onto the protein but do not adsorb onto the nitrocellulose membrane. The membrane was dried and placed adjacent to a photoflash. The INS was then obtained as for example 5 except that each membrane was subjected to the photoflash three times with an integration time of six milliseconds for each photoflash and the INS values reported are the sum of the three INS values. Results of the summed INS values for the membrane with different quantities of adsorbed BSA are shown in table 3 below.
As can be seen, different concentrations of proteins can be readily discerned.
Nitrocellulose membrane disks (6 mm diameter) were punched out of Millipore™ GSWP04700 nitrocellulose membranes. The disks were placed in a solution of a protein peroxidase conjugate (goat anti-rabbit peroxidase conjugate product number 0545, 2 ml, diluted 500:1 ratio in PB, Sigma Chemical Company) and gently mixed for 30 minutes at room temperature. The disks were removed from the protein peroxidase conjugate solution and washed twice with PB for 3 minutes. In the present example the analyte is 3,3′-diaminobenzidine, which forms a brown coloured precipitate through the action of the enzyme. Analyte solution was prepared by diluting a stock solution of 3,3′-diaminobenzidine (0.7 mg/ml) with deionized water. The analyte stock solution also contained urea hydrogen peroxide (0.2 mg/ml). Separate disks were then placed in the solutions containing different amounts of analyte for 5 minutes. After 5 minutes the disks were then placed in water to stop further enzyme reaction. The disks were dried and placed adjacent to a photoflash. The INS was then obtained as for example 7. Results of the summed INS values for the disks with different quantities of 3,3′diaminobenzidine are shown in table 4.
Nitrocellulose membrane disks (6 mm diameter) were punched out of Millipore™ GSWP04700 nitrocellulose membranes. The disks were placed in a solution of goat anti-rabbit antibody (0.1 mg/ml, 2 ml in PB, Sigma Chemical Company, product number R2004) and gently mixed for 30 minutes at room temperature. The disks were rinsed once with PB and non-specific binding sites were then blocked with casein solution (0.5% wt/vol in PB, 2 ml) for 90 minutes. As a model analyte, rabbit anti-BSA antibody (Sigma Chemical Company, product number 1520) was prepared at different concentrations in PB. Concentration ranged from 2 to 200 ng/ml. Separate disks were incubated with the rabbit anti-BSA antibody solutions for 30 minutes at room temperature. The disks were washed in PB for two minutes. The disks were then incubated with goat anti-rabbit antibody peroxidase conjugate (diluted 500:1 ratio in PB, Sigma Chemical Company product number 0545) for 10 minutes with gentle mixing. The disks were then washed in PB for three minutes. This wash was repeated twice more. Then a solution (0.7 ml) of 3,3′-diaminobenzidine (0.7 mg/ml), urea hydrogen peroxide (0.2 mg/ml) in TRIS buffer (0.06M) was added to each disk and the disks were incubated for two minutes. The disks were then added to deionized water for 2 minutes. The disks were then dried and placed adjacent to a photoflash. The INS was then obtained as for example 5 except that the total integration time was six milliseconds after the photoflash was activated. Results of the INS for the disks with different quantities of the analyte (rabbit anti-BSA antibody) are shown in table 5.
The Au16-goat anti-rabbit antibody conjugate was made according to the method of Wang et al J. Virological Methods, 132 (2006) 212-215. To a solution of Au16 gold nanoparticles (2 ml) was added potassium carbonate buffer (2% wt/vol) to increase the pH of the solution to 8.6. A solution of goat anti-rabbit antibody (ICN#55602, 30 μl, 500 μg/ml) was then added to the Au16 gold nanoparticle solution and the solution was allowed to stand for 60 minutes at room temperature to form the Au16-goat anti-rabbit antibody conjugate. The solution was then purified from excess antibody by centrifugation (14,000 rpm, 20 minutes), followed by resuspension of the Au 16-goat anti-rabbit antibody conjugate pellet in PB containing 1% wt/vol of BSA.
Nitrocellulose membrane disks (6 mm diameter) were punched out of Millipore™ GSWP04700 nitrocellulose membranes. The disks were placed in a solution of goat anti-rabbit antibody (0.1 mg/ml, 2 ml in PB, Sigma Chemical Company, product number R2004) and gently mixed for 30 minutes at room temperature. The disks were rinsed once with PB and non-specific binding sites were then blocked with casein solution (0.5% wt/vol in PB, 2 ml) for 90 minutes. As a model analyte, rabbit anti-BSA antibody (Sigma Chemical Company, product number 1520) was prepared at different concentrations in PB. Concentration ranged from 2 to 200 ng/ml. Separate disks were incubated with the rabbit anti-BSA antibody solutions for 30 minutes at room temperature. The disks were washed in PB for two minutes. The disks were then incubated overnight with solutions (100 microliters) of Au16-goat anti-rabbit antibody conjugate, made according to example 10. The disks were then washed with deionized water. The disks were then dried and placed adjacent to a photoflash. The INS was then obtained as for example 5 except that the total integration time was six milliseconds after the photoflash was activated. Results of the INS for the disks with different quantities of the analyte (rabbit anti-BSA antibody) are shown in table 5.
A solution of Au16 gold nanoparticles (10 ml) was treated with cystamine hydrochloride (100 ul, 0.1M aqueous solution). Rapid cross-linking of the nanoparticles occurred as evidenced by the rapid colour change from burgundy-red to blue-black. Aliquots of this solution were diluted with water as required and were then filtered through Millipore™ GSWP04700 membranes. These membranes are derived from nitrocellulose and have a nominal pore size of 0.22 um. The filter area used was approximately 0.28 cm2. After filtration the cross-linked nanoparticles formed a thin film on and in the pores of the membrane. The membrane segments containing the cross-linked nanoparticle films were then placed in a solution of LI silver enhancer solution from Nanoprobes Inc, NY for 15 minutes. The LI silver enhancer solution consists of a two part electroless plating solutions that autocatalytically plate silver onto gold nanoparticles, thereby increasing the size and optical density of the nanoparticles. The membrane segments were washed with deionized water. The membrane segments were then dried and placed adjacent to a photoflash. The INS was then obtained as for example 5. Results of the INS for the membrane segments that had been treated with the silver enhancer solution according to the present example are compared to the INS for membrane segments that had not been treated with the silver enhancer solution according to example 5, are shown in table 6.
As can be clearly seen there is a significant increase in the INS for the nanoparticles material on the nitrocellulose membrane once treated with silver enhancer solution.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed anywhere before the priority date of each claim of this application.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005905353 | Sep 2005 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2006/001417 | 9/28/2006 | WO | 00 | 5/18/2009 |
