GB2278447A - Dielectric porosity change immunoassay - Google Patents
Dielectric porosity change immunoassay Download PDFInfo
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- GB2278447A GB2278447A GB9311206A GB9311206A GB2278447A GB 2278447 A GB2278447 A GB 2278447A GB 9311206 A GB9311206 A GB 9311206A GB 9311206 A GB9311206 A GB 9311206A GB 2278447 A GB2278447 A GB 2278447A
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- 230000008859 change Effects 0.000 title claims abstract description 14
- 238000003018 immunoassay Methods 0.000 title claims abstract description 13
- 108010046334 Urease Proteins 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 10
- 229920000642 polymer Polymers 0.000 claims abstract description 8
- GAMPNQJDUFQVQO-UHFFFAOYSA-N acetic acid;phthalic acid Chemical compound CC(O)=O.OC(=O)C1=CC=CC=C1C(O)=O GAMPNQJDUFQVQO-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229920002301 cellulose acetate Polymers 0.000 claims abstract description 3
- 230000003197 catalytic effect Effects 0.000 claims abstract 12
- 239000012491 analyte Substances 0.000 claims description 27
- 108090000790 Enzymes Proteins 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 16
- 229920000623 Cellulose acetate phthalate Polymers 0.000 claims description 14
- 229940081734 cellulose acetate phthalate Drugs 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 230000015556 catabolic process Effects 0.000 claims description 12
- 239000003792 electrolyte Substances 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 claims description 2
- 230000009870 specific binding Effects 0.000 claims description 2
- 230000035699 permeability Effects 0.000 claims 4
- 239000003446 ligand Substances 0.000 claims 2
- 239000004382 Amylase Substances 0.000 claims 1
- 102000013142 Amylases Human genes 0.000 claims 1
- 108010065511 Amylases Proteins 0.000 claims 1
- 102000004533 Endonucleases Human genes 0.000 claims 1
- 108010042407 Endonucleases Proteins 0.000 claims 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims 1
- 102000004882 Lipase Human genes 0.000 claims 1
- 108090001060 Lipase Proteins 0.000 claims 1
- 239000004367 Lipase Substances 0.000 claims 1
- 102000004316 Oxidoreductases Human genes 0.000 claims 1
- 108090000854 Oxidoreductases Proteins 0.000 claims 1
- 235000019418 amylase Nutrition 0.000 claims 1
- 239000012911 assay medium Substances 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 239000000470 constituent Substances 0.000 claims 1
- 230000000984 immunochemical effect Effects 0.000 claims 1
- 235000019421 lipase Nutrition 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 claims 1
- 150000007523 nucleic acids Chemical class 0.000 claims 1
- 102000039446 nucleic acids Human genes 0.000 claims 1
- 108020004707 nucleic acids Proteins 0.000 claims 1
- 238000006479 redox reaction Methods 0.000 claims 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 230000027455 binding Effects 0.000 description 6
- 238000000576 coating method Methods 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 229920003141 Eudragit® S 100 Polymers 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- FLKPEMZONWLCSK-UHFFFAOYSA-N diethyl phthalate Chemical compound CCOC(=O)C1=CC=CC=C1C(=O)OCC FLKPEMZONWLCSK-UHFFFAOYSA-N 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 239000002702 enteric coating Substances 0.000 description 3
- 238000009505 enteric coating Methods 0.000 description 3
- 239000004014 plasticizer Substances 0.000 description 3
- 229920006254 polymer film Polymers 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 229910021607 Silver chloride Inorganic materials 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000008364 bulk solution Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000005595 deprotonation Effects 0.000 description 1
- 238000010537 deprotonation reaction Methods 0.000 description 1
- 238000007812 electrochemical assay Methods 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007650 screen-printing Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/002—Electrode membranes
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A homogeneous immunoassay in which the detectable signal is produced by the effect of a reactive or catalytic species which, during the course of the reaction of the ingredients of the immunoassay, becomes immobilised at or very near to a dielectric layer covering an electrode. Said reactive or catalytic species directly or indirectly effects a reaction with the said dielectric layer wherein the dielectric layer becomes porous causing a measurable change in electrical properties at the electrode surface. The electrical property may be resistance or capacitance. The dielectric layer may be of a pH-sensitive polymer, panticularly cellulose acetate hydrogen phthalate and the species preferably causes a change in pH. The species may be urease.
Description
Dielectric Breakdown Immunoassay
Background Of the Invention
This invention relates to a novel homogeneous immunoassay format. Immunoassays may be divided into 'homogeneous' and 'heterogeneous' categories. In a heterogeneous immunoassay the analyte in the sample is used to bind a certain amount of a labelled species to a solid phase via antibody-antigen interaction. A wash step then separates the bound label from the free label. One or another of these label populations may then be quantitated, the amount of label measured bearing a known relationship to the concentration of analyte in the sample. In a homogeneous immunoassay no wash step is used: rather binding of analyte in the sample to its specific binding partner (antigen or antibody) causes a change in the measurable activity of the label used.The advantage of the homogeneous assay is that it is easier to perform, requiring less operations than its heterogeneous counterpart.
In operation, however, those few homogeneous formats that have been demonstrated lack sensitivity, often because the change in activity of the label is not great. For example, one assay format known commercially as EMIT form Syva relies on binding of enzyme-labelled antigen to an antibody, in competition with the analyte: binding of the enzyme changes its specific activity, but only by a factor of approximately 50 %. Thus there is a need of simple homogeneous immunoassays in which the activity of the label undergoes a quantitative change.
Background Of the Invention
In the present invention, a novel homogeneous immunoassay format is described based on the impedance analysis of dielectric coatings of electrodes. The impedance of an electrode is sensitive to a number of factors. Changes in electrode impedance can usefully be measured by changes in double layer capacity. The double layer capacity (Cdl) arises from the separation at the surface of an electrode between the electronic charges in the metal and the (mobile) ionic charges in the solution in contact with the metal. For a metal (e.g. gold) in contact with a solution of aqueous potassium chloride, this separation is due to the presence of a layer of water molecules on the metal surface.This double layer capacity may be calculated from the standard formula for a parallel plate condenser,
Cdl =0ear A/d where Er is the effective dielectric constant of the water layer,
A is the electrode area and d is the diameter of a water molecule.
It has now been found that minute imperfections in a dielectric layer give rise to a dramatic increase in double layer capacity which can easily be measured.
Summary Of Invention
The invention relies on the immunochemically modulated binding of an effector species to a dielectric coated on an electrode where the effector species is capable of affecting the dielectric properties of the electrode coating. Preferably but not exclusively, the effector species would cause the localised breakdown of the dielectric layer and this breakdown would be measured by a change in the double layer capacity at the electrode surface. However, other measures of dielectric breakdown could be envisaged, such as increasing current at a polarised electrode. The effector species is preferably an enzyme or catalyst that produces a product capable of reacting with the dielectric, or an enzyme that hydrolyses the dielectric directly.
For example, it is possible to use pH changing enzymes in connection with a dielectric that becomes soluble when the pH is changed.
One of many appropriate combinations of an enzyme with a dielectric is the combination of urease with materials such as those used in enteric coatings for tablets. Enteric coatings work by being insoluble at the low pH of the stomach, but are soluble at the pH in the intestine (pH 6 and above). One example of such a coating material is cellulose acetate hydrogen phthalate. Further examples are methyl vinyl ether-maleic anhydride copolymer esters, anionic polymerisates of methacrylic acid and esters of methacrylic acid (EudragitB of Röhm-Pharma, Darmstadt, Germany).
Urease catalyses the reaction
H20 + urea --- > 2NH3 + CO2.
The resulting increase of the pH value leads to a solubilisation of said materials.
If, for example, an antibody immobilised onto said pH-sensitive materials captures an urease-labelled conjugate in an immunoassay, the urease produces a local pH change that leads to the solubilisation of the dielectric layer at the point of conjugate capture. Most importantly, it turned out that the local solubilisation even occurs in solutions where because of the buffering capacity of the bulk solution a significant pH change in the solution does not take place. Since the assays according to this invention do not require a wash step they can be characterized as homogeneous. However, as presumably the reactions do not take place entirely in solution it appears to be more correct to call them pseudo-homogeneous.
Additionally, this method can be enhanced by polarising the working electrode at a potential where reactive species can be produced when the electrode comes into contact with the electrolyte. For example, it is advantageous to polarise the electrode at 1V vs Ag/AgCl. No reaction takes place until some small amount of electrolyte comes into contact with the electrode, as the dielectric layer is just starting to break down. Oxygen reduction at the electrode surface then produces OH which increases the local pH further and accelerates the breakdown of the dielectric. This amplifies the original signal considerably.
The invention further can be reduced to practice by the use of an H202-producing enzyme such as glucose oxidase in conjunction with Fenton's reaction. This reaction is commonly used in synthetic organic chemistry to hydroxylate aromatics, and works by producing the extremely reactive radical OH.; e.g.
H2O2 + Fe2+ ---- > Fe3+ + OH + HO The hydroxyl radical is so reactive that it survives only until it encounters an organic species. In this case a dielectric coating is chosen which contains structure elements reacting with the HO radicals. The introduction of hydroxyl groups enhances the solubility of the coating giving rise to a significant increase of the double layer capacity.
Examples
General mechanism
Planar carbon electrodes (1 mm diameter) formed by screen printing were coated with cellulose acetate phthalate (CAP) (Kodak) as follows: 70 mg CAP was mixed with 30 mg diethyl phthalate plasticiser and added to 400 mg of acetone to form a viscous solution. 5 Fl of this solution was then placed onto the working electrode and allowed to dry in air at room temperature.
The capacitance of the coated electrodes in 150 mmol L-1 sodium chloride, pH 4, was measured using a frequency response analyser (Schlumberger) and compared with that of bare carbon electrodes.
Coated electrodes were then exposed to 150 mmol L-1 sodium chloride, pH 6.5, for 15 minutes and the capacitance remeasured.
Electrode Conditions: Capacitance:
Bare electrode capacitance 24.6 pF cam~2 Coated electrode capacitance, pH 4 0.002 pF cam~2 Coated electrode after 15 minutes at pH 6.5 0.331 AF cm'2 Exposure of the coated electrode to a solution at pH 6.5 causes a 160 fold increase in the capacitance of the electrode.
Gold rod electrodes (4 mm diameter) were coated with cellulose acetate phthalate (CAP) (Kodak) as follows:
The electrode was dip coated once using a 1:8 w/w CAP / 30 % diethyl phthalate plasticiser to acetone mixture and allowed to dry for 30 minutes at room temperature.
The capacitance of the coated electrodes in 140 mmol L-1 sodium chloride, pH 4, was measured using a frequency response analyser (Schlumberger) in a three electrode configuration using a silver/silver chloride reference and gold counter electrode.
The capacitance of the coated electrode was measured after 5 minutes and 1 hour. The pH 4 solution was removed from the cell and replaced with pH 6.5, 140 mmol L-1 sodium chloride solution and the capacitance measured at 30 minute intervals.
Electrode conditions: Capacitance (EF cm-2):
Uncoated electrode 12.2
Coated electrode, pH 4, 5 minutes 0.002
Coated electrode, pH 4, 1 hour 0.002
Coated electrode, pH 6.5, 30 minutes 1.176
Coated electrode, pH 6,5, 1 hour 1.584
Coated electrode, pH 6.5, 1.5 hours 1.944
Coated electrode, pH 6.5, 2 hours 2.224
These results are represented graphically in Figure 1.
The pKa values of enteric coatings, CAP and Eudragit S100 polymers, have been calculated by titration of the solid in aqueous solution against dilute base. As both polymers' pH sensitivity is a direct result of the deprotonation of acid ester groups, the pKa value corresponds to the breakdown pH of the polymer.
The pKa of CAP is approximately 6.0, and the pKa of Eudragit
S100 approximately 7.0.
Breakdown of polymer films using urease
CAP polymer breakdown
A gold rod electrode (4 mm diameter) was coated with CAP as previously described and inserted into a glass cell. The buffer solution (1 mL) was EDTA (0.5 mM), sodium chloride (140 mM), urea (16 mM) adjusted to pH 4.8 using hydrochloric acid (0.1 M). Capacitance values were obtained at pH 4.8 after 30 and 60 minutes and then urease (20 L of a 1 mg mL-l solution) was added and the capacitance measured at 90, 120 and 150 minutes.
Time (minx Capacitance (F cm'21; 30 0.0004
60 (urease added) 0.0004
90 0.0133
120 5.048
150 6.544
These results are represented graphically in Figure 2 and confirm that it is possible to breakdown the CAP polymer film by the use of the enzyme urease causing a change in pH and resulting a a 4 orders of magnitude change in electrode capacitance.
Eudragit S100 polymer breakdown
The gold rod electrode (4 mm diameter) was dip coated twice using 20 % w/w Eudragit S100 (Röhm-Pharma) in acetone containing 20 % w/w dibutyl phthalate plasticiser and 5 % w/w Tween 80 and then placed in an atmosphere saturated with acetone for 15 minutes. The electrode was then removed and allowed to dry for 30 minutes at room temperature.
The glass cell was used containing 1 mL of buffer solution (described above) and capacitance values were obtained after 30 and 60 minutes. Urease (20 L of a 1 mg mL'1 solution) was added and the capacitance measured at 75 and 90 minutes.
Time (mien) : Capacitance (F cm~2): 30 0.0005
60 (urease added) 0.0006
75 1.488
90 9.760
The results are represented in Figure 3 and show that the film is stable in the buffer solution after 1 hour. 45 minutes after the addition of urease the electrode capacitance had returned to its uncoated value due to total breakdown of the polymer film.
Immunoassay protocols resulting in binding of an enzyme such as urease to the dielectric layer turn out to represent a highly sensitive method when used in conjunction with capacitance measurements.
The electrochemical assay according to this invention can be used in many different formats known to the man skilled in the art.
So it is possible to immobilise in any manner known to the man skilled in the art an antibody at the electrode surface for which the analyte to be measured and an analyte enzyme conjugate or an analyte analogue enzyme conjugate compete.
For a sandwich format a first antibody against the analyte to be measured is immobilised at the electrode surface and a second antibody labelled with an enzyme is present in the solution.
In a competition format the solution contains a biotin labelled analyte or a biotin labelled analogue of the analyte. An enzyme conjugate with avidin is also present in the solution or may be added after the capture reaction of the biotin labelled analyte or analyte analogue and the analyte to be measured with the immobilised antibody. Other binding pairs in place of avidin/biotin, e.g. IgG-aIgG may be used equally.
It is also possible to use a competitive assay where an analyte or an analogue of the analyte is conjugated with an anti-enzyme antibody. Also present in solution is the analyte to be measured and free enzyme, where the signal generated is inversely proportional to the analyte concentration being measured.
In a further competitive format, the competition between the analyte and an enzyme conjugate of the analyte or an analogue of the analyte with an enzyme can be performed in a wick (bibulous layer) or a capillary channel in which an antibody against the analyte is immobilised on the surface. After having passed the wick or capillary channel, the unbound enzyme conjugate of the analyte comes into contact with the electrode where an antienzyme antibody is immobilised. The signal generated is proportional to the concentration of analyte present.
Claims (12)
1. A method for determining the presence of an analyte in a
sample suspected of containing said analyte, said analyte
being a member of a specific binding pair consisting of
ligand and anti-ligand, wherein the amount of a detectable
signal is a function of the amount of analyte in the assay
medium, said detectable signal being detected at an
electrode, where
a) the electrode is covered with a thin layer of an
electrically resistive material that separates the
electrode from an electrolyte;
b) the detectable signal is produced by the effect of a
reactive or catalytic species that during the course
of the reaction becomes immobilised at or very near to
the dielectric electrode covering layer;;
c) the said reactive or catalytic species directly or
indirectly effects a reaction with the said dielectric
layer wherein the dielectric layer becomes porous or
more porous to the electrolyte, causing a measurable
change in electrical properties at the electrode
surface.
2. A method according to claim 1 where the electrically
resistive material is a pH-sensitive polymer and where
the reactive or catalytic species causes a change in pH
sufficient to increase the permeability of the electrode
covering layer.
3. A method to claim 1 in which the polymer is cellulose
acetate hydrogen phthalate.
4. A method according to claim 2 in which the reactive or
catalytic species is urease.
5. An immunoassay method in which a urease-antibody or
urease-antigen conjugate becomes bound at or very near a
layer of cellulose acetate phthalate via a specific
immunochemical reaction where the layer of cellulose
acetate phthalate covers an electrode and where the
urease activity of the conjugate leads to an increased
permeability of the cellulose acetate phthalate layer to
electrolyte, measured by a change in capacitance or
current passed by the underlying electrode.
6. A method according to claim 1 in which the electrode is
polarised at a potential at which a redox reaction can
occur with a constituent of the electrolyte such that
breakdown of the dielectric layer becomes accelerated
once the electrode first becomes exposed to the
electrolyte.
7. A method according to claim 1 where the activity of the
catalytic species results in the formation of a hydroxyl
radical, which is able to react with the dielectric
layer resulting in an increase in the permeability to
electrolyte of said layer.
8. A method according to claim 7 where the catalytic
species is a hydrogen peroxide-producing oxidase and the
electrolyte contains species able to promote Fenton's
reaction.
9. A method according to claim 1 in which the catalytic
species is a polymer-degrading enzyme which acts
directly on the dielectric layer to increase the
permeability of the layer.
10. A method according to claim 9 in which the catalytic
species is amylase or amyloglucosidase.
11. A method according to claim 9 in which the catalytic
species is an endonuclease and the dielectric comprises
or contains a nucleic acid.
12. A method according to claim 9 in which the catalytic
species is a lipase and the dielectric comprises or
contains lipid.
Priority Applications (11)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9311206A GB2278447A (en) | 1993-05-29 | 1993-05-29 | Dielectric porosity change immunoassay |
| DK94918378.4T DK0700520T3 (en) | 1993-05-29 | 1994-05-26 | Sensors based on polymer conversion |
| PCT/EP1994/001714 WO1994028414A1 (en) | 1993-05-29 | 1994-05-26 | Sensors based on polymer transformation |
| DE69404653T DE69404653T2 (en) | 1993-05-29 | 1994-05-26 | SENSORS BASED ON POLYMER CONVERSION |
| EP94918378A EP0700520B1 (en) | 1993-05-29 | 1994-05-26 | Sensors based on polymer transformation |
| US08/549,800 US5846744A (en) | 1993-05-29 | 1994-05-26 | Sensors based on polymer transformation |
| JP50022895A JP3713516B2 (en) | 1993-05-29 | 1994-05-26 | Sensors based on polymer transformations |
| AT94918378T ATE156270T1 (en) | 1993-05-29 | 1994-05-26 | SENSORS BASED ON POLYMER CONVERSION |
| ES94918378T ES2107833T3 (en) | 1993-05-29 | 1994-05-26 | SENSORS BASED ON TRANSFORMATION OF POLYMERS. |
| GR970402851T GR3025214T3 (en) | 1993-05-29 | 1997-10-29 | Sensors based on polymer transformation |
| HK98113863A HK1012708A1 (en) | 1993-05-29 | 1998-12-17 | Sensors based on polymer transformation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9311206A GB2278447A (en) | 1993-05-29 | 1993-05-29 | Dielectric porosity change immunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB9311206D0 GB9311206D0 (en) | 1993-07-14 |
| GB2278447A true GB2278447A (en) | 1994-11-30 |
Family
ID=10736397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9311206A Withdrawn GB2278447A (en) | 1993-05-29 | 1993-05-29 | Dielectric porosity change immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2278447A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2350677A (en) * | 1999-06-04 | 2000-12-06 | Cambridge Life Sciences | Enzyme detection |
| WO2007049269A1 (en) * | 2005-10-27 | 2007-05-03 | Bio-Rad Haifa Ltd. | Binding layer and method for its preparation and uses thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4352884A (en) * | 1980-02-19 | 1982-10-05 | Kuraray Co., Ltd | Carrier having acrylate copolymer coating for immobilization of bioactive materials |
| US4402819A (en) * | 1980-03-17 | 1983-09-06 | University Of Delaware | Antibody-selective membrane electrodes |
| EP0193154A2 (en) * | 1985-02-25 | 1986-09-03 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Potential-causing element for immunosensor |
| EP0525723A2 (en) * | 1991-07-29 | 1993-02-03 | Mochida Pharmaceutical Co., Ltd. | Process and device for specific binding assay |
-
1993
- 1993-05-29 GB GB9311206A patent/GB2278447A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4352884A (en) * | 1980-02-19 | 1982-10-05 | Kuraray Co., Ltd | Carrier having acrylate copolymer coating for immobilization of bioactive materials |
| US4402819A (en) * | 1980-03-17 | 1983-09-06 | University Of Delaware | Antibody-selective membrane electrodes |
| EP0193154A2 (en) * | 1985-02-25 | 1986-09-03 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Potential-causing element for immunosensor |
| EP0525723A2 (en) * | 1991-07-29 | 1993-02-03 | Mochida Pharmaceutical Co., Ltd. | Process and device for specific binding assay |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2350677A (en) * | 1999-06-04 | 2000-12-06 | Cambridge Life Sciences | Enzyme detection |
| WO2007049269A1 (en) * | 2005-10-27 | 2007-05-03 | Bio-Rad Haifa Ltd. | Binding layer and method for its preparation and uses thereof |
| US9040309B2 (en) | 2005-10-27 | 2015-05-26 | Bio-Rad Haifa Ltd. | Binding layer and method for its preparation and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9311206D0 (en) | 1993-07-14 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |