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WO1997045741A1 - Immunoelectrode dans l'ecoulement du fluide pour immunodosage amperometrique rapide - Google Patents

Immunoelectrode dans l'ecoulement du fluide pour immunodosage amperometrique rapide Download PDF

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Publication number
WO1997045741A1
WO1997045741A1 PCT/US1997/008587 US9708587W WO9745741A1 WO 1997045741 A1 WO1997045741 A1 WO 1997045741A1 US 9708587 W US9708587 W US 9708587W WO 9745741 A1 WO9745741 A1 WO 9745741A1
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WIPO (PCT)
Prior art keywords
flow
immuno
electrode
cell
analyte
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Application number
PCT/US1997/008587
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English (en)
Inventor
Ebtisam S. Wilkins
Andrey L. Ghindilis
Plamen B. Atanasov
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Priority to AU31360/97A priority Critical patent/AU3136097A/en
Publication of WO1997045741A1 publication Critical patent/WO1997045741A1/fr
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    • 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

Definitions

  • This invention relates to sensors for detecting and quantifying a target analyte via immunological based reactions, and more particularly to a flow through amperometric sensor.
  • Immunoassay techniques are based on the ability of antibodies to form complexes with the corresponding antigens. This property of highly specific molecular recognition of antigens by antibodies leads to high selectivity of assays based on immune principles. The high affinity of antigen-antibody interactions allows very small quantities of a target analyte to be determined. Contemporary immunological techniques allow production of antibodies against a great number of antigens, and enables immunoanalysis of many substances such as low molecular weight drugs, short peptides, toxins, as well as polypeptides, proteins, viruses and bacteria.
  • Immunoassay techniques are used mainly in clinical analyses and medical diagnostics. Immunoassay techniques could, in principle, be utilized in many non-clinical applications if assay systems that were better adapted for field conditions were available.
  • an immuno-sensor (biosensor) consists of a signal transducer and a biochemically interactive system employing principles of biological molecular recognition. Based on the nature of the physical detection used in the transducer, immuno-sensing systems can be classified as optical, gravimetric and electrochemical. In optical transducers, detection is based on light-sensitive elements. The optical signal detection can be conducted by spectrophotometric, spectrofluorimetric, hemiluminometric, reflectometric or other related techniques.
  • Gravimetric transducers are based on sensitive detection of mass changes following antigen-antibody complex formation.
  • Piezoelectric detectors are typically based on acoustical resonators having resonant frequencies that are altered by the change in mass of a layer which is in contact with the resonator. This layer typically includes one member of an antigen- antibody complex. When the other member attaches to the layer, the resonance frequency shifts. These transducers cannot distinguish between specific binding and non-specific binding.
  • Electrochemical transducers are based on detection of changes in electro transfer caused by the immuno-interaction. In particular, this detection is brought about using amperometric, poteniometric, conductometric (at constant voltage) or impedimetric (at alternative voltage) devices.
  • Flow-injection principles can be used to enhance the efficiency of the immuno- interaction.
  • Prior art flow-injection immuno-sensing systems are based on a principle of displacement.
  • the immunoassay system is arranged as a column containing immobilized antibodies.
  • the column is saturated with a solution containing labeled antigen.
  • the column contains a solid carrier with immobilized antibody-labeled antigen complexes.
  • the affinity of antibodies for labeled antigens is usually significantly lower than their affinity for unlabeled (free) antigen due to stearic factors. Therefore, injection of free antigen into the column results in displacement of the labeled by the unlabeled antigen. Labeled antigen then is detected at the outlet of the column.
  • a similar scheme can be realized based on the use of immobilized antigen. In this case, injection of the analyte leads to replacement of antibody conjugated complex.
  • Another object of the invention is to provide a portable immuno-sensor capable to detect both (i) high molecular weight and (ii) low molecular weight target analytes under non- laboratory conditions.
  • the present invention is an electrode and measuring cell for fast flow immunoassay of a wide range of analytes.
  • the high surface area electrode comprises an electroconductive material with immobilized immuno-species deposited on glass, paper or plastic filter.
  • the solution containing the target analyte and/or the enzyme labeled immunoconjugate flows through the electrode.
  • the assay procedure is based on a competitive or 'sandwich' scheme.
  • the immuno-interaction between the immobilized immunospecies target analyte and the enzyme labeled immunoconjugate results in binding of the enzyme label to the electrode surface.
  • the assay of the enzyme label is performed by flow injection of the corresponding enzyme substrate, and amperometric measurement of the product of the enzymatic reaction.
  • the measuring cell is equipped with an inlet, outlet, a reference electrode, a counter electrode, and a current collector attached to a disposable flow immuno-electrode.
  • Figure 1 is a schematic drawing of a flow-through sensor system according to the present invention.
  • Figure 2 is a cross-sectional view of an assay apparatus according to the present invention.
  • Figure 3 is an exploded cross-sectional view of assay probe 50 shown in Figure 2.
  • the present invention comprises a flow-through system in which the immuno- electrode utilizes a highly dispersed electro-conductive immuno-sorbent which also provides the immuno-electrode function. That is, the immuno-column doubles as the electrochemical transducer.
  • the highly dispersed material comprising the immuno-sorbent provides a high surface area-to- volume ratio which, in turn, provides a high rate of immuno-complex formation.
  • the high conductivity of the immuno-sorbent material allows the electrochemical detection of the enzyme label directly in the column. Therefore, the present invention provides the advantages of both the flow-immunoassay techniques and techniques that utilize immuno-electrodes.
  • FIG. 1 is a schematic drawing of a flow-through sensor system 10 according to the present invention.
  • System 10 utilizes a miniature immuno-column 17 which includes a highly dispersed immuno-sorbent 15 which acts as a working (measuring) electrode.
  • the current generated by the electrodes discussed below is measured by measurement assembly 16 which also includes conventional control circuitry for operating the various other components of the system. To simplify the drawing, the connections between measurement assembly 16 and the various other components have been omitted from the drawing.
  • Immuno-column 17 serves as both an immuno-reactor and an electrochemical measuring cell.
  • Immuno-column 17 includes an inert body which supports a porous filter membrane 18.
  • the immuno-sorbent 15 is deposited upon the filter membrane 18.
  • Filter membrane 18 is preferably constructed from glass, paper, or plastic.
  • the manner in which the system shown in Figure 1 is used may be more easily understood with reference to performing a conventional 'sandwich' immuno-analysis for a target analyte, X.
  • the immuno-sorbent 15 is assumed to be loaded with antibodies to X which are immobilized on the particles of the immuno-sorbent at the start of the assay.
  • the sample is injected into immuno-column 17 via sample port 13 and valve assembly 12 in a suitable carrier liquid.
  • Valve Assembly 12 may include a pump for moving the various fluids through the column. If molecules of X are present in the sample, they are bound to the immuno-sorbent by the antibodies attached to the particles. A second solution containing antibody to X is then introduced into the immuno-column from reservoir 22.
  • These antibodies are labeled with an enzyme that catalyzes a reaction involving a substrate Y.
  • an enzyme that catalyzes a reaction involving a substrate Y.
  • the immuno-column 17 is washed with carrier liquid from reservoir 24 to remove any unbound labeled antibody.
  • a solution containing substrate Y is then introduced into immuno-column 17 from reservoir 23.
  • the amperometric output from the electrodes is measured by assembly 16. The output is the current between the working and counter electrodes generated when the potential between the working and reference electrodes is maintained at a constant value.
  • the amperometric output is proportional to the concentration of the product of enzymatic reaction and therefore, proportional to the amount of the enzyme label bound to the immuno-sorbent.
  • the bound concentration of bound enzyme is proportional to the amount of X bound to the column.
  • the present invention may also be used to perform assays based on competitive inhibition of the antibody binding.
  • the immuno-sorbent is loaded with X', a compound that binds antibody to X via a group that is similar in configuration to X.
  • the presence of X will inhibit the binding of antibody to X'.
  • X and antibody to X' are introduced into the column.
  • the antibody to X' is labeled with the substrate. After the washing step described above, the amount of labeled antibody is measured as described above by introducing Y into the column and measuring the output from the electrodes.
  • a variant of the sensor arrangement involves a current collector 19 affixed above the immuno-sorbent layer 15 in intimate contact with the later.
  • the current collector 19 is used to uptake the amperometric signal from the immuno-sorbent layer 15 which may be viewed as an immuno-electrode, polarized as working electrode in amperometric mode.
  • the current collector 19 consists of a conductive probe made from electrochemically inert material (such as carbon, graphite, gold, etc.).
  • the immuno-column is a disposable assembly which is connected to the various electrodes when the column is attached to a non-disposable portion of the apparatus.
  • Figures 2 and 3 Such an embodiment is shown in Figures 2 and 3 at 50.
  • Figure 2 is a cross-sectional view of an assay apparatus according to the present invention.
  • Figure 3 is an exploded cross-sectional view of assay probe 50.
  • Assay probe 50 is constructed from three sub-assemblies shown at 60. 70, and 80, respectively.
  • the immuno-column sub-assembly 60 is a disposable unit which includes the immuno-sorbent 63 on a filter membrane 62 which is supported on an insulating plastic subort 61.
  • the immuno- column assembly fits into a reference electrode assembly 70 which includes the reference electrode 65 and counter electrode 64.
  • the working electrode assembly 80 fits into the immuno-electrode assembly and provides the working electrode 66 which is preferably constructed from carbon.
  • a flow tube 67 provides the connection through which the sample is pumped through the immuno-column.
  • the flow tube may be constructed from a glass capillary tube.
  • the electrodes contributing to the amperometric signal transduction: the working (measuring) electrode represented by flow immuno-sorbent layer 15, the reference electrode 20 and (or) the counter electrode 21 are in contact with the liquid flowing through the sensor assembly. Both a three electrode circuit scheme (with working electrode 15. counter 21 and reference electrodes 20) and a two electrode circuit scheme (with working electrode 15 and reference electrodes 20 where the reference electrode serves as a counter electrode as well) for the amperometric signal transduction can be employed.
  • Example 1 Determination of rabbit IgG utilizing a 'sandwich' assay scheme.
  • Immunoassay procedure The assay system was assembled by connecting the immuno-column to the liquid stream. The current collector was attached to the immuno- electrode. Then a sample containing target analyte (rabbit IgG) is introduced into the liquid stream formed by 0.01 M Na-phosphate buffer (pH 7.4), containing 0.15 M NaCl and 0.05% Tween-20 (PBST) and flows (for 2 minutes) through immuno-column. PBST containing peroxidase labeled antibodies against rabbit IgG (1 :50 diluted) is then allowed to flow through the immuno-column for 5 minutes. Then washing solution (PBST alone) is then allowed to flow through the immuno-column in order to remove unbound labeled antibodies.
  • PBST peroxidase labeled antibodies against rabbit IgG
  • Example 2 Determination of rabbit IgG utilizing a competitive binding protocol.
  • Immuno-sorbent preparation The immobilization of rabbit IgG on ULTI carbon was performed using Woodward's reagent by the method described above with reference to Example 1 to form the immuno-sorbent. Immuno-electrode was prepared by deposition of the immuno-sorbent suspension in the immuno-column and centrifugation.
  • Immunoassay procedure The assay system was assembled by connecting the immuno-column to the liquid stream.
  • the current collector was attached to the immuno- electrode.
  • a sample containing mixture of a target analyte (rabbit IgG) and peroxidase labeled antibodies against rabbit IgG (1 :400 diluted) was introduced into the liquid stream formed by 0.01 M Na-phosphate buffer (pH 7.4), containing 0.15 M NaCl and 0.05% Tween- 20 (PBST) and was allowed to flow for 8 minutes through immuno-column.
  • PBST 0.15 M NaCl and 0.05% Tween- 20
  • the solution containing 1 mM sodium iodide in of 0.1 M sodium acetate buffer containing 0.05% Tween-20 and 1 mM H 2 0 2 was allowed to flow through immuno-column.
  • the amperometric output was measured while the sodium iodide solution flowed through the immuno-column.
  • the flow rate at all the stages of the assay was 0.09 ml/min.
  • the sensor response is obtained by the method described above (see Example 1).
  • the immuno-electrode response was found to be inversely proportional to the concentration of rabbit IgG as expected.
  • Example 3 Determination of antibodies against Hantavirus in human blood plasma.
  • the recombinant nucleocapsid protein was formed from expression of plasmid constructs with the complete nucleocapsid gene in E. coli.
  • the product is a fusion protein with matose binding protein that is purified by passage through amaltose affinity column.
  • the immobilization of recombinant protein of Hantavirus on ULTI carbon was performed using Woodward's reagent by the method described above with reference to Example 1.
  • the immuo-electrode was prepared by deposition of the immuno-sorbent suspension in the immuno-column and centrifugation.
  • Immunoassay procedure The assay system was assembled by connecting the immuno-column to the liquid stream. The current collector was attached to the immuno- electrode. Then a blood plasma sample (diluted by PBST 1 :2) was introduced into the liquid stream and allowed to flow for 2 minutes through the immuno-column. PBST containing peroxidase labeled antibodies against human IgG (1 :50 diluted) was then allowed to flow through the immuno-column for 5 minutes. The immuno-column was then washed (PBST alone) for 2 minutes.
  • the sensor response was then measured while a solution containing 1 mM sodium iodide in of 0.1 M sodium acetate buffer containing 0.05% Tween-20 and 1 mM H 2 0 2 flowed through the immuno-column.
  • the flow rate of all the stages of the assay was 0.09 ml/min.
  • the sensor response to negative blood sample is practically the same as the control background response obtained for buffer solution.
  • the Hantavirus positive blood sample demonstrates an electrode response about 5 times higher than the background sensor response.
  • immuno-columns can be provided as disposable units.
  • the cost of such columns is minimal, since the structural members and electrodes may be constructed from inexpensive materials and the amount of material is small.
  • Systems that are partially disposable will also be apparent to those skilled in the art from the above discussion.
  • a disposable element comprising the immuno-electrode element comprising the immuno-sorbent coated particles on a filter membrane may be utilized.
  • the actual connection to current collector 19 may be made by a contact in the wall of the immuno-column.
  • a more complex disposable unit that includes electrodes 20 and/or 21 may also be provided.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
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  • Urology & Nephrology (AREA)
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  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne une électrode (15, 19) ainsi qu'une cellule de mesure (17, 50) destinée à l'immunodosage rapide en écoulement d'une vaste gamme de produits à analyser. L'électrode de grande superficie (15, 19) est constituée d'un matériau électroconducteur. Des immuno-espèces immobilisées sont déposées sur un filtre en verre, en papier ou en plastique (18, 62). La solution contenant le produit cible à analyser et/ou l'immunoconjugué s'écoule par l'électrode (15, 19). Le dosage se fait en mode compétitif ou sandwich. Il résulte de l'interaction immunitaire entre le produit cible à analyser contenant les immuno-espèces immobilisées et l'immunoconjugué marqué par une enzyme une liaison de l'enzyme de marquage sur la surface d'électrode. Le dosage de l'enzyme de marquage se fait selon la technique FIA pour le substrat de l'enzyme correspondante, à la suite de quoi on effectue une mesure ampérométrique du produit de la réaction enzymatique.
PCT/US1997/008587 1996-05-29 1997-05-20 Immunoelectrode dans l'ecoulement du fluide pour immunodosage amperometrique rapide Ceased WO1997045741A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU31360/97A AU3136097A (en) 1996-05-29 1997-05-20 Flow-through immuno-electrode for fast amperometric immunoassays

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1856696P 1996-05-29 1996-05-29
US60/018,566 1996-05-29

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WO1997045741A1 true WO1997045741A1 (fr) 1997-12-04

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027626A3 (fr) * 1999-10-01 2001-10-18 Biopreventive Ltd Moyens permettant d'effectuer des dosages immunologiques et systeme comprenant ces moyens
WO2009036931A1 (fr) 2007-09-18 2009-03-26 Eads Deutschland Gmbh Dispositif et procédé pour régénérer des biocapteurs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5281539A (en) * 1989-10-02 1994-01-25 The Regents Of The University Of Michigan Immunoassay device for continuous monitoring

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5281539A (en) * 1989-10-02 1994-01-25 The Regents Of The University Of Michigan Immunoassay device for continuous monitoring

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027626A3 (fr) * 1999-10-01 2001-10-18 Biopreventive Ltd Moyens permettant d'effectuer des dosages immunologiques et systeme comprenant ces moyens
WO2009036931A1 (fr) 2007-09-18 2009-03-26 Eads Deutschland Gmbh Dispositif et procédé pour régénérer des biocapteurs
EP2191270B1 (fr) * 2007-09-18 2013-07-24 EADS Deutschland GmbH Dispositif et procédé pour régénérer des biocapteurs

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Publication number Publication date
AU3136097A (en) 1998-01-05

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