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WO1998037409A1 - Procede de detection electrochimique de macromolecules immunoactives - Google Patents

Procede de detection electrochimique de macromolecules immunoactives Download PDF

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Publication number
WO1998037409A1
WO1998037409A1 PCT/GB1998/000548 GB9800548W WO9837409A1 WO 1998037409 A1 WO1998037409 A1 WO 1998037409A1 GB 9800548 W GB9800548 W GB 9800548W WO 9837409 A1 WO9837409 A1 WO 9837409A1
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WIPO (PCT)
Prior art keywords
solution
potential
electrochemical
electrode
membrane
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PCT/GB1998/000548
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English (en)
Inventor
Dmitri Alexandrovich Farmakovski
Yevgeni Yurevich Milanovski
Vladimir Rurikovich Cherkasov
Yuri Sergeyevich Biryukov
Boris Vladimirovich Komarov
Original Assignee
Biosensor Technology Limited
Cross, Rupert, Edward, Blount
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Biosensor Technology Limited, Cross, Rupert, Edward, Blount filed Critical Biosensor Technology Limited
Priority to AU63005/98A priority Critical patent/AU6300598A/en
Publication of WO1998037409A1 publication Critical patent/WO1998037409A1/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

  • the invention relates to the sphere of medicine, pharmacology, biotechnology, agriculture, ecology, and more particularly to methods of laboratory investigation and analysis of various biological fluids, such as blood serum, lymph, urine, saliva, etc. undertaken for the purpose of medical diagnostics, industrial and environmental monitoring of both natural and man-made subjects .
  • immuno-active macromolecules or antigens (Ag)
  • macromolecules or antigens (Ag)
  • Ab antigens
  • Antibodies can also be antigens. Schematically, the antigen-antibody interaction can be represented as follows:
  • a further drawback of this method is the need for significant quantities of specialised apparatus and reagents: micro-titration plates, washers, thermostats, stainers, instruments for recording enzyme marker activity, etc., which makes the test more time consuming and costly.
  • potentiometric indication methods measuring the electrochemical potential
  • aqueous solutions proteins are polyelectrolytes, and therefore antigen-antibody interaction should alter their charge.
  • potentiometric sensors modified by antibodies or antigens for direct detection of immuno- active macromolecules in solutions has shown that such detection systems are characterised by low sensitivity and extremely low specificity, which is attributable to the high level of non-specific interactions between the components of the test solutions and the sensor surfaces [3, 4].
  • the most promising techniques are electrochemical methods of detecting macromolecules in solutions based on recording the change in isoelectric point of the antibodies or antigens as a result of the formation of Ab-Ag complexes during the immunochemical reaction.
  • the isoelectric point of a protein is taken to mean the pH value of a protein solution at which the protein molecules have a zero total charge.
  • the pH of the solution moves away from the value corresponding to the isoelectric point of the protein, into the more acidic or more alkaline range, the protein molecule will accordingly become positively or negatively charged.
  • the interacting antigen and antibody normally have different isoelectric points the Ag-Ab complex formed during the immunochemical reaction will have a different isoelectric point from the original values of its components.
  • the change in the isoelectric point of antibodies and antigens as a result of immunochemical reactions can be recorded by the ion-step procedure, as detailed in [4], In this method, use is made of an ion- sensitive sensor modified with antibodies or antigens.
  • This method is based on the detection of displacement of the isoelectric point of a membrane placed on an electrochemical sensor and incorporating appropriate receptors, following interaction between the latter and the macromolecules. In so doing, it is assumed that the membrane charge is directly determined by the charge of the receptors contained in it, and that for a fixed interaction time the amount by which the isoelectric point is displaced is proportional to the content of macromolecules in the solution under test.
  • This method involves the following:
  • An ion-permeable membrane made from latex and agarose is formed by chemical synthesis on the surface of an electrochemical sensor - an ion- selective field effect transistor (ISFET) .
  • ISFET ion- selective field effect transistor
  • Appropriate receptors are immobilised in the membrane by placing the ISFET complete with membrane in contact with a solution of the receptor molecules, thereby producing an im uno-sensor.
  • An electrochemical measuring cell is assembled by placing the immuno-sensor and a reference electrode interconnected by means of an electrical measuring instrument into a vessel through which the working solution is circulated.
  • the pH of the working solution is varied in an even manner by means of a gradient mixer, shifting the pH from the acid or neutral region into the alkaline region.
  • stage 4 the ionic strength of the working solution is periodically subjected to a step change by adding a saline solution with an ionic strength greater than that of the working solution.
  • stages 4 and 5 the voltage across the electrochemical cell is measured using the electrical instrument, and the pH of the working solution is measured using a pH-meter.
  • stage 6 a continuous plot of electrochemical cell potential versus working solution pH is taken with the aid of a two- coordinate chart recorder connected to the measuring device.
  • the point of intersection with the abscissa is found on the graph obtained in stage 7 , this being defined as the isoelectric point of the membrane with immobilised receptors.
  • the immuno-sensor and membrane containing the receptors is incubated for a fixed period of time in a solution containing a known quantity of receptor-specific macromolecules.
  • the immuno-sensor and membrane containing the receptors is incubated for a fixed period of time in a solution containing a known quantity of receptor-specific macromolecules .
  • stages 3 to 8 are repeated. 11. The amount by which the isoelectric point of the membrane is displaced as a result of interaction between the receptors and the macromolecules is determined.
  • Stages 3 to 11 are performed successively using solutions with different concentrations of specific macromolecules, and a calibration curve is plotted for displacement of the membrane isoelectric point versus the concentration of macromolecules in the solution.
  • the calibration curve obtained is then used to determine the content of macromolecules in different test solutions.
  • the invention provides a method of electrochemical indication of immuno-active macromolecules which is to a large extent free of the shortcomings inherent in the method described above, being simpler and cheaper, requiring less time to carry out, exhibiting greater sensitivity, and enhanced reliability of the results obtained.
  • the essence of the invention lies in the achievement of the above technical result in a method of electrochemical detection of immuno-active macromolecules in test solutions, involving the formation of an immuno-specific sensor with a membrane containing immobilised specific receptors; arrangement of an electrochemical measuring cell using the immuno- sensor and a reference electrode connected by means of a measuring device; immersion of these in a working solution; determination of the isoelectric point displacement of the membrane by measuring the cell potential with step changes in the ionic strength of the working solution; in which the membrane is formed from an electro-conductive polymer by electrochemical synthesis from a monomer solution containing specific receptors on the surface of a potentiometric electrode; and to determine the isoelectric point displacement of the membrane a test solution of ionic strength greater than that of the working solution is added to the working solution with the latter at constant pH.
  • a process exemplifying the invention consists of the following:
  • a potentiometric measuring electrode is used as the electrochemical sensor.
  • the electrode may be of random shape and may be made of any material having metallic conductivity and resistance to aqueous media This makes it possible to substantially reduce the cost of the test, by using a less expensive electrochemical sensor compared with the ISFET, and also by the fact that it allows the use of standard and inexpensive potentiometry instruments as the measuring device.
  • the membrane is formed on the surface of the electrochemical sensor by electrochemical synthesis of an electrically conductive polymer.
  • the electro-conductive polymer membrane performs a dual function, serving both to bind the receptors at the surface of the electrochemical sensor and render the electrochemical sensor sensitive to variation in ionic strength of the solution.
  • Immobilisation of the receptors in the membrane is carried out simultaneously with electrochemical synthesis of the membrane, providing controllability of the binding process.
  • a solution containing macromolecules specific to the bioreceptors on the electrochemical sensor is used as the solution with ionic strength greater than that of the working solution.
  • the isoelectric point displacement of the membrane as a result of interaction between the receptors and macromolecules is determined in relation to the change in the electrochemical cell potential over time and following interaction between the electrochemical sensor and the solution of greater ionic strength than that of the working solution, containing macromolecules specific to the receptors on the sensor.
  • the invention also provides a method of electrochemical detection of immuno-active macromolecules in a sample, comprising the steps of: a) preparing a sensing electrode having an electroconductive polymer coating, the coating having immobilised therein receptors which are specific to a desired macromolecule to be detected in the sample, b) treating the sensing electrode by immersion in a test solution containing the sample so that said desired macromolecules bind to said specific receptors, c) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when immersed in an electrolyte, and d) determining the change in the electric potential difference resulting from a change in ionic strength of the electrolyte at constant pH.
  • test solution used in step (b) is an electrolyte comprising a buffer solution of predetermined pH and the sample, and steps (c) and (d) are performed by monitoring and determining the change in electric potential difference when the treated sensing electrode is transferred from said test solution to electrolyte comprising pure buffer solution of said predetermined pH.
  • the invention further provides a method of producing a sensor for electrochemical detection of biological material, the sensor comprising an electrically conductive electrode coated with an electroconductive polymer with desired biomaterial immobilised therein, the method comprising the steps of: a) preparing an isotonic solution containing a monomer of the polymer to form the coating and the biomaterial to be immobilised therein, b) immersing the electrode to be coated in the isotonic solution, and c) applying a cyclic electric potential between the electrode and the solution to coat the electrode by electrochemical synthesis of the polymer from the solution, said cyclic electric potential being applied for at least one full cycle and having a peak value applied to the electrode which is less than +2 volts.
  • the invention still further provides apparatus for electrochemical detection of immuno-active molecules in a sample, comprising a sensing electrode having an electroconductive polymer coating, the coating having immobilised therein receptors which are specific to a desired macromolecule to be detected in the sample, means arranged to treat the sensing electrode by immersion in a test solution containing the sample so that said desired macromolecules bind to said specific receptors, means arranged to monitor the electric potential difference between the treated sensing electrode and a reference electrode when immersed in an electrolyte, means arranged to change the ionic strength of the electrolyte in which the sensing electrode and reference electrode are immersed while maintaining the pH constant, and means to determine the change in said monitored potential difference resulting from said change in ionic strength.
  • Fig.l An ion-sensitive membrane made of electroconductive polymer (2) and receptors (3) is formed on the surface of the electrochemical sensor (1) .
  • Fig.2 Formation of an electrochemical measuring cell, where (4) - electrochemical sensor with membrane;
  • Fig. 4 Diagram of variations in electrochemical cell potential caused by the immunosensor interaction with solutions containing sensor specific macromolecules in various concentrations (11-13) and with a solution not containing sensor specific macromolecules (14).
  • Fig. 5 A flow diagram illustrating software for controlling an assay procedure.
  • Fig. 6 A Gaussian plot of the statistical distribution of results obtained during calibration.
  • Fig. 7 A calibration curve for use in quantitative results.
  • a membrane made from an electroconductive polymer is formed on the surface of a potentiometric measuring electrode.
  • the basic requirements for the electrode used to implement the declared method are that it should possess metallic or quasi-metallic conductivity and that it should be stable in aqueous media.
  • the electrode may take the form of standard potentiometric electrodes as used in biochemical research [8], or may be designed specifically for use in the declared method.
  • the membrane is formed by electrochemical synthesis from a monomer solution (aniline, thiofene, furan, pyrrole) in a polar solvent (water, acetonitrile) . Electrochemical synthesis techniques suitable for the purposes of the declared method are well known and are described in [9].
  • receptors are incorporated into the membrane.
  • the receptors are dissolved in the electrochemical synthesis solution.
  • the receptors used may be monoclonal and polyclonal antibodies (for antigen detection) , protein antigens (viral lysates, recombinant proteins, synthetic peptides, hormones) (for antibody detection) , single-strand DNA molecules (for DNA detection), fragments of bacterial, plant and animal cells, intact bacterial cells, various chemical compounds conjugated with inert proteins (haptens) .
  • the concentration of receptors in the solution may vary in the range 0.1 - 10000 ⁇ g/ml.
  • the presence of receptors in the electrochemical synthesis solution may cause some variation in the synthesis parameters (synthesis time, current strength, applied potential) compared with those obtaining when a solution containing only monomer is used.
  • these parameters are specially selected in relation to receptor type, receptor concentration, etc.
  • the immunosensor is thus made.
  • the choice of electroconductive polymers as the material for the immunosensor membrane is dictated by their amenability to processing, low cost, excellent ion- exchange and ion-selective properties imparting sensitivity to changes in the ionic strength of the solution, and the ability of electroconductive polymers to hold a significant quantity of protein material [9] .
  • An electrochemical cell is set up by immersing the immunosensor and reference electrode linked together by a measuring device in vessel filled with working solution at a fixed pH.
  • the reference electrode may be of a standard commercially- available type [10], or may be of the type specially designed for use in medical measurements [8].
  • the measuring device may be a standard potentiometric measuring instrument [8] or a potentiostat [11]. Measuring devices specifically designed for implementation of the declared method may also be used.
  • the working solution may take the form of aqueous buffer solutions with high buffering capacity ensuring constant pH values, suitable both for electrochemical measurements and for immunological investigations: phosphate-saline, Tris-HCl, carbonate-bicarbonate, acetate, borate , etc. [12].
  • the container for the working solution in the declared method may be any vessel of suitable size made of a material with minimal adsorption properties, for example the well of a standard micro-titration plate.
  • the volume of working solution in the electrochemical cell may be 50 - 5000 ⁇ l depending on the type of test. 4.
  • the electrochemical cell potential is recorded for a fixed period of time (100-300 seconds depending on the nature of the test) using a recording device (chart recorder or special PC add-on card) linked to the measuring device.
  • Step changes are made to the ionic strength of the solution in the electrochemical cell and the immunosensor is simultaneously brought into contact with the macromolecules under test, by adding to the electrochemical cell a solution of ionic strength greater than that of the working solution with a known concentration of macromolecules under test.
  • these same fluids may be used as the higher ionic strength solution.
  • the use of these fluids is possible since their ionic strength is normally considerably greater than that of buffer solutions used as the working medium [8].
  • the dilution factor may range from 1:10 to 1:1000.
  • these fluids are used as the higher-ionic-strength medium, and their ionic strength is boosted artificially (if needed) by the addition of base electrolyte which does not alter the pH and does not impede the immuno-chemical reaction (for example NaCl, KC1 or Na : S0 4 ) .
  • base electrolyte which does not alter the pH and does not impede the immuno-chemical reaction
  • Solution may be introduced into the electrochemical cell either by direct addition into the vessel containing working solution, or by transferring the immunosensor and reference electrode from a vessel with clean working solution to a vessel containing working solution and higher-ion-strength solution.
  • the latter is preferable, as it allows precise control of the dilution of the higher-ion-strength solution and, consequently, increases the accuracy of the test.
  • test macromolecules in the higher- ion-strength solution may vary, depending on the nature of the test, in the range 0 to 10000 ⁇ g/ml.
  • the electrochemical cell potential and the nature of the change in potential in the presence of the higher-ion-strength solution are recorded over a fixed period of time (as in step 4) .
  • the time spent by the higher-ion-strength solution in the electrochemical cell, and hence the time spent recording potential values and potential variation, may be between 30 and 1200 seconds depending on the nature of the test.
  • the criterion is the minimum time taken for the reaction between the immunoactive molecules and the receptors in the sensor membrane to produce a noticeable change in the isoelectric point of the membrane.
  • step 6 the higher-ion-strength solution in the electrochemical cell is replaced by working solution.
  • the substitution is made either by drawing off the higher-ion-strength solution from the vessel containing the working solution, or by transferring the immunosensor and reference electrode from the vessel with the higher-ion- strength solution into a vessel with working solution.
  • step 7 the electrochemical cell potential and the variation of potential in the working solution are recorded (as in step 6) .
  • the time period during which recording is carried out may be 60 - 1200 seconds depending on the nature of the test, and is determined by the desorption from the membrane surface of the immunosensor of macromolecules which have not reacted with the receptors, and by the establishment of ionic equilibrium between the membrane and working solution.
  • a final value for cell potential is thus obtained following interaction between the immunosensor and the solution of greater ionic strength than the working solution.
  • the isoelectric point displacement of the membrane is determined with reference to the change in the electrochemical cell potential during and after interaction between the immunosensor and the higher-ion-strength solution. This is possible with the working solution at fixed pH because displacement of the isoelectric point of the membrane caused by the immunochemical reaction produces a change in the response of the immunosensor to a change in the solution ionic strength at practically all pH values.
  • Steps 4-9 are repeated using solutions of greater ionic strength than the working solution with different known concentrations of test macromolecules .
  • step 11 Using the data obtained in step 10, a calibration curve is plotted for the variation in electrochemical cell potential versus concentration of test macromolecules in the solution of greater ionic strength than the working solution.
  • the resultant calibration curve is used to determine (indicate) the content of macromolecules in test solutions (with unknown macromolecule content) .
  • steps 4-8 are carried out using the solutions under test as the higher- ion-strength media.
  • the test solutions must be similar in terms of ionic strength, pH and ion composition to the solutions used during step 11.
  • the declared technique can be illustrated by the following examples.
  • Milli-Ro/Milli-Q equipment (Millipore, USA) ;
  • electrochemical cell 2210 two-channel recorder (LKB - Instrumentation, Sweden) .
  • Membrane electrochemical synthesis was carried out on pyrrole aqueous solution electrode with base electrolyte and dedicated receptors. Pyrrole, base electrolyte and dedicated receptors concentrations were 0.10 N, o.l5 N and 50 mg/1, respectively.
  • the electrochemical synthesis was carried out in electrode potential cyclic sweep mode in the range -800 to 1200 mV (wrt Ag/AgCl - reference electrode) at a sweep rate of 100 mV/sec. As soon as the required membrane thickness was achieved ( ⁇ l ⁇ m) the process was terminated.
  • the electrode with the membrane was removed from the cell, washed in base electrolyte solution and placed in the electrochemical synthesis cell filled with pure base electrolyte, where it was maintained at static potential + 500 mV (wrt Ag/AgCl - reference electrode) until base current stabilisation was achieved.
  • the immunosensor thus produced was stored at +4°C in saline phosphate buffer solution to which was added 0.01% sodium azide.
  • a series of specimen test solutions with a Human Chorionic Gonadotropin level of 0.1 - 10 IU/ l were prepared.
  • An electrochemical cell was made up by placing the immunosensor and silver chloride reference electrode in the working solution and connecting them to the input of a digital millivoltmeter connected to the recorder.
  • the immunosensor was placed into a microtitration plate containing lOO ⁇ l of working solution, whilst the reference electrode was placed into a larger vessel also containing working solution.
  • the microtitration plate and the vessel with the reference electrode were connected were connected by means of a saline bridge filled with 0.1 N KC1 solution.
  • step 1.9 Potential change on the cell was recorded for 400 seconds. 1.10 After step 1.9, the immunosensor and saline bridge were transferred into a plate with pure working solution and potential on the cell was recorded for 600 seconds to obtain the final electrochemical cell potential.
  • Steps 1.7 - 1.10 were repeated successively, each time using a sample of test solution with higher HCG concentration.
  • the cell potential offset pattern was quantitatively estimated by the difference between base and final cell potential in millivolts.
  • HIV-1 p24, gp41 and gpl20 (American Bio- Technologies Inc. , USA) stored in saline phosphate buffer solution at +4°C;
  • - reference electrode for electrochemical synthesis see 1.1
  • - reference electrode for electrochemical cell - purpose made small Ag/AgCl reference electrode, 3mm in diameter, filled with KC1 saturated agarose (Serva, West Germany) ;
  • the electrode with the membrane was removed from the cell, washed in base electrolyte solution and placed in the cell for electrochemical synthesis filled with base electrolyte, where it was maintained at static potential +700 mV (wrt Ag/AgCl reference electrode) until base current stabilisation was achieved.
  • the immunosensor thus prepared was stored at +4°C in saline phosphate buffer solution with added 0.01% of sodium azide.
  • An electrochemical cell was made up by placing the immunosensor and reference electrode onto a microtitration plate filled with lOO ⁇ l of working solution and connecting them to the meter input.
  • step 2.9 the immunosensor and reference electrode were transferred onto a plate with pure working solution and cell potential modulation was recorded for 900 seconds - to obtain the final electrochemical cell potential value.
  • Steps 2.7-2.10 were repeated in succession, each time using a test solution sample with a higher concentration of antibodies.
  • HBsAg hepatitis B lyophite surface antigen
  • Lavsan film substrates measuring 60 x 48mm were washed in hot isopropanol (Merck, West Germany) and dried in isopropanol vapour phase.
  • a 0.05 ⁇ m chromium coating was applied to the substrates by the magnetron spray method.
  • Photoresist was applied by centrifugation onto the chromium coating and dried at +80°C for 20 minutes.
  • the photoresist coating was exposed to ultraviolet light through a patterned template, the developed in KOH solution and dried at +100°C for 20 minutes.
  • a chromium pattern was obtained by etching in cerium sulphate solution.
  • the photoresist was removed using dimethyl formamide (Merck, West Germany) , washed and dried in isopropanol vapour phase.
  • a 0.50 ⁇ m coating of gold was applied by galvanic deposition from auric chloride solution, then washed and dried in isopropanol vapour phase.
  • the potentiometric electrode was thus prepared.
  • Membrane electrochemical synthesis was carried out on an electrode of pyrrole aqueous solution with base electrolyte and dedicated receptors. Pyrrole, base electrolyte and dedicated receptor concentrations were 0.15 N, 0.10 N and 5 mg/1, respectively. The electrochemical synthesis and further treatment of the electrode were carried out as described in 2.3.
  • Electrochemical cell potential was recorded for 100 seconds to obtain the base cell potential value.
  • step 3.11 After step 3.10, the immunosensor and reference electrode were transferred onto a plate with pure working solution and cell potential was recorded for 300 seconds - to obtain the final electrochemical cell potential value.
  • Steps 3.8-3.11 were repeated in succession, each time using test solution samples with a HBsAg higher concentration.
  • the pattern of cell potential offset was estimated quantitatively by the size (in arbitrary units) of the area under the curve of cell potential change versus time as recorded in step 3.10, and by the initial rate (in mV/sec) of potential modulation.
  • a calibration curve was plotted showing the correlation between the area under the curve of cell potential change versus time and the initial rate of potential change and the concentration of HBsAg in the test solution. 3.15 The resultant calibration curve was used to determine HBsAg concentration in blood serum samples from patients with hepatitis B.
  • a polypyrrole membrane incorporating bioreceptors is formed on the surface of a planar electrode.
  • the basic requirements for the electrode are that it should possess metallic conductivity and should be stable in aqueous media.
  • gold planar electrodes on a ceramic substrate are used for this purpose.
  • the electrodes are thoroughly cleaned and washed. (The procedure for preparing the electrodes before polypyrrole coating will be supplied in due course) . Maximum storage time of newly washed electrodes - 3-4 hours. The clean electrodes are kept in a special holder with a capacity of 25-30 electrodes, from where they are transferred directly to the polypyrrole electrochemical synthesis process .
  • Pyrrole (Merck) is distilled in standard water-cooled apparatus at atmospheric pressure at 135-140°C. The freshly distilled pyrrole is kept in a sealed dark-glass vessel under 2 at -20 to -5°C. Maximum storage period
  • the proportions of the components may vary wichin the indicated limits according to specific requirements as to the thickness and ionic sensitivity of the polypyrrole film.
  • the following solution composition is recommended for sensors intended for HBsAg diagnostics:
  • the vessel containing the mixture is exposed to medium- intensity ultrasound for 2-4 minutes to ensure that all components are completely dissolved. Avoid heating the solution over 40 °C during the dissolving process.
  • the prepared solution may be stored in a sealed dark-glass vessel for not more than 2 hours.
  • the biomaterial incorporated into the polymer membrane may take the form of antibodies, antigens, fragments or whole molecules of single-strand DNA, etc.
  • Biosensors for HBsAg diagnostics are made using mouse monoclonal HBsAg antibodies (Calbiochem, cat . # 398557). Storage and handling instructions for the biomaterial are given on the pack.
  • a solution of antibodies is added to the synthesis solution such that the final concentration of antibodies is 130 ⁇ g/ml .
  • the finished solution may be stored for a period not exceeding 30 minutes. It should be noted that this final concentration is specified for antibodies of particular origin; when using antibodies from a different source, this figure may vary in the range 5-30 ⁇ g/ml.
  • the final concentration of this material in the electrochemical synthesis solution may also vary considerably.
  • the final concentration of HIV-l recombinant proteins should be 100-150 ⁇ g/ml
  • the final concentration of Human Chorionic Gonadotropin monoclonal antibodies should be 35-50 ⁇ g/ml.
  • the polymer membrane is produced in a triple-electrode small -volume cell.
  • the electrode inserted into the holder is used as the main electrode; gold or platinum wire is used as the auxiliary electrode; and a micro silver chloride electrode is used as the reference.
  • the complete electrode array is assembled on a single mounting.
  • the wells of a standard polystyrene micro-titration tray (volume 200 ⁇ l) are suitable for use as the cell.
  • the micro-titration wells are treated with a water-soluble silicone solution. A maximum of 3 polypyrrole synthesis operations can be carried out in each of these wells.
  • a potentio-dynamic regime should be employed with a continuous voltage sweep at the electrode from 0 to 1200 mV (relative to the Ag/AgCI reference electrode) and a sweep rate of 100 mV/sec.
  • each sweep cycle adds an average of 0.20-0.25 ⁇ m to the membrane thickness.
  • the recommended number of cycles is 6 (membrane thickness 1.3 ⁇ m) .
  • the process of polypyrrole film formation should be monitored with reference to the shape of the voltampere curve and the total quantity of electricity Q passing through the electrode. This can be achieved with the aid of a chart recorder or other recording device. In so doing, the Q value for the first and subsequent cycles should not differ by more than 15%. If this is not the case, membrane growth will tend to be uneven, and such sensors should be scrapped.
  • the parameters of the electrochemical synthesis process may vary within the following limits:
  • the resultant biosensor is carefully rinsed with distilled water followed by a 0.1 M phosphate-salt buffer solution, then immersed in buffer solution for Composition of storage buffer solution
  • the biosensors should be stored at a temperature of 2-8°C.
  • o_.r ⁇ ei ⁇ pecrirtrrood ⁇ e ⁇ --s ⁇ f-.o w r electrochemical polymerisation of polypyrrole
  • the metal electrodes are first washed twice in a 2-5% KOH solution for 30 minutes, then rinsed with deionised water and washed in acetone for 5 minutes, following which they are air dried at room temperature for 20 minutes.
  • the electrodes are then placed in a fluoroplastic holder and placed in a Soxhlet apparatus where they are bathed in hot isopropyl alcohol for 0.5 - 2 hours.
  • the electrodes and holder are then removed from the Soxhlet apparatus and the electrodes are dried in isopropyl alcohol vapour.
  • the electrodes are placed in an enclosed sealed vessel where they may be kept for an hour and a half before commencing the process of electrochemical polymerisation of polypyrrole.
  • the volume of working solution in the well should be 200 ⁇ l.
  • the biosensor is removed from the holding buffer solution and placed in the appropriate slot in a special holder ;
  • a miniature silver chloride reference electrode is connected to the holder ;
  • the biosensor and reference electrode are placed in a micro-titration well filled with working buffer solution.
  • the composition of the working buffer solution is designed to minimise non-specific adsorption of components of the sample under test onto the biosensor Where the biosensors are used for HBsAg diagnostics, the working buffer solution will be made up as follows
  • EDTA ethylenediamine tetra-acetic acid
  • the measuring process is divided into 3 stages, each stage being carried out in its own micro-titration well
  • the first and third wells are filled with clean working solution
  • the second well contains working solution and the sample under test
  • the proportions of working solution and test sample will be 160 ⁇ l and 40 ⁇ l respectively
  • the potential of the biosensor is measured relative to the reference electrode in clean working buffer solution
  • the purpose of this first stage measurement is to determine the initial potential of the sensor before incubation with serum, and to evaluate the dnft (r) of the biosensor zero line
  • the duration of the first measuring stage may be 20 - 100 seconds
  • the biosensor and reference electrode are transferred to the micro-titration well containing the test sample diluted with working solution, and the biosensor potential is measured relative to the reference electrode.
  • the duration of the second measurement stage is determined by the time required to allow the immuno-chemical reaction with the bioreceptors immobilised on the surface of the biosensor to proceed to the fullest extent possible.
  • the duration of the measurement may be 200 to 600 seconds.
  • the curve of biosensor potential variation in the presence of a test sample is normally parabolic in form and represents the response of the biosensor to the change in ion composition of the ambient solution when the test sample is added to it. This response is modulated by the change in total charge (isoelectric point) of the polymer membrane. As a change in the isoelectric point of the membrane may be induced both by a specific immuno-chemical reaction
  • the signal obtained may vary in both form and amplitude .
  • the biosensor and reference electrode are transferred to the next micro-titration well containing clean working buffer solution of the same composition as that in the first well. In so doing, the potential of the biosensor will tend to revert to its original value before incubation with the test sample (allowing for zero point drift) .
  • stage 3 The duration of stage 3 is longer or the same as that of stage 2, and where the biosensors are used for HBsAg As in stage 2 , the value recorded at the third measurement stage is an integral delineated by the 'biosensor potential - time' curve, S 3 :
  • the above described assay procedure may be performed under software control
  • FIG. 5 A block-diagram of the Biosensor assay procedure is given in Figure 5 Ail of the operations descnbed are performed under the control of an IBM-compatible computer using a specially developed software package running under Microsoft Windows 3 xx or Windows 95 Minimum requirements for the PC system resources are defined solely by the minimum requirements of the operating system used
  • the program After starting the software, the program performs an initial test of the special hardware card fitted in the computer If the card is absent or faulty, the program displays a message to this effect and exits
  • the Biosensor System (hereinafter-System) initiates the mode selection sequence Measurement', 'Work with database' or 'Finish'
  • the first two modes may be run simultaneously and will function normally together
  • the program displays the database browser window showing full details of measurements completed, including results, date of test, etc (see below)
  • the program includes a range of basic features for the manipulation of database entries move to particular records (samples) in the database, sort and retrieve on specific options or keywords, display measuring curves for selected records (samples), convert to numeric format for subsequent mathematical processing (e g Microsoft Excel), create standard reports on the results of measurements carried out, and print out to any printer available to the operating system
  • Some of the keywords that can be used to search the database include: operator name, sample number, sampling date and blood sample analysis date, memo, assay result in both quantitative (arbitrary units) and qualitative form (" + ", "-” or " + /-").
  • the program also permits connection to external databases held on other computers.
  • the database format is compatible with all well-known commercial database formats (e.g. Microsoft Access, Borland Paradox, Corel Paradox, etc.) .
  • Measurement mode is one option in the range of operating modes of the system, which can be run simultaneously with the database search mode. These modes are displayed separately in standard windows, and switching from one to the other is achieved in the manner customary for the particular interface concerned.
  • the serum (plasma) sample is transferred to the well in the micro-titration tray, where it is diluted with a special buffer solution.
  • a window is displayed on the screen for entry of the operator's name, sample number and sampling date. These details are subsequently entered in the database and serve as search parameters for the retrieval and manipulation of database records. Any combination of characters and numbers may be In addition to the above details, the date of the test is automatically assigned to each sample (current system time) .
  • the program After entering the sample number, the program proceeds to measure the biosensor potential relative to that of the reference electrode, at pre-set time intervals (usually every 5 seconds). The result of each measurement is displayed on the screen in the form of points on the coordinates " biosensor signal - time". The duration of the first measurement interval is seconds, following which the computer displays a message for the sensor to be moved to another well
  • the purpose of the first interval of measurement is to determine the initial sensor potential before incubation with the serum, and also to determine any datum-line drift (r) of the biosensor, which is calculated by linearising the curve obtained by the least squares method.
  • the biosensor and reference electrode are transferred to a micro-titration well containing blood serum diluted with buffer solution.
  • the measurement time is seconds, on completion of which the computer displays a message to this effect.
  • the time taken to perform this stage has been specified with so as to ensure that the immuno-chemical reaction with the antibodies immobilised on the surface of the biosensor takes place to the fullest extent possible consistent with the desire to minimise the overall duration of the test.
  • the signals generated are recorded in the same manner as described above.
  • the curve obtained is usually parabolic in form and represents the response of the biosensor to the variation in ion composition of the sample, which is modelled by the variation in total charge (isoelectric pcint) of the polymer biomembrane.
  • the signal obtained can vary both in shape and amplitude.
  • the surface area (integral) S 2 bounded by the curve obtained is determined, and the origin of the coordinates is taken to be the first point obtained in the 2 nd measurement interval
  • the biosensor and electrode are transferred to the next micro-titration well with clean buffer solution of the same composition as the first well.
  • the biosensor potential will tend to revert to its initial potential before incubation with the test sample (allowing for datum-line drift)
  • the composition of the buffer solution is specified so as to minimise residual non-specific adsorption of serum constituents on the bio-membrane, therefore after placement in the clean buffer solution the isoelectric point of the membrane will revert to its initial value, and the sensor potential will tend to revert to the potential value obtaining at the 1 st measurement interval
  • the total charge of the membrane will change only by an amount matching the non-specific component of the signal, and the charge attributable to antibody-antibody interaction will remain unchanged Consequently, it is to be expected that, following incubation with a sample containing specific material, the sensor will not assume its initial condition (before incubation with sample), and therefore the final potential of the biosensor will be significantly different from the initial value
  • the quantity recorded in the 3 d measurement interval is an integral S 3 bounded by the "biosensor signal - time" curve
  • recording of the biosensor signal takes place at discrete time intervals, and the result of each measurement is displayed on screen
  • the values obtained for ⁇ , S 2 and S 3 are transmitted for processing in the pre-treatment unit
  • the integral values are recalculated to allow for the datum line drift ⁇ obtained in the first interval, S ⁇ 2 , S v 3
  • the ratio (R) of these corrected integrals is used as the analytical result forming the basis for determination of the presence or absence of a specific component in the sample under test
  • the data are passed to the Final Signal Treatment unit where they are compared with the calibration data and a final determination is made on the nature of the sample "- 1 -" - where a specific component is present, "-" - where there is no specific component, or "- 1 - '- " where the result falls into the "grey zone”
  • the calibration data are stored in the computer on initial installation of the software and cannot be altered without specialist intervention See below for details of the calibration method and procedure
  • the System includes an initial calibration stage, which is performed on blood serum samples of known characteristics.
  • the calibration data are permanently stored in the computer and can be altered only by entering a special password giving access to the calibration data.
  • the sequence of actions required to calibrate the system is outlined below.
  • the calibration process itself closely resembles the procedure described above for testing a sample of unknown characteristics (see above and fig 5 - 'measurement' block) , differing only in that the operator is required to enter specific characteristics when entering the sample details.
  • the operator may indicate specificity as " + " (for a sample containing a specific component) or "-".
  • it will also be necessary to indicate the content (for positive samples) of the specific component (in ng/ml).
  • Each value of the integral ratio R obtained in the calibration stage is held in a special calibration database. These stored values are used to plot a Gaussian distribution of positive and negative results, and a calibration curve is also plotted where a statistically significant sample is obtained from the quantitative data.
  • a Gaussian distribution (fig.6) is plotted in ail cases, irrespective of whether or not quantitative data are required. Based on statistical analysis of this distribution, the criterion for assessment of the measurement results is determined ("+" or "-"), and the boundaries of the so-called “grey zone” are identified, i.e. the region in which a correct decision cannot be reached with the specified confidence interval. This curve is plotted using conventional statistical methods, and the confidence interval is taken as the value generally accepted in medical practice, i.e. 3y . If desired, the value of the confidence interval may be adjusted.
  • the observed signal from the biosensor depends directly on the isoelectric point of the bio-membrane (membrane with antibodies immobilised in it), and hence on its total charge.
  • the immuno-chemical reaction proceeds at the surface of the biosensor, formation of the antigen-antibody complex is accompanied by a corresponding change in the total charge of the biomembrane, and will therefore lead to a change in the biosensor response.
  • the quantity of complex formed (and hence the concentration of a specific component) will be proportional to the change in the biosensor signal, i.e. the higher the concentration of a component (e.g. antigen) in the sample, the higher the value of the integral ratio R.

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Abstract

La présente invention se rapporte à un procédé de détection électrochimique destiné à détecter les macromolécules immunoactives dans des solutions d'essai. L'invention consiste d'abord à produire un capteur immunologique renfermant une membrane modifiée par capteur spécifique, puis à former une cellule de mesure électrochimique à partir du capteur immunosensible et d'une électrode de référence liés par un instrument de mesure. On place ce dernier à l'intérieur de la solution de travail, puis on détermine en fonction de la concentration de macromolécules dans la solution d'essai, le déplacement du point isoélectrique de la membrane, en mesurant le potentiel de la cellule quant aux changements par étapes de la force ionique de la solution de travail; la membrane étant formée d'un polymère électroconducteur par synthèse électrochimique d'une solution monomère renfermant des récepteurs spécifiques sur la surface de l'électrode potentiométrique. Enfin, on détermine le déplacement du point isoélectrique de la membrane, et on introduit dans la solution de travail une solution d'essai ayant une force ionique plus grande que celle de la solution de travail à pH constant.
PCT/GB1998/000548 1997-02-20 1998-02-20 Procede de detection electrochimique de macromolecules immunoactives WO1998037409A1 (fr)

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AU63005/98A AU6300598A (en) 1997-02-20 1998-02-20 Method of electrochemical detection of immunoactive macromolecules

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RU97102274A RU2107296C1 (ru) 1997-02-20 1997-02-20 Способ электрохимической индикации иммунохимически активных макромолекул в исследуемых растворах
RU97102274/14 1997-02-20

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002523746A (ja) * 1998-08-24 2002-07-30 センサー−テック・リミテッド 分析物の電気化学的分析法
WO2003019171A1 (fr) * 2001-08-24 2003-03-06 Sensor-Tech Limited Procedes de production de capteurs potentiometriques a haute sensibilite
WO2003081244A3 (fr) * 2002-03-26 2003-12-31 Sensor Tech Ltd Procede pour l'analyse electrochimique de cellules
US7756560B2 (en) * 2002-06-03 2010-07-13 Siemens Aktiengesellschaft Sensor arrangement and method for operating a sensor arrangement
CN113219025A (zh) * 2021-05-07 2021-08-06 安徽大学 一种定量检测溴酸钾的方法
CN113219027A (zh) * 2021-05-07 2021-08-06 安徽大学 一种定量检测碘酸钾的方法
US20230280300A1 (en) * 2022-03-02 2023-09-07 Clifford H. Kern, III Device, procedure and system for detecting bacterial pathogens including methicillin-resistant staphylococcus aureus or clostridium difficile

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RU2266959C2 (ru) * 2004-01-15 2005-12-27 Сорокин Виктор Фёдорович Способ получения биосенсоров (варианты)
RU2252411C1 (ru) * 2004-04-09 2005-05-20 Общество с ограниченной ответственностью "Институт рентгеновской оптики" Флюоресцентный сенсор на основе многоканальных структур
RU2271006C1 (ru) * 2004-07-19 2006-02-27 Липецкий Государственный Технический Университет (Лгту) Способ детектирования сульфаметоксазола с помощью пьезокварцевого иммуносенсора
GB2420180A (en) * 2004-11-11 2006-05-17 Sensor Tech Ltd Method of electrochemical analysis of an analyte
RU2320990C1 (ru) * 2006-06-07 2008-03-27 ГОУ ВПО "Казанский государственный университет им. В.И. Ульянова-Ленина" Способ определения антител в сыворотке крови
RU2375705C1 (ru) * 2008-08-22 2009-12-10 Учреждение Российской Академии наук Институт физической химии и электрохимии им. А.Н. Фрумкина Российской Академии наук Способ электрохимического мультисенсорного обнаружения и идентификации алкалоидов
RU2466680C1 (ru) 2011-10-03 2012-11-20 Общество С Ограниченной Ответственностью "Витацел" Способ диагностики состояния кожи пациента (варианты)

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WO1996002001A1 (fr) * 1994-07-07 1996-01-25 Leaver, Jonathan Dosage immunologique par procede electrochimique
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EP0193154A2 (fr) * 1985-02-25 1986-09-03 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Elément potentiélaire pour un immunosenseur
US5312762A (en) * 1989-03-13 1994-05-17 Guiseppi Elie Anthony Method of measuring an analyte by measuring electrical resistance of a polymer film reacting with the analyte
DE3916432A1 (de) * 1989-05-20 1990-11-22 Cammann Karl Verfahren und vorrichtung zur konzentrationsbestimmung eines antikoerper-antigenpaares
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WO1996002001A1 (fr) * 1994-07-07 1996-01-25 Leaver, Jonathan Dosage immunologique par procede electrochimique

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002523746A (ja) * 1998-08-24 2002-07-30 センサー−テック・リミテッド 分析物の電気化学的分析法
JP4774150B2 (ja) * 1998-08-24 2011-09-14 センサーテック・リミテッド 分析物の電気化学的分析法
AU2002321531B2 (en) * 2001-08-24 2007-12-13 Sensortec Limited Methods for producing highly sensitive potentiometric sensors
CN100430721C (zh) * 2001-08-24 2008-11-05 传感技术有限公司 高敏感性电位传感器的制造方法
WO2003019171A1 (fr) * 2001-08-24 2003-03-06 Sensor-Tech Limited Procedes de production de capteurs potentiometriques a haute sensibilite
US8404096B2 (en) 2001-08-24 2013-03-26 Sensortec Limited Methods for producing highly sensitive potentiometric sensors
WO2003081244A3 (fr) * 2002-03-26 2003-12-31 Sensor Tech Ltd Procede pour l'analyse electrochimique de cellules
US7756560B2 (en) * 2002-06-03 2010-07-13 Siemens Aktiengesellschaft Sensor arrangement and method for operating a sensor arrangement
CN113219025A (zh) * 2021-05-07 2021-08-06 安徽大学 一种定量检测溴酸钾的方法
CN113219027A (zh) * 2021-05-07 2021-08-06 安徽大学 一种定量检测碘酸钾的方法
CN113219027B (zh) * 2021-05-07 2023-07-21 安徽大学 一种定量检测碘酸钾的方法
CN113219025B (zh) * 2021-05-07 2023-07-25 安徽大学 一种定量检测溴酸钾的方法
US20230280300A1 (en) * 2022-03-02 2023-09-07 Clifford H. Kern, III Device, procedure and system for detecting bacterial pathogens including methicillin-resistant staphylococcus aureus or clostridium difficile

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