WO1998037409A1 - Method of electrochemical detection of immunoactive macromolecules - Google Patents

Abstract

A method of electrochemical detection of immunoactive macromolecules in test solutions, which involves producing an immunosensor comprising a specific-receptor-modified membrane; forming an electrochemical measuring cell from the immuno-sensitive sensor and a reference electrode linked by a measuring instrument; placing the latter into the working solution, and determining the displacement of the isoelectric point of the membrane in relation to the concentration of macromolecules in the solution under test, by measuring the cell potential with step-changes in the ionic strength of the working solution, in which the membrane is formed from electroconductive polymer by electrochemical synthesis from a monomer solution containing specific receptors on the surface of the potentiometric electrode; to determine the isoelectric point displacement of the membrane, a test solution with an ionic strength greater than that of the working solution at constant pH is added to the working solution.

Description

METHOD OF ELECTROCHEMICAL DETECTION OF IMMUNOACTIVE MACROMOLECULES

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 .

The term immuno-active macromolecules (hereinafter macromolecules) , or antigens (Ag) , is taken to mean macromolecules of natural and man-made origin which, on entering the organism of higher animals (including humans) , induce the formation of special protein molecules - antibodies (Ab) - which have the ability of specifically binding the antigen to form a weak- dissociation complex. Antibodies can also be antigens. Schematically, the antigen-antibody interaction can be represented as follows:

Ab + Ag AbAg .

The equilibrium constant of this reaction is defined as:

K = [AbAg] / [Ab] [Ag] . Hence, for a fixed antibody concentration, the equilibrium ratio of the bonded and free antigen concentrations is quantitatively related to the total concentration of antigen. This ratio underpins all immuno-assay methods for antigen and antibody detection. One of the basic methods of detection is Enzyme Linked Immune Solid-Phase Assay (ELISA) , which is based on the interaction between an antigen (or antibody) immobilised in solid phase and a specific antibody (antigen) , with subsequent separation of the bonded and free components of the immuno-chemical pair, and the use of antibodies tagged with an enzyme marker to identify the bonded component .

A significant drawback of this method is the length of the analytical procedure. The majority of ELISA tests take 2 hours or more to complete, requiring the performance of a series of successive immuno-chemical reactions and logging of enzyme activity.

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.

Similar shortcomings also affect methods based on other forms of tagging: fluorescent, chemi-luminescent, radio- nuclide markers, etc. Therefore, various lines of research have been pursued in recent times in an attempt to combine immuno-chemical indication methods with electro-chemical detection techniques, given their relative simplicity and low cost [1].

The essential problem encountered by these researchers was the fact that formation of the antigen-antibody complex is not accompanied by any transference of electricity capable of being recorded using simple amperometric systems (measuring the current strength) .

Therefore, most of these investigators were obliged to introduce enzyme-tagged antibodies into the electrochemical measuring system and to use amperometric detection on the enzyme reaction products following formation of the antigen-antibody complex. It should be noted that the amperometric methods are affected by the same shortcomings as the conventional enzyme-linked immuno-assay (ELISA) method [2].

The use of potentiometric indication methods (measuring the electrochemical potential) is based on the premise that in aqueous solutions proteins are polyelectrolytes, and therefore antigen-antibody interaction should alter their charge.

However, the use of 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. When 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. As 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.

There is a known method of electrochemical indication of immuno-active macromolecules in solutions using an immuno-sensitive sensor [5, 6, 7],

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:

1. 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) .

2. Appropriate receptors (antigens or antibodies) 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.

3. 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.

4. 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.

5. During 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.

6. During 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.

7. During 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.

8. 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.

9. 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 .

10. On completion of stage 9, 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.

12. 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.

13. The calibration curve obtained is then used to determine the content of macromolecules in different test solutions.

The method described above has a number of drawbacks:

- High cost of the test, attributable to the use of an ISFET as the electrochemical sensor, with the consequent need for complex apparatus to measure the ISFET cell potential, and other costly items of equipment, including a circulation vessel of complex construction, peristaltic pumps for the delivery of working solution and high ionic strength solution, pH gradient vessel, and pH- meter;

- Time-consuming and unwieldy process for deposition of the ion-permeable membrane on the ISFET gate;

- High probability of obtaining non-reproducible results through lack of control over the properties of the membrane in the process of chemical synthesis and receptor immobilisation;

- Substantial prolongation of the test owing to the fact that the process of interaction between the receptors and macromolecules is temporally and spatially separate from the actual electrochemical measuring process.

All of these drawbacks render this method of little use as an alternative to conventional methods of detecting macromolecules .

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:

1. 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.

2. The membrane is formed on the surface of the electrochemical sensor by electrochemical synthesis of an electrically conductive polymer. In so doing, 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. 3. Immobilisation of the receptors in the membrane is carried out simultaneously with electrochemical synthesis of the membrane, providing controllability of the binding process.

This makes it possible to greatly improve the technical viability of the manufacturing process for the electrochemical sensor, to simplify its construction and reduce costs, and the use of electrochemical synthesis to form the membrane makes it possible not only to closely monitor but also to control its properties (for example, ion permeability and sensitivity) . All of these factors are ultimately conducive to reducing the cost of the test and enhancing the repeatability of the results obtained. 4. Measurements of the voltage across the electrochemical measuring cell are carried out for a fixed pH of the working solution, using solutions with a high buffering capacity for this purpose. In the disclosed method, this effectively dispenses with the need for costly apparatus for the measurement and control of the pH of the working solution and circulation cells, thereby increasing the process efficiency and speed of the test, substantially reducing its cost and increasing the reliability of the results obtained by eliminating the error associated with pH measurement and control.

5. 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.

This has the effect of considerably reducing the time taken to complete the test by temporally and spatially combining the process of interaction between the receptors and macromolecules and the electrochemical measuring process.

6. 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.

Conveniently, the 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.

Brief description of the drawings.

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;

(5) - reference electrode; (6) - meter; (7) - logger; (8) - working solution. Fig. 3 Injection of solution (9) containing macromolecules (10) specific to the electrochemical sensor into the electrochemical cell.

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.

The method shown is carried out as follows: 1. 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].

2. Simultaneously with the electrochemical synthesis process, receptors are incorporated into the membrane. For this purpose, the receptors are dissolved in the electrochemical synthesis solution. Depending on the type of test required, 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 μg/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. In each particular case, 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] .

3. 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]. In contrast to the prototype of the declared method, in which a purpose-made circulation-type cell was used exclusively, 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 μl 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.

The base potential of the cell for a fixed pH value of the working solution is thus obtained.

5. 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.

Where the declared method is used for the detection of immuno-active macromolecules in biological fluids (blood serum, lymph, urine, saliva, semen) , 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]. In order to reduce the natural variability in ion composition and pH of the biological fluids, they are used at maximum possible dilution in the working fluid. Depending on the type of test, the dilution factor may range from 1:10 to 1:1000.

Where the declared method is used to investigate other fluids of natural or man-made origin, 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_:S0₄) .

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.

The content of test macromolecules in the higher- ion-strength solution may vary, depending on the nature of the test, in the range 0 to 10000 μg/ml.

6. 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.

7. On completion of 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. 8. On completion of 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. 9. 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.

A number of parameters may be used for a quantitative evaluation of the potential change:

• the difference between the base and final values of the electrochemical cell potential;

• the difference between the maximum potential recorded in step 6 and the base potential;

• the difference between the maximum potential recorded in step 6 and the final potential;

• the size (in arbitrary units) of the area under the curve of potential variation versus time obtained in step 6;

• the initial, final, average or maximum rate of electrochemical cell potential change during step 6.

It is also possible to use a combination of the above parameters to increase the reliability of the results obtained. 10. Steps 4-9 are repeated using solutions of greater ionic strength than the working solution with different known concentrations of test macromolecules .

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.

12. The resultant calibration curve is used to determine (indicate) the content of macromolecules in test solutions (with unknown macromolecule content) . For this purpose, steps 4-8 are carried out using the solutions under test as the higher- ion-strength media. In so doing, 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.

Example 1.

1.1. Reagents and materials: - monomer - pyrrole (Pyrrole > 98%, Merck, West Germany) twice treated by vacuum distillation and stored under N in an opaque vessel at +4°C;

- base electrolyte for electrochemical synthesis - KC1 (99%, Sigma, USA); - dedicated receptors - Mouse Monoclonal antibodies Anti-Chorionic Gonadotropin Human, Affinity constant 1 x lθ^y L/mol, Calbiochem, USA, stored in a phosphate saline buffer solution at +4°C;

- for aqueous solution preparation - deionised water (reagent grade, resistance > 18 MOhm) , produced on

Milli-Ro/Milli-Q equipment (Millipore, USA) ;

- working solution prepared from Phosphate Buffered Saline Tablets (Sigma, USA) ;

- electrode for potentiometric measurements - length of platinum wire (csa 0.07cιrr) soldered within a glass tube, polished with abrasive paste (particle diameter 0.3 μm) and washed in deionised water;

- reference electrode - commercially available standard silver chloride reference electrode (Ag/AgCl/3 M KC1, Radelkis, Hungary);

- vessels for process solution - polystyrene microtitration plates (Linbro, Great Britain) ;

- solutions to be tested with known content of macromolecules - samples of female human urine with a standard level of chorionic gonadotropin (Chorionic Gonadotropin, Female Human Urine, > 3000 IU/mg, Calbiochem, USA) diluted by working solution to various degrees of concentration;

- solution to be tested with unknown macromolecules content - samples of urine from various people. 1.2 Equipment

- for electrochemical synthesis - a teflon cell, 100 ml, and PI-50.1 potentiostat-galvanostat (Russia);

- for working solution pH control - Checkmate pH- meter (Mettler, Switzerland) ; - for potential measurements in electrochemical cell OP 211/1 digital millivoltmeter (Radelkis, Hungary) ;

- for electrochemical synthesis monitoring and potential registration on electrochemical cell 2210 two-channel recorder (LKB - Instrumentation, Sweden) .

1.3 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 (^~ lμm) 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.

1.4 Working solution with pH 7.4 was prepared.

1.5 A series of specimen test solutions with a Human Chorionic Gonadotropin level of 0.1 - 10 IU/ l were prepared. 1.6 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 lOOμl 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.

1.7 Potential was recorded on the electrochemical cell for 100 seconds to estimate base potential on the cell. 1.8 20μl of solution to be tested with the lowest HCG level was added to the microtitration plate from where 20μl of working solution had been removed.

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.

1.11 Steps 1.7 - 1.10 were repeated successively, each time using a sample of test solution with higher HCG concentration.

1.12 The cell potential offset pattern was quantitatively estimated by the difference between base and final cell potential in millivolts.

1.13 A calibration graph was plotted showing HCG concentration differential ratio of the base and final cell potential in the test solution.

1.14 The plotted calibration graph was then used to determine the HCG concentration in the urine samples from different patients. To this end, the urine samples were used as test solution in steps 1.7 - 1.10 and 1.12.

Example 2.

2.1 Reagents and materials:

- monomer - see 1.1; - base electrolyte for electrochemical synthesis - Na^SO,, (ACS Reagent, Sigma, USA) ;

- dedicated receptors - recombinant envelope proteins HIV-1 p24, gp41 and gpl20 (American Bio- Technologies Inc. , USA) stored in saline phosphate buffer solution at +4°C;

- aqueous solutions preparation - see 1.1;

- working solution preparation - see 1.1;

- potentiometric electrode - planar electrode 0.5 x 10 mm, made of gold, set on a ceramic base plate, 8.0 x 30 mm, degreased in hot isopropanol (Merck, West Germany) , and dried in isopropanol vapour phase ;

- 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) ;

- working solution vessel - see 1.1; - solutions to be tested with known macromolecule concentration - a mixture of monoclonal antibodies to p24, gp41 and gpl20 (Orbit Enterprises, USA), diluted with normal human serum (Scientific Cardiological Centre, Academy of Medical Sciences of the Russian Federation, Russia) to various concentrations;

- solutions to be tested with unknown concentration of macromolecules - samples of blood serum taken from HIV-1 positive patients. 2.2 Equipment

- for electrochemical synthesis - see 1.1;

- for pH monitoring in the working solution - see 1.1;

- for electrochemical cell potential measurement and registration - a purpose designed meter based on an

IBM-compatible computer was used. The meter comprised an amplifier and analog-to-digital converter and was computer controlled by custom software. 2.3 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 0.30 N, 0.15 N and 100 mg/1, respectively. The electrochemical synthesis was carried out in potentiostatic mode at +950 mV (wrt Ag/AgCl - reference electrode) . As soon as the required thickness of the membrane was achieved (" 3μm) the process was terminated. After that 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.

2.4 Working solution with pH 7.4 was prepared. 2.5 A series of samples of the solution to be tested with antibodies concentration in the range 0-10 μg/ml were prepared.

2.6 An electrochemical cell was made up by placing the immunosensor and reference electrode onto a microtitration plate filled with lOOμl of working solution and connecting them to the meter input.

2.7 Electrochemical cell potential was recorded for 200 seconds to obtain the base cell potential value.

2.8 5μl of solution to be tested, with the lowest antibodies concentration, was added to the microtitration plate from where 5μl of working solution had previously been removed. 2.9 The variation in cell potential was recorded for 900 seconds.

2.10 After 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.

2.11 Steps 2.7-2.10 were repeated in succession, each time using a test solution sample with a higher concentration of antibodies.

2.12 The pattern of cell potential offset was estimated quantitatively on the basis of the maximum cell potential value recorded in step 2.9.

2.13 A calibration curve was plotted showing the correlation between maximum cell potential and the concentration of antibodies in the test solution.

2.14 The resultant calibration curve was used to determine antibody concentration in the blood serum of HIV-1 positive patients.

Example 3.

3.1 Reagents and materials:

- monomer - see 1.1; - base electrolyte for electrochemical synthesis - NaCl (ACS Reagent, Sigma, USA) ;

- dedicated receptors - Mouse Monoclonal Anti-HBsAg, Affinity constant 1 x 10'°L/Mol (Calbiochem, USA) stored in saline phosphate buffer solution with 0.01% sodium azide at +4°C; - aqueous solution preparation - see 1.1;

- working solution - see 1.1;

- potentiometric electrode - lavsan film, 500μm, (Vladimir Chemical Plant, Russia) , vacuum-dispersed chromium target, auric chloride solution (Sigma, USA), FP-383 photoresist (NIIPIK Institute, Russia) , cerium sulphate solution (Sigma, USA) , KOH aqueous solution (Sigma, USA) ;

- reference electrode for electrochemical synthesis - see 1.1;

- reference electrode for electrochemical cell - see 2.1;

- working solution vessel - see 1.1;

- samples of test solution with known macromolecules concentration - hepatitis B lyophite surface antigen (HBsAg) (State Institute for Standardization and Control attached to the Prophylactic-Epidemiological Committee of the Russian Federation, Russia) , diluted with normal human serum to various concentrations;

- test solutions with unknown concentration of macromolecules - samples of blood serum from patients with various forms of hepatitis B.

3.2 Equipment - for potentiometric electrode preparation - type UVN vacuum deposition unit (Russia) , PNF-6TS photoresist deposition system (Russia) , photo- template, STP-300 photo-exposure unit (Russia) , EU 18 dry heat cabinet (Jouan, France) , a cell similar to that used in electrochemical synthesis, PI-50.1 potentiostat (Russia) ; - for electrochemical synthesis - see 1.1;

- for pH monitoring in the process solution - see 1.1;

- for electrochemical cell potential measurement and registration - see 2.2;

3.3 Lavsan film substrates measuring 60 x 48mm were washed in hot isopropanol (Merck, West Germany) and dried in isopropanol vapour phase. A 0.05μm 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 0.50μm 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.

3.4 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.

3.5 Process solution with pH 7.4 was prepared. 3.6 A series of samples of the solution to be tested with HBsAg concentration in the range 0-1.00 μg/ml were prepared.

3.7 An electrochemical cell was prepared as described in 2.6.

3.8 Electrochemical cell potential was recorded for 100 seconds to obtain the base cell potential value.

3.9 lOμl of test solution, with the lowest HBsAg concentration, was added to the microtitration plate from where lOμl of process solution had previously been removed.

3.10 The variation in cell potential was recorded for 300 seconds.

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.

3.12 Steps 3.8-3.11 were repeated in succession, each time using test solution samples with a HBsAg higher concentration.

3.13 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.

3.14 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.

There follows by way of example a detailed description of a process for producing a biosensor.

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. At the present time, gold planar electrodes on a ceramic substrate are used for this purpose.

Preparation of electrodes for polypyrrole deposition

Before the polypyrrole is applied, 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

1 month ,

Preparation of solutions to obtain polypyrrole As one of the essential requirements for polypyrrole film used as the base material for the manufacture of biosensors is a high level of sensitivity to the ionic composition of the solutions, it is advisable to use sodium sulphate, sodium phosphate or sodium dodecyl sulphate (SDS) as the background electrolyte in the electrochemical synthesis of polypyrrole. Selection of a particular background electrolyte will be dictated by the requirements for specific ionic sensitivity of the polypyrrole film in relation to the particular type of biosensor concerned.

Typical composition of solution for electrochemical polymerisation of polypyrrole:

Pyrrole (Merck) - 2-4 vol . %

Sodium sulphate (Sigma) , 0.3M solution - 9-15 vol . %

Deionised water

(Millipore, resistance not less than 18 MOhm) - 89 - 71 vol . %

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. For example, the following solution composition is recommended for sensors intended for HBsAg diagnostics:

Pyrrole (Merck) - 2.5 vol.'

Sodium sulphate (Sigma), 0.3M solution - 11.1 vol

Deionised water (Millipore) - 86.4 vol.

After mixing the above components together, 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.

Immediately prior to the electrochemical synthesis of polypyrrole membrane, a solution of antibodies is added to the synthesis solution such that the final concentration of antibodies is 130 ~μg/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 μg/ml.

Where a different biomaterial is used, the final concentration of this material in the electrochemical synthesis solution may also vary considerably. Thus, for example, the final concentration of HIV-l recombinant proteins should be 100-150 μg/ml, and the final concentration of Human Chorionic Gonadotropin monoclonal antibodies should be 35-50 μg/ml.

Electrochemical synthesis

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. For convenience, the complete electrode array is assembled on a single mounting.

The wells of a standard polystyrene micro-titration tray (volume 200μl) are suitable for use as the cell. To prevent sorption of biomaterial onto the polystyrene during synthesis, 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.

For the production of sensors for HBsAg diagnostics, 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.

Depending on the desired thickness of the membrane, between 2 and 10 sweep cycles may be applied, on the basis that each sweep cycle adds an average of 0.20-0.25μm to the membrane thickness. Where the sensors are intended for HBsAg diagnostics, the recommended number of cycles is 6 (membrane thickness 1.3μm) . 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. Sensors in which the aggregate Q value exceeds the average figure by more than 10% should also be rejected. Note that the absolute value for the quantity of electricity Q will vary according to the geometrical dimensions and shape of the cell used for electrochemical polymerisation, as well as the geometrical dimensions and shape of the electrode on which the synthesis is being performed. A typical average Q value for a cell based on a micro-titration well and a 2mm² gold planar electrode is approximately 600 mQ.

When making biosensors using a different background electrolyte and/or different biomaterial, the parameters of the electrochemical synthesis process may vary within the following limits:

lower limit of potential sweep - 800 to +600 mV

upper limit of potential sweep - +1200 to +1800 mV

potential sweep rate - 50 to 150 mV/sec

(Potential values are given in relation to an Ag/AgCl reference electrode) .

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

Sodium chloride (Sigma) 8.0 g

Tris (hydroxymethyl) aminomethane (Sigma) 12.165 g

Potassium chloride 0.2 g

Deionised water

(Millipore, resistance not less than 19 MOhm) 1 litre

After preparing the solution, its pH is carefully adjusted to 8.0 using a 0. IN solution of HCl.

The biosensors should be stored at a temperature of 2-8°C.

_Preparat.i.on o_.r= βeiιpecrirtrroodαeι--sι f-.o^wr 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. On completion of these procedures, 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.

There follows by way of example a detailed description of an assay procedure.

For measuring purposes it is advisable to use the wells of a standard polystyrene microtitration tray. The volume of working solution in the well should be 200μl.

The following operations are involved in the performance of measurements using the biosensor^:

- 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

double-substitution sodium phosphate (Sigma) - 0 1-0 2 M

single-substitution sodium phosphate (Sigma) - 0 1-0 2 M

potassium chloride (Sigma) - 0 05-0 1 M

sodium chloride (Sigma) - 0 1-0 2 M

sodium azide (Sigma) - 0 02%

ethylenediamine tetra-acetic acid (EDTA) (Sigma) - 0 373 g/L

tween-20 (Merck) - 0 05-0 1 vol %

deionised water (Mil pore, resistance not less than 18 MOhm)

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, and the second well contains working solution and the sample under test Where the biosensor is used for HBsAg diagnostics, the proportions of working solution and test sample will be 160μl and 40 μl respectively

In the first measunng stage, 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 On completion of the first measurement stage, 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.

In the case of biosensors for HBsAg diagnostics, 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

(interaction between the biomaterial immobilised on the biosensor and the corresponding antigen in the sample under test) , and by non-specific physical adsorption of test sample components onto the biomembrane, and also as a result of natural variability in the ionic properties of different test samples, the signal obtained may vary in both form and amplitude .

On completion of the second measurement stage, the area (integral) described by the resultant curve of biosensor potential variation is determined as S₂, and the origin of the coordinates is taken as the first point (first value of biosensor potential) obtained in the 2^nd measurement stage-. s₂ = J7₂ (t) dt , ^T2

where: T₂ - duration of second measurement stage (220 sec) ; f₂ - curve of 'biosensor signal (mV) versus time'; t - current time.

On completion of the second measuring stage, 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) .

Because non-specific adsorption of test sample components onto the biomembrane is minimal as a result of correctly specifying the composition of the buffer solution, when the membrane is introduced into clean buffer solution its isoelectric point will revert to its initial value and the sensor potential will tend towards its observed value at the end of the first measuring stage. In the case of a specific immuno-chemical reaction, after the biosensor in placed in clean working buffer solution the total membrane charge will change only by an amount matching the non-specific signal component, and the charge attributable to antigen/bioreceptor interaction in the biomembrane will remain unchanged. Therefore, following contact between the biosensor and a test sample containing a specific antigen, the biosensor will not revert to its initial state (before incubation with the sample) , and therefore the final potential of the biosensor will differ noticeably from its original value .

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, S₃:

S₃ = J7₃(t) dt,

where: T₃ - duration of 3rd measurement stage (400 sec) ; ₃ - curve of 'biosensor signal (mV) versus time'; t - current time.

On completion of measurement stage 3 , the values obtained for S₂ and S₃ are adjusted to allow for the zero line drift γ obtained in the first stage. The ratio (R) of these corrected integrals is used as the analytical result on the basis of which a determination is made as to the presence or absence of a specific component in the sample under test :

R =

The above described assay procedure may be performed under software control

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

A step-by-step description of the assay procedure is presented below

Initialisation Block

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

Mode Selection

On successful completion of the self-test, the Biosensor System (hereinafter- System) initiates the mode selection sequence Measurement', 'Work with database' or 'Finish' In a multi-tasking environment (Windows 95), the first two modes may be run simultaneously and will function normally together

Mode: Working with Data Base

In this mode, 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. To facilitate this, the database format is compatible with all well-known commercial database formats (e.g. Microsoft Access, Borland Paradox, Corel Paradox, etc.) .

Mode: Measurement

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.

Before selecting this mode, the Biosensor System must be fully assembled and ready to run. To prepare the system for operation, it will be necessary to carry out the following operations:

• Connect the biosensor holder to the hardware card using the attached cable;

• Remove the silver-chloride mini-electrode from its container and insert it into the special slot in the holder;

• Remove the biosensor from its package and insert into the appropriate slot in the holder;

• Lower the holder until the tip of the biosensor and reference electrode are inserted into the well in the micro-titration tray below the level of the working buffer solution.

Sample Preparation

For test purposes, it is essential to use a sample of serum or blood plasma prepared by a standard procedure. Immediately before the test, the serum (plasma) sample is transferred to the well in the micro-titration tray, where it is diluted with a special buffer solution.

Sample ID input

When the program is run in measurement mode, 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) .

1 ^st Interval of Measurement

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.

2^nd Interval of Measurement

After the first measurement stage, 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. As a change in the isoelectric point of the membrane can be induced both by a specific immuno-chemical reaction (interaction between the antibodies immobilised on the sensor with the corresponding antibody in the sample under test) and by non-specific adsorption of serum components on the biomembrane, and also by virtue of the natural variability in the ion composition of serum from different individuals, the signal obtained can vary both in shape and amplitude. As a result of the 2^nd measurement stage, the surface area (integral) S₂ 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

3^rd Interval of Measurement

On completion of the 2^nd measuring 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 On doing this, the biosensor potential will tend to revert to its initial potential before incubation with the test sample (allowing for datum-line drift)

In so doing, 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

However, in the case of a specific immuno-chemical reaction, even after placing the biosensor in the buffer solution 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 As in the 2^nd interval, the quantity recorded in the 3^d measurement interval is an integral S₃ bounded by the "biosensor signal - time" curve

As in the first and second intervals, recording of the biosensor signal takes place at discrete time intervals, and the result of each measurement is displayed on screen

Result Pretreatment

On completion of the 3^rd measurement interval, the values obtained for γ, S₂ and S₃ are transmitted for processing in the pre-treatment unit As a first step, the integral values are recalculated to allow for the datum line drift γ obtained in the first interval, S^γ 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

Final Signal Treatment

Following computation of the corrected integral ratio R, 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

Mode: Calibration Procedure

To facilitate a qualitative evaluation of the specific characteristics of the blood sample under test, and to obtain quantitative test data, 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.

• To calibrate the Biosensor Analyser, it is necessary to run the system in calibration mode. On entering this mode, a message is displayed on-screen requesting the entry of a calibration data access password. If the user does not have the password, it will not be possible for that person to change the system calibration, ensuring on one hand that the unit will function correctly and, on the other, provides the facility - if required - to adjust the system calibration in situ.

• 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. In cases where a qualitative analysis only is required (of the "yes" or "no" kind), the operator may indicate specificity as " + " (for a sample containing a specific component) or "-". However, where quantitative measurements are required, it will also be necessary to indicate the content (for positive samples) of the specific component (in ng/ml).

• At least 200 points are required on average to achieve reliable calibration, and the percentage of positive results must be under 5% or over 30%. The calibration procedure is performed automatically, and where there are insufficient calibration data to proceed with the test in "Measurement" mode, a message to this effect is displayed.

• 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.

Having plotted this graph, two values can then be obtained: R₀ - cut off and 2_A ^s3y - width of grey zone. Thus, a resutt is taken as positive (containing a specific component) if the value R obtained in the test (see above) is greater than the value (R + d), and negative if its value is below (R₀ + d). If the value of R falls in the interval +/-A, the specificity of the sample is doubtful and requires further investigation using alternative diagnostic systems.

• Plotting of calibration curve. Where there is a sufficient volume of quantitative data for assessment of the content of a specific component, it is possible to compare the integral value R at the 2^nd and 3"¹ measurement intervals with the declared concentration of the component. To do this, a calibration curve is plotted (fig.7) ,which is then used to obtain the quantitative data, the result of the analysts.

It is possible to obtain quantitative data because, as previously mentioned, 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. As 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. Thus, 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.

Clearly, it will only be possible to obtain reliable qualitative data using this technology provided that the properties of the biosensor are fully reproducible (for example, properties like antibody concentration, sensitivity to variation in the ion strength of the solution, etc.). The results of laboratory testing of biosensors by the authors, combined with the changeover to using single-use (disposable) biosensors, indicate that the possibility of obtaining reliable quantitative data may be viewed with a high degree of optimism. Result of Current Measurement

On completion of the third cycle of measurements and processing of the results obtained, the following information is displayed on-screen

• R value in arbitrary units

• indication of the presence or otherwise of a specific component in the sample (in the form "+", "-" or "+/-") To make the results easier to read, these symbols are highlighted in different colours red, blue and green respectively

• Where a quantitative interpretation of the results is possible, the concentration of the specific component in the sample is also displayed (in ng/ml)

Data Base Updating

Once the analysis results have been displayed on the screen, the user is given the option of saving the results obtained in the database andl if the answer is yes, a new record is added to the database, including all of the measurement attributes described above

References :

[1] Grin M.J., New approaches in electrochemical immuno-assay.// In Biosensors: Fundamental Principles and Applications. M. : Mir, 1992 (rus.).

[2] Aizawa M. et al, Enzyme immunosensor, III.

Amperometric determination of human chorionic gonadotropin by membrane bound antibody.//

Analytical Biochemistry, 94, 22-8, 1979.

[3] Yamamoto N et al., Potentiometric investigations of antigen-antibody and enzyme-enzyme inhibitor reactions using chemically modified metal electrodes.// J. of Immunology Methods, 22, 309-17, 1978.

[4] Bergveld P., A critical evaluation of direct electrical protein detection methods.// Biosensors & Bioelectronics, 6, 1991).

[5] Schasfoot R.B.M., Bergveld P., Bomer J., Kooyman R.P.H., Greve J. , Modulation of the ISFET response by an immunological reaction.// Sensors and Actuators, 17, 1989.

[6] Schasfoot R.B.M., Keldermans C.E.J.M., Kooyman

R.P.H., Bergveld P., Greve J. , Competitive immunological detection of Progesterone by means of the ion-step induced response of an immunoFET.// Sensors and Actuators, Bl, 1990.

[7] Schasfoot R.B.M., Kooyman R.P.H., Bergveld P., Greve J. , A new approach to immnoFET operation.// Biosensors and Bioelectronics, 5, 1990. [8] Havash E. , Ion- and molecular-selective electrodes in biochemical systems, M.. : Mir, 1988 (rus.) .

[9] Electrochemistry of polymers, M. : Nauka, 1990 (rus. ) .

[10] Organic electrochemistry, m. : Chi ia, 1988 (rus.).

[11] Methods of measurement in electrochemistry, M. : Mir, 1977 (rus.).

[12] Immunological methods of research, M. : Mir, 1988 (rus. ) .

Claims

1. Method of electrochemical detection of immunoactive macromolecules in test solutions, which involves producing an immunosensor comprising a specific- receptor-modified membrane; forming an electrochemical measuring cell from the immuno-sensitive sensor and a reference electrode linked by a measuring instrument; placing the latter into the working solution, and determining the displacement of the isoelectric point of the membrane in relation to the concentration of macromolecules in the solution under test, by measuring the cell potential with step-changes in the ionic strength of the working solution, distinguished by the fact that the membrane is formed from electroconductive polymer by electrochemical synthesis from a monomer solution containing specific receptors on the surface of the potentiometric electrode; to determine the isoelectric point displacement of the membrane, a test solution with an ionic strength greater than that of the working solution at constant pH is added to the working solution.

2. Method according to claim 1, in which aniline, thiophene, furan or pyrrole are used as the monomer for electrochemical synthesis of the membrane.

3. Method according to claim 1, in which monoclonal and polyclonal antibodies, viral lysates, recombinant proteins, synthetic peptides, hormones, single-strand DNA molecules, fragments of bacterial, plant and animal cells, intact bacterial cells, various chemical compounds conjugated with inert proteins (haptens) , are used as specific receptors.

4. Method according to claim 1, in which biological fluids such as blood serum, lymph, urine, saliva and semen are used as the test solution with an ionic strength greater than that of the working solution.

5. Method according to claim 1, in which the introduction of test solution with an ionic strength greater than that of the working solution into the electrochemical cell is carried out by direct addition to the vessel with the working solution.

6. Method according to claim 1, in which the introduction of test solution with an ionic strength greater than that of the working solution into the electrochemical cell is carried out by transferring the immunosensor and reference electrode from a vessel containing pure working solution to another vessel containing working solution and the solution with greater ionic strength.

7. Method according to claim 1, in which the difference between the values of base and final potentials across the electrochemical cell is used for quantitative assessment of the nature of the change in potential in the electrochemical cell.

8. Method according to claim 1, in which the difference between the maximum potential on the electrochemical cell recorded on contact between the immunosensor and the test solution and the value of base potential in the electrochemical cell is used for quantitative assessment of the nature of the change in potential in the electrochemical cell.

9. Method according to claim 1, in which the difference between the maximum potential on the electrochemical cell recorded on contact between the immunosensor and the test solution and the value of final potential in the electrochemical cell is used for quantitative assessment of the nature of the change in potential in the electrochemical cell.

10. Method according to claim 1, in which the value (in arbitrary units) of the area under the curve of the variation in electrochemical cell potential versus time, obtained on contact between the immunosensor and the test solution, is used for quantitative assessment of the nature of the change in potential in the electrochemical cell.

11. Method according to claim 1, in which the initial, final, average or maximum rate of change in the value of electrochemical cell potential on contact between the immunosensor and the test solution, are used for quantitative assessment of the nature of the change in potential in the electrochemical cell.

12. 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.

13. A method as claimed in Claim 12, wherein the 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.

14. 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.

15. A method as claimed in Claim 14, wherein the cyclic electric potential has a sawtooth form.

16. A method as claimed in either of Claims 14 or 15, wherein the cyclic electric potential is applied for at least two cycles.

17. 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.