The present invention relates to a method and a device for analytical determination of molecules by means of electrochemical analysis and admittance measurement.

It is of interest to be able to measure the presence and content of different biologically active substances in many different fields such as environmental management, clinical examinations and biotechnical processes. Many different measuring methods in which the measuring signal is based on different chemical or physical properties, directly for the substance to be analyzed or indirectly, for example at a chemical reaction, have consequently been developed. One method which has gained a great and general use is electrochemical determination wherein a voltage is applied over electrodes and a resultant current is analyzed.

An enzymatically catalysed chemical reaction where the specificity of an enzyme or an antibody is combined with the more general electrochemical measuring method, which measures a property of the enzyme substrate or the enzyme product, is used in many bioanalytical applications.

In other applications the binding/adsorption/ of a biological substance to a surface is measured directly. The detection can be based on differences in amounts, for example at the binding of antigens to antibodies which have been applied to a surface.

When a voltage is applied over a pair of electrodes potential jumps are formed at the phase transitions. In most systems the voltage difference leads to a transport of chemical substances by the electrostatic field of force and formation of a coating which additionally complicates the potential jumps. Several attempts have been made to study these complicating factors, see for example the problems of overvoltage which have been described in the book

Electrochemical Kinetics: Theoretical Aspects by Klaus J. Vetter (Academic Press, 1967) .

CONFIRMATION COPY

At measurements when electrodes are used in different solutions it is also necessary to consider that different reactions take place on the electrode surfaces. The molecules present in the solution can be adsorbed on the electrode surface and thus change the phase transition between the electrode and the solution.

If the electrode voltage exceeds the oxidation/reduction-potential for any of the chemical substances which are present reduction and oxidation reactions (redox-reactions) will occur on the electrode surface. This often occurs at the usually used electrode voltages of 0.1 to 1 V and can lead to permanent modifica¬ tion of the electrode surfaces.

One method which has been found advantageous in measurements where electrodes are used is the use of alternating voltage which can reduce the adsorption of molecules to the electrode surfaces considerably. Alternating voltage also gives more information since current intensity and phase shift are measured simultaneously, or, in other words, conductance and capacitance are measured simultaneously and this is generally termed impedance spectroscopy or admittance spectroscopy. The method is described for example in the book Impedance spectroscopy by J.R. MacDonald (John Wiley & Sons, New York, 1987) . The problem with redox-reactions at electrode voltages of 0.1 to 1 V remains, but no permanent modifications of the electrode surface are formed since the direction of the reaction changes with each cycle of the alternating voltage. The object of the present invention was to provide an improved method and device for analytical determination of a biologically active substance.

The object of the invention is achieved by the method and device as claimed in the claims. According to the invention a method for qualitative or quantitative determination of a biologically active substance by means of measurement of admittance with alternating voltage between two electrodes is obtained. The method is

characterised in that the measurement is carried out at an electrode voltage low enough to minimise unwanted electrode reactions while at the same time the field strength is kept high. According to the present invention it was found that the above mentioned problems of electrode reactions are efficiently overcome if the measurements are made with electrodes positioned with a short electrode distance so that a high field strength is obtained even at low electrode voltage. A high field strength is necessary with regard to noise since the relative noise level (S/N or signal to noise ratio) is inversely proportional to the field strength. Low electrode voltage should be selected so low that unwanted reactions in the case in question are avoided. What actually is low electrode voltage expressed in volt is thus dependent on the chemical composition in the observed system. Empirical data show that electrode voltages lower than or equal to 100 mV may be satisfactory, preferably lower than or equal to 50 mV, while 20 and 10 mV function more generally. With a high electric field strength is meant a field strength higher than or equeal to 50 kV/m.

The demands on high field strength and low electrode voltage can be combined only if the distance between the electrodes is short. The relation between the geometry of the electrodes and electrical field strength for a given electrode voltage is apparent from the following formulae. If two flat, parallel electrodes, each with a surface, A, and separated by a distance, d, are considered the admittance Y can be expressed as

Y = G + jB (1) where j = -l och G is the conductance in the medium between the electrodes defined as

G = σ • A/d (2) where σ is the conductivity in the medium. B is the susceptance. Provided that the inductance can be ignored, B can be expressed as B = ωC, where C is the capacitance and ω =2πf is the angular velocity and f is the frequency. The

capacitance can be expressed as a function of the permittivity (or the dielectric constant),ε , and the susceptance can thus also be expressed as a function of the permittivity. B = ω C = ω ε • A/d (3) and also the admittance:

Y = (σ + j ω ε) • A/d (4) Since the permittivity is a complex quantity, defined as ε = ε' -j ε" (5) a combination of 4 and 5 gives

Y = (σ + ω ε ^■ ' + j ω ε ^■ ) • A/d (6) where the properties of the material, σ and ε, and the geometry of the cell, A/d, have been separated.

The values of the real and imaginary parts of the permittivity, ε ' and ε ' ' , of a dielectric media do generally vary with frequency, giving rise to either a relaxation or a resonance phenomena. By studying the frequency spectrum of ε ' and ε ' ' it is possible to deduce whether the dielectrica show relaxation or resonance behaviour. From such spectra it is possible to determine the value of the frequency (relaxation frequency) where the system relaxes, defined as the frequency where ε ¹ falls slowly and ε ' ' exhibits a broad peak. In the case of a resonance behaviour it is also possible to deduce the value of the frequency (resonance frequency) where the system goes into resonance, defined as the frequency where ε ' shows a rapid fall and ε ' ' shows a narrow peak.

The electric field between the electrodes follows the equation E = V/d where E is the electric field strength and V is the difference in voltage between the electrodes. In a high electric field regime, non-linear dielectric behaviour may be observed. This regime can either be a DC or AC field, and makes it possible to shift relaxation frequencies upwards or downwards in frequency, respectively. These shifts are specific for the dielectric media between the electrodes. Thus, according to a preferred embodiment the

method according to the invention is characterised in that the electric alternating field strength or the static field strength shifts the relaxation frequency as a function of the field strength. According to a further aspect of the invention a device is obtained for qualitative or quantitative analytical determination of a biologically active substance by means of admittance measurement between two electrodes. The device is characterised in that the electrode pattern has been defined in such a manner that the distance between the electrodes is 150 nm or less.

The electrodes ^' can be designed in different manners. Some examples are: Two separate wires arranged in parallel or in other manner. Finger-structures where each electrode has been branched into a number of separate, parallel wires (fingers) which can lie in a pattern as a linear lattice or as a circular structure with concentric rings.

Four-electrode systems where the outer electrodes are connected to the voltage source and the two inner electrodes are connected to the analysis instrument.

The electrodes can be made of different metals or metal systems such as for example: gold, titanium, copper, palladium, chromium, silver, platinum, nickel, aluminium, wolfram or iridium or alloys of these metals.

The electrode patterns can be defined on different surfaces such as semi-conductors (eg silicon, gallium arsenide, indium phosphide) , oxides (eg silicon dioxide) , nitrides (eg silicon nitride) , polymers (eg polystyrene, polymethylmethacrylate) according to conventional lithographic methods using a mask of photoresist material obtained by exposure to visible light, UV-light, electronic beam, X-ray or ion-ray or other methods for transfer of patterns. To bind suitable biomolecules to the surface where the electrodes have been applied it is possible to use any of the methods known from different chromatographic techniques. A silicon dioxide surface can for example be

modified with aminopropyl triethoxysilane, carboxypropyl triethoxysilane or epoxypropyl triethoxysilane in order to obtain a surface with amino-, carboxy- and epoxy groups, respectively. The biomolecule in question can then be adsorbed with electrostatic bonds to a charged surface of amino or carboxy groups or be bound covalently be means of CNBr, triazine, carbodiimide, carbonyl diimidazole or formation of a Schiff base.

The analytical method is based on a combination of the specificity of a biochemical reaction, eg binding of a substrate to an enzyme or binding of an antigen to an antibody, and a general physical method of measurement: admittance measurement at one or several frequencies.

The measurement can be directed to adsorption to the surface with the electrode pattern. What is adsorbed may be biomolecules such as for example antibodies, or virus or whole cells, eg bacteria.

The measurement can also be directed to enzymatic activity, where an enzyme has been bound to the surface and is allowed to catalyse a chemical reaction.

Some non-limiting examples of different ways of producing structured surfaces follows below.

1. For a silicon wafer with a thickness of about 300μm a surface layer with a thickness of about 300 nm is oxidised to silicon dioxide with water vapour at 1100°C. The surface is then coated with a thin layer of photoresist, consisting of polymethylmethacrylate, and the wafer is then placed in an oven at 160°C for 24 hours. The surface is then exposed to electron beam lithography and developed in a mixture of methyl isobutylketone:isopropanol 1:1, whereby the exposed pattern becomes apparent. Gold is volatilised in a metallizer to cover the whole surface to a thickness of 20 nm. Remaining photoresist is washed away with acetone whereby the part of the gold which is on top of the photoresist comes along ("lift-off") . Thereby the gold forms the pattern which was defined at the exposure. In this manner finger-structures with a finger breadth of

100 nm and an electrode distance of 100 nm and a total size of 100 μm in square are produced, and in addition to this comes surfaces of contact ("bond pads").

2. The surface of a silicon wafer which had been oxidised according to example 1 is coated with amino groups by submerging into a slightly alkaline aqueous solution with aminopropyl triethoxysilane and the wafer is then placed in an oven at a temperature of 120°C overnight. The wafer is then coated with an electrode pattern according to example 1.

Some non-limiting examples on binding processes follows.

3. The surface of silicon wafer which had been oxidised according to example 1 is coated with an electrode pattern according to example 1. The silicon dioxide surface is then coated with amino groups by reaction with aminopropyl triethoxysilane in gas phase.

4. The enzyme horseradish peroxidase is bound to a silicon wafer prepared according to example 2. Freeze dried peroxidase is dissolved in distilled water and 0.1 M sodium metaperiodate is then added. After 20 minutes the salt is removed by dialysis. 0.1 M sodium carbonate is then added and the mixture is applied to the electrode surface with the amino groups. After 2 hours the surface is washed with distilled water and 0.1 M sodium borohydride is then added. After additionally 2 hours the surface is washed with distilled water once more.

5. Urease is bound to a silicon wafer which has been prepared according to example 2, but with the use of carboxypropyl triethoxysilane instead of aminopropyl triethoxysilane, according to the following. l-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride is dissolved in 0.05 M sodium bicarbonate and applied to the surface with carboxyl groups and allowed to react in

darkness during 1 hour. pH is kept below 7.0. After washing of the surface urease dissolved in 0.05 M sodium bicarbonate is added.

Some non-limiting examples of measurements follow below.

6. A drop of 10 μl containing 10 mM Tris-HCl, pH 7.5, 10 mM phenol, 10 mM 4-amino-antipyrine and 2 mM hydrogen peroxide was placed on a finger-structure with peroxidase, prepared according to example 4. The change in admittance was measured as a function of the time at 10 kHz. After 100 seconds the change in conductance was -3% and the change in susceptance +20%, measured at a frequency of 10 kHz. At a field strength of 7.7 kV/m a relaxation frequency of 11000 Hz was measured and this was shifted down to 400 Hz at a field strength of 67 kV/m.

7. A solution of gamma globulin having a concentration of 10 μg/ml dissolved in 10 mM Tris-HCl buffer, pH 7.5, was applied to a finger structure with amino groups prepared according to example 2. The change in admittance was measured as a function of the time at 10 kHz at an electrode voltage of 10 mV. After 100 seconds the change in conductance was 7% and the change in susceptance was +32%, measured at 10 kHz.

8. A drop of 10 ml containing 10 mM urea in 10 mM sodium phosphate buffer, pH 7.0, was placed on an electrode structure consisting of four parallel electrodes having the enzyme urease bound thereto in correspondence with example 5. The change in admittance was measured as a function of time at 10 kHz. After 100 seconds the change in conductance was +53% and the change in susceptance was -4%, measured at a frequency of 10 kHz.