DETECTION METHOD

Abstract: Substances having specifically bonding partners, such as antibodies and antigens or nucleic acids and their complements, are assayed electrometrically by coating an electrode with a specifically bonding substance, treating the coated electrode with a test sample and exposing the treated electrode to different ion compositions. The different ion compositions are provided by exchanging salt solutions or by electrochemically producing ions using an additional metal couple. The electrode signal is indicative of the type and amount of the substance to be assayed.

Description: Detection method. The invention relates to a method for assaying a component of the reaction between two or more substances which specifically bond to each other, in a test sample, on the basis of the response of an electrode to which the product of said reaction is bound. Such a method is described in US Patent Publication 4,020,830. The method described therein is used to determine the concentrations of, inter alia, ions, enzymes, antibodies and antigens. In said method, an ion-sensitive so-called ISFET (ion-selective field effect transistor) is used as electrode. Such a method was developed in order to make it possible to carry out a direct assay in which, as a result of the interaction of the sub- stance to be detected with a measuring probe, a directly measurable physical change occurs in the probe. This is, for example, of importance for medical diagnosis. Typical examples of substances which are of importance in medical diagnosis are immunochemically active substances such as antigens or haptens. which are generally related to disease symptoms, infectious organisms and the like, and antibodies or fragments thereof directed against them. Said antigens or haptens on the one hand, and the antibodies directed against them on the other hand, can enter into a specific reaction with each other. To determine a component participating in such an immunochemical reaction, use is in general made of indirect assay methods for at least one reaction component to which a detectable substance is bonded. As detectable substance, it is then usual to take a radio-active atom (in a radio-immunoassay) , an enzyme (in a so-called enzyme immunoassay) or an erythrocyte. a latex particle, a metal sol particle or a dye particle (in an agglutination assay). The disadvantage of all these indirect methods is that they are rather time-consuming. Direct and immediate recording of the specific (immunochemical) reaction is not possible. In a direct assay method described in literature (US Patents 4.238.757 and 3.966.580) based on an ISFET. the ISFET is provided with a chemically selective membrane in the "gate" zone which contains an immunochemical component reacting with the component to be detected. The term IMFET is then used. However, it appears that such a device does not give a reliable, unambiguous and reproducible- measurement signal in relation to the quantity present of the immunochemical component to be assayed (Collins & Janata (1982), Anal. Clin. Acta 136, 93-99). An improved method has now been found for assaying a component of the reaction between two or more substances specifically bonding to each other,by means of an ion-sensitive electrode such as, for example, the above-mentioned ISFET. This method is characterized in that the compo- nent to be assayed is incubated with at least one specifically bonding substance, with either the specifically bonding substance having been bound to the electrode prior to incubation or the reaction product being bound to the electrode after incubation with the component to be detec¬ ted, whereafter the electrode is exposed successively to different ion compositions, the membrane potential change induced by the ion composi¬ tion alteration being measured, and said change being a measure of the type and quantity of the component to be assayed in the sample. The electrode is exposed to the differing ion compositions in par¬ ticular on the side to which the specifically bonding substance is bound. In a preferred embodiment, the different ion compositions are changing electrolyte solutions. Said potential change may, for example, be measured as a function of the time which has elapsed after the salt compositon change or, for example, it may be measured at a fixed instant in time after said change. It has been found, in particular, that initially the electrode is in equilibrium with the original salt solution which is located on the membrane side, i.e. on the side to which the specifically bonding substance is bound, of the electrode and that after the salt composition change, an equilibrium will again be established. However, the concen- tration of any ions to which the electrode is specifically sensitive will also preferably have to be kept constant during the whole course of the measurement. If. for example, a pH sensitive ISFET is used, this means that the pH must remain constant despite the changing composition of the liquid in other respects, whereas if, for example an Ag/AgCl electrode is used, the Cl ion concentration must be kept constant during the whole experiment. The pH will have to be constant too in some cases, for example for immunochemical reasons. It may be imagined, without therewith explaining the observed phenomenon with certainty, that in the method according to the inven- tion, the new concentration slowly homogenizes on the membrane side of the electrode while the salt concentration of the solution is being changed, but that the cations and anions do not diffuse at the same velocity into the medium surrounding the electrode, as a result of the charged state of the membrane which is characteristic of a certain specific bonding reaction which has taken place. As a result of this, a membrane potential could be produced transiently, the form of which in terms of. for example, maximum amplitude, width, rise and fall time, is typical of the charged state of the membrane and therefore of the quan¬ tity and the type of the component to be assayed. It has been found, in particular, that the form of the transient change in potential is also dependent on the concentration difference produced and the concentration ratio of the ion before and after the change to which the electrode is exposed, on the type of ions of which the concentration is altered, on the thickness of the membrane and on the pH of the test solution. Here "membrane" is understood to mean any layer which, when deposited on an electrode, is capable of conducting at least a portion of the charge carriers in the electrolyte solution and which contains a component which speciffically bonds the substance to be detected. This may be a monomolecular layer of the specifically bonding component(s) , but the layer may also consist of a mixture of the specifically bonding component(s) and inert substances. Depending on the component to be measured, the different ion con¬ centrations, the magnitude and direction of the concentration change. the buffering action of the test solution and the pH can be adjusted to the conditions and requirements with respect to measuring speed and accuracy. The change in the salt composition itself may be carried out in various ways, optionally repeatedly at a different pH. The electrode may. for example, be immersed consecutively in two or more different salt solutions after incubation in the sample or the salt solutions of different compositions may be passed over the electrode in a continuous- flow cell. The minimum dimensions of the electrodes have a favourable effect on the speed of measurement and on the required volumes of the measurement solution. The composition of the liquid may also be in¬ fluenced electrochemically by means of of a generation electrode. Thus, in the case of an ISFET, for example, a rapid local pH change may be produced coulometrically by adding a ring electrode around the gate. Such a method could also be used to neutralize the specific bonding (electrode regeneration). The ion composition change can also be effected by electrochemical means using an additional metal couple producing ions in or near the membrane, for example by short-circuiting. Such a couple, a so-called actuator, can be mounted onto the electrode. An example of such an inte- grated electrode is the Zn/Ag-AgCl couple. Such integrated electrodes are advantageous in that there is no need to use different salt solu¬ tions and that they can optionally be introduced directly into the measuring environment. The invention also relates to such an electrode to which a specifi- cally bonding substance is bound. Suitable electrodes for use in the method according to the inven¬ tion are, in particular, ion-sensitive electrodes such as, for example, the ISFET already mentioned above (US Patent 4,020,830), Ag/AgCl elec¬ trode, glass electrode, coated wire etc. A so-called REFET may also be used (US Patent 4,269,682), provided the latter achieves a stable con¬ tact with the test liquid regardless of the composition and concentra¬ tion of said liquid. In general, any electrode is suitable which is sen¬ sitive to potential and which preferably also has a stable working point. The (ion-sensitive) electrode for use in the method according to the invention may be coated with a membrane by specific adsorption, for example ^ of a certain protein, optionally after a prior treatment of the membrane. The state of this optional membrane preparation can also be tested with the detection method as described in this invention. The specific charge in or at the membrane may also be the result of a reaction between two or more substances which specifically bond to each other from a test sample, the bonding partner of the component to be detected having been bound in or to the membrane (immobilized) in a prior preparation phase. The bonding partner may also be bound directly, for example covalently, to the electrode. The method according to the invention is used advantageously for assaying an immunoreactive substance (antibody-antigen). Further, nucleic acids (DNA probes, RNA probes) and other specifically bonding or complexing substances which lend themselves to being immobilized in membrane form on an electrode or to being bonded directly thereto (co-valently) such as valinomycin, or a crown ether for the detection of specific ions, of haemoglobin for oxygen, may be used with advantage in the method according to the invention. It is also possible to detect neutral substances by providing them with a charge label. Thus, proges- terone can be assayed for example. An "electrolyte concentration change" as mentioned above will induce a potential change whose form is specific to the type and the quantity of the molecule or ion complexed by the membrane. The method according to the invention lends itself therefore both to qualitative and to quantitative assays. By using a series of elec¬ trodes, each of them treated with another specifically bonding sub¬ stance, a corresponding series of relevant substances can be assayed. The sample in which the component is determined according to the present method may be biological or non-biological in nature, for exam- pie soil samples, samples of microbial, vegetable or animal origin etc. If desired, it may be an extract or a fraction derived from one of the above-mentioned types of samples. For detecting a component of animal origin, in particular, a body fluid, cellular material or, for example, an excretion or extract thereof may be used as a sample. In the method according to the invention, a reference electrode which does not respond to the change in composition of the liquid, for example a calomel electrode, is required in the test liquid. If, how¬ ever, two measuring electrodes of the same type are used, one of which is provided with a bonding partner of one of the types described, optionally in membrane form, and the other is not (blank), a difference measurement can be carried out, in which case the requirement for the reference electrode to be stable does not apply. The latter may then consist, for example, of a more or less noble metal because the elec¬ trode potential which is then unstable is automatically suppressed as a common signal in the difference measurement. The complete electrode con¬ figuration, viz. one coated electrode, one blank electrode and one metal reference electrode, may be simultaneously incubated with the sample to be investigated and then simultaneously exposed to the solutions with different salt compositions. The method according to the invention is also useful for assaying samples which may ' also show non-specific bonding or adsorption. The potential change which is due to the component to be assayed can then be obtained by differential measurement of an electrode provided with a specifically bonding substance and an electrode provided with a non- specifically bonding substance. Example Using the method described above, a distinction was made between the presence of antibodies alone on the gate area of an ISFET and the presence of an immunocomplex of antibodies and antigen on the gate area. as set out below. 3. Detection of the antibody alone on the gate area. 10 /ul of a solution of 1.2 mg/ml of antihuman serum albumin was pipetted onto the gate area of one of two ISFETs and incubated for 1 hour at room temperature. Then 10 /ul of 2.5% (w/v) glutaraldehyde solution was pipetted onto the same gate area and incubated for 15 min. at room temperature. Then both gate areas were rinsed well with distilled water and then equilibrated in ion concentration shock medium _1 consisting of (a) 0.001 mol/1 HEPES/NaOH buffer, pH 6.45, or (b) 0.001 mol/1 HEPES/NaOH buffer, pH 7.05 for 1 hour at room tempe¬ rature. The medium J_ was then instantaneously replaced by ion concen¬ tration shock medium j^ consisting of (a) 0.1 mol/1 HEPES/NaOH buffer, pH 6.45, or (b) 0.1 mol/1 HEPES/NaOH buffer, pH 7.05. Immediately after the ion concentration shock media were changed, the potential change between the gate areas of the two ISFETs was recorded as a function of time with constant current density between source and drain (see fig. 1 (a) and (b)). Detection of an immunocomplex on the gate area of the ISFET pair. 10 /ul of a solution of 1.2 mg/ l antihuman serum albumin was pipetted onto the gate area of one of two ISFETs, followed immediate¬ ly by a further 10 /ul of a 1.2 mg/ml solution of human serum albumin. This mixture was incubated for 1 hour at room temperature, after which the same method was followed as mentioned under 1, star¬ ting from the incubation with glutaraldehyde. The potential change recorded between the two gate areas is also shown in figure 1 (a) and (b). Fig. 1. Recording of the potential difference ( Δ ) between the gate of the ISFET covered with protein and the gate of the ISFET without protein as a function of time following the ion concentration shock. (a) using the ion concentration shock medium having pH 6.45. (b) using the ion concentration shock medium having pH 7.05. continuous lines: protein = hSA/anti-hSA complex broken lines: protein = anti-hSA alone.

Claims: CLAIMS . 1. A method for assaying a component of the reaction between two or more substances which specifically bond to each other, in a test sample, on the basis of the response of an electrode to which the product of said reaction is bound, characterized in that the compo¬ nent to be assayed is incubated with at least one specifically bonding substance, with either the specifically bonding substance having been bound to the electrode prior to incubation, or the reaction product being bound to the electrode after incubation with the component to be assayed, whereafter the electrode is exposed successively to different ion compositions, the membrane potential change induced by the ion com¬ position alteration being measured, and said change being a measure of the quantity and the type of the component to be assayed in the sample. 2. The method of claim 1, characterized in that the different ion compositions are different electrolyte solutions. 3. The method of claim 1, characterized in that the different ion compositions are obtained by electrochemical production of ions. 4. The method of any of claims 1-3, characterized in that the component to be assayed is an immunoreactive component. 5. The method of any of claims 1-3, characterized in that the component to be assayed is a nucleic acid. 6. An electrode to be used in the method of claim 3, to which elec- trode a specifically bonding substance is bound and which electrode is provided with a metal couple for electrochemically obtaining different ion solutions.

Inventors: BERGVELD PIET, GREVE JAN, KOOYMAN ROB PETER HERMAN, SCHASFOORT RICHARDUS BERNARDUS, BERGVELD, PIET, GREVE, JAN, KOOYMAN, ROB, PETER, HERMAN, SCHASFOORT, RICHARDUS, BERNARDUS, MARIA
Applicants: UNIV TWENTE, UNIVERSITEIT TWENTE
Application year: 1989
Main classification: G01N27
IPC codes: G01N27, G01N33