CN111351938A - Method for electrically exciting labeled molecules and electrode coated with insulating film - Google Patents

Abstract

The present invention relates to a method for electrically exciting labeled molecules and an insulating film-coated electrode, the method comprising: at least partially immersing the electrode in an electrolyte solution containing at least one labeling molecule; exciting the label molecules by electrical pulses from the electrodes, thereby producing excited labels; and detecting luminescence emitted by the excited label; wherein the electrode comprises a p-type semiconductor material and at least said part of the electrode immersed in the solution is substantially covered with an electrically insulating film comprising the p-type semiconductor material, the electrically insulating film having a band gap equal to or greater than 5eV, wherein the electrically insulating film is covered with a shielding layer comprising an organic polymer material, thereby providing a basis for reproducible analytical applications in bioaffinity assays.

Description

Method for electrically exciting labeled molecules and electrode coated with insulating film

Technical Field

The present invention relates to a method for electrically exciting labelled molecules and to an insulating film coated electrode, in particular to the electrical excitation of a labelled species at an electrode covered with one or more insulating layers, and the use of the resulting luminescence (electroluminescence, EL) in analytical methods, in particular in bioaffinity assays.

Background

Many commercially important analytical methods are based on the following principles: the analyte can be identified and quantified from the matrix using a labeling substance. For example, in an assay based on the biological properties of an analyte, such as an immunoassay, analyte (a) may be selectively captured from a solution on a solid support by means of an antibody immobilized on the surface of the solid support, and the amount of (a) may be quantified using another antibody that selectively binds to (a) and is labeled with a suitable marker substance. Such marker substances may be, for example, radioisotopes, enzymes, molecules that absorb or produce fluorescence or phosphorescence, certain metal chelates, etc., which may be chemically coupled to the antibody. Alternatively, purified (A) may be labeled (A-L) and the amount of unlabeled (A) may be determined by an antibody immobilized on a solid support using a competition reaction between (A-L) and analyte (A). DNA and RNA detection assays are based on the principle of bioaffinity similar to immunoassays and are performed according to the relevant procedures. In addition, other chemical and biochemical analysis methods may be based on similar principles. Currently, there is an increasing need for multi-parameter assays due to the increasing demand for lower cost and/or increased ease and accuracy of assays. One solution to these problems is to use marker compounds that emit light at different wavelengths. Various methods and strategies in immunoassays are described, for example, in The Immunoassay Handbook (ed by David Wild, Stockton Press Ltd., New York,1994, pages 1-618).

It is known that organic luminophores and metal chelates suitable for labeling in analytical methods can be excited by light or by electrochemical means, resulting in specific emission of the labeling substance. Methods based on these phenomena are generally sensitive and well suited for excitation of the labeling substance. However, difficulties are encountered when the concentration of label in the actual assay is very low; for example, the presence of Tyndall, Raleigh, and Raman scattering, as well as background fluorescence that is common in biological samples, complicates the use of fluorescence. Phosphorescence in the liquid phase can be used primarily only in connection with some specifically synthesized lanthanide chelates. The long-life photoluminescence applications of these compounds are limited, mainly because of the complexity of the equipment required and the high cost of the pulsed light source.

Electrochemiluminescence can be produced in a non-aqueous solvent at an inert metal electrode with a very simple apparatus. However, bioaffinity assays of commercial importance are generally only suitable for use in aqueous solutions. The sample is virtually always aqueous, and therefore the display method of the marking substance must be suitable for use with aqueous solutions. Currently, only certain transition metal chelates are capable of acting as electrochemiluminescent labels in micellar solutions, which are in principle not completely aqueous solutions. However, these methods using conventional electrochemical and inert metal electrodes do not allow simultaneous excitation of several marker substances with sufficiently different emission spectra and/or luminescence lifetimes.

Metal (e.g., Pt and Au) electrodes or carbon electrodes, which are inactive in activity, are mainly used in conventional electrochemistry. Their application is limited to a narrow potential window due to water splitting reactions, the release of hydrogen and oxygen. At these electrodes, luminophores, which may be used as fluorescent or phosphorescent labels, are usually not electrically excited in aqueous solution, because the high anodic and cathodic potentials required to excite the reaction are not achieved. With a suitably chosen semiconductor electrode, a wider potential window can be achieved, but at this type of electrode only a few marker substances can be excited in a completely aqueous solution.

The invention provides a significant improvement in the application of active metal or semiconductor electrodes and allows simultaneous excitation of a plurality of different label substances in a complete aqueous solution. The present invention utilizes a novel electrode which is a conductor covered with an insulating film that is not useful in the conventional electrochemical field. These electrodes are hereinafter referred to as insulator electrodes or insulating film-coated electrodes.

Disclosure of Invention

The present invention relates to the use of electrical pulses at electrodes covered with an insulating film to excite marker molecules useful in chemical and biochemical analysis, and the use of such electrodes in chemical, clinical and biochemical analysis. The electrodes comprise an electrically conductive base material coated with an organic or inorganic insulating film or a multilayer of such films, such that one or more marker compounds can be excited in aqueous solution to an excited state that can be de-excited by ultraviolet, visible or infrared light emission, thereby providing a basis for reproducible analytical applications in bioaffinity assays, such as immunoassays and DNA detection assays.

Drawings

FIG. 1A shows a measurement apparatus incorporating an insulating film coated electrode for use in one embodiment of the method of the present invention.

FIG. 1B shows a measurement apparatus incorporating an insulating film coated electrode for use in another embodiment of the method of the present invention.

FIG. 2 depicts the measurement principle of the immunoassay of the present invention.

Fig. 3 shows various shapes of the insulating film coated electrode of the present invention.

FIG. 4 shows phospholipase A₂The standard curve of the immunoassay of (a), wherein the working electrode is covered with (a) a natural oxide layer, (b) an anodized oxide layer, and (c) an anodized oxide layer covered with polystyrene.

Fig. 5 depicts a standard curve for an immunoassay for TSH, wherein the working electrode is an aluminum electrode covered with (a) a native oxide layer, (b) a layer modified by anodization and by coating with an epoxy layer.

Fig. 6 shows a standard curve for a competitive assay for thyroxine (T4), where the insulating film coated electrode is an anodized aluminum electrode.

Fig. 7 shows the detection of philadelphia chromosomes by DNA hybridization, wherein the insulating film coated electrode is a polystyrene coated aluminum electrode.

Fig. 8 depicts a standard curve for an immunoassay for TSH, wherein the electrode is oxide coated aluminum (a) and (b) the surface of the oxide has undergone silanization modification.

Fig. 9 shows a standard curve of immunoassay for CRP, in which the insulating film-coated electrode is a magnesium electrode covered with oxide coated with polystyrene.

FIG. 10 shows β₂-standard curve of immunoassay of microglobulin, wherein the insulating film coated electrode is an anodized silicon electrode.

Fig. 11 depicts a standard curve for an immunoassay for TSH in which the working electrodes are (a) a silicon electrode covered with a natural oxide film (by anodizing the silicon electrode) and (c) an anodized silicon electrode covered with polystyrene.

FIG. 12 shows the detection of Philadelphia chromosomes by DNA hybridization, wherein the insulating film coated electrode is an anodized silicon electrode covered with polystyrene.

Fig. 13 shows a standard curve for a competitive assay for thyroxine (T4), where the insulating film coated electrode is an anodized silicon electrode.

Fig. 14 depicts a standard curve for an immunoassay for TSH, wherein the insulator film coated electrode is a zinc electrode that is either (a) cathodically treated or (b) directly coated with a polystyrene layer and a paraffin layer in sequence.

FIG. 15 shows β₂Standard curve for immunoassay of microglobulin, wherein the insulating film coated electrode is an ITO glass plate coated with polystyrene and paraffin in sequence.

FIG. 16 shows β₂Standard curve for immunoassay of microglobulin, where the insulating film coated electrode is an Au-PET film coated with polystyrene and paraffin in sequence.

Fig. 17 depicts a standard curve for an immunoassay for CRP, where the insulating film coated electrode is a polyaniline film coated with polystyrene and paraffin in that order.

Fig. 18 shows a standard curve for an immunoassay for CRP, where the insulating film coated electrode is steel coated with alumina and polystyrene.

Fig. 19 shows a recorded EL spectrum showing EL emission markers capable of simultaneously exciting a short lifetime and a long lifetime.

FIG. 20 depicts TSH and PLA₂A standard curve of simultaneous immunoassay, wherein the insulating film coated electrode is an anodized aluminum electrode coated with polystyrene.

FIG. 21 shows phospholipase A using latex beads₂Wherein the insulating film coated electrode is an anodized aluminum electrode coated with polystyrene.

FIG. 22 shows phospholipase A₂The standard curve of the immunoassay of (a), wherein the 5-fluorosalicylic acid label (a) is directly measured using enzymatic amplification and total EL (b) or time resolved EL detection (c) is applied with a polystyrene coated magnesium electrode by generating a ternary tb (iii) complex prior to EL measurement.

FIG. 23 depicts β₂Standard curve for immunoassay of microglobulin, wherein the insulating film coated electrode is made of anodized silicon.

FIG. 24 shows β₂Standard curve for immunoassay of microglobulin, wherein the insulating film coated electrode is made of anodized silicon and the label consists of liposomes containing luminophores.

FIG. 25 shows β₂Standard curve for immunoassay of microglobulin, wherein the insulating film coated electrode is made of anodized silicon and the label is a luminophore that can be photo-detached by UV light.

FIG. 26 depicts β based on energy transfer₂-standard curve for microglobulin immunoassay, wherein the insulating film coated electrode is made of anodized silicon.

FIG. 27 shows β₂Standard curve for immunoassay of microglobulin, in which the insulating film-coated electrode is made of anodized silicon and the label is made of polystyrene particles containing terbium chelateAnd (4) forming.

Fig. 28 shows the time versus CECL strength to explain why "Tb" (terbium) was used in lanthanide chelates to label the materials.

Detailed Description

The object of the present invention is a method and a device with which one or more different types of labeling substances can be simultaneously electrically excited, so that the resulting luminescence can be used in bioaffinity assays, such as immunoassays and DNA or RNA detection assays.

It has been experimentally observed that extremely harsh redox conditions can be generated on aluminum electrodes, and these conditions are very similar to those of water radiolysis (s. kulmala, "electrically generated and chemically transformed (III) luminescence at oxide-converted aluminum electrodes and closed reagents", Academic distribution, turuinyliopsosto, 1995). Using electrodes covered with native oxide films that produce non-reproducible results, electrically induced luminescence at aluminum electrodes has been studied for several years, as described in the following references: kankare, K.

S.kulmala and k.huaapakka, anal.chim Acta,256(1992)17 and j.kankare, k.haapakka, s.kulmala, V.

Eskola and h.takalo, anal.chim. Acta,266(1992) 205. The nature of the metal itself is presumed to be the most important component in the system, while the importance of naturally occurring 1-2nm thick oxide films is not understood. For example, in life, tantalum electrodes have been claimed to be identical to aluminum electrodes and can be used in the same applications as aluminum electrodes (british patent GB 2217007B). However, tantalum oxide is an n-type semiconductor (s. morrison, "electrochemical at semiconductor oxidized metals," Plenum Press, New York,1980, s.183) with a band gap of about 4eV, and therefore, an oxide-coated tantalum electrode cannot be used in accordance with the principles of the present invention. In the present invention, an insulator electrode is defined as an electrode on which at least one is presentThe coating is composed of a material having a band gap greater than or equal to 5 eV.

The invention is based on a thin, quality insulating film, typically near 4nm thick, on or near which bioaffinity reactions are carried out, or to which bioaffinity reaction products are brought with a suitable medium (e.g. an electrolyte solution) or on a suitable support material (e.g. the surface of magnetic latex particles). The applicability of the present invention is based in part on the fact that: the presence of the insulating film brings the fermi level of the base conductor to a high cathodic pulse potential and subsequently allows the transfer of energetic (hot) electrons into the electrolyte solution by means of tunneling through the insulating film or as a result of electron avalanches. If the fermi level of the electron-emitting substrate conductor is higher than the conduction band edge of water (-1.3 eV on the vacuum scale), thermionic electrons can be injected into the conduction band of water and thus produce hydrated electrons as the cathodic medium for the reduction reaction, as described in the case of radiative decomposition of water or photoionization of solutes.

The insulating film on the electrode also provides a basis for the fermi level of the base conductor to reach high anodic pulse potentials, which makes possible a new anodic process, i.e. the injection of holes into the valence band of water. The process is similar to injecting electrons into the conduction band of water and by injecting the H formed₂O⁺The ions (valence band holes in water) dissociate into protons and hydroxyl radicals (known from pulsed radiolysis of water) resulting in the production of hydroxyl radicals. Certain metal oxides, Al₂O₃、SiO₂And MgO, hydroxyl radicals can also be generated by other solid state mechanisms described in the following references: kulmala, T.Ala-Kleme, A.Kulmala, D.Papkovsky and K.Loikas, "clinical electronic transmitted chemistry of luminel at dispersed Oxide-converted Aluminum Electrodes", anal. chem., press; s. kumala&T.Ala-Kleme,Anal.Chim.Acta,355(1997)1-5。

Hydroxyl radicals having a strong tendency to undergo addition reactions and hydrogen abstraction can be converted into other oxidative radicals, which are reactants more suitable for generating redox luminescence. These secondary oxidative radicals can be generated by adding anions from the halide and pseudohalide series to the electrolyte solution for measurement (X ═ halide or pseudohalide ions);

OH+X^-→OH^-+X^-

if the insulating film cannot generate hydroxyl radicals by the above mechanism, or if it is desired to increase the amount of oxidizing species at the expense of reducing equivalents, hydroxyl radicals can be generated from hydrogen peroxide according to the following reaction:

H₂O₂+e^-→OH+OH^-

similar techniques also allow for the production of sulfate and phosphate radicals, which are generally oxidants that are more suitable for redox-excited pathways than hydroxyl radicals (s.kulmala t.ala-Kleme, a.kulmala, d.papkovsky and k.loikas, "catalytic electrically amplified chemistry of luminel at dispersed oxide-converted Aluminum Electrodes", anal.chem, in press.).

S₂O_s ^2-+e^-→SO₄ ^-+SO₄ ^2-

P₂O_s ^4-+e^-→PO₄ ^2-+pO₄ ^3-

Protonation of the phosphate radical affects the oxidizing power of the radical, whereas the oxidizing power of the sulfate radical is independent of pH above pH 2.

Thus, highly oxidizing and reducing conditions can be created simultaneously near the insulating film coated electrode, which is generally a prerequisite for the presence of redox luminescence in aqueous solutions.

Although the solid state phenomenon used in the present invention is known in the theory of physics, the present type of insulator electrode has not been used in analytical chemistry, except in the case of an aluminum electrode covered with a naturally occurring oxide film of poor quality and excessive thinness (british patent GB2217007B), which has not led to any practical application of such an aluminum electrode. In contrast, the present invention forms a significant improvement to the electrode by recognizing the proper function of the insulating film and its intentional preparation.

According to the invention, the electrode has an electrically conductive base layer which can Be composed, for example, of carbon (graphite, glassy carbon) or of a metal (for example Be, Mg, Al, Ga, In, Au, Pt, Cu, Fe, Ru, stainless steel, Zn, Hg, Ag, Ni, Pd, Hf, Zr) (Ta is also suitable as base conductor, but Ta₂O₅Cannot be used as an insulating film). However, the smaller the work function of the substrate conductor, the better the electrode may work.

The conductor may also be a heavily doped semiconductor or metal oxide, e.g. Si, Ge, Sr, ZnO, SnO₂And the like. The base conductor may also be composed of a conductive polymer (e.g., polyaniline, polypyrrole, polyacetylene, polythiophene) or a corresponding polymer made from substituted monomers. The resistivity of the base conductor should be<10Ωcm。

The insulating layer of the electrode may be made of: some metal oxides, e.g. SiO₂、MgO、CaO、 SrO、BaO、Al₂O₃、HfO₂(ii) a Some other inorganic insulators, such as diamond, silicates, or nitrides; some organic insulating materials, such as paraffin, other solid or liquid hydrocarbons; organic insulating polymers such as teflon, polyethylene, polypropylene, polystyrene, polyacrylamide, epoxy plastic, and the like. In general, metal oxides can only be used as insulating film materials with pulsed excitation, since DC negative polarization usually destroys the insulating properties of oxide films within a few milliseconds.

The coating film of the electrode can be produced by the following method: anodic oxidation, Atomic Layer Epitaxy (ALE), spraying a polymer material or polymerizable material onto the electrode surface, dipping the electrode into the above solution and allowing the solvent to evaporate, the Langmuir-Blodgett method, or other methods known from other coating processes. Particularly in the case of silicon, there are several alternative methods to produce good quality SiO known in the electronics industry₂And (3) a membrane.

Although it is in principle possible to use aluminium electrodes coated with naturally occurring oxide films to excite some marker substances, commercial application of these electrodes is not possible due to the poor quality of the natural oxide films and the resulting irreproducibility of the analysis results being too high (S.Kulmala, "electrically generated and chemically transformed electrodes and substrates", academic discovery, Turun-thioplast, 1995, pp.25-31 and 114-. However, by manufacturing a good-quality insulating film with an appropriate thickness, the reproducibility of analysis can be improved to a desired level. The invention is characterized in that one or more coating films are carefully layered on the base conductor, and it is noted that the total thickness of the coating layers is optimal. In the case of aluminum, such an insulating film cannot be produced by oxidizing aluminum in air, but can be produced by other methods. The preferred method is to anodize the aluminum in a suitable electrolyte solution and then coat it with an organic material to prevent the aqueous solution from destroying the insulation of the oxide film during the bioaffinity determination process, which is always somewhat unavoidable in the absence of other barrier coatings. The aluminum oxide film can also be made by coating some other material (e.g., plastic, graphite, glassy carbon, metal) with aluminum, then oxidizing the aluminum and adding a final shielding layer.

When the aluminum oxide film is immersed in an aqueous solution, various uncontrollable processes start to be performed in the aluminum oxide film from the outside of the layer depending on the solution conditions. Thus, the nature of naturally occurring very thin (1-2nm thick) spontaneously formed oxide films varies with time and causes a significant drop in EL production efficiency. As indicated in example 1, the EL production efficiency in aqueous solution decreased by 90% during coating with the protein film (necessary as the first step of any immunoassay). This disadvantage can be prevented when the oxide film has been made to an optimum thickness and is preferably covered with an additional shielding film, such as an organic insulating polymer film. Some organic polymers are also particularly advantageous because they improve the coatability of antibodies and other biomaterials onto the electrodes.

In bioanalytical methods, DNA and RNA detection techniques are practically as important as immunoassay techniques. Aluminum electrodes covered with a thin naturally occurring oxide film were unexpectedly completely unsuitable for use in nucleic acid hybridization assays. However, it has been found experimentally that even aluminum electrodes can be used for these methods if the oxide film is coated with a thin organic film (e.g., a polystyrene layer).

Suitable polymer films can be readily produced by immersing the electrode in a polystyrene solution (prepared by dissolving polystyrene in an organic solvent such as benzene or toluene). In a similar manner, many other polymers that can be dissolved as dilute solvent solutions can also be used to make thin polymer films. Among the polymers that can be used, mention may be made of: polyamides, polyamines, polybutadienes, polycarbonates, polyenes, polyesters, polyethylenes, polyethyleneimides, polyoxymethylenes, etc., as described in textbooks of polymer chemistry. The polymer films may also be made from a mixture of polymers, or they may be doped with inorganic materials. These polymer films can be made very smooth by using commercially available equipment for growing polymer films. The electrode surface need not be completely coated with an insulating film designed to be effective for activating labels, the insulating film may be in the form of very small dots or islands surrounded by a thicker insulating film that does not allow any mechanism of current transport. The electrode is not always in the form of a flat plate, and may have a mesh shape, a spike shape, a tube shape, a perforated plate shape, or the like.

Similar polymer films can also be produced by allowing polymerization to occur on the surface of a conductor or the surface of a conductor covered with an insulating film. In this case, the reactants of the polymerization reaction are separately dissolved in a suitable inert solvent, such as toluene, benzene, methylene chloride, and the like. One of these components may be deposited by a special deposition apparatus, or alternatively, the electrode is automatically immersed in a solution of the component and the solvent is allowed to evaporate. The other component may be deposited and polymerized in a similar manner. Alternatively, the reactants of the polymerization reaction are mixed in a solvent immediately prior to deposition. In all cases, the optimum thickness of the film can be found experimentally by adjusting the concentration of the coating material in the solvent or by adjusting the film thickness using a coating apparatus. In some cases, it is preferable to coat the electrodes by spraying. The polymer must be divided in the spray into droplets with a diameter of 1-1000nm in a suitable solvent, such as toluene, benzene, cyclohexane, chlorinated hydrocarbons, DMF, DMSO or alcohols.

The main disadvantage of aluminum oxide films is that they cannot withstand alkaline or acidic conditions. This disadvantage can be prevented by an additional shielding layer or by replacing the aluminum oxide film with another insulating film. MgO films are particularly suitable for alkaline conditions because these films are insoluble in alkaline aqueous solutions. The MgO films can be prepared by, for example, ALE technique, and these films can also be coated with other films as described above in the case of aluminum oxide films.

Very well known SiO₂Film contrast to Al₂O₃Coatings of MgO and other alkaline earth metal oxides have been less studied. In the electronics industry based on silicon technology, SiO₂Is the most important insulating film material. In this industrial field, there are several kinds of SiO catalysts which can be used for producing high-quality SiO₂A method of making a membrane. Therefore, in manufacturing the electrode material used in the embodiment of the present invention, a technically mature silicon technology is preferable.

If a suitable co-reactant that generates strong oxidizing radicals by single electron reduction (e.g., peroxodisulfate, peroxodiphosphate, or hydrogen peroxide) is added to generate the oxidizing species that are normally necessary to excite most tag species, the inorganic insulating film can be replaced entirely with a suitable organic insulating film. Combinations of inorganic and organic films are often preferred.

In another embodiment, sodium azide is more preferred than the peroxodisulfate, peroxodiphosphate or hydrogen peroxide. In this embodiment, when sodium azide is used as a co-reactant, the luminescent lifetime of terbium becomes the longest when terbium is used as a labeling substance, so that the ECL (electrochemiluminescence) performance of the present invention is greatly improved (Tb will be described later).

Thus, in the present invention, when sodium azide is used among the various co-reactants, the background value can be reduced compared to the other co-reactants, thereby improving the sensitivity and thus the performance of ECL.

The present invention makes a particular significant improvement over the prior art by increasing the reproducibility to the level required for practical analytical methods. Other advantages of the present invention are the firing event itself and the exact timing of firing. Furthermore, a large number of very different marker substances (which differ in their emission spectra and luminescence lifetimes) can be excited simultaneously, which allows multi-parameter measurements (example 17). They may also give the possibility of improving the accuracy of the determination by internal normalization. For example, in a homogeneous assay where unreacted excess label is not distinguishable from the immune complex, dual labeling enables simultaneous quantification of two different antibodies or their concentration ratios (example 23). This allows efficient exclusion of matrix effects originating from deviating sample compositions, which often hamper the utilization of homogeneous assays. Other major advantages of the present invention are the simplicity and low cost of the measuring instrument.

Many types of lights may serve as labels according to the present invention. For example, the following luminophores (or derivatives thereof) may be used: 9-fluorenylmethylchloroformate (emission 309nm), luminol (emission 420nm), fluorescein (emission 516nm), salicylate (emission region 400-450nm), aminonaphthalenesulfonate (emission region 400-500nm) and coumarin (emission region 450-550 nm), aromatic lanthanide (III) chelates, such as certain derivatives of complexes of terbium (III) with the following ligands: n is a radical of¹- (4-aminobenzyl) diethylenetriamine-N₁,N₂,N₃,N₃-tetraacetate (tb (iii) -1), 4- (phenylethyl) (1-hydroxyphenyl) -2, 6-diyl) bis-methylenenitro) tetra (acetate) (tb (iii) -2); 4-benzoyl (1-hydroxybenzene) -2, 6-diyl) -bis (methylenenitro) tetra (acetate) (Tb (III) -3), N²- (4-aminobenzyl) -diethylenetriamine-N¹,N¹,N³,N³-tetraacetate (tb (iii) 4); 4-methyl (1-hydroxyphenyl) -2, 6-diyl) bis (methylenenitro) tetra (acetate) (Tb (III) -5) (in the case of all Tb (III) chelates the strongest emission line is at 545 nm), derivatives of certain transition metal chelates, such as ruthenium (II) and osmium (II) -tripyridyl (trisbipyridyl) and tripeyrazinyl (trispyrazyl) complexes (emission range 550-650 nm).

As above, the labeling substance that can be used in the present invention is one of an organic light emitter, a lanthanide chelate compound, and a transition metal chelate compound, but according to one embodiment of the present invention, a lanthanide chelate compound is more preferable as the labeling substance than an organic light emitter and a transition metal chelate compound. This is because, relatively speaking, lanthanides always exhibit a certain emission due to internal f-shell electronic transitions without being affected by a solution or the like.

In a preferred embodiment, "Tb (terbium)" is used in particular in the lanthanide series. This is because Tb has a long emission lifetime. (As described above, the luminescence lifetime of Tb becomes longer by using sodium azide as a co-reactant). Long luminescence lifetime means that time resolved measurements are easy to perform. The reason for performing time resolved measurements is that a significant number of tested samples of commercial importance are biochemical and biological substances. Biochemical and biological substances have essentially an autofluorescent (autofluorescence) property. Autofluorescence is used as background, which is responsible for the reduced sensitivity. However, this autofluorescence collapses and disappears in a short time when exposed to high energy. Thus, by continuing the luminescence measurement for a relatively long time (i.e., time resolved measurement), autofluorescence and other background signals at the beginning of the measurement can be excluded to account for only later signals. The principle is shown in FIG. 28[ Soini, Erkki, Timo

and Charles B.Reimer. "Time-resolved fluorescence oflanthanide probes and applications in biotechnology."CRC Critical Reviews inAnalytical Chemistry 18.2(1987):105-154]&[Lis,Stefan,Takaumi Kimura,and ZenkoYoshida."Luminescence lifetime of lanthanide(III)ions in aqueous solutioncontaining azide ion."Journal of alloys and compounds 323(2001):125-127]As shown.

The high electric field across the insulating film also induces solid state electroluminescence in the film to some extent. Such solid state electroluminescence can also excite the light emitters by virtue of energy transfer from the intrinsic emission centers if the light emitters are located close enough to the insulating film. This effect enhances the proximity effect required for homogeneous assays.

The insulating film coated electrodes described in the present invention can be used in an EL cell comprising at least two electrodes: a working electrode covered with an insulating film, and a counter electrode.

The dielectric film coated working electrode should meet the above criteria and, depending on the optical properties and thickness of the conductive material, it may be optically transparent or opaque. Typically, the transmission of a sufficiently thin base conductor is sufficiently high in the desired optical range. The use of a transparent working electrode allows electrically excited luminescence to be measured through the working electrode.

The choice of the electrodes of the method is not critical. Conventional inert electrode materials (Pt, Au) are very suitable. Often even certain metals that are anodically dissolved can be used, as measurements are typically taken on a time scale where the anodic product from the counter or auxiliary electrode has no time to diffuse to the working electrode. Some metal oxide electrodes, such as indium tin oxide, are also well suited as anode materials. In this case, the anode material can be easily made optically transparent. Stainless steel is also an advantageous electrode material. If an opaque metal electrode acts as the counter electrode, its shape may be chosen such that the luminescence can be measured behind the counter electrode. For example, a wire electrode covering only a very small portion of the surface of the working electrode may be used, or a hole drilled through the anode material allows light to be detected behind the anode. The optically transparent counter electrode may be prepared from a suitable film, such as plastic or glass coated with a thin Au film, which may be further coated with a thin shielding film that allows electron and/or hole tunneling through the outer film.

Label excitation in the detection phase may Be accomplished by a series of cathodic voltage pulses if the thickness of the insulating film on the working electrode is appropriate, but in the case where the substrate material is an anodically oxidizable material (e.g., Si, Al, Be, or Mg), it is sometimes advantageous to thicken the oxide film growth with an oxidizing anodic pulse preceding each cathodic excitation pulse.

Depending on the desired sensitivity range of the analyte, low cost semiconductor detectors or more expensive and sensitive photomultiplier tubes are suitable light detectors for use in electroluminescent meters.

The method of the invention can be used for detecting L_n,-X_x-Y_yIn the form ofA molecule wherein L is a label or a mixture of different types of labels which are derivatives of organic luminophores, such as derivatives of fluorescein, aminonaphthalenesulfonic acid, salicylate, rhodamine, or coumarin; derivatives of lanthanide chelates, such as derivatives of Tb (III), Eu (III), Y (III), Sm (III), Dy (III), Gd (III) chelates; derivatives of transition metal chelates, such as ruthenium (II) -terpyridyl or ruthenium (II) -tripyrazinyl chelates; wherein one or more derivatives are bound directly to compound Y via suitable functional groups or are bound to compound Y via one or more linking compounds X, wherein examples of Y are proteins, antibodies, enzymes or nucleic acids which have an affinity for the analyte to be quantified, and wherein the integer indices n, X and Y denote the number L, X and Y and are equal to or greater than 1, and wherein compound Y can bind to cells, cell components, viruses, bacteria, nucleic acids, DNA, RNA, DNA fragments, RNA fragments, polysaccharides, proteins, polypeptides, enzymes, metabolites, hormones, pharmacological substances, medical drugs, alkaloids, steroids, vitamins, amino acids, carbohydrates, environmental pollutants or antibodies, and wherein linking compound X can comprise as basic linking function a chemical group, for example a ureido group, a thioureido group, a thiol, An amide, a substituted imide, a thioether, -S-, sulfonamide, or N-substituted sulfonamide, the chemical group being part of a larger molecule or polymer attached to compound Y.

The methods of the invention include competitive bioaffinity methods in which labeled analyte L is present_n-X_x-A_a(where the integer subscripts n, x, and a denote the numbers of L, X and A) and analyte A from the sample compete for binding to Y on the surface of the dielectric film coated electrode, thus enabling the concentration of the analyte to be determined with the dielectric film coated electrode. L is_n-X_x-Y_yBinding to a on the surface of the insulating film-coated electrode is likely to be inhibited by a originating from the sample.

The label L may be an enzyme capable of amplifying the luminescent emitter. For example, the enzyme may be alkaline phosphatase and the luminescent molecule may be a highly luminescent molecule or lanthanide chelate produced by the enzyme.

The method of the invention comprises the following determination: (i) wherein only the labeled molecules located in the vicinity of the insulating film-coated layer can be excited by an electric pulse, thereby enabling the analysis by the principle of homogeneous assay without the need to separate the free label L before the detection step_n-X_x-Y_y(ii) quantification by heterogeneous principle and removal of free label L from the vicinity of the electrode by a washing step prior to the detection step_n-X_x-Y_yAnd (iii) the basic immunoreaction is carried out in a separate incubation chamber with a small size solid support material (e.g. paramagnetic latex particles), whereby only label detection is carried out on the electrode surface after incubation and possible washing by bringing the solid support material close to the electrode.

The novel electrodes described in the present invention can also be used for the electrical excitation of other types of luminophores than those shown in the present description and examples, since it is clear that many other types of molecules can also be excited on the insulating film-coated electrodes with the method of the present invention. The application of the electrode covered by the insulating film is not limited to certain device configurations or to the analytical methods described herein. In principle, the reverse procedure can also be used. In this case, the labeling molecule is illuminated with light, and a photocurrent induced by light injection of carriers into the detection electrode is measured, or the potential of the detection electrode is measured, thereby allowing the quantification of the label and the analyte. In these applications, the detection electrode should preferably not be covered with an insulating film, or advantageously, the thickness of the passivation film should be less than 4 nm.

FIG. 1A shows an electrode and luminescence measurement instrument for use in one embodiment of the present invention. The working electrode is composed of a conductive base material C and an insulating film I on the surface thereof. The insulating film may be composed of one or more layers of the same or different insulating materials I₁，I₂...I_nAnd (4) forming. The working electrode may be optically transparent or opaque. Using a transparent working electrode, a detector D can be utilized₂Light is measured through the working electrode. Two-electrode cells are usually sufficient, but it is also possible to use conventional three-electrode cells, which are powered by the operating powerElectrodes and auxiliary and reference electrodes (e.g., Ag/AgCl electrodes). The working electrode (or auxiliary electrode) can be geometrically chosen such that the measurement of light is from the counter electrode (detector D)₁) Whether optically transparent or opaque to the light generated by the electrode pair. When both the working electrode and the counter electrode are sufficiently transparent, the double marking and the measurement of light of two different wavelengths can be done very simply (filter F)₁And F₂) Without the need for expensive optics or beam splitters. Photodetector D₁And D₂Which may be a photomultiplier tube, other less sensitive detectors may be used when the analyte concentration in the sample is sufficiently high. The light measuring means depending on the application are single or multi-channel gated integrators or photon counters.

Fig. 1B shows a measuring device of another embodiment, which includes an insulating film-coated electrode used in the method of the present invention. In the case of fig. 1A, the conductor may be a semiconductor or a metal oxide, and in a preferred embodiment, the semiconductor material used as the conductor is preferably a p-type semiconductor material. As previously described, when sodium azide is used as a co-reactant and Tb is used as a labeling substance, the most suitable material as an electrode (particularly, working electrode) is a p-type semiconductor. When electrons are emitted from the p-type semiconductor, holes formed at the valence band edge improve intrinsic emission (intrinsic emission) performance. This provides a more persistent luminous environment for the relatively long-emitting Tb

"Journal of electrochemical Chemistry 769(2016):11-15," Markus, et al, "Immunoassay of β 2-microobulin at oxide-coated synthesized p-silicon electrodes," Journal of electrochemical Chemistry 769(2016), and references cited therein]. Therefore, in order to improve the performance of the present invention, a p-type semiconductor capable of achieving better performance is used instead of the existing ordinary conductor or n-type semiconductor.

When a p-type semiconductor is used as described above, the electrode and the luminescence measurement device of fig. 1B different from fig. 1A can be configured. As shown in fig. 1B, since the working electrode C of the p-type semiconductor is made opaque,and the generated light is transmitted only through the right-side photodetector, it is possible to simplify the apparatus and reduce the cost since only one photodetector and one filter are required, unlike fig. 1A. In the case of p-type semiconductors, since Lumo/Homo are as SiO₂Above 9eV of the band gap of (a), the voltage applied to the working electrode may range from 9V to the breakdown voltage (V) of the semiconductor material_{Breakdown of})。

Fig. 2 shows the measurement principle of the immunochemical reaction with an insulator electrode. The working electrode is composed of a base material C (1) and an insulating film I (2). Most commonly, immunoassays are performed using immunoassay principles; then, the insulating film I (2) is coated with an antibody (4) specific to the analyte (5). An immunoassay is then performed, such that a mixture of the sample and the label (7), i.e. a labeled second antibody specific for the analyte (5), is incubated in a buffer solution in contact with the surface of the working electrode. This results in the formation of immunocomplexes I- (4) - (5) - (6) - (7) on the surface of the insulating film. If the sensitivity requirements for the determination of the analyte are not extremely high, the amount of analyte can be determined after this reaction step by exciting the labeling molecule (7), which participates in the complexes I- (4) - (5) - (6) - (7), with an electrical pulse. This so-called homogeneous assay principle is possible because excitation of the marker molecules is only possible within a certain distance (8) from the surface of the insulating film, while marker molecules further away are not excited. On the other hand, in the case of the heterogeneous principle, the labeled antibody [ i.e., entities (6) - (7) ] that is not bound to complexes I- (4) - (5) - (6) - (7) is washed away, thereby providing better sensitivity than using the homogeneous assay principle. The chemical reaction at the counter electrode (3) does not normally produce luminescence with the luminophores used in the present invention.

Fig. 3. depending on the material of the working electrode (insulator electrode), they may sometimes be molded into the shape (a) of a container or cup, in which case the insulating film must be prepared on the electrode surface after the shape of the electrode is completed. All insulator electrodes can generally be used as plate electrodes (b), but metal-based insulator electrodes can be used in many different forms, for example, the "rake electrodes" (c) used in some embodiments.

Example 1

Preparation of insulator electrodes by anodising aluminium oxide and surface modification by coating polystyrene, and phospholipase A on the surface of these modified electrodes₂And (4) performing immunoassay.

And (4) anodizing the aluminum electrode. The Al electrode (Merck art.1057) molded and cut to final shape was first washed with hexane in an ultrasonic bath. The hexane was then evaporated. First, the mixture was neutralized with ammonia in a 0.5mol/L boric acid solution at 2mA/cm²Until an anodizing voltage of 2.92V was reached. Thereafter, the anodization was continued at a constant potential until the current density was less than 10. mu.A/cm²。

The oxidized electrode was coated with polystyrene. First, the anodized electrode portion was coated with polystyrene by sonicating the electrode in a 0.7mg/mL solution of polystyrene in benzene for 10 seconds. The electrode was then slowly lifted from the solution and allowed to dry at room temperature. The dried electrode was then slowly dipped into the above-mentioned polystyrene solution twice more, and the solvent was allowed to evaporate completely at room temperature between each dipping.

The electrodes are coated with the antibody. Typically, in the presence of an antibody (10. mu.g/mL; anti-PLA; Invitrogen; antibody-based₂Clone 2E1, Labmaster Oy, Turku, Finland) in TSA buffer (0.05mol/L Tris-HCl, pH7.75, 0.9% NaCl, 0.05% NaN₃) And the electrodes were incubated overnight to coat the electrodes with monoclonal antibodies. The next day, the electrodes were washed six times with a washing solution (0.01mol/L Tris-HCl, pH7.75, 0.9% NaCl and 0.02% Tween20) and in a saturated solution (containing 1g bovine serum albumin, 60g sorbitol and 1mmol CaCl per liter)₂TSA buffer of (c). The equilibrated electrodes can be dried at room temperature without washing and stored dry for at least 3 months.

Labeled antibodies are prepared. Isothiocyanate derivatives with Tb (III) -1 chelate [ Tb ]³⁺-N' - (para-isothiocyanatobenzyl) diethylenetriamine-N¹,N²,N³Tetra acetic acid ester](WallacOy, Turku, Finland) marker human pancreatic phospholipase A₂Specific polyclonal sheep antibodyBulk (affinity purified by Labmaster Oy, Turku, finland) the procedure was as follows: the antibody was reacted with the chelate at a molar ratio of 1:60 at pH 9.5. With 1mol/L Na₂CO₃Solution conditioning pH. labeled antibody was separated from unreacted chelate by gel filtration (Sepharose 6B 1 × 50cm, Sephadex G-501 × 5cm) using TSA buffer as eluent in this way usually 5 to 10 chelate molecules could be bound to one antibody molecule, 0.1% bovine serum albumin was added to the labeled antibody for improved stability.

A standard sample was prepared. Preparation of human PLA in TSA buffer containing 7% bovine serum Albumin₂Standard (0,1, 5,9,54, 324ng/mL, Labmaster Oy, Turku, Finland).

And (4) performing immunoassay. Immunoassays are performed in wells of microtiter strips. First, 25. mu.l of standard and 175. mu.l of assay buffer (0.05mol/L Tris-HCl, pH7.75, 0.9% NaCl, 0.5% NaN) were added₃0.5% bovine serum albumin, 0.05% bovine gamma globulin, and 0.01% Tween 40). Then the electrode was added and after 1 hour of incubation, the electrode was washed with a washing solution and allowed to react with the labeled antibody (500 ng/200. mu.L) for 1 hour. After the reaction, the electrode was washed in a solution containing 0.01mol/LNaN₃Or 1mmol/L K₂S₂O₈EL was measured in a measuring solution (0.2mol/L borate buffer, pH-adjusted to 7.75 with sulfuric acid). FIG. 4 shows a typical standard curve obtained in this assay, in which (a) the electrode was air-oxidized, (b) the electrode was anodized at 2.92V, and (c) the electrode was anodized at 2.92V and coated with a thin layer of polystyrene (at 1mol/L K) as described above₂S₂O₈In the measurement solution of (1). Measurements were performed using an aluminum cup electrode and an instrument described in the academic paper of s.kulmala (University of turku, Tuku, finland, 1995, pages 34-35). In the measurement, the excitation pulse was 200ms and-10V, and the frequency was 100 Hz. The intensity of the EL is integrated over 200 excitation pulses.

Example 2

The surfaces of the insulator electrodes were modified by coating with epoxy resin, and TSH immunoassays were performed on the surfaces of these modified electrodes.

The electrodes were cleaned and anodized as described in example 1 and covered with a thin layer of epoxy.

The electrodes are coated with epoxy. Both components of a Super epoxy glue (Loctite Finland Oy, art. n: o 120-1, Finland) were dissolved in 1% (w/v) in toluene. The components were mixed 1:1 and the electrode was immersed in the solution in an ultrasonic bath and then slowly pulled out of the solution. The flowing solution from the lower edge was dried, the electrode was allowed to dry at room temperature until the toluene evaporated, and then dried in an oven at 40 ℃ for 24 hours. By repeating this process, a thicker coating layer can be prepared.

The electrodes were incubated for 3.0 hours in TSA buffer containing 30. mu.g/mL coated antibody (clone 8661, α chain specific for TSH, Pharmacia, Uppsala, Sweden), coated with antibody by physical adsorption after coating, the surface was washed with running wash solution and equilibrated overnight in TSA buffer pH7.75 containing 0.1% bovine serum albumin and 5% D-sorbitol after equilibration, the electrodes were dried and they were stable in storage for at least one year.

In this case, a monoclonal antibody specific to β chain of TSH was labeled (clone 5404, Medix Oy, Helsinki, Finland).

A standard sample was prepared. TSH standards (0,0.25,1.5,9,54, 324. mu.U/mL) were prepared by diluting stock standards (Scripps Laboratories Inc, San Diego, USA) in TSA buffer containing 7% bovine serum albumin.

And (4) performing immunoassay. TSH standards (20. mu.L) and 180. mu.l (300ng) of labeled antibody were added to polystyrene wells. After 1 hour incubation, the electrodes were washed and EL was measured as in examples 1a-1b, but using an electroluminescent meter constructed by adapting an Arcus fluorometer (Wallac, Turku, Finland). The rake electrode is shown in figure 3 c. Fig. 5 shows the management curves obtained with (a) an electrode covered with a naturally occurring oxide film and (b) an electrode covered with an anodic oxide film and an epoxy film.

Example 3

Competitive immunoassay for thyroxine (T4) in which the insulator electrode is an anodized aluminum electrode coated with polystyrene.

Labeling of thyroxine. Thyroxine was conjugated to gelatin as described below. T4-N-hydroxysuccinimide ester (1mg, Wallac Oy, Turku, finland) was dissolved in 194mL dioxane and 0.1mL of this solution was added to 1mL 0.05mol/L phosphate buffer (pH 7.3) (e.merck, Darmstadt, germany) containing 20mg gelatin. After the reaction at +4 ℃ overnight, gel filtration was performed using a PD10 column (Pharmacia, Uppsala, sweden) and the above-mentioned phosphate buffer solution as an eluent, thereby separating gelatin from the small molecule reagent. The purified conjugate was labeled with an isothiocyanate derivative of terbium chelate as in example 1, but in this case the molar ratio of Tb chelate to gelatin was 200: 1. Assuming that gelatin has a molecular weight of 1 million, about 100 Tb chelates can be bound to one gelatin molecule.

The electrodes are coated with the antibody. The electrodes were incubated overnight in 0.1mol/L phosphate buffer (pH 4.9) containing 5. mu.g/mL of antibody to coat the electrodes with rabbit anti-mouse immunoglobulin (Dako, Glostrup, Denmark). The following day, the electrode was washed with the washing solution as in example 1 and washed in a solution containing 0.1% bovine serum albumin, 6% D-sorbitol and 1mmol/LCaCl₂Was equilibrated overnight in the TSA buffer of (1). The coated electrode was dried without washing. It can be stored at room temperature for at least 3 months without loss of activity.

A standard sample was prepared. In a solution containing 0.1mol/L NaCl and 0.1% NaN₃(HEPES buffer) and 0.1% casein in 0.01mol/L HEPES-0.001mol/L sodium phosphate buffer (pH 7.4) T4 standards (0,10,50, 100,150 and 300nmol/L) were prepared.

And (4) performing immunoassay. To the wells of the microtiter plate, 20. mu.L of the standard, as well as 100. mu.L (0.5ng) of monoclonal mouse anti-T4 antibody (Medix Inc, USA) and 100. mu.L of gelatin-T4-Tb-conjugate (100ng/mL) were pipetted. Electrodes were added to the wells, the electrodes were washed 5 times after 1 hour of incubation, and measurements were performed as in example 2. The standard curve is shown in fig. 6.

Example 4

The philadelphia chromosome was detected by DANA hybridization, in which an insulator electrode was made of anodized aluminum and coated with polystyrene.

The probe was labeled with Tb chelate. Amino group-containing oligonucleotide (TTCGGGAAGTCGCCGGTCATCGTAGA- (C-NH) was labeled with Tb chelate as in example 2₂)₂₅-5', Wallac, Turku, finland) except that NAP-5 and NAP-10 columns (Pharmacia, Uppsala, sweden) were used to purify labeled nucleic acids.

The probe was labeled with biotin. For this assay, another probe (C- (NH) was also prepared₂-C) -GTCGTAAGGCGACTGGTAGTTATTCCTT-5', Wallac, Turku, Finland). The N-hydroxysuccinimide derivative of biotin was reacted with the probe (5 nmol in 50. mu.L, 50:1 molar ratio) in 3.7. mu. L N, N-dimethylformamide overnight at pH 9.5 and +4 ℃. Adding Na₂CO₃The pH was adjusted so that the final molar concentration was 50mmol L. The probe was purified as a probe labeled with Tb chelate.

The electrodes were coated with streptavidin. The electrodes were coated by incubation in TSA buffer containing 10. mu.g/mL streptavidin for 12-15 hours. The next day, the electrode was washed with a washing solution and washed in a solution containing 0.1% NaN₃0.5% bovine serum albumin and 6% sorbitol in TSA buffer overnight. The electrode was dried and stored dry.

And (4) hybridizing. From chromosome Ph by PCR¹(TYKS, Turku, Finland) A170 base pair sequence from a K562 human cell line was amplified and used as a positive sample, while distilled water was a negative control. The positive sample and negative control were kept at 100 ℃ for 10 minutes, then cooled on an ice bath, and then centrifuged with a microcentrifuge at 12000rpm for 1 minute. Samples (50. mu.l) were pipetted into disposable cuvettes, followed by addition of 200. mu.l assay buffer containing 2ng of biotinylated probe and 2ng of probe with Tb (III) chelate label. One liter of assay buffer (25mM TRIS-HCl, pH 7.75) contained 33.72g NaCl, 0.25g NaN₃2.5g bovine serum albumin, 0.25g bovine serum gamma globulin and 0.05mL Tween 40. The reaction was allowed to proceed at 50 ℃ for 2 hours. Washing the electrode 6 timesAfter that, EL was measured as in example 2. Fig. 7 shows the results obtained with an uncoated electrode (a) and an electrode coated with polystyrene (b).

Example 5

The surfaces of aluminum electrodes were modified by silanization, and TSH immunoassays were performed on the surfaces of these modified electrodes.

Silanization of aluminum electrodes. The oxide-covered aluminum electrode was first washed with toluene in an ultrasonic bath and then dried at 100 ℃ for 1 hour. Silanization was performed by sonicating the electrode twice for 30 seconds in 5% dichloromethylsilane in toluene. After silanization, the electrode was washed once with toluene and twice with methanol.

The electrodes are coated with the antibody. The electrodes were coated as in example 2.

Labeled antibodies are prepared. anti-TSH antibodies were labeled with isothiocyanate derivatives of tb (iii) -2-chelate as shown in examples 1 and 2.

A standard sample was prepared. TSH standards were prepared as in example 2.

An immunochemical assay. Immunochemical determination and measurement of EL were carried out as in example 2. The standard curve obtained in the test is shown in fig. 8.

Example 6

The magnesium oxide surface of the electrode was coated with polystyrene to modify the magnesium electrode, which was used to perform immunochemical measurements of C-reactive proteins.

The electrodes were coated with polystyrene. An appropriately sized Mg electrode (merck art.5812) was washed in hexane using an ultrasonic bath. The hexane was evaporated and the electrode was coated with polystyrene as in example 1.

The electrodes are coated with the antibody. The electrodes were coated with mouse anti-human CRP antibody (clone 7H4, Labmaster ltd., Turku) by incubating the electrodes for 1 hour in TSA buffer (pH 8.7) containing 10 μ g/mL antibody. The electrodes were washed, stabilized and stored as in example 1.

Labeled antibodies are prepared. The antibody (horse anti-human C-reactive protein, CRP, clone 5F3, Labmaster ltd., Turku) was labeled as in example 1.

A standard sample was prepared. By adding a solution containing 7% bovine serum albumin and 1mmol/L CaCl₂The standard samples (0, 5, 50, 500, 2000 and 5000ng/mL) were prepared by dissolving a stock solution (Labmaster Ltd., Turku) of CRP in the TSA buffer (standard buffer) of (1).

And (4) carrying out immunochemical detection. mu.L of standard and 200. mu.L of standard buffer were added to a disposable cuvette (Brad, Cat. 759015, Wertheim, Germany) and the volume of the cuvette was reduced to 250. mu.L with a piece of Teflon. The coated electrode was inserted into the solution and incubated for 1 hour. After washing (6 times), labeled antibody in standard buffer (200 μ L, 500ng) was added to the cuvette and incubated for an additional 1 hour. After the incubation, the electrodes were washed and EL was measured in the same manner as in example 1 except that EL mu g itself was completed using a side-type photomultiplier tube and a disposable spectrophotometer cuvette made of polystyrene. The cuvette had a teflon holder for the disk-type working electrode and for the Pt wire counter electrode. The obtained standard curve is shown in fig. 9.

Example 7

β on the surface of a pulse anodized silicon electrode₂-immunochemical determination of microglobulin.

The silicon electrode was made from antimony doped n-Si disks with orientation (III) and resistivity of 0.008-0.015 Ω cm (okmetric ltd, finland).

Anodization of the silicon electrode. The Si electrode was anodized in an electrolyte solution similar to aluminum in example 1, but using pulse anodization in a pulse train instead of DC anodization. The pulse train consists of anodic and cathodic pulses (200 mus, +5V or-5V each, frequency 100Hz) with intermittent zero levels of 10ms between pulses. After anodization, the electrode was rinsed with quartz distilled water.

The electrodes are coated with the antibody. Typically, in the presence of 10. mu.g/mL antibody in 0.2M NaH₂PO₄Incubated overnight in solution, whereupon mice were used to resist β₂Microglobulin antibody (clone 6G12, Labmaster ltd., Turku) coated silica coated with oxideThe next day, the electrode was washed six times with a washing solution (0.01M TRIS-HCl buffer, pH7.75, 0.9% NaCl and 0.02% Tween20) and saturated overnight in a saturated solution of 0.05M Tris-HCl buffer, pH7.75, containing 1g bovine serum albumin, 60g sorbitol and 1mmol CaCl per liter (0.80 × 0.005 × 5.0.0 cm)₂. After washing (6 times), the saturated electrodes can be stored dry for at least 3 months.

Labeled antibodies are prepared. By reacting the antibody with the chelate at a molar ratio of 1:60 at pH 9.5, with an isothiocyanate derivative of Tb (II) -4 [ Tb³⁺-N- (4-isothiocyanatobenzoyl) -diethylenetriamine-N¹,N¹,N³,N³-tetraacetate](Wallac Ltd., Turku) labeled secondary monoclonal antibody β₂Microglobulin antibody (clone 1F10, Labmaster ltd., Turku). With 1M Na₂CO₃The solution was adjusted to pH. TSA buffer (0.05mol/L TRIS-HCl, pH7.75, 0.9% NaCl, 0.05% NaN) was used₃) As the mobile phase, the labelled antibody was purified from the unreacted chelate by gel filtration (Sepharose 6B 1 × 50cm, Sephadex G-501 × 5 cm.) typically, 5 to 10 chelate molecules could be bound to one antibody molecule in this way, 0.1% bovine serum albumin was added to the solution of labelled antibody for improved stability.

β₂β purified from human ascites fluid (75.5mg/mL, Labmaster Ltd., Turku, Finland) in TSA buffer₂Preparation of standards for microglobulin (0.4,1.6,4.0,8.0 and 16 mg/L). The TSA buffer contained 7.5% bovine serum albumin.

An immunochemical assay. The immunochemical reaction is carried out in a disposable polystyrene cuvette (1mL, Brand, Cat. No. 759015, Wertheim Germany) whose volume has been reduced to 250. mu.l with a Teflon filler. The standards were diluted 1:50 in assay buffer (0.05mol/L TRIS-HCl, pH7.75, 0.9% NaCl, 0.05% NaN)₃0.5% bovine serum albumin and 0.01% Tween20) and added to the bottom of the cuvette (40 μ L). Then 160. mu.L of labeled antibody in assay buffer (500ng, containing 100ng of labeled antibody) was addedBody and 400ng unlabeled antibody, clone 1F 10). Finally, the antibody-coated electrode was placed in a cuvette. The immunochemical reaction was allowed to proceed for 1 hour, and the electrode was washed 6 times with a washing solution.

Measurement of EL. EL was measured using an electroluminescent meter and a cuvette specifically prepared for this purpose. The Pt wire was used as a counter electrode in the cuvette. Using a catalyst containing 2mmol/L K₂S₂O₈0.2mol/L borate buffer (pH 7.75) as a measurement buffer.

The standard curve obtained is shown in fig. 10.

Example 8

TSH immunoassay was performed using an anodized silicon electrode and an anodized silicon electrode additionally coated with polystyrene.

The electrodes were coated with polystyrene. A silicon electrode was prepared and anodized as in example 7. The anodized electrode was coated with polystyrene by sonicating the electrode in a 1.5mg/mL solution of polystyrene in benzene for 30 seconds. After that, the electrode was slowly lifted from the solution and allowed to dry at room temperature.

The electrodes were coated with antibody (clone 8661, pharmacia, Uppsala, sweden) as in examples 2 and 7. The immunoassay was performed in a cuvette similar to that in example 7, but using the labeled antibody prepared in example 2 (80ng of Tb (III) -2 labeled antibody/electrode) and the standard solution of example 2. FIG. 11 shows a standard curve obtained with potentiostatic excitation (-10V, 200. mu.s excitation pulse, 500 excitation cycles). An anodised electrode (a), an anodised electrode (b), and an anodised and polystyrene coated electrode (c).

Example 9

The DNA hybridization method was performed using anodized and polystyrene coated modified Si electrodes.

The silicon electrode was made from antimony doped n-Si disks with an orientation of (111) and resistivity of 0.008-0.015 Ω cm (okmetric ltd, finland).

And (4) anodizing the electrode. The electrode was anodized in neutral 0.5M ammonium borate buffer, firstAt constant current (1 mA/cm)²) Down to 5.2V and then at constant potential for 10 min.

Coated with polystyrene. The electrode was first sonicated in a 10mg/mL solution of polystyrene in benzene for 10 seconds, thereby coating the anodized portion of the electrode with polystyrene. After that, the electrode was slowly lifted from the solution and allowed to dry at room temperature.

Hybridization and EL measurement were conducted as in example 4 except that the electroluminescence meter and cuvette in example 7 were used. The results are shown in FIG. 12.

Example 10

T4 immunochemical detection was performed with an anodized silicon electrode.

Si electrodes were prepared and anodized as in example 9 and coated with antibody as in example 3.

And (4) carrying out immunochemical detection. mu.L of the standard, 200. mu.L of monoclonal mouse anti-T4 antibody (Medix Biotech, Inc, USA) and 200mL of gelatin-T4-Tb-conjugate (50ng/mL) were pipetted into a disposable cuvette. The electrodes were placed in cuvettes and incubated for 1.5 hours with shaking, then washed 6 times. Measurements were made as in example 7. The standard curve is shown in fig. 13.

Example 11

TSH immunochemical assays were performed using Zn electrodes coated with alternating polystyrene and paraffin layers.

First, a cup-shaped electrode (volume 450. mu.l) made of Zn (Johnson Matthey Alfa products) was washed with hexane in an ultrasonic bath. Thereafter, the electrode is cathodically treated in a peroxodisulfate solution to produce a thin oxide layer or is directly coated with an organic layer.

Cathodic oxidation of the Zn electrode. The zinc cup electrode was filled with 0.450ml of 0.01M K in 0.2M borate buffer (pH 9.2)₂S₂O₈The solution was treated and cathodically polarized with 10000 pulses (200. mu.s/pulse, -amplitude of 10V and frequency of 100 Hz). After that, the electrode was washed with quartz distilled water and dried.

The electrodes are coated with an organic layer. The oxidized or unoxidized electrode was slowly immersed in a solution of polystyrene in benzene (0.5 mg/mL). The electrode was allowed to dry at room temperature for 24 hours. Thereafter, the electrode coated with polystyrene was slowly immersed in a pentane solution of paraffin (1.0mg/mL) and allowed to dry for 8 hours. A new polystyrene layer was then added as described in example 8.

An immunoassay was performed in the same manner as in example 1, but using the reagent from example 2. The standard curve is shown in fig. 14.

Example 12

β using an insulator electrode made of a glass plate₂-a microglobulin immunoassay, said glass plate being covered with an indium tin oxide surface film and coated with alternating layers of polystyrene and paraffin.

The electrode (7.0 × 55mm) was cut from a glass plate coated with indium tin oxide (Lohja Oy, Lohja, finland) and then coated with a polystyrene-paraffin-polystyrene layer as in example 11.

Labeling second monoclonal antibody β with an isothiocyanate derivative of Tb (III) -2 chelate₂Microglobulin antibodies (clone 1F10, Labmaster ltd, Turku, finland). Labeling with isothiocyanate derivatives of Tb (III) -4 chelate was performed as in example 7. The tag was purified by gel filtration as in example 7.

Immunochemical assay and EL measurement were carried out as in example 7. The standard curve is shown in fig. 15.

Example 13

β was carried out using a polyethylene terephthalate sheet covered with a transparent gold film (Au-PET foil) and coated with alternating layers of polystyrene and paraffin₂-microglobulin immunoassay.

Au-PET foil (Intrex film, model 28FX43, Sierracin, Sylmar, CA, USA) was glued on a glass plate (8.0 × 55mm) and the resulting plate electrode was coated with a polystyrene-paraffin-polystyrene layer as in example 7 β was performed as in example 7₂Immunochemical detection of microglobulin, but using the labeled antibody prepared in example 12. The standard curve is shown in fig. 16.

Example 14

CRP immunoassays are performed using an insulator electrode based on a conductive polymer membrane coated with alternating layers of polystyrene and paraffin.

A conductive polymer layer is prepared and coated with an insulating layer. Steel electrodes were coated with polyaniline according to the paper: michaelson, a. mcevoy and n.kuramoto, fact.polym.17 (1992) 197. The electrode coated with the conductive polymer was then immersed in a hexane solution containing 1.0mg/mL paraffin, thereby further coating a paraffin layer. The electrode was allowed to dry for 12 hours. Thereafter, the electrode was coated with a polystyrene layer by dipping the electrode into a benzene solution containing 1.0mg/mL polystyrene.

The electrodes were coated with the antibody and CRP immunoassay was performed as in example 6. The resulting standard curve is shown in fig. 17.

Example 15

CRP immunoassays were performed using alumina and polystyrene coated insulated steel electrodes.

Coating of the Steel with aluminium oxide Using ALE technology, a layer of aluminium oxide is formed from Al (CH) on a steel sheet (50 × 50mm) at 200 DEG C₃)₃And growth in water. 90 cycles were used in this growth to produce an oxide layer about 5nm thick. Before the steel plate was removed from the reactor, it was cooled to 60 ℃ in a nitrogen atmosphere of 10 mbar.

The alumina coated steel was cut into strips of 7.0mm width, which were dipped twice in a benzene solution containing 0.7mg/ml polystyrene, thereby covering them with a thin polystyrene layer. The strips were allowed to dry between and after 8 hours of immersion at room temperature.

An immunoassay was performed in the same manner as in the examples, except that the TSA buffer (pH 7.8) was an incubation buffer. The standard curve is shown in fig. 18.

Example 16

Mouse IgG was detected by a label emitting short-lived and long-lived emission using oxide coated magnesium coated with polystyrene.

The magnesium electrode was coated with polystyrene as described in example 6 and with mouse IgG as in example 1.

Labeled antibodies are prepared. Rabbit antibodies against mouse IgG (Dako, Denmark) were labeled with fluorescein-5-isothiocyanate (Sigma, F-7250) as was used to label the antibody with Tb (III) -1-isocyanate in example 1. The molar ratio of antibody to label was 1: 200. Accordingly, the same antibody was labeled with Eu (III) -3-isothiocyanate. The molar ratio of antibody to label was 1: 100.

Mouse IgG was detected. The immunoreactions were carried out as in example 6. Labeled antibody (Eu (III) -3-label is added in excess, in a total amount of 200ng/mL) at a molar ratio of 1: 01. After 30 minutes of incubation, the electrodes were washed with distilled water and transferred to the cuvette of a time-resolved spectrometer and the EL spectra were recorded in a 500 μ s time slice (fig. 19). The nm spectrum shows that the simultaneous short-lived (fluorescein-labeled) and long-lived (Eu (III) -3-labeled) emissions can be separated by time-resolved detection. When the electrode was coated with rabbit IgG, no spectrum of either label was obtained. The spectra were recorded as in example 1, using a spectrometer (S) described elsewhere.

And j.kankare, j. anal. instrum.18(1986) 171).

Example 17

Simultaneous measurement of TSH and PLA using anodized polystyrene coated aluminum electrode₂。

And (4) labeling the antibody. Monoclonal antibodies specific for human TSH were labeled with Aminohexylethylisobutanol (AHEI) using the method of Schroeder et al (Methods in Enzymology, Vol 57, M.Deluca (Ed.,), Academic Press, N.Y.,1978) (clone 5404, Medix Oy, Helsinki).

And (4) performing immunoassay. Mixing PLA₂And TSH standards (25. mu.L each) and 175. mu.L of antibody cocktail in TSA buffer (500ng anti-PLA)₂Antibody and 300ng anti-TSH antibody) was added to the cuvette, the electrode (7.0mm × 0.3mm × 50mm) was inserted into the cuvette and incubated for 1 hour with shaking, the electrode was washed with a washing solution, and el-standard curve was measured with an electroluminescence meter as in example 16 and is shown in fig. 20.

Example 18

PLA Using latex particles as solid support and polystyrene coated anodized aluminum cups₂And (4) performing immunoassay.

The aluminum electrode was anodized and coated with polystyrene as in example 1.

The latex particles were coated with the antibody. Stock latex particle suspension (Sigma, LB-8) was diluted to 1:100 with TSA buffer. To this dilution (100. mu.L) was added 100. mu.L of anti-PLA in TSA buffer at 5.7mg/ml₂A solution of antibody (clone 2E1, Labmaster Oy, Turku, Finland) and the mixture was incubated overnight. The particles were separated by centrifugation and washed with a washing solution. After that, the latex particles were saturated with 0.1% bovine serum albumin solution as in example 1. The tb (iii) -1 chelate-antibody preparation was the same as in example 1.

And (4) performing immunoassay. Approximately 1.8 million coated latex particles (20. mu.l of 1:20 dilution) were applied to 1.5mL polypropylene centrifuge tubes saturated with bovine serum albumin. To this suspension 20. mu.l of the standard and 20. mu.l of Tb (III) -1-labeled antibody (500ng) were added and incubated for 20 minutes at room temperature. The particles were then recovered by centrifugation and washed 2 times with the wash solution and 1 time with the EL assay solution, to which 0.02% Tween 40(w/v) was added. The particles were suspended in an EL measurement solution, and EL was measured by using a cup-shaped electrode. Fig. 21 shows the standard curve obtained.

Example 19

Use of polystyrene coated magnesium electrodes in PLA₂Enzymatic amplification is performed in immunoassays, where latex particles are used as the solid support.

The magnesium electrode was coated with a polystyrene film as in example 6. Latex particles were coated with antibody as in example 18.

The antibodies were labeled with alkaline phosphatase. Labeling polyclonal sheep anti-PLA with alkaline phosphatase (ALF) and maleimide₂Antibodies (e.ishikawa, m.imagawa, s.hashida, s.yoshitake, y.hamuguchi and t.ueno, j.immunassay, 4(1983) 209).

And (4) performing immunoassay. Approximately 1.8 million coated latex particles (20. mu.l of 1:20 dilution) were applied to 1.5mL polypropylene centrifuge tubes saturated with bovine serum albumin. To this suspension was added 20. mu.L of the standard and 20. mu.L of ALF-labeled antibody (600ng), and the mixture was incubated at room temperature for 15 minutes. The particles were then separated by centrifugation and washed 2 times with the washing solution. The particles were suspended in 200. mu.l of a solution containing 1mmol/L of substrate (phosphate ester of 5-fluorosalicylic acid, FSAF, Kronern Systems Inc., Mississauga, Ontario Canada). After 15 minutes incubation, the supernatant was separated and 100. mu.L of it was transferred to an EL cuvette. It contained a disposable coated Mg working electrode and 2 non-disposable platinum wire counter electrodes. Then 150. mu.l of 0.1M NaOH containing 1.0mmol/L potassium peroxodisulfate was added. EL was measured through a 420nm interference filter. The standard curve (a) is shown in fig. 22. Alternative detection methods are as follows: mu.L of the supernatant was added to 100. mu.L of a solution of 0.5mM Tb (III) -EDTA in 0.2M NaOH. The solution was pipetted and reacted for 15 minutes. The solution was transferred to an EL cuvette and 25. mu.L of 0.01M potassium peroxodisulfate was added. After mixing with a pipette, the EL signal was measured. The standard curve is shown in fig. 22. The total signal (b) and the time resolved signal (c) (8ms window and 50 μ s delay) were measured with a 545nm interference filter by integrating over 10000 excitation pulses.

Example 20

β on a microtiter strip by separating Tb ions from antibodies after immunoreaction and measuring free Tb ions with the new complex using an anodized silicon electrode₂-microglobulin immunoassay.

The wells of the microtiter strips were coated for use in the electrodes as described in example 7. The antibody was labeled with T (III) -1 chelate as in example 7 and the immunoassay was still performed as in example 7. After 1 hour incubation, the wells were washed with wash solution and 200. mu.L of 0.1M Glycine-H was added₂SO₄Buffer (pH 2.5) and then incubation for 15 minutes, then 150. mu.L of the incubated solution was added to the cuvette, followed by 45. mu.L of 5 × 10-containing solution^-40.5mol/L Na of mol/L ligand 5₂CO₃. The solution was mixed well and 205. mu.L of measurement buffer was added. Silicon cell coated with oxide as in example 7The Tb (III) -5 chelate complex thus produced was very quantitatively determined. The standard curve is shown in fig. 23.

Example 21

β was performed using anodized silicon electrodes with liposomes as the carrier for labeling₂-microglobulin immunoassay.

Liposomes containing Tb-5 complexes were prepared by mixing them with antibodies (anti- β) according to OP.Vonk, B.Wagner, Clin.chem.,37(1991)1519₂-microglobulin, clone 6G12, Labmaster Oy, Turku, finland.) immunoreaction was performed as in example 20 except that the buffer contained no detergent, after washing, 230 μ L of 0.1M glycine buffer (pH 3.2) containing 0.1% Triton X-100 was added and incubated for 10 minutes, 60 μ L of a solution containing 5 × 10 was added to the solution (200 μ L)^-40.5mol/L Na of mol/L ligand 5₂CO₃. Measurement buffer (240 μ L) was added to the cuvette and EL was measured using a 5v (dc) anodized silicon electrode as in example 20. The standard curve is shown in fig. 24.

Example 22

β was performed by UV light chemical stripping of the mark and then measuring using an anodized silicon electrode₂-microglobulin immunoassay.

The microtiter strips were coated as in example 20.

And (4) labeling the antibody. Antibodies (clone 1F10, Labmaster Oy, Turku, Finland) were labeled with a light-releasing label (rhodamine-administered sulfosuccinimidyl esters, Molecular Probes, R-7091, Eugene, USA) according to the manufacturer's instructions.

Immunoassays were performed in wells of microtiter plates as in example 20, except that 200. mu.L of 0.05M Na was pipetted after washing₂B₄O₇Added to the wells.

The label was detached with uv radiation and EL measurements were performed. The microtiter strips were exposed to UV light (Philips HPLR) from the top for 4.0 minutes. Remove 180. mu.L of solution from each well and transfer to a cuvette, add 320. mu.L of measurement solution. The total emission was measured as in example 20, but using a 520nm bandpass filter. The standard curve is shown in fig. 25.

Example 23

β is carried out using energy transfer from donor to acceptor (which enables observation of delayed light emission from the acceptor, whose lifetime is related to the emission lifetime of the donor)₂Homogeneous immunoassay of microglobulin.

And (4) labeling the antibody. The antibody for coating (clone 6G12, Labmaster Oy, Turku, Finland) was labeled with Tb (III) -2-isothiocyanate as in example 2 for labeling the anti-TSH antibody. The second monoclonal antibody (clone 1F10, LabmasterOy, Turku, Finland) was labeled with rhodamine B isothiocyanate (Sigma, R1755) in a 90:1 label/antibody ratio. Rhodamine B-labeled antibody was diluted after purification in 0.2M borate buffer (pH 7.8) containing 4% bovine serum albumin.

And (4) coating the electrode. The silicon electrode was coated with tb (iii) -2 labeled antibody as was done with unlabeled antibody in example 7.

And (4) performing immunoassay. Standard solutions (40. mu.L; the same standards as in example 7, at a dilution of 1:50 in 0.2M borate buffer (pH 7.8) containing 4% bovine serum albumin and 0.005% Tween20) and 160. mu.l of rhodamine B-labeled antibody (800ng) were added to the cuvette. The electrodes were inserted into a cuvette containing a disposable steel counter electrode. After incubation for 20 minutes with shaking, the cuvette was closed and placed in the electroluminometer. The EL signal ratio at 490nm and 670nm was measured (delay time 500. mu.s; measurement window 3.0 ms). The results are shown in FIG. 26.

Example 24

β Using latex particles containing Terbium (III) chelate and anodized silicon electrode₂Homogeneous immunoassay of microglobulin.

Latex particles were prepared and coated with antibody. Polystyrene was dissolved in a saturated solution of Tb (III) -5 chelate in benzene to obtain a concentration of 20 mg/mL. The mixture (400 μ L) was added to 15mL pentane in an ultrasonic bath with a pipette and sonicated for 5 minutes. Water (15mL) was added and the mixture was shaken for 2 minutes. The latex particles were centrifuged and suspended in 0.5mL of TSA buffer.

And (4) labeling the antibody. The antibody for coating (clone 6G12, Labmaster Oy, Turku, Finland) was labeled with 9-fluorenylmethylchloroformate (FMOC, Aldrich, No.16,051-2) according to the manufacturer's instructions using a label/antibody ratio of 80: 1.

And (4) coating the electrode. A silicon electrode was prepared and anodized as in example 9. The electrodes were coated with FMOC-labeled antibody as was done with unlabeled antibody in example 7.

And coating latex particles. The latex particles were coated with the antibody (clone 1F10, Labmaster Oy, Turku, Finland) as in example 10.

And (4) performing immunoassay. Standard solutions (40. mu.L; the same standard solutions as in example 7, diluted 1:50 in 0.2M borate buffer (pH 7.8) containing 4% bovine serum albumin and 0.005% Tween20), 160. mu.L containing 1mmol/L K₂S₂O₈The aforementioned buffer and 20. mu.L of the particle suspension were mixed in a cuvette. The reaction was allowed to proceed for 20 minutes with shaking and then 10 minutes without shaking. Finally, EL was measured as in example 7. The standard curve is shown in fig. 27.

Claims (26)

1. A method of electrically exciting a labeled molecule, the method comprising:

at least partially immersing the electrode in an electrolyte solution containing at least one labeling molecule;

exciting the label molecules by electrical pulses from the electrodes, thereby producing excited labels; and

detecting luminescence emitted by the excited labels;

wherein the electrode comprises a p-type semiconductor material, and at least a portion of the electrode immersed in the solution is substantially covered with an electrically insulating film comprising an oxide of the p-type semiconductor material, the electrically insulating film having a band gap of 5eV or more,

wherein the electrically insulating film is covered by a shielding layer comprising an organic polymer material.

2. The method of claim 1, wherein the resistivity of the electrode is less than 10 Ω cm.

3. The method of claim 1, wherein said electrode of p-type semiconductor material is a heavily doped semiconductor material selected from the group consisting of Si and Ge.

4. The method of claim 1, wherein the electrode of p-type semiconductor material is composite silicon, or silicon that is semi-conductive by being doped with other materials.

5. The method of claim 1, wherein a voltage between 9V and the breakdown voltage of the p-type semiconductor material is applied to the electrode.

6. The method of claim 1, wherein the electrode is sufficiently transparent in the emission wavelength range of the label to enable measurement of luminescence through the electrode.

7. The method of claim 1, wherein the electrode is substantially opaque in an emission wavelength range of the indicia.

8. The method of claim 1, wherein the label is in accordance with formula L_n-X_x-Y_yA portion of the detectable molecule of (1), wherein

L is the label;

x is a linking compound;

y is a compound having affinity to the analyte to be quantified and is a member selected from the group consisting of a protein, an antibody, an enzyme, and a nucleic acid; and wherein the integer subscripts n, x, and Y represent the number of L, X and Y and are equal to or greater than 1.

9. The method of claim 8, wherein the compound Y is capable of binding to at least one member selected from the group consisting of cells, cellular components, viruses, bacteria, nucleic acids, DNA, RNA, DNA fragments, RNA fragments, polysaccharides, proteins, polypeptides, enzymes, metabolites, hormones, alkaloids, steroids, vitamins, amino acids, carbohydrates, and antibodies.

10. The method of claim 8, wherein X comprises a chemical linking functionality selected from the group consisting of urea groups, thiourea groups, amides, substituted imides, thioethers, -S-, sulfonamides, and N-substituted sulfonamides, and wherein said chemical linking functionality is part of a larger molecule or polymer attached to Y.

11. The method of claim 1, wherein the lanthanide chelate is a member of the group consisting of tb (iii), eu (iii), y (iii), sm (iii), dy (iii), and gd (iii) chelates.

12. The method of claim 1, wherein the lanthanide chelate is terbium (Tb).

13. The method of claim 8, wherein the electrode further comprises a compound Y 'bound directly or indirectly to a surface of the electrode, the compound Y' having a specific affinity with Y for the same analyte, and wherein, prior to the detecting step, an affinity reaction occurs on a surface of the insulating film.

14. The method of claim 8, wherein competition for binding to Y on the surface of the electrically insulating film occurs between labeled analytes that conform to formula L_n-X_x-A_aWherein

A is the analyte of interest and,

the integer subscripts n, x, and a denote the number of L, X and A.

15. The method of claim 14, wherein L_n-X_x-A_aBinding to a on the surface of the electrically insulating film is inhibited by a derived from the sample.

16. The method of claim 8, further comprising, prior to the detecting step, a washing step that removes free label L from the vicinity of the electrode_n-X_x-Y_y。

17. The method of claim 8, further comprising an incubation step in which the detectable molecule is bound to a solid support in particulate form prior to being excited.

18. The method of claim 17, wherein the luminophore is detached from the detectable molecule bound to the solid support prior to quantifying the luminescent signal.

19. The method of claim 1, further comprising adding at least one co-reactant capable of converting at least one primary reducing or oxidizing component produced by the electrode such that the at least one primary reducing or oxidizing component is converted to a secondary oxidizing or reducing component more suitable for exciting the label.

20. The method of claim 19, wherein the co-reactant is selected from the group consisting of peroxydisulfate, peroxydiphosphate, and hydrogen peroxide.

21. The process of claim 19, wherein the co-reactant is sodium azide.

22. An insulating film coated electrode comprising:

a p-type semiconductor material;

an electrically insulating film comprising an oxide of said p-type semiconductor material covering substantially all of said p-type semiconductor material, said film having a band gap equal to or greater than 5 eV; and

a compound that binds directly or indirectly to the surface of the electrode, the compound being capable of binding to a member of the group consisting of a cell, a cellular component, a virus, a bacterium, a nucleic acid, DNA, RNA, a DNA fragment, an RNA fragment, a polysaccharide, a protein, a polypeptide, an enzyme, a metabolite, a hormone, an alkaloid, a steroid, a vitamin, an amino acid, a carbohydrate, and an antibody;

wherein the electrically insulating film is covered by a shielding layer comprising an organic polymer material.

23. The insulator film coated electrode of claim 22, wherein said electrode of p-type semiconductor material is a heavily doped semiconductor material selected from the group consisting of Si and Ge.

24. The insulating film coated electrode as claimed in claim 22, wherein the electrode of p-type semiconductor material is composite silicon, or silicon having semiconductivity by being doped with other materials.

25. The insulator film coated electrode of claim 22, wherein a band gap of the electrically insulating film covering the p-type semiconductor material is greater than 5 eV.

26. The insulator coated electrode of claim 22, wherein said electrically insulating film has an average thickness of 2 to 5 nm.

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