FI111663B - Use of time-delayed measurement system for measuring electrochemiluminescence for analytical purposes - Google Patents

Description

111663 t

The invention relates to so-called electrochemiluminescence measurement method for analytical purposes. excitation of long-lasting electrochemiluminescence-emitting labels using electrical pulses applied to a measuring solution through specially designed electrodes. After tuning, the electromagnetic radiation emitted from the labels is measured after a desired time after tuning with a measuring device.

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Immunodiagnostic and hybridization-based measurement methods are almost exclusively based on the labeling of certain molecules with other easily measurable molecules, i.e., labels. Such labels may be radioisotopes, radiation-absorbing or fluorescent compounds, enzymes, or certain metal chelates covalently attached to, for example, an antibody. Most commonly, analyte (A), assayed by a solid-bound antibody, is selected from an extremely large mixture of other molecules. In most cases, the antibody-folded molecules are measured by another labeled antibody, also selectively bound to analyte (A). Analyte (A) can also be labeled to determine the concentration of analyte (A) in the unknown sample by competitive reaction. The so-called 5-parameter multi-parameter analysis utilizes several different labels and antibodies, allowing the concentration of several analytes to be measured in the same sample.

A wide variety of commercially important methods have been developed, with particular emphasis on labeling technology and measurement of the label. Prior art 10 is described, for example, in The Immunoassay Handbook, Edited by David Wild, Stockton Press, New Yourk, 1994, pages 1-618.

Many organic compounds and so-called metal chelates give off strong radiation when tuned to a higher energy state in appropriate ways. Luminescence-based methods are thus generally extremely sensitive and are therefore well suited, for example, for diagnostic applications. The lower limit of sensitivity is often determined by the properties of the antibodies used and not by the measurement itself. Many other physical and chemical factors can also degrade measurement results.

20 There are many ways in which the excitation of the labels can be achieved. Light is most commonly used, but excitation can also be achieved electronically. This is done in a simple cell with only suitable electrodes to be applied. The hardware required is much simpler and less expensive than light tuning, since no expensive and sensitive optical components are needed. Electrical tuning can be carried out both in aqueous solutions, where bioaffectivity determinations are almost always performed, and in nonaqueous solvents.

In anhydrous solvents, active metal electrodes (eg, Pt and Au) are usually used for electrical excitation, which, due to the relatively low potential oxygen and hydrogen evolution reactions, are practically non-usable for excitation of anthrophores in aqueous solutions. Instead, insulating metal electrodes (e.g., oxide-coated aluminum electrode) are well suited for this purpose, «3 111663 because the insulating oxide layer allows the simultaneous production of highly oxidizing and reducing conditions in the aqueous solution, which enables the (electrochemistry) luminescence (ECL). Such electrodes are described in more detail in Kulmala et al. FI 970593. It is then possible to excite luminophores of very different optical and redox properties simultaneously, and to use wavelength discrimination for detection. In this way, after electron excitation, different labels can be measured simultaneously if they emit at different wavelengths. Often, however, the emission belts of the labels are relatively wide, which greatly limits the use of multiple labels.

In accordance with the present invention, it has been found that, if the luminophores used as labels are suitably selected, the insulating surface electrodes described above can also produce long-term luminescence of these luminophores. In this case, a time-resolved measurement method can be applied in which the specific, long-lived luminescence emitted by the luminophores is recorded only after the short-lived background luminescence has ceased. This usually results in very low detection limits and an excellent signal to noise ratio.

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By time difference in the present invention, it is meant that, after an electrical excitation pulse, the emulsion pulse is obtained from different labels at different times and of different lengths with different molecules. The present invention does not contemplate whether delayed emission is due to a slow chemical reaction or a slow discharge of the excited state by the excited molecule. Only the latter phenomenon occurs when luminophores are excited with light, and even in very rare cases.

Time difference measurement is known per se for chelating lanthanide ions using photoexcitation (I. Hemmilä, "Applications of 30 Fluorescence in Immunoassays", John Wiley & Sons, New York, 1991).

Here, the goal has been to achieve a low background signal by excluding the non-specific luminescence component of the sample. Previously, it has been believed that the 4,116,663 time-resolved detection method is only suitable for the determination of electrically excited Tb (III) and Eu (HI) chelates, since only lanthanide chelates have a sufficient lifespan for time-resolved detection (Kankare et al., UK Patent GB 2 217 007 B). ). However, contrary to theory, we have surprisingly found that several organic luminophores and transition metal organometallic compounds produce long-lived luminescence in the case of electric excitation, even if long-term luminescence is not produced by light excitation of this luminophore.

According to the present invention, other types of lanthanide chelates other than those mentioned above can be used as luminophores in time-resolved measurements when electrically excited.

Another very significant advantage of the invention is that by using both wavelength discrimination and time resolution, about twice the amount of labeled material from the same sample can be used. Thus, for example, instead of three labels, six different labels can be used and six analytes can be measured from the same sample. This will significantly reduce the cost of analysis currently pursued in healthcare. On the other hand, the advantage obtained can also be utilized by various internal assurances of the measurement so that, for example, one label is used as an internal control. In multiparameter measurements, it is also possible to determine the ratio of different analytes, thereby providing a significant increase in measurement reliability and / or obtaining qualitatively new data from the measurement as disclosed, for example, in Katroukha et al. EN 960167 in the case of troponin I in two different forms.

The use of electrical tuning in the manner described in the present invention also provides significant technical advantages. According to the invention, it is easy to tune all the labels simultaneously with a simple and inexpensive device. Since the excitation · is performed by electrical pulses, the measurement of the emitted light can also be easily and accurately synchronized with the emitted light measurement. This is important in 30 time-resolved measuring mode, where a certain, well-defined time after the electrical excitation pulse must be measured.

i 5 111663 detection methods, in particular for multiparametric assays. By using the time-resolved measurement method of the present invention, significantly lower detection limits and wider linear response ranges are achieved than the detection methods described above.

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It is an object of the invention to provide an apparatus utilizing a time-resolved method of measuring which allows different types of labels to be tuned to be used in bio-affinity analyzes.

This object is achieved by excitation of the label molecules with previously known dielectric metal or semiconductor electrodes, but also utilizing a time-resolved measurement method characterized by the principles set forth in claims 1-2.

Experimentally, it has been found experimentally with an oxide-coated aluminum electrode to achieve extremely strong oxidizing and reducing conditions in the presence of suitable compounds when applying strong cathodic polarization pulses through the electrode. These phenomena have been described e.g. In the dissertation of S. Kulmala (Electrogenerated lanthanide (III) luminescence at oxide-covered aluminum electrodes, University of Tumn, 1995) and in J. Kankare, K. Fälden, S. Kulmala and K. Haapakka, Anal. Chim. Acta, 1992, 256, 17 and S. Kulmala, T. Ala-Kleme, L. Heikkilä, and L. Väre, J. Chem. Soc., Faraday Trans. 1997, 93, 3107. Although in the first studies aluminum metal itself was thought to be the most important factor in the formation of luminescence, it subsequently became apparent that the 2-3 nm thick oxide layer on the metal surface allowed energy intermediates such as hydrated electrons and sulphate radicals to form luminescence electrodes. during. Later, it has also been found that the oxide layer formed on the surface of silicon electrodes of the n- and p -... type acts similarly, allowing the formation of luminophoric EKLs also with these electrodes, which are quite commonly used in the electronics industry and therefore well known. However, the oxide layer formed on the surface of all metals or semiconductors does not function as such an insulating layer through which the so-called. the transfer of hot electrons to the aqueous solution would be possible. For example, tantalum oxides 6111663

However, the oxide layer formed on the surface of all metals or semiconductors does not function as such an insulating layer through which the so-called. the transfer of hot electrons to the aqueous solution would be possible. For example, tantalum oxide coated tantalum electrodes cannot be used as working electrodes in such applications, although this is incorrectly claimed in UK 5 Patent GB 2 217 007 B, because tantalum oxide can be classified as an n-type semiconductor (S. Morrison, Electrochemistry at Semiconductor , 1980, p.183), and thus does not function as an insulating electrode.

The usefulness of insulating surface electrodes is mainly based on the fact that the base metal of the electrode can be placed at a very cathodic or anodic potential. tunneling of hot electrons, either directly or indirectly, e.g. as a result of an electron shear, from the conductive belt of the oxide layer to the conductive belt of water, and the subsequent generation of hydrated electrons is possible. These hot and hydrated electrons are the major primary intermediates for cathodic electrochemiluminescence in aqueous solutions (S. Kulmala, A. Kulmala, T. Ala-Kleme and J. Pihlaja, Anal. Chim. Acta 1998, 367, 17; S. Kulmala, T.). Ala-Kleme, M. Latva, K. Loikas and H. Takalo, J. Fluores 1998, 8, 59). Such energetic redox reactions cannot be achieved with any active metal electrode in aqueous solution. In addition to the electrons hydrated in these reactions, the key intermediates include the sulfate radical from the peroxodisulfate by one-electron reduction, hydrogen peroxide, or solid state electroluminescence: 'The hydroxyl radical generated by the mechanism Of these, however, the sulfate radical is more useful for excitation of luminophores,. since it does not have a very high tendency to side reactions, while the hydroxyl radical is quite easily involved in the addition and hydrogen removal reactions.

In the invention, conductive metals or semiconductors such as Be, Mg, Al, Ga, In, Au, Pt, Cu, Fe, Ru, Stainless Steel, Zn, Hg, are used as base materials for the working electrodes.

Ag, Ni, Pd, Hf, Zr or Si, Ge or a metal oxide. In addition to alumina, the thin film on the working electrode surface may be, for example, SiO2, MgO, CaO, SrO, BaO, H2O2, or any other inorganic dielectric such as a diamond, a silicate or a nitride. Organic insulators such as paraffin, Teflon, polyethylene, polystyrene and epoxy resins etc. are also suitable as insulating films over conductive electrode material. If metal oxide is used as the insulating material on the electrode surface, pulsed voltage must be used for excitation of the luminophores, since the cathodic dc voltage destroys the insulating properties of the oxide layer already within a few milliseconds.

Although, for example in the case of aluminum, an oxide layer of about 2-3 nm already self-formed in air is sufficient in itself to excite the luminophores of many different types of labels when high cathodic polarization is applied to the electrode, such insulating films can be improved and made thicker. Effective methods include e.g. anodic oxidation (if the base metal is anodically oxidizable), ALE (Atomic Layer Epitaxy), CVD (Chemical Vapor Deposition), MBE (Molecular Beam Epitaxy), Langmuir-Blodgett coating, and various spraying and other techniques. The inorganic films described above can also be replaced entirely by organic insulating films, provided that the formation of the radicals described above which are essential for the formation of ECL is ensured. This can be done by adding peroxodisulfate, peroxodiphosphate, hydrogen peroxide or other compound to the solution which produces reactive intermediates by single-electron reduction.

• · *

Time-resolved measurement has been applied to immunoanalysis for certain Tb (III) and Eu (III) chelates producing long-lived luminescence, but in the present invention it is applicable to the detection of organic luminophores and transition metal chelates. In this case, the luminescence emitted by the luminophore is only recorded when.! a certain delay has elapsed since the potential pulse is exhausted. In this case, the short-lived background luminescence has expired so that it does not interfere with the analyte-specific luminescence. In this case, very low detection limits for many luminophores are usually achieved.

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In principle, alternative methods for performing bioaffinity assays include assays on disposable electrodes or on other solid supports, such as magnetic latex particles, which are introduced onto the electrode producing electrochemiluminescence only at or just before the detection step of the labels.

Useful labels for use in the invention include, for example, derivatives of the following organic luminophores: luminol (emission 420 nm), eosin and fluorescein (emission 516 nm), derivatives of certain transition metal chelates such as ruthenium (II) and osmium (II) trispyridyl (emission range 550-650 nm) derivatives, Pt and Pd porphyrins and their derivatives, salicylate derivatives (emission range 400-450 nm), aminonaphthalene sulfonate derivatives (emission range 400-500 nm) and coumarins (emission range 450 nm-550 nm) ), luminescent proteins, etc.

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In the case of a cathodic excitation mode, the working electrode must meet the requirements described above and, depending on the optical properties and thickness of the conductive material, may be either optically transparent or opaque. Generally, the transmittance of metal films of sufficiently thin base material is high enough in the desired optical region. The transparent working electrode enables the measurement of electrically excited luminescence through the working electrode. 1

The selection of the counter electrode material in the method is not critical. Conventional inert electrode materials (Pt, Au) work well. Often, metals that are slightly anodically soluble can also be used, since measurements are usually made in a time scale where anodic products from the counter or auxiliary electrode do not have time to diffuse to the working electrode. Also, some metal oxide films, such as indium tin oxide, are well suited as an anode material. This makes the anode material optically transparent. Stainless steel is also a preferred electrode material. If 30 opaque metal electrodes are used as the counter electrode, its shape can be selected so that luminescence can be measured behind the counter electrode. For example, a wire electrode is used which covers only a very small part of the working electrode surface or a hole (s) of suitable size (s) have been drilled into the anode material. The optically transparent counter electrode may also be made of a sufficiently thin metal film or conductive metal oxide film, such as by coating a plastic or glass sheet with a thin film of Au or indium tin oxide.

5

If the thickness of the working electrode dielectric layer is appropriate, excitation in the label detection step may be performed by a cathodic voltage pulse sequence, but if the substrate material is anodically oxidizable material such as Si, Al, Be or Mg can be reinforced with anodic pulsed anode prior to each cathode excitation pulse.

Depending on the sensitivity of the analyte assay region, either a cheap semiconductor detector or, in the case of analytes requiring extreme sensitivity, a photomultiplier tube may be used to detect light in the electrochemiluminometer. However, it is essential that the response of the 15 detectors is fast enough for time-resolved measurements.

In some situations, such as in the case of internal standardization, it is preferable to observe two labels which emit at the same wavelength but with one very luminescent and the other very long-lasting. The parameters are then selected so that short duration luminescence monitors a parameter resulting in an excitation pulse signal such that the long duration luminescence signal does not have a significant proportion of the total signal during the excitation pulse. In this case, the parameter with low signal output can be accurately determined by time-resolved luminescence measurement.

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With the device constructed according to the invention, two analytes can be measured from the same sample or one sample and one internal standard. However, it is easy to construct devices with multiple detectors and where, by means of a dispersive element such as a prism or modern nonlinear optics, different wavelength ranges are directed to 30 different detectors.

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In the following, the invention will be further illustrated by diagrams and non-limiting examples and related figures.

Figure 1 shows the electrodes and luminescence measuring apparatus 5 used in the invention in general. The working electrode consists of a conductive base material, P, and a thin insulating layer E on its surface in the case of a cathodic excitation mode. It may consist of one or more layers E}, E2 .... No. of the same or different insulating material. The working electrode may be opaque or opaque. When using light-transmitting working electrodes, light measurement can be made through the working electrode with a detector 10 ΐ> 2 · In the method, a two-electrode cell is generally sufficient, but a conventional three-electrode cell can also be used. The working electrode and auxiliary electrode and Ag / AgCl are described above. The working electrode (or auxiliary electrode) can always be chosen with its geometry such that light is measured from the direction of the counter electrode (detector Dj), regardless of whether the counter electrode (or auxiliary electrode) is light-transmitting or non-light-transmitting. When the working electrode and the counter electrode are both sufficiently transmissive, double labeling and measurement of light at two different wavelengths (filters S1 and S2) can be done extremely simply without the need for expensive optics or beam splitter. The light detectors D 1 and D 2 may be photomultiplier tubes, but other less sensitive detectors 20 are also useful when the analyte concentrations are high enough.

However, depending on the application, the light metering apparatus consists of either a single or multiple channel gateway integrator or photon counter, but it is essential that each detector can measure light in a desired time window relative to the end of the excitation pulse.

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Figure 2. Schematic side view of disposable thin layer cell. The cell is made of two optically transparent parts which include fluid transfer tubes, grooves and waste grooves. One half of the cell contains a working electrode and the other a counter electrode. (1) A conductive film that functions as a biofilm working electrode; (2) 30 working electrode electrical contacts; (3) sample and buffer feed tube; (4) pipe with waste; (5) waste space; (6) air exhaust pipe; (7) counter-electrode made of stainless steel; (8) a gap in the counter electrode for light measurement; (9) electrical u 111663 contact to counter electrode. The working electrode (1) may also be optically transparent, allowing light measurement through the working electrode, whereby the counter electrode may be made of stainless steel and does not require an opening (8) for measuring the light. The working electrode is typically an insulating, silicon, aluminum, or magnesium electrode.

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Figure 3. Calibration curve of Ru (bpy) 32+ chelate based on time-resolved electrochemical luminescence measurement method.

10 Figure 4. Eosin calibration curve based on time-resolved electrochemiluminescence measurement method

Figure 5. Calibration curve for luminol based on time-resolved electrochemical luminescence measurement method.

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Figure 6. PtCP calibration curve based on time-resolved electrochemical luminescence measurement method with oxide-coated magnesium electrodes.

Figure 7. Calibration curve of PtCP based on a time-resolved 2 0 measurement method of electrochemiluminescence with oxide-coated aluminum electrodes

Figure 8. Calibration curve for PtCP based on time-resolved electrochemical luminescence measurement method for oxide-coated silicon electrodes 2 Figure 9. Calibration curve for immunometric determination of B2 microglobulin.

Figure 10. Calibration curve for hTSH immunometric assay.

Figure 11. Calibration curve of luminol in solution containing 7-hydroxy-4-30 methyl coumarin.

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Example 1. Determination of Ruthenium (II) tris (2,2'-bipyridine) from aqueous solution

Ru (bpy) 3+ (bpy = 2,2'-bipyridine) was determined by time difference with an Arcus luminometer in 0.05 M Na2B407 buffer (pH 9.2) supplemented with 0.1 M sodium sulfate and 5 10 3 M peroxodisulfate. The working electrodes used were 12-tooth aluminum brushes, which had been vacuum-evaporated to form a uniform oxide layer. In the assays, the rake was mounted in a special plastic cell with 250 μΐ of the test solution pipetted into each sample site. For the assays, a 1.5 ms time window was used for recording electrochemiluminescence with a delay time of 1 ps after the excitation pulse. This assay method 10 gives a linear log-log calibration curve for Ru (bpy)] + over a range of about six orders with a detection limit of about 10 -10 M (Figure 3).

Example 2. Time-resolved Eosin Assay in Aqueous Solution Eosin (2 ', 4', 5 ', 7'-tetrabromofluorescein) was assayed in time in 0.05 M sodium tetraborate buffer pH 7.8 (adjusted with 0.1 M H 2 SO 4) 0.1% sodium azide added. The assay was performed on an Arcus electroluminescence with a vacuum evaporated aluminum rake as working electrode and platinum comb counter electrode. A 1.5 ms time window of 20 ms with a 1 ps delay after the excitation pulse was used to record luminescence. The calibration curve is shown in Figure 4.

Example 3. Time-resolved Luminol Assay Luminol was assayed by the time-resolved measurement of 0.05 M aqueous sodium tetraborate (pH 7.8; adjusted with 0.1 M H 2 SO 4) supplemented with 0.1% sodium azide. The assay was performed using a vacuum rake with an aluminum rake manufactured as a working electrode and a platinum wire comb as the counter electrode. The device was a 30 electroluminometer modified from an Arcus luminometer manufactured by Wallac Oyj (Finland) with a time window of 1.5 ms used for luminescence measurement and a delay of 1 ps after the excitation pulse. The calibration curve is shown in Figure 6.

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Example 4. Time-resolved Determination of Platinum Acoproporphyrin from an Aqueous Magnesium Magnesium Electrode

Platinacoproporphyrin (PtCP) was assayed in a time-varying manner in 0.05M sodium tetraborate-5 buffer (pH 9.2) containing 1x103M K2S2O8. A magnesium strip was used as the working electrode and a platinum wire served as an auxiliary electrode for the measurement waste. The electric tuner was a coulostatic pulse generator, which produced a -50V pulse polarization at a frequency of 40Hz at a rate of about 120pC / pulse. The volume of the solution in the cup was 350 µl. An interference filter was passed between the photomultiplier and the sample solution, which passed wavelengths longer than 580nm. A 500ps time window and a 20ps delay after the electrical pulse were used for the measurements. The calibration curve is shown in Figure 6.

Example 5. Time-resolved Assay of Platinum Acoproporphyrin from an Aqueous Solution with an Aluminum-15 Electrode

Platinacoproporphyrin (PtCP) was assayed in time in 0.05M sodium tetraborate buffer (pH 9.2) with 1x10 3M K 2 S 2 O 8. The working electrode used was an aluminum cup and an auxiliary electrode used for measuring reflections was a platinum wire. The electric tuning source was a coulostatic pulse generator, which produced a -50V pulse polarization at a frequency of 40Hz at a rate of approximately ΠΟμΟρηΙββΐ. The volume of the solution in the cup was 350 µl. Between the photomultiplier and the sample solution • ·: An interference filter was introduced that passed wavelengths longer than 580nm.

The measurements used a 500ps time window and a 20ps delay after the electrical pulse. 2 5 The calibration curve is shown in Figure 7.

Example 6. Time-resolved Assay of Platinumacoproporphyrin from an Aqueous Solution with a Silicon Electrode. The working electrode was anodized n-type silicon electrodes and the auxiliary electrode in the measuring jet was a steel i4111663 cylinder. The anodization was performed in 0.5M H3BO3 solution adjusted to pH 7.1 with 25% ammonia. The anodization voltage was set at 5.69V, whereby the oxide layer obtained on the silicon pile was about 2.2nm. The electrical excitation source was a coulostatic pulse generator, which produced a -50V pulse polarization at 40Hz at a frequency of about 120pC. The volume of the solution in the cup was 350 µl. An interference filter was passed between the photomultiplier and the sample solution, which passed wavelengths longer than 580 nm. The measurements used a 300ps time window and a 20μβ delay after the electrical pulse. The calibration curve is shown in Figure 8.

Example 7. Immunochemical determination of β2 microglobulin using liposomes for binding luminol to antibody and insulating electrode silicon electrodes.

Silicon electrodes were made of antimony doped n-Si plates with orientation (111) and resistivity of 0.008-0.015 Ω cm (Okmetic Oyj, Finland). The electrodes were cut to 15.0.0 x 55 mm prior to surface treatments.

Anodization of silicon electrode. The silicon electrodes were initially oxidized galvanostatically at a current density of 2 mA / cm in a 0.5 M boric acid solution neutralized with ammonia until an anode voltage of 5.2 V was reached. Thereafter, the anodization at 20 was continued potentiostatically until the current flow dropped below 10 μΑ / cm. After anodizing, the electrode was rinsed with quartz distilled water. 1)

Antibody Coating Wells of Microtiter Plate. The wells of the microtiter plate were coated with mouse anti-β-microglobulin antibody (clone 6G12, Labmaster Oy, 25 Finland) by incubation overnight in 0.2 molar NaH 2 PO 4 solution at 10 µg / mL. The next day, the well was washed six times with washing solution (0.01 M Tris-HCl, pH 7.75, 0.9% NaCl, and 0.02% Tween 20) and

·· - equilibrated overnight in 0.05 M Tris-HCl, pH

7.75 containing 1 g of bovine serum albumin per liter, 60 g of sorbitol and 1 300 mmol of CaCl2.

111663 fi2 microglobulin standard. Standards (0.4, 1.6, 4.0, 8.0, and 16 mg / L) of β2 microglobulin purified from human ascites fluid (75.5 mg / mL, Labmaster Oy, Turku) were prepared in TSA buffer supplemented with 7.5% bovine serum albumin.

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Preparation of labeled antibody. Liposomes containing luminol were prepared and then bound to the antibody (anti-β2 microglobulin, (clone 1F10, Labmaster Oy, Turku)) (GP. Vonk, B. Wagner, Clin. Chem., 1991, 37, 1519). .

10 Immunochemical Assay. The immunochemical reaction was carried out as follows. The standards were diluted 1:50 in TSA buffer supplemented with 7.5% bovine serum albumin. 20 pL standard and 80 pL liposome-labeled antibody (100 ng). The mixture was incubated for 1 h with gentle shaking. After washing, 230 µL of 0.1% Triton X-100 solution in pH 3.2 0.1 M glycine buffer was added to the well and incubated for 10 min. The solution was taken into a cuvette at 200 pL and 60 pL of a 0.5 mol / L Na 2 CO 3 solution was added. 240 µL of measuring buffer was added to the rivet and electroluminescence was measured using 5.2 volt DC anodized silicon electrodes.

Electroluminescence measurement. The electroluminescence was measured by an electroluminescent 20 in a specially prepared assay cell with a threaded steel tube anode and a seal between Teflon and the disposable silicon electrode and steel tube anode. 0.2 mol / L: 1 borate buffer, pH 7.75 with 10 mmol / L sodium azide was used as the assay buffer (350 pL).

The standard plot of the measurements is shown in Figure 10. 2 2

Example 8. Immunoassay with Oxide-Disposed Disposable Silicon Electrodes.

. Coating the electrode with the antibody, anodized silicon electrodes were prepared as in Example 7 (60 x 6.0 mm). One end of the silicon electrodes was coated with antibody in disposable spectrophotometer cuvettes (Plastibrand, Cat. No. 7590 15, i6 111663

Brand, Wertheim, Germany). Prior to plating, the antibody was treated with 0.1 M citrate (pH 2.5) for 15 min. The electrodes were coated with a monoclonal antibody by incubating the electrode in a coating buffer solution (0.1 M MES, 0.3 M H3B03, pH 6.5, 0.1% 25% pre-polymerized glutaraldehyde, 0.005% bovine gamma globulin for 4 h). 10 μg / mL (anti-TSH, Medix, Finland).

The coated electrodes were washed once with wash solution (0.01 M Tris-HCl, pH 7.75, 0.9%

NaCl and 0.02% Tween 20), and saturated with bovine serum albumin (BSA) by incubation for one hour in Tris buffer (0.1% BSA, 0.05 M Tris-H2SO4, pH 7.5, 0.1%

Tween 20, 0.1% NaN 3), followed by incubation of the electrodes in 5% 10 sorbitol solution for 2 minutes. The electrodes were dried and adhesive tape was applied to the electrodes, leaving a 10.0 mm high area at the lower end of the electrode active.

Immunochemical assay. The commercial Boehringer-Mannheim preparation of Elecsys® TSH Immunoassay was used as the labeled antibody, and TSH-15 standards (0, 0.25, 1.5, 9, 54, 324 μυ / mL) were prepared by diluting a strain standard (Scripps Laboratories, Inc.). , San Diego, USA) in incubation buffer (0.05 mol / L Tris-HCl, pH 7.75, 0.9% NaCl, 0.05% NaN3, 0.5% bovine serum albumin, 0.05% bovine serum gamma globulin) and 0.01% Tween 20). Immunochemical assay was performed in disposable spectrophotometer 20 cuvettes made of 1.5 mL polystyrene. 125 μί standard, 250 μι Ru-labeled anti-TSH antibody solution (Elecsys® TSH Immunoassay testq) and 525 μί above incubation buffer were pipetted into the bottom of the cuvette. Water-flushed electrodes were placed in the wells and incubated for 1 h with shaking, after which the electrodes were washed with running wash solution. Electroluminescence was measured in a disposable plastic fluorometer cuvette in 0.05 25 M Na2B407 buffer (pH 9.2) supplemented with 0.1 M sodium sulfate and 10'3 M peroxodisulfate. The counter electrode was a glass plate covered with indium tin oxide, and the EKL was measured through the counter electrode. Electrical contact to the silicon electrode was made by a • copper-clamping joint coated with an In-Ga eutectic alloy.

The measurement time window was 0.500 ms and the delay was 1 μβ. Figure 10 shows a standard plot of the hTSH-30 assay. 1 i7 111663

Example 9. Simultaneous detection of luminol and 7-hydroxy-4-methylcoumarin using a single detector. 7-Hydroxy-4-methylcoumarin produces electrochemiluminescence only during the cathodic pulse, and thus its derivatives can be used as a measure of the success of electrode backing when the actual amount of analyte 5 can be measured at the same wavelength based on long-term luminescence of luminol.

Saija solutions were prepared in which each had a concentration of 7-hydroxy-4-methylcoumarin of 1 x 10 7 mol / L, and the concentration of luminol was varied. As a measuring electrolyte, 0.05 M aqueous solution of sodium tetraborate (pH 7.8; adjusted with 0.1 M H 2 SO 4) supplemented with 0.1% sodium azide. Fig. 11 shows the luminescence during the cathodic pulse and in the time window 1 to 9 ms after the cathodic pulse at 450 nm. As can be seen in the figure, changing the concentration of luminol does not alter the signal during the cathodic pulse, but the intensity of the delayed luminescence follows the concentration of luminol.

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Claims (2)

Method for use in bioaffinity determination, characterized in that one or more markers of various kinds are electrically excited in a particular measuring cell with electrical pulses by means of an isolated working electrode, and the electromagnetic strain emitted is fed by means of a measuring instrument which is in synchronization. contact with pulses such that the long-lived luminescence-producing marker is used either luminol, isoluminol, eosin, fluorescein, ruthenium (II) complex, platinum or palladium porphyrin or luminescent protein, and the light emitted by the markers is measured in time delay from the desired time interval of the emission pulse and the measurement can be defined for one or more analyte from the same sample. Method according to claim 1, characterized in that the same sample is measured more than one analyte by varying the time delay of emitting light from different markers and / or by measuring light at different path lengths.