JPH0750032B2 - Luminescence-based analytical method for electrogeneration in solution - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to
It relates to a method of being excited directly by electron transfer from an electrode or indirectly by an electric pulse by an electrochemically induced mediated reaction. The light emission from the compound is detected after the end of the excitation pulse.

This new method has applications in areas with very low detection limits. For example, there is an analytical method for connecting an assay such as an immunoassay and a hybrid assay.

[Problems to be Solved by the Invention] Although various analytical methods based on luminescence are known for their sensitivity, they are disadvantageous in that the concentration of luminescent species is extremely low. Fluorescence sensitivity is limited by Rayleigh and Raman scattering as well as fluorescent impurities that increase nonspecific background emission. Phosphorescence is mainly limited to the solid state, and a very small amount of light emission of these compounds having room temperature phosphorescence in a solution state is usually extremely sensitive to oxygen, so that its practical use is hindered. The delayed fluorescence of certain lanthanide chelates has been used as a reference in immunoassays, but is very difficult to detect. Conventional fluorescence and phosphorescence based methods use excitation by light and require a suitable light source and optics. These methods based on chemiluminescence (CL) do not require optics for excitation and their instrumentation is usually very simple. However, the CL method often suffers from difficult chemical disorders.

The method presented by the present invention overcomes the drawbacks of other emission methods. The electronic instrumentation, which does not require excitation optics and which is necessary for pulse excitation by electric current, can be very easily manufactured. The core of the present invention is the total elimination of non-specific background emission by using a suitable emissive compound with a long-lived luminescence and measuring the light emitted some time after the excitation pulse. .

[Prior Art] Electrochemiluminescence (ECL) has been known for a long time. The application of this method to immunoassays has already been proposed by Byrd et al. (D. Ege, W. Becker and
A. Bard, Anal. Chem. 56 (1984) 2413, PCT Int. Appl.wo 86
/ 02734). They propose to use compounds containing ruthenium or osmium as labels when binding the assay. Platinum and glassy carbon are used as the material of the electrode in the example, and the light emission from this electrode is measured at the stage of potential pulse.

As we have previously published, electrogenerated luminescence is generated by a large number of inorganic ions in the presence of a suitable oxidant from an oxide-coated aluminum or tantalum electrode. [K, Haapakket, etc.
Anal.Chim.Acta.171 (1985) 259]. In these studies as well, the light emission from the electrode was measured when a potential pulse was applied to the electrode.

It would be highly advantageous to use economical, preferentially disposable electrodes and relatively long-lived, long-lived luminescent compounds. Such a method would have applications, for example, in combining homogeneous and heterogeneous immunoassays with fairly simple and economical instrumentation. In the case of immunoassays, or in the more general combined assay, the two components react specifically with each other and the product is quantified in a suitable and sensitive manner. If it is necessary to separate the products before measuring, the method is called heterogeneous, and if the separation step is unnecessary, it is called a homogeneous method. A homogeneous assay is preferred because it is simple. And heterogeneous assays have low detection limits. Typically, in these methods, the presence of a compound is indicated by labeling it with a quantifiable chemical moiety, such as a sensitive substance such as a radioisotope, an enzyme, a fluorescent compound. It is particularly advantageous to label with a fluorescent compound that has a slow emission decay in the excited state. Most of the samples that are immunoassayed contain naturally occurring fluorescent species, which increases background luminescence and interferes with the detection limits of conventional fluorescence measurements.

The chelates of europium and terbium have fluorescence lifetimes in the millisecond range, which are several orders of magnitude longer than the "natural" fluorescence of organic compounds of biological origin.

A luminescence-based homogeneous assay is possible if the surface-adsorbed antibody-antigen complex can excite and selectively excite the labeled compound in solution. This has already been accomplished (US Pat. No. 3,939,350 in 1976) by using a labeled antigen bound to an antibody bound to a quartz slide. The sample is excited from the other side while totally reflecting the light from the slide surface. At the time of measurement, only the solid phase binding portion is excited, so that a separation step is not necessary.
This method has limitations in use in conventional assays due to the strict optical requirements for sample slides. Furthermore, scattering and background fluorescence remain a difficult problem. Preferential excitation of the luminescent material at or near the surface can be technically more easily achieved by electroluminescence.

[Means for Solving the Problems] The present invention further provides an electric pulse by applying an electric pulse to and / or at the surface of the electrode immersed in a solution containing a chemical moiety containing terbium or europium as a solute. The present invention relates to a method for measuring the presence and / or amount of a chemical moiety containing terbium or europium by measuring delayed luminescence after being added for a while. The measured emission is considered to indicate the amount of chemical moieties present near the electrode. This measurement phenomenon is named here delayed electroluminescence, or DEL for short.

The general formula of the chemical moiety is (MZ) n-Lm-Yp. However, M is terbium or europium, n is an integer of 1 or more, m and p are integers of 0 or more, Z is a multidentate ligand, and L is a bonding group.
Y will be described later. Z, L and Y are compositions capable of causing the chemical moieties to emit light when tailored to the conditions required for delayed electroluminescence.

In the simplest example, m and p are both 0 and n is 1.
In this case, M-Z is a chelate of terbium or europium. One preferable structure of Z is as follows.

This method can be used for highly sensitive determination of terbium as described in Example 1, for example.

In more complex cases, n, m ≧ 1 and p ≧ 1.
It is said that the substance Y can be labeled with the DEL label. Among the suitable substances Y are many biological substances such as whole cells, subcellular particles, viruses, nucleic acids, nucleotides, oligonucleotides, polynucleotides, polysaccharides, proteins, polypeptides, enzymes, cellular metabolites, hormones, pharmacological agents. There are substances, alkaloids, steroids, vitamins, amino acids, carbohydrates, serum-derived antibodies or monoclonal antibodies. Also included within the scope of the invention are synthetic materials such as drugs, synthetic nucleic acids, synthetic polypeptides. The substance Y is bound to the chelate M-Z by the linking group L. The linking group can be a divalent radical, commonly used to label molecules to be analyzed with probe molecules, as is well known to those skilled in the art. Among these divalent linking groups, -CONH-, -CONM
e-like ureido, thioureido, amide; -S
A thioether such as-, --S--S--; --SO2NH--, --S
Includes sulfamides such as O2NMe-. The linking group L also contains a molecular chain called a spacer whose length and composition change. This spacer is used to keep the chelate portion and the substance Y apart from each other at an appropriate distance,
Moreover, it has the above-mentioned divalent linking group as a side group. A part of these side groups is bonded to the multidentate ligand Z,
The other part binds to the substance Y. The multidentate ligand Z is -CH2N
It may be an aromatic compound having a chelating side group such as (CH2COOH) 2. The structure of one preferred chelate is as follows; The present invention can be used to measure labeled moieties of interest, can be used to measure labeled analytes of interest, or in both competitive binding and non-competitive binding assays. In some cases it can be used to utilize a labeled analogue of the analyte of interest to determine the analyte of interest. Also, these binding assays are either heterogeneous or homogeneous. Similar binding assays can also be used for nucleic acid hybridization techniques, in which case, for example, hybridization and affinity-based hybridization assays based on PCR (polymerase chain reaction) techniques can be used for dot hybridization.
Applications include dot-blot and sandwich hybridization assays.

For example, in a competitive immunoassay, when an antibody is coated on the surface of an electrode, the antigen and an antigen having a DEL label compete with each other for the active site of the antibody. Now the antigen corresponds to substance Y and can belong to one of the types mentioned above. The amount of antibody / antigen complex on the electrode surface is DEL
Depending on the immunoreaction, the quantification may be carried out directly, or a washing may be carried out and an appropriate electrolyte solution containing, for example, peroxydisulfate may be added, followed by quantification. Alternatively, a homogeneous, non-competitive immunoassay can be performed by immobilizing the "catching" antibody on the electrode surface. Sample antigen captured with such antibodies is quantified using DEL-labeled antibody bound elsewhere on the antigen. The antigen in this case is the substance listed above in relation to the definition of Y.

A. Device Although we have not claimed the device, we will explain it in detail to give a clear image of this measurement method.

The instrument consists of a pulse generator, potentiostat, sample cell with two or three electrodes, an optional light filter or monochromator, a photodetector and a gated integrator or photon counter.
The pulse generator may be anything that produces a pulse chain with adjustable amplitude and freely programmable.

An ordinary three-electrode potentiostatic device is preferable as the potentiostatic device, but if only a two-electrode potentiostatic device can be used, a booster amplifier capable of supplying a current of several tens of milliamperes is used.

The sample cell and photodetector are housed in the same light blocking box. The cell has two or three electrodes immersed in an electrolytic solution. In the case of three electrodes, one electrode is the reference electrode, one electrode is the auxiliary electrode, and the other electrode is the working electrode.
These electrodes are connected in a conventional manner to a potentiostat. Luminescence is measured with a working electrode made of a conductive material. Preferred materials are oxide covered metals such as aluminum, tantalum, zirconium or hafnium. The reference electrode may be a conventional electrode such as a calomel electrode or an Ag-AgCl electrode. The auxiliary electrode may be any conductive material, but platinum is usually used. If only two electrodes are used, the electrodes may be made of the same material, for example aluminum, in which case light can be emitted from both electrodes. Alternatively, one electrode is made of another material. Otherwise, the sample cup itself may be made of aluminum, in which case it will act as the working electrode and the light will be emitted from it.

The intensity of light from the working electrode is measured using a photomultiplier tube or a photodiode with an optical filter or a monochromator in the middle, and the electrical signal from the photodetector is gated integrator or gated photon counter. Tell to. Gating is synchronized with the pulse from the pulse generator with an appropriate delay.

B. Method The sample for measuring DEL is a compound dissolved in a solution or adsorbed on the surface of the working electrode. The decay of the electroluminescence of the compound should be slow. The preferred compound is a luminescent lanthanide complex, preferably a chelate of eg Tb ³⁺ or Eu ³⁺ , the decay of which is on the millisecond time scale. This compound can be measured by itself, but can also be attached as a label to the material to be assayed. In addition to the compound to be measured, the electrolytic solution in the sample cell contains an electrolyte, but preferably a sulfate or acetate which increases the conductivity is used. An oxidizing agent such as peroxydisulfate, hydrogen peroxide or dissolved oxygen may be present in the solution. The function of the oxidizing agent directly or mediated electrolytic reduction reaction, for ^{_{example, S 2 O 8 2- + e}} -SO 4 2- + SO 4 - is to generate an ionic group rich in reactivity by.

These ionic groups react with a light emitting compound to emit light. As a result, after the cathodic pulse is applied to the working electrode,
Electroluminescence is observed. Anodic pulses could potentially be used for certain lanthanide compounds.

The luminescent compound provides the working electrode with a series of cathodic pulses of suitable duration and impact coefficient. The resulting emission is measured after the end of the cathodic pulse using the appropriate gate width and delay. For the preferred terbium complex, the cathodic pulse length varies from 0.2 ms to 5 ms, the post-pulse delay is 0.1 ms to 0.5 ms, and the gate width is 2 ms to 10 ms. In the case of Europium Complex, it is about 4 times shorter. The signal integrated during the open gate time is averaged over the period required to achieve the required signal to noise ratio.

Examples Example I Standard curve for terbium by electroluminescence The sample solution in this example is a 0.3 M solution of sodium sulfate,
0.001M solution of potassium persulfate and 3,6-bis-
[N, N-bis (carboxymethyl) aminomethyl] -4
A 10 ^-5 M solution of benzoylphenol, and its p
Adjust H to 11.2 with 5 × 10 ⁻⁴ Tris and NaOH. The DEL was measured in a disposable cup made of aluminum with a thickness of 0.3 mm and a purity of 99.9%. Terbium chloride was added at increasing doses and delayed electroluminescence was measured using a cathodic pulse with a continuous time of 1 ms, an amplitude of 8.5 V and an impact coefficient of 4%. The light emitted from the aluminum cup was measured with a photomultiplier tube and a two-channel photon counter [Standard Research model SR400]. The gate of one channel was open 0.2ms to 10ms after the end of the cathodic pulse, and the other channel counted "dark current" photons for 10.2ms to 20ms. After counting 100 s, the values of the two counter registers were subtracted from each other. The results are shown in Table 1 and FIG.

Example II Production of compound for indexing Synthesis of 4- (3-nitrobenzoyl) -2,6-bis [N, N-bis (methoxycarbonylmethyl) aminomethyl] phenol (1) 37% aqueous formaldehyde solution (0.81 g, 10 mmol) in methanol (20 mL). ) Was dissolved in the solution, dimethylimino diacetate (1.61 g, 10 mmol) was added. The resulting solution was concentrated in vacuo. Separately prepared methanol (25 ml) was added to the residue and the resulting solution was concentrated in vacuo. 4-hydroxy-3'-nitrobenzophenone (1.
22 g, 5 mmol) and the resulting mixture is stirred at 110
Heated at ° C for 20 hours. The product was purified by chromatography on silica gel using chloroform as the eluent. The yield of yellowish oil was 1.76 g (60%). ¹
H NMR (CDC13): δ 3.48 (1H, S), 3.58 (8H, S), 3.71
(12H, S) 4.08 (4H, S), 7.73 (2H, S), 7.56-8.58 (4H, S
m).

Synthesis of 4- (3-aluminobenzoyl) -2,6-bis [N, N-bis (methoxycarbonylmethyl) aminomethyl] phenol (2) Compound 1 (0.89g, 1.5mmol) pressure 50psi (3.52kg / cm)
Under hydrogen atmosphere, the mixture was stirred for 1 hour in methanol (50 mL) containing 10% Pd / c (90 mg). The resulting mixture was passed and evaporated in vacuo. The product was used as an eluent for gas oil (b.
Purified by chromatography on silica gel using p.50-70 ° C) / ethyl acetate (2: 5). The yield of yellowish oil was 0.40 g (48%). ¹ H NMR (CDC13): K3.
56, (1H, S) 3.59 (8H, S), 3.71 (12H, S), 4.01 (6H, broad S), 7.05-7.14 (4H, m), 7.70 (2H, S).

4- (3-isothiocyanatobenzoyl) -2,6-bis [N, N-bis (carboxylmethyl) aminomethyl]
Synthesis of Terbium Complex (3) of Phenol Compound (2) (0.40 g, 0.71 mmol) was stirred in a solution of 0.5 M KOH-ethanol (20 mL) and water (5 mL) for 3 hours.
The resulting mixture was neutralized with 1M HCl and evaporated in vacuo.
Water (15 mL) and terbium chloride (0.27 g, 0.72 mmol) were added and the pH was adjusted to 8.0 before passing the mixture. A few milliliters of acetone was added to the liquid to separate the terbium complex. A small portion (68 mg) of this complex in water (3 mL) was replaced with thiophosgene (3 mg) in CHC13.
1 μL, 0.4 mmol) and NaHCO3 (42 mg, 0.5 mmol) were added dropwise. After stirring for 1 hour, the aqueous layer was separated and washed with CHC13. After adding a few milliliters of acetone, the precipitate was separated and purified by chromatography on silica gel using CH3CN / H2O (4: 1) as eluent. Yield is
It was 15 mg [38%; based on (2)].

Example III Human Pancreatin Phosphorivase A2 Heterogeneous Sandwich Immunoassay Labeling of Sheep-Anti-Human PLA2 Antiserum: 4- (3-Isothiocyanatobenzoyl) -2,6-bis [N, N-bis (carboxy A terbium complex of (methyl) -aminomethyl] phenol [(3) of Example II] was allowed to react with the antibody overnight at pH 9.5 in a 60-fold molar excess. Labeled antibody is Sephadex G-50 (1 x 5.5 cm)
On a column packed with Sepharose 6B (1 x 5.2 cm) using 0.1 M sodium carbonate buffer pH 9.3 containing 9 g / L NaCl and 0.05% NaN3 as eluent Separated from excess free terbium complex.

Aluminum cup coating: Aluminum cup (thickness 0.3 mm, made of 99.9% pure aluminum foil), 0.5M Tris pH 7.5 containing 9g / L NaCl and 0.05% NaN3 (TSA-buffer). Coated with anti-human PLA2 antiserum by physical adsorption overnight at room temperature in HCl buffer. After coating the aluminum cup was washed with wash solution (9g / L NaCl, 0.01% NaN3 and 0.2g / L Tween20) and saturated overnight with 0,1% bovine serum albumin (BSA) and at + 4 ° C. Stored wet.

Immunoassay: The aluminum cup was washed once with 500 μL of wash solution. 0,9,54 and 324 ng in TSA-buffer (0.1% BSA)
25 μL of standard containing / mL of phospholipase A2 was added to the cup, then 175 μL of Tb in 0.05 M Tris-H2SO4 buffer at pH 7.8 containing 5 g / L BSA, 0.5 g / L NaN3. Anti-PLA2 antibody labeled with (570 ng / mL) was added. After incubating for 3 hours with continuous shaking, the cup was washed 6 times with the washing solution. Aside from the fact that the counting time was only 3 seconds, as in Example I, 0.3 mol / L Na2SO4 and 0.001 m
450 μL of 0.001M of pH 8.7 containing ol / L of K2S2O8
Electroluminescence was measured in cups after adding Tris-H2SO4 buffer. The results of the assay are shown in Table 2 and FIG.

Example IV Homogeneous sandwich of human pancreatic PLA2 in serum
Immunoassay Coating of cups and labeling of sheep-anti-human PLA2 antiserum was performed as in Example III.

Immunoassay: The aluminum cup was washed once with 500 μL of wash solution. 25 μL standard containing 0,9,54 and 324 ng / mL phospholipase A2 in human serum was added to the cup, then 5 g / L BSA, 0.5 g / L NaN3, 0.3 ml / L Na2SO4, 0.001 mol /
Anti-PLA2 antibody (235 ng / mL) labeled with 425 μL Tb in 0.05 M Tris-H2SO4 buffer at pH 8.7 containing L K2S2O8
Was added. After incubation for 3 hours with continuous shaking.
Apart from the fact that the counting time was only 3 seconds,
Electroluminescence was measured directly in a cup as in Example I. The results of the assay are shown in Table 3 and FIG.

[Brief description of drawings]

1 is a graph showing the measurement results of Example I, FIG. 2 is a graph showing the measurement results of Example III, and FIG. 3 is a graph showing the measurement results of Example IV.

 ─────────────────────────────────────────────────── --Continued from the front page (56) Reference JP-A-1-153940 (JP, A) JP-A-57-186170 (JP, A) JP-A-56-155834 (JP, A) US Pat. No. 4,280815 (US) , A) U.S. Pat. No. 4374120 (US, A) Ehlektrokhimiya, Vol. 14, No. 2 (dew) P.I. 246-250 (1978)

Claims (18)

[Claims] 1. The presence and / or amount of a chemical moiety containing television or europium is measured by applying an electrical pulse to an electrode immersed in a solution and measuring delayed emission some time after the end of the pulse. In the method of understanding, the chemical moiety is bound to the electrode and / or is present in the solution and the emission is indicative of the amount of chemical moiety present in the vicinity of the electrode. A method characterized by being considered to be. 2. The method of claim 1, wherein the chemical moiety has the general formula: (MZ) n-Lm-Yp. Where: M is terbium or europium; Z is a multidentate ligand of M; L is an ureido such as -CONH-, -CONMe, thiuredo, amide; -S-, -S- There is a linking group such as a thioether such as S-; a sulfonamide such as SO2NH-, -SO2NMe-, and L can contain a molecular chain of various compositions and lengths, and this molecular chain is a divalent group as described above. Is bound to the multidentate ligand Z by a part of the group
It is also bonded to the substance Y by another part. Then, Y is a substance that binds to Z via one or more linking groups L; n is an integer of 1 or more; p is an integer of 0 or more; m is an integer of 0 or more. 3. The method according to claim 1, wherein the chemical moiety is capable of binding to a chemical substance. 4. In a competitive binding method for determining the presence of an analyte of interest, the analyte and the chemical moiety competitively bind to the chemical material, while the chemical moiety has the general formula: (MZ ) N-Lm-Yp, where M is terbium or europium; Z is a polydentate ligand of M; L is an ureido such as -CONH-, -CONMe, thioureido, amide. A thioether such as -S-, -SS-; a linking group such as a sulfonamide such as SO2NH-, -SO2NMe-, and L can include molecular chains of various compositions and lengths; Moreover, this molecular chain is bound to the multidentate ligand Z by a part of the divalent group and is bound to the substance Y by another part. Then, Y is a substance that binds to Z via one or more linking groups L; n is an integer of 1 or more; p is an integer of 1 or more; m is an integer of 1 or more. The method comprises: a) adding the chemical material, chemical moiety and analyte as described above,
Contacting under suitable conditions to form a reagent mixture; b) inducing luminescence of a chemical moiety by applying an electric pulse to an electrode immersed in a solution; c) applying the electric pulse; It is characterized in that it consists in detecting the light emitted a little later and thereby measuring the analyte in question. 5. Y is whole cell, subcellular particle, virus, nucleic acid, polysaccharide, protein, polypeptide, enzyme, cell metabolite,
Method according to claim 2, 3 or 4, characterized in that it is a hormone, pharmacological substance, drug, alkaloid, steroid, vitamin, amino acid or carbohydrate. 6. The method according to claim 2, wherein Y is a nucleotide, an oligonucleotide or a polynucleotide.
The method described. 7. The method according to claim 5, wherein Y is an antibody.
The method described. 8. The chemical substance is whole cell, subcellular particle, virus, nucleic acid, polysaccharide, protein, polypeptide, enzyme, cell metabolite, hormone, pharmacological substance, drug, alkaloid, steroid, vitamin, amino acid. The method according to claim 3, which is also a carbohydrate. 9. The method of claim 3, wherein the chemical is immobilized on the surface of at least one electrode. 10. The method according to claim 3, wherein the chemical substance is an antibody. 11. The analyte is whole cell, subcellular particle, virus, nucleic acid, nucleotide, oligonucleotide, polynucleotide, polysaccharide, protein, polypeptide, enzyme, cell metabolite, hormone, pharmacological substance, drug, Method according to claim 4, characterized in that it is an alkaloid, steroid, vitamin, amino acid or carbohydrate. 12. The method of claim 11, wherein the analyte is an antibody. 13. The method of claim 4, wherein the chemical material is an antibody. 14. The analyte is an analyte-specific antibody immobilized on the surface of an electrode, Y is an antibody to another or the same antigenic determinant on the analyte, and the analyte is between the antibodies. The method of claim 3, wherein the method is associated with However, this method is a non-competitive assay. 15. The analyte is whole cell, subcellular particle, virus, nucleic acid, oligonucleotide, polynucleotide, polysaccharide, protein, polypeptide, enzyme, cell metabolite,
15. The method according to claim 14, characterized in that it is a hormone, a pharmacological substance, an alkaloid or a carbohydrate. 16. The method is a homogeneous method, and the appropriate conditions are such that the bound chemical moiety and the unbound chemical moiety are not separated before luminescence by electric pulse is detected. Claims 3, 4, 14 or
Method described in 15. 17. The method is a heterogeneous method, and the appropriate conditions are to separate the bound and unbound chemical moieties prior to applying electrical pulses to the electrodes and measuring luminescence. Claim 3, characterized in that it consists of
The method described in 4, 14, or 15. 18. The method of claim 1, wherein at least one of the electrodes is made of aluminum.