JP3342065B2 - Agglutination reaction method and agglutination reaction device, and concentration measurement method and concentration measurement device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for agglutinating a substance in a sample liquid with an active substance, in particular, a biological substance which selectively binds to a trace substance in the sample liquid.
Furthermore, the present invention relates to a method and an apparatus for measuring the concentration of a substance in a sample liquid by using them.

[0002]

2. Description of the Related Art Conventionally, an agglutination reaction is caused by the action of a substance in a sample liquid and a biological substance, and the degree of the aggregation is measured to determine the degree of aggregation of the substance, for example, an immunologically active antigen. There is a method of measuring the concentration.

As such a method, the above-mentioned immunological method is applied to a dispersion liquid (latex reagent) in which fine particles such as polystyrene carrying an antibody that selectively binds to an antigen are dispersed in a liquid medium such as water. Latex agglutination immunoassay (LAIA) for measuring the concentration of antigen in a sample solution by observing the agglutination caused by the action of a sample solution having an active antigen on the surface of the sample solution is described in JM Singer et al. Singeret. Al.) [Am. J. Med. , 21888 (1956)], and thereafter, various studies have been made on the method. And, as a method of determining the degree of the aggregation,
Although quantitative measurement is difficult, there is an advantage that the result is simple and the result can be obtained in a short time. Therefore, a visual judgment method is widely used practically.

[0004] In recent years, attempts have been made to optically measure the degree of agglomeration.
ure et. al. ) Proposes a method of optically measuring the change in turbidity due to the agglutination reaction and performing quantitative analysis from kinetic analysis [A. Feature et. al. ; P
rotides Biol Fluids, Proc.
Colloq. , 2589 (1972)].

In an inspection utilizing such an agglutination reaction,
A method has been performed in which a latex reagent and a sample solution are mixed, left to stand, and the degree of aggregation is measured.

[0006]

However, in such a method in which the reagent and the sample solution are mixed and allowed to stand, the reaction rate depends only on the natural diffusion of the latex reagent, so that the reaction time is short. There are problems that measurement is difficult and measurement data is inaccurate.

[0007] Therefore, in the method utilizing these agglutination reactions, the necessity of a short-time inspection is required to carry out the agglutination reaction between a trace substance and a biological substance in a liquid medium as described above.
Agitation of the liquid medium is performed to promote the reaction.

Conventionally, a method of mechanically agitating or applying vibrations by a stirrer has been used for stirring the liquid medium.

However, these methods have problems that a large amount of liquid medium, reagents and sample liquids are required, and that the apparatus itself is large.

In view of the above problems of the prior art, an object of the present invention is to provide a method and an apparatus for easily performing an agglutination reaction in a shorter time and using a small amount of liquid (dispersion medium, sample liquid, etc.). Another object of the present invention is to provide a method and an apparatus for measuring the concentration of a substance in a sample solution.

[0011]

SUMMARY OF THE INVENTION The present invention, which has been made to achieve the above object, provides a method for converting a substance in a sample solution and a biological substance which selectively binds to the substance into an ionic conductive material. A method for causing an agglutination reaction in a liquid medium having a liquid medium having an ion conductivity in which the sample liquid and the biological substance are dispersed between an electrode pair, and a positive side is provided between the electrode pair. And applying an alternating voltage that periodically fluctuates to the negative side to agitate the liquid medium, and an apparatus for performing the agglutination reaction method, wherein the electrode pair And a reaction cell provided with at least one set of the electrode, and periodically fluctuates between the electrode pair between the positive side and the negative side.
An agglutination reaction device comprising power supply means for applying an alternating voltage.

Furthermore, the present invention is a concentration measuring method characterized by detecting a degree of agglutination of a reaction mixture generated by the agglutination reaction method and measuring a concentration of a substance in the sample solution, Further, there is provided an apparatus for carrying out the concentration measuring method, wherein a reaction cell provided with at least one or more pairs of the electrodes is periodically changed between a positive side and a negative side between the electrode pairs.
A concentration measuring device comprising: a power supply unit for applying an alternating voltage; and a detection unit for detecting the degree of aggregation of the reaction mixture in the reaction cell.

The substance in the sample solution and the biological substance that can be used in the present invention are not particularly limited as long as they are substances that selectively bind to each other.

Examples of the substance in the sample liquid include proteins, sugars, hormones, viruses, DN,
A, RNA and the like.

On the other hand, the above-mentioned biological substance as a reagent is
Those exhibiting biological specificity for the above substances can be selected.

Here, the term "biological specificity" means that a specific bond such as an antigen-antibody reaction, DNA and RNA hybridization, and avidin-biotin bond is formed.

The biological substances referred to in the present invention include natural or synthetic peptides, proteins, enzymes, saccharides, lectins,
Includes viruses, bacteria, nucleic acids, DNA, RNA, antigens (including, for example, recombinant antigens) and antibodies. The following substances are particularly useful in clinicopathology.

[0018] IgG, IgM, immunoglobulin, such as IgE, complement, CRP, ferritin, alpha _{1-microglobulin,} plasma proteins and their antibodies such as beta _{2-microglobulin,} alpha-fetoprotein, carcinoembryonic antigen (C
EA), prostatic acid phosphatase (PAP), CA
Tumor markers such as 19-9, CA-125 and their antibodies, luteinizing hormone (LH), follicle stimulating hormone (FSH), human ciliary gonadotrobin (hCG), hormones such as estrogen, insulin and their antibodies; HBV-related antigen (HBs. HBe. HBc),
Virus infection related substances such as HIV and ATL and their antibodies, bacteria such as diphtheria, botulinum, mycoplasma, treponema pallidum and their antibodies, protozoa such as toxoplasma, trichomonas, leishmania, trivanozoma, malaria parasite, and the like Antiepileptic drugs such as antibodies, phenytoin, phenobarbital, cardiovascular drugs such as quinidine and digoxinine, antiasthmatic drugs such as theophylline, antibiotics such as chloramphenicol and gentamicin, and drugs such as antibodies and other enzymes, There are bacterial exotoxins (such as styrrelidine O) and antibodies thereof, and a substance that causes an antigen-antibody reaction with the substance in the sample solution is appropriately selected and used.

In general, in the antigen-antibody reaction or the like, a substance in a sample solution and a biological substance as a reagent undergo an agglutination reaction. At this time, depending on the type and amount of the substance, a bio-related substance can be carried on the fine particle surface to cause an agglutination reaction.

The fine particles used in the present invention include:
Examples of the fine particles include biological fine particles, inorganic fine particles, and organic fine particles. Examples of the microparticles derived from the organism include bacteria such as erythrocytes, dispersed staphylococci, and streptococci. Examples of the inorganic fine particles include silica, alumina, bentonite and the like. Further, as the organic fine particles, for example,
Homopolymers and / or copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates and methacrylates, styrene-butadiene copolymer, methyl methacrylate-butadiene copolymer Examples include butadiene-based copolymers such as coalesced particles and fine particles such as lipid bilayer liposomes.

The binding of the bio-related substance to the fine particles is performed physically and / or chemically as described later. In the physical bonding, the surface of the fine particles is preferably hydrophobic, and styrene is preferably used. Particularly preferred are homopolymer fine particles, vinyl copolymer fine particles containing styrene as a main component, or styrene-butadiene copolymer containing styrene as a main component. The particle size of the above-mentioned fine particles is preferably in the range of 0.05 μm to 10 μm, particularly preferably in the range of 0.2 μm to 5 μm, in any case of fine particles derived from living organisms, inorganic fine particles, and organic fine particles. It is. When the particle diameter is less than 0.05 μm, it becomes difficult to disperse a biological substance as a reagent on the surface thereof, and
Above μm, the stability of the dispersed reagent becomes poor.

In the present invention, the following known method can be used to carry (immobilize) the above-mentioned biological substance on the surface of the fine particles.

For example, i) an ion bonding method, ii) a physical adsorption method, and iii) a covalent bonding method.

The ion binding method is used for proteins, DNA, RNA
And the like, such that biologically-related substances such as are electrostatically bound to the surface of the fine particles.

The physical adsorption method is a bonding method utilizing a hydrophobic bond between a lipophilic portion on the surface of fine particles and a lipophilic portion of a protein.

In the ion bonding method and the physical adsorption method, the bonding reaction step is simple, but the bonding force between the fine particles and the biological substance is weak.

On the other hand, in the covalent bonding method, a highly reactive functional group is bonded to at least one of the fine particle surface and the biological substance, and the two are covalently bonded via the functional group, and a strong bonding force can be obtained. . When the microparticles and the biological substance are bound by the covalent bonding method, the functional groups that can participate in the binding in the biological substance include free amino groups, hydroxyl groups, phosphate groups, carboxyl groups, cysteine sulfhydryl groups, and histidine. Imidazole group, tyrosine phenol group, serine and threonine hydroxyl group.

These functional groups react with various diazonium salts, acid amides, isocyanates, active alkyl halide groups, active ester groups, and the like. Therefore, by introducing these functional groups onto the surface of the fine particles, a bio-related substance can be supported on the surface of the fine particles by various methods. On the other hand, higher-order structures of biological substances, particularly biological substances including proteins, are fragile because they are held by relatively weak bonds such as hydrogen bonds, hydrophobic bonds, and ionic bonds. It is desirable to carry out the treatment under mild conditions, avoiding treatments such as strong acids and strong alkalis.

As one method for carrying out the immobilization reaction under mild conditions, a bifunctional crosslinking agent which reacts with the fine particles and the functional group of the biological substance can be used. Examples of the bifunctional crosslinking agent include carbodiimides represented by the general formula RN = C = NR 'and general formulas CHO-R-CHO
A dialdehyde represented by the formula: O = C = NRN = C =
And diisocyanates represented by O (in these general formulas, R and R 'are the same or different and substituted or unsubstituted alkyl, aryl, alkylaryl or arylalkyl groups).

In the present invention, the sample liquid containing the substance to be measured and the biological substance as the reagent or the fine particles carrying the substance are dispersed in a liquid medium having ionic conductivity.

Examples of the liquid medium having ionic conductivity include water in which an electrolyte is dissolved or water and alcohols;
A mixed solvent of an organic solvent compatible with water such as ketones is used. It is necessary for the ionic conductivity that part or all of the electrolyte in these liquids be ionized to ions. Further, a pH buffer, a protein, a surfactant, a water-soluble polymer compound and the like are appropriately added to the liquid medium.

Generally, the antigen-antibody reaction and the hybridization are easily affected by the pH of the solvent, and thus a pH buffer is added to adjust the pH to an optimum value. For example, phosphate, Tris, HCl buffer and the like are used. Protein is added to prevent non-specific reactions,
For example, bovine serum albumin, gelatin and the like are used.
Surfactants and water-soluble polymer compounds are effective as a dispersing aid for reagents. For example, nonionic surfactants such as Tween 20 and anionic surfactants, polyvinyl alcohol, polyacrylamide, polyacrylate, hydroxyacrylate, etc. Ethyl cellulose or the like is used. However, these additives are used in a range that does not inhibit the agglutination reaction.

Further, the reagent is appropriately diluted and adjusted by the liquid medium according to the kind and amount of the substance in the sample solution.
The solid concentration varies depending on the type or size of the reaction cell or the like to be used, but is generally preferably from 0.01 to
It is adjusted in 5%, more preferably in the range of 0.05 to 2%.

In the present invention, a sample liquid containing the substance to be measured and a liquid medium having ionic conductivity in which the reagent is dispersed are introduced between the electrode pairs, and a voltage that fluctuates periodically between the electrode pairs is applied. The liquid medium is agitated by applying an electromagnetic force to the ions in the vicinity of the electrodes.

For this reason, the conductivity of the liquid medium having ionic conductivity is desirably large, and is preferably in the range of 10 ⁻⁵ S / cm or more, more preferably 10 ⁻⁴ S / cm or more.
That is all. When the electric conductivity is small, the current flowing between the electrode pair decreases, and at the same time, the magnetic field generated in the vicinity of the electrode decreases, so that the electromagnetic force acting on the ions decreases and the flow of the liquid medium becomes difficult.

The electrodes used in the present invention include metals,
Conventionally known electrode materials such as carbon, polymer materials in which these are dispersed as conductive fillers, and conductive polymer materials such as polypyrrole can be used. Generally, the choice of these electrode materials depends on the voltage applied between the electrode pairs. That is, if a direct current (DC) component is present in the applied voltage, depending on the electrode material, anodization occurs, and the electrode is dissolved or an oxide film is formed, which may adversely affect the flow of the liquid medium and its durability stability. . In such a case, it is necessary to select an electrode material such as platinum that is not easily subjected to anodic oxidation. Although it is conceivable to lower the applied voltage, the electromagnetic force acting on the ions is limited to a low value, so that the upper limit of the flow speed of the liquid medium may be suppressed low or the liquid medium may not flow.

In consideration of these points, the voltage applied between the electrode pairs is preferably a periodically fluctuating voltage.
In this case, the waveform is not particularly limited, and a rectangular wave, a sine wave, a triangular wave, or the like can be used.

As the voltage which fluctuates periodically, for example, there is an alternating (AC) voltage as shown in FIG. Since the time average of the applied voltage is 0, the alternating voltage is particularly preferable because the anodic oxidation of the electrode and the electrolysis of the liquid hardly occur.

If necessary, a voltage obtained by combining a plurality of alternating voltages or a voltage obtained by superimposing a DC voltage on an alternating voltage as shown in FIG. 5B may be used.
It is necessary that the thickness be in a range where the above-described anodic oxidation does not occur.

Further, in the case of an applied voltage whose duty ratio can be changed like the rectangular wave voltage shown in FIG. 5C, the preferable duty ratio is 10 to 90%, more preferably, 20%.
~ 80%. When the duty ratio is smaller than 10% or larger than 90%, the responsiveness of ions in the liquid to the electric field becomes poor, and the applied electromagnetic force becomes small, so that the flow becomes weak.

The frequency and amplitude voltage value are related to the presence or absence of the above-described anodic oxidation of the electrode and the electrolysis of liquid (generation of bubbles, reaction, etc.). In general, the lower the frequency and the larger the amplitude voltage value, the more. Anodizing and electrolysis are likely to occur. For example, in the case of an aqueous electrolyte solution having a conductivity of about 1 mS / cm, a preferable frequency is 100 KHz or more, more preferably 1 MHz or more. Also preferred amplitude voltage value changes by the conductivity of the distance and the electrode shape and the liquid medium between the electrode pair, but generally 10 ⁴ to 10 as a field value ⁶
V / m.

Although there are many unclear points about the principle of the flow of the liquid medium in the present invention, it is considered as follows if one of the electrode pairs is described as a working electrode and the other as a counter electrode.

When a voltage is applied between the working electrode and the counter electrode, an electric field is generated between these electrodes. The ions in the liquid medium move due to this electric field, and at this time, a current flows between both electrodes. At the same time, a magnetic field is generated near the working electrode by the current flowing through the working electrode. Ions moving by an electric field near the working electrode receive an electromagnetic force (Lorentz force) from a magnetic field. This Lorentz force seems to be the flow mechanism in the present invention. When the voltage applied between the working electrode and the counter electrode is an alternating voltage, the movement of ions due to the electric field is practically negligible when averaged over time, and only the electromagnetic force acts.

In the configuration of the electrode of the present invention, it is preferable that the magnitude of the movement of the ions (the magnitude of the current) due to the electric field and the strength of the magnetic field near the electrode are large.

Therefore, it is preferable that the electrode is shaped so that the electric field is concentrated on the working electrode so that both the electric field and the magnetic field near the working electrode are large.

Also, by using such a working electrode, the application of a magnetic field and an electric field can be performed simultaneously and simply, and it is not necessary to provide a means for applying a magnetic field and a means for applying an electric field independently.

Hereinafter, a configuration example of the electrode used in the present invention will be described with reference to the drawings.

In FIG. 1, 1 is a working electrode having a conical tip, and 4 is a counter electrode. When a voltage is applied between these electrodes, the surrounding ions move along the electric field (lines of electric force 3) (i direction in the figure), and a current I flows through the working electrode 1. This current I forms a magnetic field 2 concentrically about the conical axis of the working electrode. At this time, the ions in the vicinity of the working electrode are moving by receiving a force from the electric field 3 formed between the opposing electrodes 4, and thus receive an electromagnetic force F from the magnetic field 2, and the direction is almost the conical surface at the tip of the working electrode Along the direction. This direction does not change even if the polarity of the applied voltage is reversed, and does not change whether the ions are positive or negative.

Also, as shown in FIG. 2, an electrode pair can be formed by facing two conical electrodes shown in FIG.

In this case, the two electrodes are mutually
And the counter electrode 4. When a voltage is applied between the electrodes, a liquid flows as shown by arrows in FIG.

Thus, in the agglutination reaction method of the present invention,
The liquid medium is agitated by applying electromagnetic force to ions in the liquid medium in a minute area near the electrode, so that the aggregation reaction can be promoted and a small amount of liquid (dispersion medium, sample liquid, etc.) can be used. Aggregation reaction can be easily performed.

Next, the agglutination reaction device of the present invention will be described with reference to the drawings. FIG. 3 shows one configuration example of the agglutination reaction device of the present invention having the electrode configuration as shown in FIG. In the figure, 6 and 6 'are glass substrates, 7 is a spacer, 10 is a reaction cell composed of these, 8, 8' are electrodes having a conical tip, and 9 is a liquid injection part.

In the present apparatus, a liquid medium having ionic conductivity in which a sample liquid containing a substance to be measured and a biological substance as a reagent are dispersed is introduced into a liquid injection section 9, and electrodes 8 and
An alternating voltage is applied between 8 'by power supply means (not shown). At this time, the liquid medium in the vicinity of the electrodes 8, 8 'moves as shown in FIG. 2, and as a result, the liquid medium is agitated, and the agglutination reaction is promoted. Also, by changing the voltage value and frequency applied between the electrodes, the efficiency of stirring can be changed, and the reaction speed of the agglutination reaction can be controlled.

Next, a method and apparatus for measuring the concentration using the agglutination reaction method and agglutination reaction apparatus of the present invention will be described.

In the concentration measuring method of the present invention, the degree of agglutination of the reaction mixture generated by the agglutination reaction is detected. As a specific measuring method, when measuring the time change of the output detected with the progress of the agglutination reaction,
It is conceivable to measure the output when the agglutination reaction is completed. It is also possible to perform a quantitative test by measuring a known sample and obtaining a calibration curve.

At this time, the region for detecting the degree of aggregation of the reaction mixture is preferably a stirring region between the electrode pairs disposed in the reaction cell.

Further, it is also possible to perform measurement while changing the efficiency of stirring by changing the voltage value and frequency applied between the electrodes.

In the concentration measuring apparatus of the present invention, as means for measuring the degree of aggregation in the reaction mixture, optical means such as measuring the transmitted light intensity and scattered light intensity of the reaction cell may be used in addition to visual observation. Electrical means such as impedance measurement is used. In the case of optical means, a light source such as a halogen lamp, a Xe lamp, etc.
Laser light sources such as Ne lasers and semiconductor lasers can also be used. Further, as the light receiving element, a photoelectron amplifier tube, a photodiode, a phototransistor, or the like can be used, and the output is amplified using an operational amplifier or the like as necessary.

[0059]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments.

Example 1 A dispersion of polystyrene latex particles (particle size: 1.2 μm, manufactured by Denka Seiken Co., Ltd.) sensitized with human CRP antibody in PBS (solid content: 1.2 mg / ml) was used as a sample solution. Standard CRP serum (manufactured by Kyowa Yuka) was mixed to a concentration of 5 μg / ml, and 1 μl was added to the reaction cell having the structure shown in FIG.
Injected.

The reaction cell is constituted by forming a liquid injection section 9 (injection port 250 μm × 500 μm) by sandwiching a spacer 7 between two slide glasses 6 and 6 ′ having a thickness of 1 mm. The electrodes 8, 8 'were gold-tungsten wires (50 μm in diameter, 10 μm or less in diameter) having a conical tip, and were opposed to each other in the reaction cell at a tip interval of 100 μm. After the mixture was injected into the reaction cell from the injection port, the aggregation state was detected and the CRP concentration was measured by a measuring device as shown in FIG.

In FIG. 4, 10 is a reaction cell, 11 is a halogen lamp, 12 and 12 'are lenses, 13 is a photodiode, 14 is a stage with a heater, 15 is a high frequency power source,
Reference numerals 6, 16 'denote electrodes, and reference numeral 17 denotes a pinhole.

In the present embodiment, the mixture in the reaction cell is maintained at 37 ° C. by the heater 14 and the high-frequency power source 1
5, a 1 MHz, ± 10 V rectangular wave was applied between the electrodes, and the liquid mixture between the electrodes was stirred for 100 seconds. At this time, the mixture between the electrodes flowed in the direction indicated by the arrow in the reaction cell of FIG. 4 and was stirred. After stirring, the transmitted light intensity between the electrodes was measured, and the state of aggregation was measured. By comparing this data with calibration curve data prepared in advance, the CRP concentration in the sample solution could be measured.

Comparative Example 1 Measurement was performed in the same manner as in Example 1 except that no electric field was applied between the electrodes. After 100 seconds, the state of aggregation of the mixed solution was detected. However, the degree of aggregation was small, and the CRP concentration was estimated to be low from a calibration curve prepared in advance, and an accurate value could not be obtained.

[0065]

As described above, according to the present invention, the following effects can be obtained. (1) A sample liquid containing a substance to be measured and a biological substance that selectively binds to the substance are dispersed in a liquid medium having ionic conductivity, and the liquid medium is introduced between an electrode pair. The agglutination reaction method and the agglutination reaction device of the present invention, which stirs the liquid medium by applying an alternating voltage that periodically fluctuates between the positive side and the negative side between Agglomeration reaction can be promoted because the liquid medium is agitated by applying force, and can be easily agglomerated in a shorter time and with a smaller device using a small amount of liquid (dispersion medium, sample liquid, etc.). The reaction can be performed. (2) The concentration measuring method and apparatus of the present invention using the agglutination reaction method and apparatus of the present invention can accurately measure the concentration of a substance in the sample solution in a shorter time.

[Brief description of the drawings]

FIG. 1 is a diagram for explaining a configuration example of an electrode that can be used in the present invention and a principle of stirring a liquid medium.

FIG. 2 is a diagram showing another configuration example of an electrode that can be used in the present invention.

FIG. 3 is a schematic configuration diagram of an agglutination reaction device used in an example of the present invention.

FIG. 4 is a schematic configuration diagram of a concentration measuring device used in an embodiment of the present invention.

FIG. 5 is an example of a periodically varying voltage waveform.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Working electrode 2 Magnetic field 3 Electric force line 4 Counter electrode 6,6 'Glass substrate 7 Spacer 8,8' Gold-tungsten wire electrode 9 Liquid injection part 10 Reaction cell 11 Halogen lamp 12,12 'Lens 13 Photodiode 14 With heater Stage 15 High frequency power supply 16, 16 'Electrode 17 Pinhole

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/50-33/98 G01N 21/82 G01N 27/28

Claims (7)

(57) [Claims] 1. A method of causing an agglutination reaction between a substance in a sample solution and a biologically-related substance that selectively binds to the substance in a liquid medium having ionic conductivity, wherein the method comprises the steps of: A liquid medium having ionic conductivity in which a sample liquid and the biological substance are dispersed is introduced, and an alternating voltage that periodically fluctuates between the electrode pair on the positive side and the negative side is applied. An agglutination reaction method characterized by stirring. 2. The agglutination reaction method according to claim 1, wherein the biological substance is carried on the surface of fine particles. 3. An apparatus for performing the agglutination reaction method according to claim 1 or 2 , wherein the reaction cell includes at least one pair of the electrode pairs , and a pair of the positive electrode and the negative electrode periodic between the pair of electrodes . An agglutination reaction device comprising a power supply means for applying an alternating voltage that fluctuates. 4. A detecting a degree of aggregation of claim 1 or 2 the reaction mixture resulting from the agglutination method described, concentration measuring method and measuring the concentration of a substance in the sample liquid. 5. The method according to claim 4 , wherein
A concentration measuring method, comprising detecting a degree of aggregation of a reaction mixture between the electrode pairs. 6. An apparatus for performing the concentration measuring method according to claim 4 or 5 , wherein a reaction cell having at least one pair of the electrode pairs is provided, and a positive electrode and a negative electrode are periodically arranged between the electrode pairs. A power supply means for applying a fluctuating alternating voltage; and a detecting means for detecting the degree of aggregation of the reaction mixture in the reaction cell. 7. The concentration measuring apparatus according to claim 6 , wherein said detecting means is an optical detecting means.