JPH06201698A - Method and device for detecting trace substance - Google Patents

Abstract

PURPOSE:To provide a method by which a substance which is selectively and specifically linked with a biological substance and contained in a sample liquid can be detected. CONSTITUTION:In this method, a trace substance is detected by forming a reaction link between a solid-phase reagent carrying a biological substance which is selectively and specifically linked with the trace substance contained in a sample liquid and the trace substance and one or more kinds of detection reagent and both the sample liquid and detection reagent solution have ionic conductivity. The reaction among the solid-phase reagent, trace substance, and detection reagent is caused by introducing the liquids having ionic conductivity between a pair of electrodes and stirring the liquids by applying a periodically fluctuating voltage across the electrodes. Since the reaction is caused while the sample liquid and reagent solution are stirred in a small area near the electrodes, the trace substance can be detected in a shorter time even when the amount of the sample liquid is small.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and a device for detecting trace substances related to living bodies such as sugars and proteins.

[0002]

2. Description of the Related Art Conventionally, various methods and devices have been used for analysis of nucleic acids, immunologically active antigens and other trace substances related to living bodies. In these analyses, a reagent that selectively binds to a trace substance is allowed to exist in a solid material such as a filter, latex particles, glass beads, etc., and a trace substance is bound to the solid material from the test sample solution, and the obtained conjugate is Radioactive and optical detection methods are known. Specifically, "Inspection and Technology vol. 16 NO. 7 1988
According to "Year", RIA (Radioimmunoassay), EIA
(Enzyme immunoassay), FIA (fluorescent immunoassay) and the like.

In these methods, the conjugate is labeled with a radioactive isotope, a dye, or a reagent labeled with an enzyme for optical detection such as detection of enzyme activity, detection of radiation dose, absorption, and luminescence. Is done. Generally, these methods require a washing step for removing excess test sample or labeling reagent that has not bound to the reagent on the solid material from the solid material, so-called B / F separation.

The various reaction steps and washing steps in the analysis of the above trace substances are generally carried out in a liquid medium,
Agitation of a liquid medium or a solid material (such as beads) is important for improving the efficiency of reaction and washing.

As a stirring method, a method of using magnetic force by using a solid material such as magnetic beads or magnetic fine particles, and a method of using a stirrer when a solid material such as a filter is used are known.

[0006]

However, in the method using magnetic fine particles, it is difficult to adjust good magnetic fine particles, and when the particle size of the magnetic fine particles becomes very small (for example, submicron), the magnitude of magnetic force becomes large. There is a drawback that it becomes small and the stirring efficiency becomes poor. In addition, when agitating a small amount of liquid, there is a problem that since the voids of the housing (cell) that holds the liquid are narrow, magnetic beads and magnetic fine particles are clogged in the voids, making it impossible to agitate.

Further, the method using the stirring bar has a problem that it is generally unsuitable for a very small amount of liquid, because carry-over is likely to occur due to insufficient cleaning of the stirring bar.

The present invention has been made in view of the above problems of the prior art.
A trace substance detection method that enables efficient agitation even for minute amounts of liquids (sample solutions, reagent solutions, etc.) to efficiently perform reaction steps and washing steps and shorten measurement time. And to provide a detection device.

[0009]

MEANS TO SOLVE THE PROBLEMS [Means and Actions for Solving the Problems] The present invention, which has been made to achieve the above-mentioned object, provides a solid material in which a biological material that selectively and specifically binds to a trace substance in a sample solution is supported on a solid material. A method for detecting a trace substance by forming a reaction conjugate with the phase reagent by the trace substance and / or one or more detection reagents, wherein the sample liquid and the detection reagent liquid are ionic conductive A liquid having ionic conductivity is introduced between electrode pairs, and a voltage that fluctuates cyclically is applied between the electrode pairs to stir the liquid while immobilizing the solid phase reagent and the trace substance. And a method for detecting a trace substance, which comprises performing a reaction step of a detection reagent, and in the above-mentioned detection method, after the reaction step, there is a washing step with a washing solution having ion conductivity, and the washing solution is Introduced between the pair of electrodes A method for detecting trace substances, characterized in that a cleaning step is performed while stirring a cleaning liquid by applying a voltage that fluctuates cyclically between the electrode pairs, and further, for implementing those detection methods. An apparatus comprising: a reaction cell provided with at least one pair of electrode pairs; a power supply means for applying a cyclically varying voltage between the electrode pairs; and a labeling substance in a reaction conjugate in the reaction cell. A trace substance detection device comprising means for detecting.

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

Examples of substances in the above-mentioned sample liquid include proteins, sugars, hormones, viruses and DNs in vivo.
A, RNA, etc. are mentioned.

On the other hand, the above biological substance as a reagent is
It is possible to select one that exhibits biological specificity for the above substances.

The term "biological specificity" as used herein means that a specific bond such as an antigen-antibody reaction, DNA or RNA hybridization, or avidin-biotin bond is formed.

The biological substance referred to in the present invention is a natural or synthetic peptide, protein, enzyme, saccharide, lectin,
It includes viruses, bacteria, nucleic acids, DNA, RNA, antigens (including recombinant antigens) antibodies, and the like. The following are examples of substances that are particularly useful clinically.

Immunoglobulins such as IgG, IgM and IgE, plasma proteins such as complement, CRP, ferritin, α ₁ microglobulin and β ₂ microglobulin and their antibodies, α-fetoprotein, carcinoembryonic antigen (C
EA), prostatic acid phosphatase (PAP), CA
19-9, tumor markers such as CA-125 and their antibodies, luteinizing hormone (LH), follicle stimulating hormone (FSH), human ciliated gonadotrobin (hCG), estrogen, hormones such as insulin and their antibodies, HBV-related antigen (HBs.HBe.HBc),
HIV, ATL, and other viral infection-related substances and their antibodies, bacteria such as diphtheria, botulinum, mycoplasma, Treponema pallidum, and their antibodies, protozoa such as Toxoplasma, Trichomonas, Leishmania, Trivanosomes, and malaria parasites, and their Anti-epileptic drugs such as antibodies, phenytoin and phenobarbital, cardiovascular drugs such as quinidine and digoxinine, anti-asthmatic drugs such as theophylline, drugs such as antibiotics such as chloramphenicol and gentamicin, and their antibodies, other enzymes, There are extracellular toxins (such as styrelidine O) and their antibodies, and the substances that cause an antigen-antibody reaction with the substances in the above sample solution are appropriately selected and used.

In the present invention, the bio-related substance is supported (immobilized) on a solid material to form a solid phase reagent. As the solid material, any material may be used as long as it can immobilize a biological substance by the method described below. For example, a glass or plastic material forming the reaction cell may be used, or a filter such as glass fiber or cellulose, or fine particles may be used.

Examples of the solid material in the form of fine particles include fine particles of biological origin, inorganic fine particles, and organic fine particles. As the fine particles derived from the organism,
For example, erythrocytes, dispersed staphylococci, streptococci, and other bacteria can be used. Examples of the inorganic fine particles include silica, alumina, bentonite and the like. Further, as the organic fine particles, for example, styrene, vinyl chloride, acrylonitrile, vinyl acetate,
Homopolymers of vinyl monomers such as acrylic acid esters and methacrylic acid esters and / or copolymers thereof, butadiene-based copolymers such as styrene-butadiene copolymer, methyl methacrylate-butadiene copolymer, and lipids Examples include fine particles such as bilayer liposomes.

In the present invention, in order to immobilize the above-mentioned biological substance on the surface of the above solid material, the following known methods can be used.

For example, i) ionic bond method, ii) physical adsorption method, iii) covalent bond method and the like can be mentioned.

The ionic bond method is used for proteins, DNA, RNA
A biological material such as is electrostatically bound to the surface of a solid material.

The physical adsorption method is a binding method utilizing a hydrophobic bond between the lipophilic portion of the surface of the solid material and the lipophilic portion of the protein.

In the ionic binding method and the physical adsorption method, the binding reaction step is simple, but the binding force between the solid material and the biological substance is weak.

On the other hand, the covalent bond method is a method in which a highly reactive functional group is bonded to at least one of the surface of a solid material and a bio-related substance, and the both are covalently bonded via this, so that a strong bonding force can be obtained. To be When binding a biomaterial with a solid material by a covalent bond method, as a functional group that can participate in the bond in the biomaterial, a free amino group, a hydroxyl group, a phosphate group, a carboxyl group, a sulfhydryl group of cysteine, There are imidazole group of histidine, phenol group of tyrosine, hydroxyl group of serine and threonine.

These functional groups react with various diazonium salts, acid amides, isocyanates, activated halogenated alkyl groups, activated ester groups and the like. Therefore, by introducing these functional groups onto the surface of the solid material, the bio-related substance can be supported on the surface of the solid material by various methods. On the other hand, the higher-order structure of a bio-related substance, particularly a bio-related substance including a protein, is fragile because it is held by a relatively weak bond such as hydrogen bond, hydrophobic bond, and ionic bond. It is desirable to avoid treatment with strong acid, strong alkali, etc., and perform under mild conditions.

As one method for carrying out the immobilization reaction under mild conditions, it is possible to use a bifunctional crosslinking agent that reacts with the functional group of the solid material and the biological substance. Examples of the bifunctional cross-linking agent include those represented by the general formula RN = C = NR '.
A carbodiimide represented by the general formula: CHO-R-CH
Dialdehyde represented by O, O = C = N-R-N = C
A diisocyanate represented by ═O and the like (in these general formulas, R and R ′ are the same or different substituted or unsubstituted alkyl groups, aryl groups, alkylaryl groups or arylalkyl groups).

Examples of the detection method that can be used in the present invention include the following methods. ("Inspection and Technology vol.16
NO. 7 pp. 591, 1988) (Sandwich method) Detecting a trace substance by forming a reaction conjugate in which a solid-phased reagent and a detection reagent are bound via a trace substance and detecting the reaction conjugate. You can

In the case of the sandwich method, as the detection reagent, a trace substance and a biologically-relevant substance exhibiting biological specificity can be used as in the solid-phased reagent.

(Competitive Method) Two types of reagents, a solid phase reagent and a trace substance, and a solid phase reagent and a detection reagent, are allowed to react competitively with the solid phase reagent and the trace substance in the sample solution. A trace substance can be detected in a complementary manner by forming the reaction conjugate of (1) and detecting the reaction conjugate composed of the solid phase reagent and the detection reagent.

In the case of the competitive method, the detection reagent is a trace substance (need to be labeled in order to distinguish it from the trace substance to be measured), or likewise a solid phase reagent and a biological specificity. A substance showing

It is necessary to detect the reaction conjugate containing the detection reagent by both the sandwich method and the competition method. This can be generally carried out by introducing into the detection reagent a labeling substance that can be detected by detection means such as optical or radiation. The labeling substance is introduced into the detection reagent described above.
The labeling substance may be chemically bound in advance, or it may be selectively and specifically bound to the detection reagent in the reaction conjugate and further labeled with another detection reagent. As the labeling substance, various conventionally used radioisotopes, dyes, enzymes and the like can be used.

In the present invention, the sample solution and / or the detection reagent solution have ionic conductivity, and these liquids are introduced between the electrode pair, and a cyclically varying voltage is applied between the electrode pair. Then, the liquid is stirred by applying an electromagnetic force to the ions near the electrode.

For this reason, it is desirable that the conductivity of the liquid having ionic conductivity is high, and the preferable range is 10 ⁻⁵ S / cm or more, and more preferably 10 ⁻⁴ S / cm.
That is all. When the conductivity is small, the current flowing between the electrode pair becomes small, and at the same time, the magnetic field generated in the vicinity of the electrodes also becomes small, so that the electromagnetic force acting on the ions becomes small and the flow of the liquid 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, conductive polymer materials such as polypyrrole, and the like can be used. Generally, the selection of these electrode materials depends on the voltage applied between the pair of electrodes. That is, when a direct current (DC) component is present in the applied voltage, it may undergo anodic oxidation depending on the electrode material, which may cause dissolution of the electrode or formation of an oxide film, which may adversely affect the flow of the liquid and its durability stability. In such a case, it is necessary to select an electrode material, such as platinum, which is hard to undergo anodic oxidation. It is also possible to reduce the applied voltage, but since the electromagnetic force acting on the ions is limited to a low level, there is a possibility that the upper limit of the flow speed of the liquid may be suppressed to a low value or the liquid may not flow.

Considering these points, the voltage applied between the pair of electrodes is preferably a voltage that fluctuates periodically.
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.

An example of the periodically varying voltage is an alternating (AC) voltage as shown in FIG. The alternating voltage is particularly preferable because the time average of the applied voltage is 0, and thus anodic oxidation of the electrodes and electrolysis of the liquid are less likely to occur.

If necessary, a voltage obtained by combining a plurality of alternating voltages or a voltage obtained by superposing a direct current voltage on the alternating voltage as shown in FIG. 15 (b) may be used. It should be within the range where the above-mentioned anodic oxidation or the like 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. 15C, the preferable duty ratio is 10 to 90%, more preferably 2
It is 0 to 80%. When the Duty ratio is less than 10% or more than 90%, the responsiveness of the ions in the liquid to the electric field deteriorates, and the electromagnetic force that acts becomes small, so that the flow becomes weak.

The frequency and amplitude voltage value are related to the presence or absence of anodic oxidation of the electrodes and electrolysis of liquid (generation of bubbles, reaction, etc.) described above. Generally, the lower the frequency and the larger the amplitude voltage value are. 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, the preferable frequency is 100 KHz or higher, more preferably 1 MHz or higher. Further, the preferable amplitude voltage value changes depending on the distance between the electrode pair, the shape of the electrode and the conductivity of the liquid medium, but is generally 10 ⁴ to 10 ⁶ as the electric field value.
V / m.

Although there are many unclear points about the principle of liquid flow in the present invention, it is considered as follows when 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. Ions in the liquid 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 due to the electric field near the working electrode are converted from the magnetic field to electromagnetic force (Lorentz force).
Receive. This Lorentz force is considered 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 the ions due to the electric field is practically almost zero on time average, and only the electromagnetic force is acting.

As the constitution of the electrode of the present invention, it is preferable that the magnitude of the movement of the ions by the electric field (the magnitude of the current) and the strength of the magnetic field in the vicinity of the electrode are large.

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

By using such a working electrode, the magnetic field and the electric field can be applied simultaneously and easily, and it is not necessary to provide a magnetic field applying means and an electric field applying means independently.

An example of the structure of the electrode used in the present invention will be described below 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 (electrical force line 3) (i direction in the figure), and a current I flows through the working electrode 1. Due to this current I, a magnetic field 2 is formed concentrically around the conical axis of the working electrode. At this time, the ions in the vicinity of the working electrode move by receiving a force from the electric field 3 formed between the counter electrodes 4, and therefore receive an electromagnetic force F from the magnetic field 2, the direction of which is approximately the conical surface of the tip of the working electrode. The direction is along. This direction does not change even when the polarity of the applied voltage is reversed, and it does not change whether the ions are positive or negative.

Further, as shown in FIG. 2, it is also possible to construct an electrode pair by facing two conical electrodes shown in FIG.

In this case, the two electrodes interact with each other and the working electrode 1
And the counter electrode 4. When a voltage is applied between the electrodes, the liquid flows as shown by the arrow in FIG.

As described above, in the method for detecting a trace substance according to the present invention, the liquid is agitated by applying an electromagnetic force to the ions in the liquid in the minute region near the electrode, so that the reaction can be carried out efficiently. The time required for the step can be shortened, and the reaction step can be easily performed using a small amount of liquid (sample solution, reagent solution, etc.).

Further, in the present invention, when a washing step is provided after the above reaction step, assuming that the washing solution used for the washing step also has ionic conductivity, stirring is carried out in the same manner as the sample solution and the reagent solution described above. As a result, cleaning can be performed efficiently and the time required for the cleaning process can be shortened.

Next, a trace substance detection device of the present invention will be described with reference to the drawings. 3A and 3B show an example of the structure of a reaction cell used in the detection device of the present invention. FIG. 3A is a perspective view and FIG. 3B is a sectional view.

In the figure, a reaction cell 5 having a cylindrical liquid holding portion 6 in the center has a filter 7 as a solid material at the bottom of the liquid holding portion 6, and an upper surface of the filter 7 is filled with the sample liquid. The reagent portion region 8 that binds to the trace substances of Further, the liquid holding unit 6 is provided with needle-shaped electrodes 1 and 4 for stirring the liquid as shown in FIG. 2, and a voltage is applied by an external power source (not shown).

As the material of the reaction cell 5, conventionally known materials such as glass, synthetic resin material, metal and the like, and composite materials thereof can be used. The reaction cell 5 depends on the type of the reagent part region 8. When it is replaced with a synthetic resin material, a synthetic resin material that is lightweight and can be produced at low cost is preferable. The reagent part region 8 is composed of a reagent that binds to a trace substance, and this reagent may be directly present on the upper surface of the filter 7, or once it is supported on an intermediate medium such as a glass fiber material or polymer fine particles, These may be arranged on the filter 7.

As described above, the bio-related substance that selectively and specifically binds to the trace substance in the sample solution is supported on the filter 7 to form the solid phase reagent, and the sample solution is stored in the liquid holding part 6. The reaction step and the washing step are performed by supplying the detection reagent solution, the washing solution and the like. The supply of various liquids to the liquid holding unit 6 is performed by dropping a necessary amount by a dispenser 9 as shown in FIG. 4, for example.

The liquid is discharged from the liquid holding portion 6 by suction through the filter 7.

The dispenser 9 is properly used depending on the type of the sample liquid, the detection reagent liquid, the washing liquid, etc., dropped on the liquid holding portion 6. If desired, the tip dispenser tip may be a disposable pipette.

For example, in the reaction step in the case of the sandwich method, first, the liquid holding portion 6 is dispensed by the dispenser 9.
The sample solution is dropped onto the filter 7 so that the entire surface of the reagent area 8 on the filter 7 and the electrodes 1 and 4 are immersed in the sample solution (in this case, the back surface of the filter is not sucked). Next, an alternating voltage is applied between the electrodes to stir the sample solution, so that the trace substance in the sample solution and the reagent in the reagent part region 8 are bonded. At this time, the temperature of the reaction cell 5 can be controlled to promote the binding reaction. The desirable temperature varies depending on the type of binding reaction, but is 5 ° C to 100 ° C, more preferably 20 ° C to 60 ° C. Generally, the time required for the binding reaction depends on the reagent in the reagent part region 8 to be used, the analyte, the pH,
The temperature varies depending on the temperature and the like, for example, about several minutes in the case of immunoreaction and about several hours in the case of DNA hybridization. Therefore, it is preferable to select these conditions so that the binding time is as short as possible.

Next, the excess sample liquid is removed by suctioning from the back surface of the filter.

Next, in a state where the suction is stopped again, a labeling reagent solution which is bound to a trace substance and labeled with a fluorescent dye or the like is dropped onto the liquid holding portion 6 by the dispenser 9 and is bound in the same manner as the sample solution described above. Reaction and removal are repeated.

Similarly in the washing step, the washing liquid is dropped into the liquid holding portion by the dispenser 9 to remove the excess sample liquid and labeling reagent liquid. The process of such washing is
You may repeat multiple times as needed.

As a result, if a trace substance is present in the sample solution, a reaction complex in which the labeling reagent is bound to the reagent portion region on the filter via the trace substance can be obtained. The presence of the obtained reaction complex is detected by irradiating the reagent part region on the filter with light from the light source 10 and detecting fluorescence or reflected light from the reagent part region by the photodetector 11, as shown in FIG. 5, for example. It is confirmed that the trace substance in the sample liquid can be detected. At this time, the stirring region between the electrode pairs arranged in the reaction cell is preferable as the detection region to be irradiated with light. In the case of the competitive method, the washing step and the detection are the same except that the sample solution and the labeling reagent solution are simultaneously dropped onto the liquid holding section 6 to cause a competitive reaction.

Further, if a calibration curve such as fluorescence intensity is obtained from a standard sample solution in which the concentration of the trace substance is known, it is possible to quantify the trace substance in the unknown sample liquid.

The light source 10 may be a non-coherent light source such as a xenon lamp, a halogen lamp, a tungsten lamp, an LED, or a coherent light source such as a laser beam. As the photodetector 11, a photomultiplier, a photodiode, a photocell, or the like can be used.

[0063]

EXAMPLES The present invention will be described in detail below with reference to examples.

Example 1 In this example, the detection apparatus of the present invention was constructed using the reaction cells shown in FIGS. 6 and 7.

FIG. 6 is a perspective view thereof, and FIG. 7 is a sectional view. First, a method for manufacturing the reaction cell in this example will be described.

A hole 15 having a diameter of 8 mm was made in an acrylic plate 15 having a thickness of 3 mm and a size of 20 mm × 30 mm, and a filter 16 made of cellulose (thickness: 500 μm, diameter: 1
0 mm, pore diameter: 0.2 μm) was adhered and stuck. At this time, the cellulose filter 16 is preliminarily set to the human CRP.
Sensitization with an antibody (IgG fraction) was carried out in advance to form a reagent part region composed of a solid-phased reagent.

Next, the electrodes 17 and 17 'made of a gold-tungsten wire having a conical tip (diameter: 50 μm, tip diameter: 10 μm or less) are opposed to each other on the filter 16 at the center of a hole having a diameter of 8 mm at a tip interval of 200 μm. And an acrylic plate 18 having a hole of 3 mm in thickness and 8 mm in diameter thereon.
Were bonded by sandwiching the electrodes 17 and 17 '.

Next, an acrylic plate 19 having a hole of 0.5 mm thickness and a diameter of 4 mm was placed thereon as a lid plate.

Using the reaction cell manufactured as described above, a detection device having a structure as shown in FIGS. 8 and 9 was manufactured.

In these figures, 20 is a reaction cell, 21 is an arm, 22 is a dispenser tip, 23 is an Ar ion laser, 24 is a photomultiplier tube, 25 is a rubber sheet, 26
Is a condenser lens, 27 is a dichroic mirror, 28 is a liquid suction port, 29 is an electrode outlet, 30 and 31 are fans, 3
2 is a temperature sensor.

The measuring device has a working space 33 in which work such as holding of the reaction cell 20 is carried out, in which air of a constant temperature from the air conditioner is circulated by the fans 30 and 31, and a signal from the temperature sensor 32 is sent. By controlling the temperature and circulation amount of the air from the air conditioner, the desired temperature can be achieved.

FIG. 8 is an explanatory diagram in the case of dropping a sample liquid containing a trace substance, a labeling reagent liquid, and a washing liquid into the reaction cell 20. The tip of the dispenser is composed of a dispenser tip 22 of a removable synthetic resin molded body, sucks and discharges the liquid, and replaces the dispenser tip 22 according to the type of the liquid to prevent contamination between a plurality of liquids. I'm losing. Further, the dispenser can be moved between the upper portion of the reaction cell and the container (not shown) in which the liquid to be dropped is present by the arm 21.

FIG. 9 is an explanatory diagram for measuring the fluorescence intensity from the reagent section region on the filter in the reaction cell. A
The laser light from the r-ion laser 23 is irradiated onto the reagent portion area by the condenser lens 26. The scattered light of fluorescence and laser light from the reagent area is collected again by the condenser lens 26, reflected only by the dichroic mirror 27 and separated from the laser light, and then incident on the photomultiplier tube 24 and detected. It

In this example, first, the human CRP antibody (I
The reaction cell in which the (GG fraction) is sensitized on the filter is set on the rubber sheet 25 of the detection device. At this time, the electrode in the cell is connected to the connector 29 for voltage application.
Next, the lid plate of the reaction cell is slid to open the liquid holding portion, and the sample liquid 200 containing a trace substance is dispensed with a dispenser.
μl was dropped on the liquid holding portion of the reaction cell, and then the lid plate of the reaction cell was slid and closed.

While maintaining the temperature of the working space 33 of the measuring apparatus at 37 ° C., 1 MHz between the electrodes in the reaction cell, ± 12
A rectangular wave voltage of V was applied to stir the sample solution on the filter for 15 minutes.

After that, the voltage application was stopped, and suction was applied from the back surface of the filter to remove the excess sample liquid on the filter.

Next, a fluorescent labeling reagent and a FITC-labeled human CRP antibody (IgG fraction) were added by a dispenser.
200 μl of a labeling reagent solution dissolved in phosphate buffer solution-physiological saline (hereinafter referred to as PBS) having a pH of 7.2 to a concentration of 2 mg / ml was dropped in the same manner as the sample solution.
The steps up to removal are repeated. Next, the cleaning liquid (PB
150 μl of S) is dropped and the washing solution is similarly removed from the filter. This process using the washing solution is repeated 3 times for washing, and finally, after sliding the lid plate open,
Move the condenser lens to the reagent area on the filter, and
^{+ When} irradiated with laser light, fluorescence was obtained from the reagent area, and the substance to be measured in the sample solution could be detected.

Comparative Example 1 In Example 1, except that no voltage was applied to the electrodes in the reaction cell when the sample liquid was allowed to act on the reagent part region to cause a reaction.
The same operation was performed. When the fluorescence intensity from the reagent area on the filter was measured in the same manner, it was 1/2 of that of Example 1.
Only the strength of was obtained. In order to obtain the same fluorescence intensity, the retention time during the reaction was required to be twice that of Example 1.

Example 2 As shown in FIG. 10, an electrode pattern 3 was formed on a styrene sheet 35 (30 mm × 40 mm) having a thickness of 500 μm.
6, 37, 38 and 39 were created by the printing method.

Next, in the region shown by the dotted line in FIG. 10, human β
A 2-microglobulin antibody was immobilized to form a reagent part region composed of a solid-phased reagent.

Next, the electrode patterns 36, 37, 38, 3
Gold-tungsten wire (diameter 50 μm, tip diameter 10 μm or less) electrodes 41, 42, 43, 44 (distance between opposing electrodes 150 μm) were connected to the end of 9 and, as shown in FIG. An acrylic plate 40 (30 mm × 30 mm) having a thickness of 3 mm, in which a 6 mm square hole was formed, was placed in the plate, and these were adhered to each other to prepare a reaction cell as shown in FIG.

13 and 14 show the structure of the detection device used in this embodiment.

The reaction cell 46 is set on the moving stage 47. On the moving stage 47, there are connectors that connect to the four electrode terminals of the reaction cell.
A cable 53 is connected from 7 to an external power source (not shown). In addition, since a heater is provided inside the moving stage 47, it can be maintained at a constant temperature.

FIG. 13 shows an arrangement for injecting a liquid into the reaction cell 46. A dispenser 52 injects a required amount of liquid (sample solution, reagent solution, etc.).

FIG. 14 shows the arrangement for optically detecting the fluorescence intensity from the reagent section region of the reaction cell. The light from the Xe lamp 48 has only the wavelength required by the bandpass filter 54. Is selected, and the condenser lens 49
The reagent area is irradiated with. The transmitted light and fluorescence from the reagent area are collected by the condenser lens 49 ', and only the fluorescence is transmitted by the bandpass filter 50, and then the photomultiplier tube 5
Detected by 1.

The following experiment was conducted using this detector.

First, 100 μl of the sample liquid containing the substance to be measured is injected into the reaction cell in the arrangement state as shown in FIG. 13, and then the reaction cell is set on the moving stage 47 of the detection device to face the inside of the reaction cell. 1MHz between two pairs of electrodes
A rectangular wave voltage of 10 V was applied for 8 minutes to perform stirring (at this time, the temperature of the moving stage was maintained at 37 ° C).

Next, the voltage application is stopped and the dispenser 5
Excess sample solution was removed by 2 and subsequently 100 μl of human β2-microglobulin antibody (polyclonal antibody IgG fraction) enzymatically labeled with alkaline phosphatase
(Concentration: 0.2 mg / ml) was added dropwise, and voltage was applied again and held for 15 minutes. After that, the excess labeling reagent solution is removed by the dispenser 52 in the same manner, and the washing with PBS is similarly performed twice.

After washing, 1 ml of a substrate solution containing 4-methylumbelliferyl phosphate (4-MUP) was dropped, and a voltage was applied for 3 minutes, and then the moving stage was moved to show the condition shown in FIG. In such an arrangement state, 4-methylumbelliferone (4
-MU) was detected by using the above-mentioned optical detection means for fluorescence from the reagent part region, whereby the substance to be measured in the sample liquid could be detected.

COMPARATIVE EXAMPLE 2 Except that no voltage was applied to the electrodes in the reaction cell when the sample liquid was made to act on the reagent part region and reacted in Example 2,
When the same experiment was performed, the detected fluorescence intensity was 3/4 of that in Example 2.

In order to obtain the same fluorescence intensity, the holding time during the reaction was 1.5 times that in Example 2.

[0092]

As described above, according to the present invention,
In the reaction step of binding a solid phase reagent, a trace substance in a sample solution or a detection reagent, and further in a washing step with a washing solution, ions in a liquid in a minute region (sample solution, reagent solution, washing solution, etc.) near the electrodes Since the liquid is agitated by applying an electromagnetic force, the reaction and the washing can be efficiently performed, the time required for the reaction step and the washing step can be shortened, and the trace substance can be detected in a shorter time. In addition to being able to do so, there is an effect that a trace substance can be detected even with a very small amount of sample liquid.

[Brief description of 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 a liquid stirring method.

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

FIG. 3 is a diagram showing a configuration example of a reaction cell used in the detection apparatus of the present invention.

FIG. 4 is a diagram showing an example of liquid supply and discharge in the detection device of the present invention.

FIG. 5 is a diagram showing an example of an optical detection method in the detection apparatus of the present invention.

6 is a perspective view of a reaction cell used in Example 1 and Comparative Example 1. FIG.

7 is a cross-sectional view of the reaction cell of FIG.

FIG. 8 is a diagram showing an arrangement configuration of the detection apparatus of the present invention used in Example 1 and Comparative Example 1 during liquid supply.

FIG. 9 is a diagram showing an arrangement configuration during fluorescence detection of the detection device of the present invention used in Example 1 and Comparative Example 1.

10 is an electrode pattern diagram for the reaction cell used in Example 2 and Comparative Example 2. FIG.

11 is a configuration diagram of a reaction cell used in Example 2 and Comparative Example 2. FIG.

12 is an overall perspective view of a reaction cell used in Example 2 and Comparative Example 2. FIG.

FIG. 13 is a diagram showing an arrangement configuration of the detection apparatus of the present invention used in Example 2 and Comparative Example 2 during liquid supply.

FIG. 14 is a diagram showing an arrangement configuration during fluorescence detection of the detection apparatus of the present invention used in Example 2 and Comparative Example 2.

FIG. 15 is an example of a voltage waveform that periodically changes.

[Explanation of symbols]

 1 Working electrode 2 Magnetic field 3 Electric force line 4 Counter electrode 5 Reaction cell 6 Liquid holding part 7 Filter 8 Reagent part area 9 Dispenser 10 Light source 11 Photodetector 15 Acrylic plate 16 Cellulose filter 17, 17 'Needle electrode 18 Acrylic plate 19 Lid Plate 20 Reaction Cell 21 Arm 22 Dispenser Chip 23 Ar Ion Laser 24 Photomultiplier Tube 25 Rubber Sheet 26 Condensing Lens 27 Dichroic Mirror 28 Liquid Suction Port 29 Electrode Outlet 30, 31 Fan 32 Temperature Sensor 33 Working Space 35 Polystyrene Sheet 36 , 37, 38, 39 Electrode pattern 40 Acrylic plate 41, 42, 43, 44 Gold-tungsten wire 45 Detector 46 Reaction cell 47 Moving stage 48 Xe lamp 49, 49 'Focusing lens 50, 54 Bandpass Iruta 51 photomultiplier 52 dispenser 53 cable

Claims (9)

[Claims] 1. A solid-phased reagent carrying on a solid material a bio-related substance that selectively and specifically binds to a trace substance in a sample liquid, the trace substance and / or one or more detection reagents A method of detecting the trace substance by forming a reaction conjugate, wherein the sample solution and the detection reagent solution have ionic conductivity, and the liquid having ionic conductivity is introduced between the electrode pair, A method for detecting a trace substance, characterized in that a reaction step of the solid phase reagent, the trace substance, and the detection reagent is performed while agitating the liquid by applying a voltage that fluctuates cyclically between the pair of electrodes. 2. After the reaction step, there is a cleaning step with a cleaning liquid having ionic conductivity, the cleaning liquid is introduced between the electrode pairs, and a cyclically varying voltage is applied between the electrode pairs. The method for detecting a trace substance according to claim 1, wherein the washing step is performed while stirring the washing liquid. 3. The method for detecting a trace substance according to claim 1, wherein at least one of the detection reagents is a labeling reagent having a labeling substance. 4. The method for detecting a trace substance according to claim 3, wherein the labeling substance in the reaction conjugate bound on the solid phase reagent between the electrode pair is detected. 5. The method for detecting a trace substance according to claim 1, wherein at least one of the detection reagents is a reagent that selectively and specifically binds to the trace substance. 6. The method for detecting a trace substance according to claim 1, wherein at least one of the detection reagents is a reagent that selectively and specifically binds to the solid phase reagent. 7. The method for detecting a trace substance according to claim 1, wherein the periodically varying voltage is an alternating voltage. 8. An apparatus for carrying out the method for detecting a trace substance according to claim 1, comprising a reaction cell provided with at least one or more pairs of electrodes, and between the pair of electrodes. A trace substance detection apparatus comprising: a power supply unit that applies a voltage that fluctuates cyclically; and a unit that detects a labeling substance in a reaction conjugate in the reaction cell. 9. The trace substance detection device according to claim 8, wherein the detection means is an optical detection means.