JP7130223B2 - Antigen-antibody reaction detector using charge transition difference - Google Patents

Description

The present invention relates to an apparatus for electrically detecting or measuring the presence of a target substance (target antigen) in a test sample.

Various methods have been developed for the detection of biomolecules, but most of them label substances that show specific reactions to the target of detection by some means (e.g. fluorescence, enzymes, radioisotopes). is. In recent years, methods have also been developed for detection without using labeling means, using quartz crystal oscillators (QCM), surface plasmons (SPR), and the like.

However, the method using fluorescence or SPR requires an optical device, which makes it difficult to reduce the size of the device, resulting in cost problems. In addition, methods using enzymes or radioactive isotopes require dedicated reagents and measurement equipment, and must be handled with care. Furthermore, the method using QCM detects the attachment of a substance to the sensor surface as a change in mass, and therefore does not require a labeling reagent, but has the problem of low detection sensitivity.

In order to solve these problems, a method of electrically detecting the presence of biomolecules using a field effect transistor (FET) has been proposed (eg, Patent Document 1). The method using the FET does not require an optical device because it electrically detects the charge of the object, and is expected to reduce the cost and size of the device. However, the method using the FET has a specific problem of limitation of the detection area (Debye length) existing on the surface of the sensing portion. When the biomolecules to be detected are located beyond this Debye length, the counter ions in the solution cancel the charge of the target, making detection impossible. In particular, this problem occurs remarkably when trying to detect an antigen-antibody reaction. While the typical size of an antibody is about 10 nm, the Debye length on the surface of the gate insulating film in a physiological salt solution is about 1 nm. The portion (antigen-binding site) that binds to the antibody sometimes exceeds the Debye length of the surface of the sensing portion, making stable detection difficult.

In response to the above problems, the present inventors expanded the Debye length of the sensing part surface by using pure water as a measurement solvent in an FET sensor for electrically detecting an antigen-antibody reaction, thereby dramatically improving detection sensitivity. It is proposed to raise it exponentially (Patent Document 2).

JP 2009-133800 WO2017/033922

The invention described in Patent Document 2 is a useful technique in that the detection limit concentration of the target substance is extremely low and the detection sensitivity is extremely high as compared with the prior art. However, when the present inventors further studied the method described in Patent Document 2, the method described in Patent Document 2 uses pure water (or low ionic solution) as a measurement solvent, so it is generally used as a measurement solvent. The present inventors have found that there is a further problem that the antigen-antibody complex to be measured is destabilized, compared to the case of using a buffer solution that is less complex.

Generally, it is known that the Debye length is inversely proportional to the ionic strength (concentration) in the measurement solution. To increase the sensitivity by increasing the Debye length, the ionic strength (concentration) of the measurement solution should be lowered. . However, since the three-dimensional structure of the protein is maintained by the ionic charge of the solution, a decrease in ionic strength (concentration) will destroy the three-dimensional structure of the protein. In particular, the bonds involved in the antigen-antibody reaction are non-covalent bonds that are strongly influenced by the ionic strength (concentration) of the surroundings, so lowering the ionic strength (concentration) to increase the Debye length changes the three-dimensional structure of the antibody. As a result, the binding between the antigen and the antibody is broken, and the antigen to be detected is separated from the antibody.

That is, the invention described in Patent Document 2 achieves a low detection limit concentration and high detection sensitivity by using pure water (low ion solution) as a measurement solvent, but on the other hand, using pure water as a measurement solvent As a result, there was a technical dilemma that the antigen-antibody complex to be measured was destabilized.

The present inventors have extensively studied methods for solving the above technical dilemma while achieving a low detection limit concentration and high detection sensitivity. As a result, the present inventors focused on the destabilization of the antigen-antibody complex itself in a low-ion solution, and by sequentially measuring the surface charge of the antigen-antibody complex in a low-ion solution, the subject The inventors have found that it is possible to determine the presence or absence of antigen-antibody reaction in , and have completed the present invention.

That is, in one embodiment, the present invention is an apparatus for electrically measuring a specific antigen in a test sample,
The device comprises an electrode, a measuring solution providing means, and a measuring means,
The electrodes are electrically connected to the measuring means,
A sample liquid containing a test sample can be placed on the surface of the electrode, and when the test sample contains an antigen, at least part of the antigen specifically recognizes the antigen. conjugated to an antibody or antibody fragment that
The measurement liquid providing means includes a measurement liquid,
The measurement liquid providing means can provide the measurement liquid to the surface of the electrode so that the sample liquid placed on the electrode can be at least partially replaced by the measurement liquid,
The measuring means is characterized in that it measures changes in the potential or current of the electrodes during the replacement over time.

Further, in one embodiment of the present invention, the antibody is bound to magnetic particles in the sample liquid.

Moreover, in one embodiment of the present invention, the apparatus further comprises a magnetic force source, and the magnetic force source is provided so as to attract magnetic particles in the sample liquid to the electrode surface by magnetic force. Characterized by

Further, in one embodiment of the present invention, the magnetic force source is a paramagnetic magnet or an electromagnet.

Moreover, in one embodiment of the present invention, the antibody is directly bound to the surface of the electrode.

Further, in one embodiment of the present invention, the provision of the measurement liquid by the measurement liquid providing means is performed continuously or temporarily.

In one embodiment of the present invention, the measurement liquid providing means further includes a pump, and the measurement liquid is provided by the measurement liquid providing means by the pump.

In one embodiment of the present invention, the device further comprises a flow path, the electrodes are present in the flow path, and the measurement liquid provided by the measurement liquid providing means passes through the flow path. and passes through the surface of the electrode.

Further, in one embodiment of the present invention, the measurement liquid is a solution having a specific resistance of 50 kΩcm or more.

Further, in one embodiment of the present invention, the measurement liquid is the same solution as the solvent of the sample liquid, the concentration of which is diluted by at least 100 times.

In one embodiment of the present invention, the measurement solution is 1 μM to 100 μM phosphate buffer or Good's buffer, and the solvent of the sample solution is 10 mM to 500 mM phosphate buffer or Good's buffer. It is characterized by

Also, in one embodiment of the present invention, the device further comprises a sample liquid providing device,
The sample liquid providing device is characterized in that the sample liquid can be placed on the surface of the electrode.

Further, in one embodiment of the present invention, the measurement results obtained when the sample solution is used are replaced with the measurement results obtained when one or a plurality of control solutions having a known concentration of the antigen are used in place of the sample solution. and measuring the concentration of the antigen in the sample liquid, or detecting the presence of the antigen in the sample liquid.

Further, in one embodiment of the present invention, the one or more control solutions include those with a concentration of 0 for the antigen.

Further, in one embodiment of the present invention, the measurement result to be compared is the potential or current at a predetermined time, the time to reach the predetermined potential or current, or the potential or current in a predetermined time range times the integral of time. value.

Moreover, in one embodiment of the present invention, the electrodes are arranged in parallel or integrated.

Moreover, one embodiment of the present invention is characterized in that the antigen is a molecule that can exist in vivo.

Further, in one embodiment of the present invention, the electrode surface further comprises a three-dimensional structure for controlling the arrangement of the magnetic particles, and/or the surface is modified for controlling the arrangement of the magnetic particles. It is characterized by

In one embodiment of the present invention, the three-dimensional structure is a hole, groove, pillar, or wall structure, and the surface modification is hydrophilic treatment or hydrophobic treatment.

Also, in one embodiment of the present invention, the measuring means includes a field effect transistor or a signal amplifier.

It should be noted that inventions in which one or more of the above-described features of the present invention are arbitrarily combined are also included in the scope of the present invention.

Compared with the invention described in Patent Document 2, the present invention is advantageous in that stable measurement is possible while realizing highly sensitive measurement by using a low ion solution as a measurement solvent.
In addition, the measurement of the target substance according to the present invention does not require labeling of the target substance (radioisotope, enzyme, fluorescent/luminescent reagent, etc.). Compared to methods of detecting target substances using radioactive isotopes (RI), the present invention is easy to handle and easy to treat after use. In addition, compared with measurement methods using enzymes, fluorescence, luminescence reagents, etc., this method is advantageous in that it does not require a large-sized optical device for measurement.
Furthermore, compared to methods using quartz crystal oscillators (QCM) and surface plasmons (SPR), the present invention does not require a large optical system device and does not require special pretreatment of the electrode surface. is advantageous in that

FIG. 1 is a schematic diagram of the apparatus (whole) of the present invention. FIG. 2 is a schematic diagram of the measuring device portion of the apparatus of the invention. FIG. 3 shows the results of continuous measurement of the surface potential of the gate electrode on which the sample was placed using the apparatus of the present invention. FIG. 4 is a graph showing the amount of change in gate surface potential at 250 seconds in the graph of FIG.

The present invention utilizes the difference in physicochemical states that antigen-antibody complexes are stable in a high ion environment and unstable in a low ion environment to measure a specific antigen in a test sample. It is an invention based on the principle of
Therefore, the "measurement liquid" in the present invention is preferably a low ion solution. The low-ion solution used as the measurement solution of the present invention is not limited as long as it has an ion concentration that destabilizes the antigen-antibody complex. As described above, a solution having a resistance of 500 kΩcm or more, 600 kΩcm or more, 700 kΩcm or more, 800 kΩcm or more, 900 kΩcm or more, or 1 MΩcm or more may be used.

In addition, for example, the solvent (usually buffer solution) of the sample solution in which the test sample is stored before the measurement using the present invention is diluted at least 100 times or more. good. Specifically, the solvent of the "sample solution containing the test sample" in the present invention is a 10 mM to 500 mM buffer solution (e.g. , 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM buffer), and the "measurement solution" in the present invention is a buffer solution of 1 μM to 100 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM , 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM buffer). In addition, the solvent of the "sample solution containing the test sample" in the present invention and the "measurement solution" in the present invention may preferably be phosphate buffer or Good's buffer, and both are different types of solutions (or different type of buffer). In general, the term "specific resistance" may also be used as the term "electrical resistivity."

In the present invention, an antibody or antibody fragment against a specific antigen to be measured is used to detect the antigen. The antibody or antibody fragment may be used in any manner as long as it can be used in a method using the device of the present invention. For example, as shown in the examples of the present application, magnetic particles having antibodies against specific antigens bound to their surfaces are brought into close contact with the surface of the gate electrode by magnetic force, so that changes in the antigen-antibody complexes on the surfaces of the magnetic particles are observed. The resulting change in potential (or change in current) can be measured. For such techniques, see WO2017/033922, for example. A similar measurement can also be performed, for example, by directly binding an antibody against a specific antigen to the surface of the gate electrode of the present invention.

When magnetic particles are used in the present invention, various types of materials can be used for the magnetic particles. For example, magnetite (Fe ₃ O ₄ ), various ferrites (XFe ₂ O ₄ , X=Mn, Co, Ni, Mg, Cu, Li _0.5 , Fe _0.5 etc.), diiron trioxide (Fe ₂ O ₃ ) and so on. Also, the type of magnetic force source for attracting magnetic particles is not limited, but may be, for example, a paramagnetic magnet or an electromagnet.

By using the device of the present invention, various trace substances detectable by antibodies can be detected. For example, the device of the present invention can be used to detect substances of biological origin, substances in the environment, or substances in food. In particular, the present invention enables detection even when the concentration of a substance to be detected in a test sample is extremely low. , semen, vaginal fluid, amniotic fluid, milk, etc.). Examples of substances in body fluids include body fluid components (eg, alkaline phosphatase, AST, ALT, lactate dehydrogenase, leucine aminopeptidase, γ-GTP, creatine kinase, cholinesterase, bilirubin, bile acid, albumin, urea nitrogen, , creatinine, uric acid, HDL cholesterol, LDL cholesterol, triglyceride, glucose, amylase, lipase, sodium, potassium, chloride, calcium, inorganic phosphorus, magnesium, zinc, iron, ferritin, C-reactive protein, β2-microglobulin, Hemoglobin A1C, glycoalbumin, ammonia, various hormones, various neurotransmitters (e.g., monoamines such as catecholamine, serotonin, melatonin, histamine; amino acids such as aspartic acid, glutamic acid, γ-aminobutyric acid, glycine, taurine; acetylcholine, various Neuropeptides such as hormones), components that are test items for general blood biochemical tests), disease-related biomarkers (e.g., tumor markers, autoimmune disease markers, central nervous system disease markers, heart disease biomarkers, etc.) ), pathogens (eg, viruses, bacteria, fungi, parasites, etc.) and their associated agents, drug molecules of previously administered drugs.

As the antibody or antibody fragment used in the present invention, a commercially available antibody or antibody fragment may be used, or a self-made antibody or antibody fragment may be used. Preferably, a commercially available antibody or antibody fragment known to bind to the antigen to be measured is used.

The measuring means in the apparatus of the present invention is not limited as long as it can measure the electrical change of the object to be measured as a change in potential or a change in current. For example, it includes a field effect transistor (FET). good. A field effect transistor is formed by forming a drain and a source with an n-type semiconductor on a p-type semiconductor, and forming a gate by insulating a silicon oxide on the channel of the p-type semiconductor. It is a type of transistor with a MOS) structure. An FET can change the current flowing between the drain and the source by generating an electron depletion layer in the channel by applying a potential to the gate. A field effect transistor for use in the present invention may be, for example, an Ion Sensitive Field Effect Transistor (ISFET).

In one embodiment of the invention, an Ion Sensitive FET can be used as part of the measurement means. In the Ion Sensitive FET, as a premise of its measurement principle, it is necessary to form an ion layer on the surface of the gate electrode. When a substance (having charged particles) comes into contact with the layer of ions formed on the surface of the gate electrode, an electrical change occurs on the surface of the gate electrode. The electrical change that occurs on the surface of the gate electrode varies depending on the electrical state of the contacting substance (for example, the presence or absence of bonding with other substances). By comparing the values measured before the change in electrical state occurred (ie, the control), the presence or absence of a change in the electrical state of the substance can be detected.

When a field effect transistor is used as part of the measuring means of the present invention, the surface of the gate electrode may be processed in any way as long as it can be used in the method of the present invention. Those surface-treated so as to generate surface functional groups in a solution (for example, in water or in a buffer solution) can be preferably used. Examples of such surface treatments include surface coating with oxides or nitrides (more particularly metal oxides or metal nitrides), more preferably tantalum oxide, aluminum oxide, nitride A surface coating of silicon, titanium oxide, or silicon oxide can be used.

In addition, among metals generally used as the gate surface of field effect transistors, even when using metals that do not substantially generate surface functional groups in solution (for example, gold, platinum, etc.), hydroxyl groups, carboxyl It is used in the present invention by further modifying the surface with functional groups such as groups, or by further coating with substances that generate surface functional groups in solution (e.g., organic substances such as oxides, nitrides, polymers, etc.). be able to. The method for modifying the metal surface with functional groups is not particularly limited, and can be appropriately carried out using methods known to those skilled in the art.

Also, for example, the measurement aimed at by the present invention can be performed by connecting an electrode for electrically changing the object to be measured to a signal amplifier (eg, a vacuum tube, a transistor, an operational amplifier, etc.).

The electrodes used in the device of the present invention may be paralleled or integrated for more accurate measurements. Further, when magnetic particles having surfaces bound to antibodies against the antigen are used for the detection of a specific antigen, on the electrode surface used in the device of the present invention, there is a method for controlling the arrangement of the magnetic particles. A three-dimensional structure may be provided, and surface modification may be performed to control the arrangement of the magnetic particles. Examples of conformations for controlling the placement of magnetic particles on the electrode surface include holes, grooves, pillars, or wall structures. Examples of surface modification for controlling the arrangement of magnetic particles on the electrode surface include hydrophilic treatment and hydrophobic treatment.

Terms used herein are used to describe particular embodiments and are not intended to be limiting of the invention, unless otherwise defined.

In addition, the term "comprising" used in this specification means that the stated items (members, steps, elements, numbers, etc.) are present, unless the context clearly requires a different understanding. It does not exclude the existence of other matters (members, steps, elements, numbers, etc.).

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used herein are to be construed as having a meaning consistent with that in this specification and related art, unless a different definition is expressly provided, and are idealized or overly formal. should not be interpreted in any meaningful sense.

In the following, the invention will be explained in more detail with reference to examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Electrical detection of BNP (brain natriuretic peptide) antigen-antibody reaction

(1) Measuring Apparatus of the Present Invention FIG. 1 shows a conceptual diagram of the measurement performed in this example, and FIG. 2 shows a schematic diagram of the measuring device portion of the measuring apparatus that is one embodiment of the present invention. 1 and 2 show an example of a device using an ion sensitive field effect transistor (ISFET) as part of the measuring means.

The apparatus shown in FIGS. 1 and 2 includes a gate electrode (114) for measuring the charge of the object to be measured, measurement liquid providing means (measurement liquid tank (102), liquid feeding tube (106), pump (101)). , and an ISFET (113) and a potential measuring device (111) that constitute measuring means, and the gate electrode (114) is electrically connected to the ISFET (113) and the potential measuring device (111). The surface of the gate electrode (114) is configured so that the sample liquid containing the test sample introduced from the sample inlet (120) can be placed thereon.

As will be described later, the antigen to be detected in this example is BNP (brain natriuretic peptide), and "anti-BNP antibody-coated magnetic particles" in which the surface of the magnetic particles is coated with an anti-BNP antibody are reacted with BNP. was used for the test as the magnetic particle sample (108). A magnet (115) is installed below the electrode (114) in the apparatus, and when a magnetic particle sample (108) is placed on the electrode (114), the sample (108) is attracted to the electrode surface by magnetic force. be done.

The measurement liquid reservoir (102) contains a measurement liquid (103), which is a low ionic solution, and the measurement liquid (103) is applied by the pump (101) to the measurement solution inlet (121) of the device. The measurement solution (103) applied to the measurement solution inlet (121) is discharged from the measurement solution outlet (122) through the channel formed on the measurement channel jig (118) of the apparatus. Note that the channel is configured to pass over the electrode (114).

By configuring the device as described above, the solvent (usually a buffer solution that is a high ionic solution) of the sample solution containing the test sample placed on the electrode (114) is replaced with the measurement solution that is a low ionic solution ( 103), the charge of the object to be measured can be measured over time.

(2) Preparation of anti-BNP antibody-coated magnetic particles Dynabeads MyOne Tosylated (Φ1 μm, 100 mg beads/mL, Veritas Co., Ltd.) was used as the magnetic particles used in this example, and the surface was modified with an anti-BNP antibody by the following method. let me
1. A 75 μL aliquot from the original vial was washed according to the product protocol.
2. To the washed magnetic particles, 100 μg of dialyzed anti-BNP antibody (ab20984, abcam) was added and reacted by slowly rotating and stirring at 37° C. for 24 hours.
3. After the reaction, discard only the supernatant using a magnet, add blocking buffer (PBS pH 7.4 with 0.5% BSA and 0.05% Tween 20), and further rotate and stir at 37°C overnight. A blocking reaction was performed.
4. The supernatant was discarded and washed three times with wash buffer (PBS pH 7.4 with 0.1% BSA and 0.05% Tween20).
5. The washed magnetic particles were dispersed in 150 μL of storage buffer (PBS pH 7.4 with 0.1% BSA, 0.05% Tween 20 and 0.02% sodium azide) to obtain stock anti-BNP antibody-coated magnetic particles. (50 mg beads/mL) and stored at 4°C.

３．良く分散させた後、３０℃で１時間、回転撹拌することで反応を行った。
４．反応後、上澄み液のみを破棄しＰＢＳ（－）で３回良く洗浄した。
５．洗浄されたそれぞれの磁性粒子を５μＬのＰＢＳ（－）でよく分散した後、１μＬずつ小分けにして測定まで４℃で保管した。 (3) Preparation of magnetic particles subjected to BNP antigen-antibody reaction The antigen used in this example was a brain natural peptide ((1-32), human, 3522, TOCRIS), and the antigen-antibody reaction was carried out by the following procedure. rice field.
1. From the anti-BNP-coated magnetic particles for stock, 1 μL each for antigen detection/control was separated into separate Eppendorf tubes, and then thoroughly washed three times with PBS(−).
2. After discarding the supernatant, a reaction solution was added to each magnetic particle according to the table below.

3. After well dispersed, the reaction was carried out by rotating and stirring at 30° C. for 1 hour.
4. After the reaction, only the supernatant was discarded and well washed with PBS(-) three times.
5. Each washed magnetic particle was well dispersed in 5 μL of PBS(−), divided into 1 μL aliquots, and stored at 4° C. until measurement.

Antigen-antibody complexes on the surface of magnetic particles are stable in PBS(-), which is a highly ionic environment.

(4) Washing of Detection Electrode The detection electrode used in the measurement was an ISFET (Ice Fetocom Co., Ltd.), and the sensor surface was made uniform by washing the sensor as follows after each measurement.
1. After vigorously spraying 70% ethanol on the sensor portion, it was rinsed with running ultrapure water.
2. Ultrasonic cleaning at 40 kHz was performed for 10 seconds each with acetone, methanol, and ultrapure water. At this time, the sensor can be efficiently washed by inserting and withdrawing it from each liquid.
3. After removing moisture with a blower, UV irradiation was performed for 2 minutes with a UV/O3 cleaning and modifying device (SKB2001N-01, Sun Energy Co., Ltd.).
4. It was immersed in ultrapure water and kept in this state until measurement.

(5) Electrical detection of BNP antigen-antibody reaction Electrical detection of the BNP antigen-antibody reaction is performed by a measurement system (Fig. 1) was used to perform real-time measurements. The measurement conditions are Vref: 2.5 V, Is: 100 μA, the measurement liquid (103) flowing in the measurement flow path jig (118) is 10 μM phosphate buffer (low ionic solution), and the flow rate is about 275 μL/min. set to
1. After confirming that the drift of the measured potential obtained from the potential measuring device (111) had subsided, the pump (101) was stopped to stop the movement of the liquid to be measured flowing through the channel.
2. The magnetic particles prepared in (3) above and stored at 4° C. were well dispersed and added to the gate portion (114) of the ISFET from the magnetic particle sample inlet (120) using an automatic pipetman.
3. After 10 to 15 seconds, the pump (101) was restarted to generate a flow of the measurement solution in the channel, thereby washing away the PBS (-) surrounding the added magnetic particles and measuring the potential.

By performing the above measurements on all the prepared magnetic particle samples and control samples and comparing the obtained potential measurement results, the presence or absence of BNP antigen-antibody reaction can be electrically distinguished as a difference in the transition of the gate surface potential. rice field.

FIG. 3 shows the results of the measurement experiment. The vertical axis indicates the amount of potential change (mV) on the gate surface of the ISFET due to the added magnetic particles, and the horizontal axis indicates the measurement time (sec). Also, the results of the magnetic particles subjected to the BNP antigen-antibody reaction are indicated by "BNP", and the results of the control sample are indicated by "No-BNP".

As shown in FIG. 3, a clear difference in transition of the gate surface potential was confirmed between the results of the magnetic particles subjected to the BNP antigen-antibody reaction and the results of the magnetic particles not subjected to the antigen-antibody reaction.

FIG. 4 shows the average amount of change in the gate surface potential at N=5 in 250 seconds (the above evaluation time) in the above graph. The vertical axis indicates the amount of potential change (mV) on the gate surface of the ISFET due to the added magnetic particles, and the horizontal axis indicates "BNP" for the magnetic particles used in the measurement that were subjected to the BNP antigen-antibody reaction, and "Control" for " No-BNP”.

As shown in FIG. 4, the presence or absence of antigen-antibody reaction using 1 μg/mL BNP antigen could be clearly confirmed as an electrical difference of about 80 mV.

As described above, by using the apparatus of the present invention, it was demonstrated that the antigen-antibody reaction can be stably and highly sensitively measured while using a low-ion solution as the measurement liquid.

100: Measuring device part 101: Pump 102: Measuring liquid tank 103: Measuring liquid 104: Waste liquid tank 105: Waste liquid 106: Liquid sending tube 107: Lead wire 108: Magnetic particle sample 109: Noise cut shield case 110: Wiring 111: Potential measurement Instrument 112: PC with measurement software
113: ISFET
114: Gate portion of ISFET 115: Magnet 116: ISFET fixing jig 117: Silicon rubber 118: Measurement channel jig 119: Reference electrode 120: Magnetic particle sample inlet 121: Measurement solution Inlet
122: Measurement solution Outlet
123: jig fixing screw

Claims (18)

A device for electrically measuring a specific antigen in a test sample,
The device comprises:
electrode,
measurement liquid providing means,
a sample liquid providing device, and
comprising measuring means electrically connected to the electrodes;
The sample liquid providing device is
magnetic particles;
an antibody that specifically recognizes the specific antigen, bound to the magnetic particles;
placing a sample solution containing the antigen in the test sample specifically recognized by the antibody and forming an antigen-antibody complex with the antibody on the surface of the electrode;
The measurement liquid providing means provides a measurement liquid having an ion concentration lower than that of the sample liquid on the surface of the electrode, at least partially replaces the sample liquid with the measurement liquid, and displaces the antigen-antibody complex. stabilize,
The measurement means measures changes in potential of the electrode over time due to destabilization of the antigen-antibody complex.
Device. 2. The device of claim 1, wherein
The device further comprises a magnetic force source,
The magnetic force source is provided so that the magnetic particles in the sample liquid can be attracted to the electrode surface by magnetic force,
Device. 3. The apparatus of claim 2, wherein
The magnetic force source is a paramagnetic magnet or an electromagnet,
Device. A device according to any one of claims 1 to 3,
The provision of the measurement liquid by the measurement liquid providing means is performed continuously or temporarily,
Device. 5. A device according to any one of claims 1 to 4,
The measurement liquid providing means comprises a pump,
The provision of the measurement liquid by the measurement liquid providing means is performed by the pump,
Device. 6. A device according to claim 5, wherein
The device further comprises a channel,
the electrode is present in the channel,
The measurement liquid provided by the measurement liquid providing means passes through the surface of the electrode via the channel,
Device. 7. A device according to any one of claims 1 to 6,
The measurement solution is a solution having a specific resistance of 50 kΩ cm or more,
Device. A device according to any one of claims 1 to 7,
The measurement solution is the same solution as the solvent of the sample solution, the concentration of which is diluted at least 100 times or more,
Device. A device according to any one of claims 1 to 7,
The measurement solution is 1 μM to 100 μM phosphate buffer or Good's buffer,
The solvent of the sample solution is 10 mM to 500 mM phosphate buffer or Good's buffer,
Device. 10. A device according to any one of claims 1 to 9,
said measuring means comprises a field effect transistor or a signal amplifier,
Device. 11. A device according to claim 10, wherein
the change in potential of the electrode is measured as a change in potential of the gate electrode of the field effect transistor or a change in current flowing between the drain and source ;
Device. 12. A device according to any one of claims 1 to 11,
By comparing the measurement results when using the sample solution with the measurement results when using one or more control solutions whose concentration of the antigen is known in advance instead of the sample solution,
measuring the concentration of the antigen in the sample liquid, or detecting the presence of the antigen in the sample liquid,
Device. 13. A device according to claim 12, wherein
characterized in that the one or more control solutions include those with a concentration of 0 for the antigen,
Device. 14. The device of claim 13, wherein
The measurement result to be compared is the potential or current at a predetermined time, the time to reach the predetermined potential or current, or the potential or current in a predetermined time range x time.
Device. 15. A device according to any one of claims 1 to 14,
characterized in that the electrodes are parallelized or integrated,
Device. 16. A device according to any one of claims 1 to 15,
characterized in that the antigen is a molecule that can exist in vivo,
Device. 4. A device according to claim 3, wherein
The electrode surface further comprises a three-dimensional structure for controlling the arrangement of the magnetic particles, and/or the surface is modified for controlling the arrangement of the magnetic particles,
Device. 18. A device according to claim 17, comprising:
the three-dimensional structure is a hole, groove, pillar, or wall structure;
The surface modification is hydrophilic treatment or hydrophobic treatment,
Device.

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