JP5683216B2 - Target substance detection method and apparatus - Google Patents

Description

  The present invention relates to a target substance detection method and apparatus. The present invention is useful for detecting various substances such as narcotics and environmental substances.

  In recent years, crimes related to the use of narcotics such as cocaine have attracted great social interest, and a rapid analysis method for such drugs is required. In cocaine analysis so far, mainly, 1) a simple determination method in which a body fluid such as urine of a subject is used as a sample and judged by an immunochromatography method which is a kind of immunoassay using an anti-cocaine antibody, and 2) a test Two methods of high-precision measurement using a gas chromatography apparatus using human hair as a sample are performed.

  Although the method 1) is simple but has low detection sensitivity, detection is difficult unless the sample has been collected within a short period of time from several minutes to several days after taking the narcotic. In addition, in the analysis of 2), which is much more accurate, it is easy to collect samples, and even if the elapsed time after taking the narcotic is longer than 1), it can be detected, and detailed analysis It has been implemented in cases that require However, a gas chromatography device is required, and analysis takes a long time.

  On the other hand, the inventors of the present application have previously succeeded in holding a channel protein in a lipid bilayer and forming an ion channel via the membrane (Non-patent Document 1). However, it is not described at all that this is applied to the detection of a substance.

R. Kawano, T. Osaki, H. Sasaki, S. Takeuchi Small 2010, 6, 2100-2104. L. Gu, J. Shim Analyst 2010, 135, 441-451. M. Stojanovic, P. Prada, D. Landry J. Am. Chem. Soc., 2001, 123, 4928-4931. Tuerk, C. and Gold L. (1990), Science, 249, 505-510

  An object of the present invention is to provide a target substance detection method and apparatus for detecting a target substance to be detected simply, rapidly and with high sensitivity.

  As a result of intensive research, the inventors of the present invention can pass a specific binding substance such as an aptamer that specifically binds to the target substance, but the bound substance that specifically binds to the target substance is because of its size. By using a membrane with a through-hole that cannot pass through and using as an index whether or not the through-hole is blocked by the binding substance of the target substance and the specific binding substance, it is simple, quick, and highly sensitive. The present inventors have found that a target substance can be detected and completed the present invention.

That is, the present invention is a method for detecting a target substance,
Contacting a specific binding substance that specifically binds to a target substance to be detected with a test sample that may contain the target substance;
Contacting the mixture obtained in the above step with one side of a membrane having pores of a predetermined size;
Examining whether or not the through hole is closed,
Wherein the predetermined size is the said specific binding substance not bound to the target substance can pass, said specific binding substance bound to the target substance Ri Ah size that can not pass through,
The through hole is a channel of a channel protein;
It said specific binding substance is, at one end, the same bases Ru aptamer der having repeat region of 15 to 50 contiguous identical bases, provides a method of detecting a target substance.

The present invention also provides a target substance comprising a membrane having a pore having a size that allows a specific binding substance that is not bound to a target substance to be detected to pass through, but cannot pass a specific binding substance that is bound to the target substance. In the detection apparatus , the through-hole is a channel of a channel protein, and the specific binding substance is an aptamer having a repeating region of the same base in which 15 to 50 identical bases are continuous at one end thereof. An apparatus for detecting a target substance is provided.

  According to the method of the present invention, a wide range of desired substances such as narcotics such as cocaine and environmental substances such as ATP can be detected simply, rapidly and with high sensitivity.

It is a schematic cross section of one preferable specific example of the apparatus used for the method of the present invention. It is a schematic diagram for demonstrating the principle of the preferable aspect of this invention. It is a figure for demonstrating the manufacturing process by the photolithography of the self-supporting film of this invention performed in the Example of this invention. It is a figure which shows the measurement result of the spike current obtained in the Example of this invention. It is a figure which shows the measurement result of cocaine obtained in the Example of this invention. It is a figure which shows the measurement result of ATP obtained in the Example of this invention.

10 Film made of paraxylene polymer (trade name Parylene) 12 Lipid bilayer membrane 14 Channel protein 16 Chamber 18 Channel 20 Electrode 22 DC power supply 24 DNA aptamer 26 Target substance

  The target substance detected by the method of the present invention is not limited as long as it is a substance capable of producing a specific binding substance such as an aptamer that can specifically bind to the target substance. For example, a narcotic such as cocaine , Environmental substances such as nucleotides such as ATP, ADP and AMP (these concentrations in the environment are used as indicators of biological pollution), various pathogens measured in diagnosis of infectious diseases and evaluation of environmental pollution, various Various biological substances and the like measured in the diagnosis of illness can be exemplified, but are not limited thereto.

  In the method of the present invention, a substance that specifically binds to a target substance (referred to as “specific binding substance” in the present invention) is used. Here, “specifically binds” means that it binds to a target substance but does not bind to a substance other than the target substance, or at least a substance other than the target substance and exists in the test sample. This means that it does not bind to other substances that have sex. The target substance includes a group of similar substances that are not intended to be distinguished from each other.

Specific binding substance, the target substance and can specifically bind, Ru larger substance der than the size of the specific binding substance before the size of the binding material after binding the binding.

The specific binding substance is an aptamer . The aptamer is composed of a polynucleotide (preferably DNA from the viewpoint of stability) such as DNA or RNA, and specifically binds to a specific substance. Aptamers are usually polynucleotides consisting of several tens to one hundred and several tens of nucleotides, and those having an arbitrary base sequence can be easily chemically synthesized using a commercially available nucleic acid synthesizer. It can be manufactured quickly. For this reason, research has been actively conducted in recent years as an alternative to antibodies in various immunoassays.

  Aptamers that specifically bind to any substance can be produced by a method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) (Non-patent Document 4). The SELEX method itself is already well known, and based on this, aptamers that specifically bind to various substances have already been obtained. In addition, various improved methods of the SELEX method have been proposed for efficiently producing an aptamer that specifically binds to a desired substance. The SELEX method uses a nucleic acid automatic synthesizer to form a library of a large number of polynucleotides having random base sequences, reacts the target substance bound to a solid phase with this library, and binds to the target substance. The process of collecting the obtained polynucleotide, amplifying it by PCR and adding it again to the solid phase on which the target substance is immobilized is repeated about 10 to several tens of times to concentrate the polynucleotide having a high binding force to the target substance. Since this is a method that actively uses chance, it is considered that aptamers that can specifically bind to almost all substances can be produced. After an aptamer that specifically binds to a target substance is obtained and its base sequence is determined, a polynucleotide having the base sequence can be easily chemically synthesized by an automatic synthesizer. In addition, according to the SELEX method, a plurality of aptamers that specifically bind to a desired substance are usually obtained. From these, select one that can pass through a through-hole described later in an unbound state with a target substance. It is also possible to do.

The aptamer used as a specific binding substance has a repeating region of the same base in which 15 to 50, more preferably 20 to 40, the same base continues at one end. Such repeated regions of the same base rarely hybridize with other regions in the aptamer molecule or other aptamer molecules, and exist linearly after binding to the target substance. This region is preferable because it pierces a later-described through-hole, and the through-hole is likely to be blocked by a combination of the target substance and the aptamer. As the same base, cytosine (c) is particularly preferable. This is because polyguanine (g) is not chemically synthesized, so if the same base is cytosine, it is possible to eliminate the possibility that the repeated region hybridizes with the region in the same molecule or in another molecule. An aptamer having such a repeating region at one end can be usually obtained by simply adding such a repeating region to one end of an aptamer that specifically binds to the target substance, and can be used in the live SELEX method described above. It can also be created by using a rally having a repeating region at one end.

In the method of the present invention, a membrane having a pore having a size that allows the specific binding substance that is not bound to the target substance to pass therethrough but cannot pass the specific binding substance bound to the target substance is used. For use aptamers as specific binding substance, the inner diameter of the hole is approximately 1nm~5nm are preferred. However, the size of the through-hole can be appropriately selected depending on the type of specific binding substance or target substance to be used, and is not necessarily limited to the above range.

A channel protein channel is used as the through- hole . A channel protein is a protein having a pore called a channel in a molecule, and various ions and the like are transported through the channel in a living body. Examples of channel proteins include α-hemolysin, Outer membrane protein F (OmpF), Mycobacterium smegmatis porin A (MspA), streptridine O, and the like. It is not limited to.

The channel protein can preferably be retained in the lipid bilayer. And it is preferable from a stability viewpoint that this lipid bilayer membrane is formed in the form which plugs up the fine through-hole provided in the self-supporting film. That is, a lipid bilayer membrane is formed so as to block the minute through-holes of the self-supporting film, the channel protein is retained in the lipid bilayer membrane, and the channel of the channel protein is used as a through hole in the present invention. Is preferred.

  A method of forming a lipid bilayer membrane in a form that closes the microscopic through-holes of the self-supporting film and retaining the channel protein in this lipid bilayer membrane has already been filed as Japanese Patent Application No. 2010-012300. 1 is disclosed, but will be described below.

  The material of the self-supporting film is not limited as long as it can open a through hole having a pore diameter of about several μm and can deposit a compound (described later) by vapor deposition. For example, nanometer-sized micropores can be produced by vapor deposition in small holes of glass or metal. It is preferable that the film is made of the same material as the compound to be deposited because the film can be formed integrally and the durability and handling of the film are increased. However, it is also possible to form a film with a material other than the compound to be deposited, and examples of the material in this case include a spin coatable polymer material (polyolefin type, polyethylene type) and the like. .

  The self-supporting film can be produced by the following method.

  First, a self-supporting film having a through hole having a pore diameter of several μm, preferably about 2 μm to 8 μm is prepared. If it is a through-hole of such a hole diameter, it can be formed by a known method such as photolithography, and this method will be specifically described in the following examples. In the next step, when vapor deposition is performed on the entire film, the film thickness of the film before vapor deposition is usually about 1 μm to 20 μm, preferably about 2 μm to 10 μm.

  Next, a compound that can be deposited on the self-supporting film is deposited on at least the inner wall of the through hole by vapor deposition, thereby reducing the diameter of the through hole to less than 1 μm. The compound to be vapor-deposited may be any compound as long as it is a compound that can be deposited on the self-supporting film having a through-hole prepared previously by vapor deposition, and a preferred example is a paraxylylene-based polymer. be able to. Among these, the paraxylene-based polymer can be isotropically vapor-deposited, and has the property that the polymer is uniformly deposited (deposited) on both the inner wall of the through hole and on both sides of the film. Since it is excellent in heat resistance and chemical resistance, it is preferable. Furthermore, as described above, if the self-supporting film to be subjected to vapor deposition is also formed of the same kind of para-xylene polymer, the self-supporting film after vapor deposition is integrated, and has excellent durability and handleability. Yes.

  The paraxylene-based polymer is a polymer composed of repeating units represented by the following general formula, and is commercially available under the trade name of Parylene, so that a commercially available product can be preferably used.

(In the formula, X represents a hydrogen atom or a fluorine atom, and R ¹ and R ² each independently represent a hydrogen atom or a chlorine atom).

  When the paraxylene-based polymer is vapor-deposited on a support in a vacuum chamber, polymerization occurs on the support to become a polymer. The molecular weight of the polymer varies depending on the deposition amount and the deposition time, and is about 500,000 at the maximum. The monomer can be deposited (deposited) on a support made of various materials by vapor deposition, and not only when the support is made of a paraxylylene polymer, but also on the support made of various materials described above. It can be attached to.

  The vapor deposition may be performed by depositing at least the compound to be vapor deposited on the inner wall of the through hole, but it is simple and preferable to deposit the entire self-supporting film before vapor deposition. When vapor deposition is performed on the entire self-supporting film, the compound is deposited on the inner wall of the through-hole, so that the diameter of the through-hole is reduced, and at the same time, it is also deposited on both surfaces of the self-supporting film. Therefore, the film thickness increases simultaneously.

  When paraxylene monomer is deposited on a self-supporting film in a vacuum chamber, deposition can be performed according to the method described in the instruction manual of Parylene (trade name). The deposition temperature is usually room temperature and the deposition time is Usually, it is 30 minutes to 180 minutes, preferably 30 minutes to 60 minutes.

  The lipid bilayer membrane of the present invention is a lipid bilayer membrane in which the peripheral edge is in contact with the inner wall of a through hole having a pore diameter of less than 1 μm and closes the through hole. The through hole is not necessarily a through hole formed in the self-supporting film, but may be a layer supported on another support. But if it is a through-hole provided in the above-mentioned self-supporting film of this invention, this film can be moved independently to arbitrary places, and the incorporation to the flow-path device mentioned later etc. will be performed easily. Is preferable.

  A lipid bilayer membrane whose peripheral edge is in contact with the inner wall of the through-hole and plugs the through-hole can be formed by simply applying a lipid solution constituting the lipid bilayer membrane to the through-hole. As the lipid constituting the lipid bilayer membrane, a lipid bilayer membrane, that is, a lipid molecule having a hydrophilic region and a hydrophobic region in one molecule, has a hydrophobic region on the inside and a hydrophilic region on the outside. The lipid is not particularly limited as long as it is a lipid capable of forming a membrane arranged in layers, but in order to simulate a reaction in a biological membrane, the same or similar one as that of a biological membrane is preferable. Lipids such as diphytanoyl phosphatidylcholine (DPhPC), dipalmytoyl phosphatidylcholine, palmitoyl oleoylphosphatidylcholine (POPC), POPC A preferred example is oil phosphatidylcholine (DOPC). Since many of these are commercially available, commercially available products can be preferably used.

  The concentration of the phospholipid in the solution used for forming the lipid bilayer membrane is not particularly limited as long as the lipid bilayer membrane can be formed, but is usually about 5 g / L to 20 g / L, preferably 7 g / L. It is about L-15g / L. The solvent of the phospholipid solution is not particularly limited, but an organic solvent is preferable, and an aliphatic hydrocarbon solvent such as n-decane is preferable. Phospholipids may also form liposomes in this solution, in which case the liquid used is a liposome suspension.

  The lipid bilayer membrane holding the channel protein can be formed by dissolving the channel protein in the phospholipid solution described above, or the lipid bilayer membrane is formed as described above, and then the channel protein This solution can also be formed by applying the solution of (1) to the lipid bilayer membrane. When the phospholipid solution is a liposome suspension containing liposomes, it can be formed by using a suspension of liposomes retaining proteins. The concentration of the channel protein in these solutions is not particularly limited and can be appropriately selected, but is usually about 1 nM to 1 mM, preferably about 0.1 μM to 100 μM.

In addition, by adjusting the vapor deposition conditions of parylene (trade name), it is possible to form through holes having a hole diameter of about 1 to 900 nm .

The present invention also provides an apparatus for detecting a target substance comprising a membrane having through holes. It is preferable to form a plurality of the above-mentioned through holes in one film because a plurality of test samples can be detected simultaneously. Further, since the through-hole is a channel protein channel, it preferably has a power source for applying a DC voltage to both sides of the membrane and a current measuring means for measuring a current passing through the membrane. If such an apparatus is used, it can be investigated whether the through-hole was obstruct | occluded by the patch clamp method mentioned later.

  The apparatus is in the form of a channel chip having at least one channel, the channel is separated from the second channel or chamber by the membrane, and the two channels or the flow are separated by the through holes. It is preferable that the channel and the chamber communicate with each other.

  As a through-hole, a membrane in which a channel protein is held in a lipid bilayer formed in a through-hole provided in a parylene film is used, and one channel is separated from the chamber by this membrane. A schematic cross-sectional view of an apparatus in which the chamber communicates is shown in FIG. In FIG. 1, 10 is a parylene film, 12 is a lipid bilayer membrane, 14 is a channel protein, 16 is a chamber, 18 is a flow path, 20 is an electrode, and 22 is a DC power source.

  The procedure for carrying out the method of the present invention using such an apparatus will be described below.

  First, a specific binding substance is brought into contact with a test sample that may contain the target substance. This can usually be performed by mixing a solution of a specific binding substance and a test sample solution. Examples of test samples include various body fluids that may contain the target substance and dilutions thereof, river water, lake water, environmental water such as seawater, and tap water, but are not limited thereto. Absent. The final concentration of the specific binding substance is not particularly limited, but can bind to the total amount of the upper limit value of the assumed target substance concentration range in order to promote the blockage of the pores when the target substance is present. An amount is preferred. The specific concentration range of the final concentration of the specific binding substance is appropriately set on a case-by-case basis, but is usually about 1 μM to 1 mM. The binding between the specific binding substance and the target substance usually occurs rapidly at room temperature.

  Next, the mixture obtained in the first step is brought into contact with one side of the membrane. This can be performed, for example, by putting the mixture in the chamber 16 when using the apparatus shown in FIG.

  As described above, as a method for retaining the channel protein in the lipid bilayer membrane, there is a method in which a lipid bilayer membrane is first formed and a channel protein solution is added thereto. It is also possible to add a channel protein to the above mixture. When this is brought into contact with the lipid bilayer membrane, the channel protein is retained in the lipid bilayer membrane, and at the same time, the channel is blocked by the binding substance of the specific binding substance and the target substance.

Next, it is examined whether or not the through hole is closed. This can be done preferably by examining whether current flows through the through holes. In the case of using an apparatus as shown in FIG. 1, this can be done by applying a DC voltage to both sides of the membrane by a DC power source 22 and measuring the current flowing through the membrane. For using channel protein, if the channel is not closed, the spike current occurs every time the specific binding substance in the channel passes. When the channel is blocked, this spike current does not occur. Therefore, by measuring this current, it is examined whether or not the target substance is present in the test sample, that is, in the test sample. The target substance can be detected. This method is a method called a patch clamp method, and is a method widely used in the field of electrophysiology (for example, Non-Patent Document 2).

  Note that the time required to close the through-hole varies depending on the concentration of the target substance in the test sample, so create a calibration curve using various known concentrations of the target substance solution. For example, it is possible to quantify the target substance in the test sample by measuring the time required until the through hole is closed. Since the target substance is inevitably detected when the target substance is quantified, the case of quantifying the target substance is also included in the “detection method” of the present invention.

  The principle of the above method is schematically shown in FIG. Each reference number in FIG. 2 indicates the same as each reference number in FIG. Reference numeral 24 denotes an aptamer, and 26 denotes a target substance. The diagram on the left side of FIG. 2 shows a case where the target substance is not present in the test sample. In the absence of the target substance, the linear aptamer passes through the channel of the channel protein 14 and a spike current is generated. On the other hand, when the target substance 26 is present in the test sample, the aptamer 24 and the target substance 26 are bound to each other, and the size of the bound substance is larger than the inner diameter of the channel. It will not be done.

Incidentally, as schematically shown in FIG. 2, in order to use the channels of the channel protein as holes, aptamers are specific binding substance, to make it easier to pass through the channel, a linear shape shown in FIG 2 What has the shape of this is preferable. In addition, since it is desired that the binding substance bound to the target substance sticks firmly into the channel and closes the channel for a long time, the binding part bound to the target substance is also inserted into the channel, that is, It is preferable to have a linear portion at one end. As described above, this can be achieved by adding a repeating region of the same base to one end of the aptamer .

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1
1. Production of self-supporting film having through-holes A self-supporting film made of a paraxylylene-based polymer having through-holes having a pore diameter of 800 nm was produced by a photolithography method schematically shown in FIG.

First, a silicon wafer is placed in a vacuum chamber, and a paraxylylene monomer (trade name Parylene C, in the above general formula, X is H ₂ , R ¹ is Cl, and R ² is H) is deposited on the silicon wafer. A film composed of 5 μm paraxylylene-based polymer was formed. The deposition temperature was 24 ° C. and the deposition time was 50 minutes. Next, aluminum was vacuum-deposited on the formed paraxylylene polymer film (deposition temperature: room temperature, deposition time: 1 minute) to form an aluminum layer having a thickness of 5 μm (1 in FIG. 3).

A photoresist was spin-coated on the aluminum layer, and the photoresist was exposed to UV light using a CAD-designed mask and then developed, and a hole having a diameter of 5 μm was formed (2 in FIG. 3). The UV exposure conditions were a UV irradiation energy amount of 24 mW / cm ² (@ 405 nm) and an exposure time of 6.5 seconds. Thereafter, a hole having a diameter of 5 μm was formed in the aluminum layer by performing a mixed acid treatment.

  Next, the parylene layer was etched by oxygen plasma (3 in FIG. 3). Specific conditions were as follows. Oxygen amount 10mL / min, 25W.

  Next, the aluminum layer was dissolved and removed by mixed acid treatment (60 ° C., 1 minute) (4 in FIG. 3).

  One end of the self-supporting film was grasped with tweezers and peeled from the silicon wafer to obtain a self-supporting film having a through hole having a hole diameter of 5 μm (5 in FIG. 3). Thus, the film was self-supporting because it was able to grip and peel off one end and move it. The obtained self-supporting film had a size of 3 mm × 3 mm.

  The peeled self-supporting film was placed in a vacuum chamber, and the same paraxylylene monomer as above was deposited (room temperature, 40 minutes). As a result, the paraxylylene polymer was further deposited on the entire surface including the inner wall of the through hole of the self-supporting film, and the hole diameter of the through hole was reduced (6 in FIG. 3). By this method, a paraxylylene polymer film (parylene film) having a through-hole having a diameter of 400 nm was obtained.

2. Device fabrication
(1) Processing of plastic plate An apparatus schematically shown in Fig. 1 was fabricated, in which the obtained parylene film was incorporated. In other words, a design designed by CAD with a small NC was cut into a polymethylmethacrylate (PMMA) plate. Thereafter, Cr / Ag was wired on the upper PMMA resin by vacuum deposition for use as the electrode 20. Finally, all the layers were bonded by thermocompression bonding and an adhesive, and a tube was attached to the flow path 18 and used as a device. The width and depth of the flow path 18 were 400 μm, respectively.

(2) Formation of lipid bilayer The upper chamber 16 and lower channel 18 in the device are filled with 1 M KCl PBS buffer, and lipid molecules (DPhPC: diphytanoyl phosphatidylcholine) are passed from the lower channel 18 using a microsyringe. A lipid membrane was formed in the parylene pore by sequentially flowing the n-decane solution in which the solution was dissolved in the order of “buffer → lipid solution → buffer”. The lipid solution was 10 mg of DPhPC (Avanti Polar Lipids, USA) dissolved in 1 mL of n-decane. The buffer was 1.0 M KCl, 10 mM PBS, pH 7.4.

(3) α-hemolysin (0.3 nM) was used as a channel membrane protein. A change in channel current was measured by filling a DNA aptamer (non-patent document 3) that forms a complex with hemolysin and cocaine in the upper chamber, a cocaine solution, and applying a voltage above and below the lipid membrane. There were two DNA aptamers, and the base sequences were 5′-atccttcaatgaagtgggtcgaca-3 ′ (SEQ ID NO: 1) and 5′-gggagacaaggaacccccccccccccccccccccccccccccccc-3 ′ (SEQ ID NO: 2), respectively. These two DNA aptamers simultaneously bind to cocaine (see the right figure in FIG. 2). This DNA aptamer is a cocaine-binding aptamer described in Non-Patent Document 3 with a 30-base poly-c region added to one 3 ′ end. The final concentration of DNA aptamer was 50 μM, and the final concentration of cocaine was 1 μM to 100 μM. For comparison, a solution containing no cocaine and a solution containing aminobenztropin which is a cocaine derivative instead of cocaine were also measured in the same manner. The applied voltage was +100 mV. The current between the electrodes arranged in the chamber 16 and the flow path 18, that is, the current flowing through the lipid bilayer membrane (membrane current) was measured. The membrane current was measured using a patch-clamp amplifier (CEZ-2400, manufactured by Nihon Kohden Co., Ltd.) connected to both electrodes. The Ag / AgCl electrode disposed in the chamber 16 was a recording electrode, and the Ag / AgCl electrode disposed in the flow path 18 was a ground electrode. The current was recorded using a digital data acquisition system (Digidata 1322A and pCLAMP ver.9, manufactured by Molecular Devices, USA).

  The results are shown in FIGS. As shown in the upper diagram of FIG. 4, when the solution does not contain cocaine, the DNA aptamer can pass through the channel, so that spike current is measured. On the other hand, when the solution contains cocaine, no spike current is generated for a long time (particularly, the right half of the lower figure). Thereby, it was possible to detect cocaine. Measurement was possible in the final cocaine concentration range of 1 μM to 100 μM. The upper right graph in FIG. 5 plots the measurement results for the case where the final concentration of cocaine is 10 μM, with the contact time between the solution and the channel protein as the horizontal axis and the occlusion probability as the vertical axis. After 25 seconds, channel blockage was observed with a probability of 1.0 (ie 100%). The bar graph in FIG. 5 shows the channel blocking probability when the contact time between the solution and the channel protein is 30 seconds. When cocaine was included, channel clogging was observed after 30 seconds with a probability of 1.0 (ie 100%), but with cocaine derivatives and without cocaine (ie with no DNA aptamer bound). Case) the channel blockage probability was almost zero. As a result, it was shown that according to the method of the present invention, cocaine can be detected with high sensitivity in as little as 30 seconds (or even 25 seconds).

Reference example 1
The same experiment as in Example 1 was performed using ATP instead of cocaine. As an ATP-binding DNA aptamer, 5′-tttttttttcactgacctgggggagtattgcggaggaaggt-3 ′ (SEQ ID NO: 3) was used. For comparison, GTP was also measured as a similar substance to ATP.

  The results are shown in FIG. As shown in a) of FIG. 6, in ATP, the channel blocking time was 10 times longer than in GTP. Further, the frequency of occurrence of spike current decreased depending on the concentration of ATP as shown in b) of FIG. As shown in c), when the ATP concentration is taken on the horizontal axis and the reciprocal event rate is taken on the vertical axis, it changed depending on the concentration of ATP.

In Example 1, so that it can close the channel over a longer period of time, since the addition of the poly c region of 30 bases at the 3 'end side of one of the aptamer, obstruction of the channel is more than Reference Example 1 It was certain.

Claims (14)

A method for detecting a target substance,
Contacting a specific binding substance that specifically binds to a target substance to be detected with a test sample that may contain the target substance;
Contacting the mixture obtained in the above step with one side of a membrane having pores of a predetermined size;
Examining whether or not the through hole is closed,
Wherein the predetermined size is the said specific binding substance not bound to the target substance can pass, said specific binding substance bound to the target substance Ri Ah size that can not pass through,
The through hole is a channel of a channel protein;
It said specific binding substance is, at one end, the same bases Ru aptamer der having repeat region of 15 to 50 contiguous identical bases, the method for detecting a target substance. The channel protein The method of claim 1, wherein in a state held in a lipid bilayer membrane. The method according to claim 1 or 2 , wherein the step of checking whether or not the through hole is closed is performed by a patch clamp method. The method according to any one of claims 1 to 3 , wherein the channel protein is α-hemolysin. The method according to claim 1, wherein the same base is cytosine. The method according to any one of claims 1 to 5 , wherein the target substance is a narcotic. The method of claim 6 , wherein the narcotic is cocaine. The method according to any one of claims 1 to 5 , wherein the target substance is a nucleotide. The method of claim 8 , wherein the nucleotide is ATP. A target substance detection apparatus comprising a membrane having a pore having a size capable of passing a specific binding substance that is not bound to a target substance to be detected, but not allowing a specific binding substance bound to the target substance to pass through , The target substance detection device, wherein the through-hole is a channel of a channel protein, and the specific binding substance is an aptamer having a repeating region of the same base consisting of 15 to 50 identical bases at one end thereof. . The device according to claim 10 , wherein the channel protein is held in a lipid bilayer membrane. The apparatus according to claim 10 or 11 , further comprising: a power source that applies a DC voltage to both sides of the membrane; and a current measuring unit that measures a current passing through the membrane. The device according to claim 10 , wherein the channel protein is α-hemolysin. The channel is in the form of a channel chip having at least one channel, the channel is separated from the second channel or chamber by the membrane, and the two channels or the channel and the chamber are separated by the through-hole. The apparatus of any one of Claims 10-13 which are communicating.

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