JP2016003935A - Method for quantitatively determining specimen, and device for quantitatively determining the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method for quantitatively determining a specimen in a short time and with high sensitivity, and preferably at a countable level.SOLUTION: A device used for quantitative determination includes: a first base plate; a second base plate bonded to the first base plate; a flow channel formed on a bond surface between the first and second base plates and communicating with the outsides of the first and second base plates; a specific binding substance immobilization region which is a partial area of the flow channel and in which a first specific binding substance specifically bound to a specimen is immobilized. The flow channel has a height of 10 nm to 10 μm, and the specific binding substance immobilization region has a length of 1 μm to 10 cm. A method for quantitatively determining a specimen includes: causing a sample to flow through the flow channel and bind a specimen in the sample to the first specific binding substance; after cleaning, circulating a second labelled specific binding substance to be bound to the specimen through the flow channel to thereby bind the specimen bound to the first specific binding substance to the second specific binding substance; and, after cleaning, quantitatively determining the label bound to the specimen.

Description

  The present invention relates to an ultrasensitive analysis method that can be applied to immunoassays and the like, and realizes quantification of a test substance at a count level, and a quantification device used therefor.

  Conventionally, quantifying various substances present in living bodies and pathogens such as bacteria and viruses by immunoassay has been widely used for diagnosis of various diseases. As an immunoassay method, a first antibody is immobilized on a solid phase, and this is reacted with an antigen in a test sample to bind the antigen to the first antibody. After washing, the antigen reacts with the antigen. An enzyme-linked immunosorbent assay (ELISA) that reacts with an enzyme-labeled second antibody, and after washing, develops color using the enzyme reaction of the labeled enzyme, and measures the degree of color development. The so-called method is widely used. As a solid phase used for ELISA, wells or beads of a microplate are widely used. However, the conventional ELISA using microplates or beads requires a large amount of test sample, and the amount of test substance possessed by one cell is measured or the test substance is quantified at a count level. You ca n’t. The reaction time required for the antigen-antibody reaction is usually as long as several tens of minutes to several hours.

  On the other hand, recently, various chemical reactions have been widely studied in a fine channel provided on a substrate. Such micro-scale space is expected to be used in fields such as diagnosis and analysis as a means of shortening mixing and reaction times, greatly reducing the amount of samples and reagents, and making small devices. ing. For example, a microchannel (microchannel) composed of a groove with a depth of several hundred μm or less is formed on a glass substrate (microchip) of several centimeters square, and this microchannel is bonded to another substrate. Allows liquid to flow without leakage. It has also been proposed and put into practical use that various functional systems such as biological materials, catalysts, and electrodes are partially modified on the inner surface of the channel to provide desired functions and integrate various chemical systems. . As a substrate material constituting the microchannel, a glass material having high strength, solvent resistance, and optical transparency for detection is desired. However, as described later, glass requires high-temperature conditions (1000 ° C or higher for quartz glass) for bonding between substrates, so all biological materials, catalysts, and electrodes modified to provide functions are not limited to thermal damage. It will burn out. For this reason, another substrate such as an elastomer that can be easily bonded is conventionally used for one of the substrates, and it has been difficult to form a channel using only a glass substrate.

  The inventors of the present application have previously found that glass substrates can be firmly bonded to each other at a low temperature of 100 ° C. or lower using oxygen plasma irradiation and methane tetrafluoride. A functional device having a fine flow path formed on the joint surface was invented and a patent application was filed (Patent Document 1). However, Patent Document 1 does not describe a specific method for measuring a test substance with high sensitivity, preferably at a count level.

WO 2014/051054 A1

  An object of the present invention is to provide a novel quantification method capable of quantifying a test substance at a high sensitivity, preferably count level, in a short time and a quantification device used therefor.

  As a result of diligent research, the inventors of the present application have optimized the size of a fine channel formed on a substrate and the length of a region where a specific binding substance such as an antibody is immobilized, and distributes the sample to the channel. The present inventors have found that the test substance can be quantified at a count level by flowing the sample at a specific flow rate corresponding to the diffusion coefficient of the test substance, and the present invention has been completed.

That is, the present invention includes a first substrate, a second substrate bonded to the first substrate, and a bonding surface of the first and second substrates, and the first and second substrates. And a specific binding substance-immobilized region in which a first specific binding substance that specifically binds to a test substance is immobilized. The sample is circulated through the channel of the quantitative device for the test substance in the sample, wherein the channel has a height of 10 nm to 10 μm and the length of the specific binding substance immobilization region is 1 μm to 10 cm. Binding the test substance in the sample to the first specific binding substance;
After washing the specific binding substance-immobilized region, a labeled second specific binding substance that specifically binds to the test substance is circulated through the flow path and bound to the first specific binding substance. Binding the test substance and the second specific binding substance;
And a method of quantifying a label bound to the test substance after washing the specific binding substance-immobilized region.

  The present invention also includes a first substrate, a second substrate bonded to the first substrate, and a bonding surface of the first and second substrates, the first and second substrates. And a specific binding substance-immobilized region in which a first specific binding substance that specifically binds to a test substance is immobilized. Provided is a device for quantifying a test substance in a sample used in the quantification method of the present invention, wherein the flow path has a height of 10 nm to 10 μm and the specific binding substance immobilization region has a length of 1 μm to 10 cm. .

  According to the method of the present invention, it is possible to reliably capture a test substance in a specific binding substance-immobilized region in a short time of within a few seconds using a very small amount of test sample. Then, it is possible to quantify at the count level. Therefore, according to the method of the present invention, the measurement sensitivity can be increased by orders of magnitude compared to conventional immunoassays, and the time required for measurement and the amount of the test sample can be less than picoliter (femtoliter). To at liters). It is considered that the microminiaturization is very suitable for the analysis of a single cell having a sample amount of picoliter level.

It is a figure which illustrates typically the method of selectively fixing an antibody on the glass substrate performed in the following Example 1. FIG. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which illustrates typically the immunoassay performed in the following Example 1. FIG. It is a figure which shows the relationship between the photograph (a) of the device used for the method performed in the following Example 1, VUV irradiation time and the contact angle of water (b), and the relationship between irradiation time and binding energy (c). . In the extended nanochannel obtained in Example 1 below, the fluorescence image of the molecular capture region and the intensity curve (a) in the longitudinal direction and the protein adsorption in the capture region and the non-capture region visualized by the fluorescently labeled antigen (B) which shows the effect of PEG with respect to FIG. 2 is a diagram (a) showing the change over time in the amount of bound antigen obtained in Example 1 below for each antigen concentration, and (b) showing the relationship between the number of antigen molecules introduced and the capture rate. Black circles show results for PBS, and white circles show results for 10-fold diluted bovine serum. Figure (a) schematically showing the intensity calibration experiment conducted in Example 1 below, calibration curve (b) showing the relationship between antigen concentration and fluorescence intensity, and time-dependent changes in the number of antigen molecules introduced and captured antigen molecules (C) (antigen concentration is 7.6 nM), and (d) is a graph showing the change in capture efficiency with time calculated based on the number of introduced and captured molecules. It is a figure which shows typically the method of sandwich ELISA performed in the following Example 2, (a) shows typically the reaction after injection | pouring of a sample liquid, (b) shows typically the enzyme reaction of a label | marker. . The lower graph is a diagram schematically showing the change with time of the TLM signal when (a) two molecules of antigens are captured. It is a figure which shows the time-dependent change of the TLM signal obtained as a result of sandwich ELISA performed in the following Example 2 for every number of the introduced antigen molecules.

  The quantitative device used in the method of the present invention is formed on the first substrate, the second substrate bonded to the first substrate, and the bonding surface of the first and second substrates, And a flow path connected to the outside of the second substrate. The first substrate and the second substrate are preferably glass substrates from the viewpoints of high strength, solvent resistance, and optical transparency for detection. As the glass substrate, for example, a widely used slide glass or a conventionally used glass substrate of a microchannel chip can be used, but the glass substrate is not limited thereto. The glass substrates can be firmly bonded at low temperature by using oxygen plasma irradiation and methane tetrafluoride (patent document 1) previously invented by the inventors of the present invention (see the following example). The bonding method is not limited to a method, and any bonding method that can maintain the function of a chemical substance modified in advance can be widely used. The flow path is formed on the bonding surface of the first and second substrates. This is because (1) the flow path is formed on the surface of the first substrate and the second substrate is smooth. (2) forming a flow path on the surface of the second substrate and bonding the first substrate with a smooth surface; or (3) connecting the flow path to the surface of both the first and second substrates. They may be formed at corresponding positions, and the two substrates may be bonded together so that a single flow path is formed on the bonding surface. The flow path may be only one as specifically described in the following examples, but as described in Patent Document 1, a plurality of (usually about 2 to several hundreds) flow paths are provided. It may be formed.

  In the method of the present invention, the height (depth) of the flow path is important, and is about 10 nm to 10 μm, preferably about 10 nm to 1000 nm, and more preferably about 20 nm to 400 nm. At this time, the lower limit of 10 nm indicates an indication of the boundary from liquid to solid in an aqueous solution surrounded by glass, and it also depends on the material of the liquid and the wall surface. A channel having a channel height (depth) of 1000 nm or less may be referred to as an “extended nanochannel” in this specification. A channel having a channel height exceeding 1000 nm may be referred to as a “micro channel” in the present specification. If a sample is circulated through such a height channel, preferably an extended nanochannel, under the condition satisfying formula [I] described later, it becomes possible to measure a test substance with extremely high sensitivity. . It was also found that the reaction time, which conventionally took 10 minutes or more, can be completed in a few seconds, and that a very large effect can be obtained, such as measurement with a sample volume of a single cell volume of picoliter or less. The width of the channel is not particularly limited, but it is usually 10 nm to 100 μm, preferably about 1 μm to 10 μm, from the viewpoint of enabling measurement with a very small amount of sample and saving the amount of a specific binding substance such as an antibody. It is. The length of the channel is not particularly limited, and is usually about 10 μm to 100 mm. The flow path communicates with the outside of the substrate. When the channel is an extended nano channel, it is preferably communicated with the outside of the substrate through a micro channel as described later.

  A first specific binding substance that specifically binds to the test substance is immobilized in a partial region of the flow path to form a “specific binding substance immobilization region”. Here, the specific binding substance is a substance that specifically binds to a test substance in a sample. For example, when the test substance is an antigen, an antibody or antigen-binding fragment thereof that reacts with the antigen with an antigen antibody In the case where the test substance is an antibody, an antigen that undergoes an antigen-antibody reaction with the antibody can be mentioned. However, the method of the present invention is not limited to immunoassay. For example, a ligand, a receptor, or a nucleic acid such as DNA is immobilized as a specific binding substance, and specific binding between a ligand and a receptor, or complementary strands of nucleic acids are interlinked. It is also possible to quantify ligands, receptors, nucleic acids and the like using the binding of

  The length of the “specific binding substance immobilization region” is about 1 μm to 10 cm, preferably 1 μm to 1000 μm, and more preferably about 50 μm to 300 μm, from the viewpoint of achieving highly sensitive quantification in a short time. In addition, a preferable method for immobilizing the first specific binding substance only on a partial region of the flow channel, particularly the extended nano flow channel is known (Patent Document 1), and is specifically described in the following examples. Yes. That is, before bonding the substrates, the entire surface of the substrate on which the extended nanochannels are formed is coated with APTES (aminopropyltriethoxysilane: silane coupling agent), and a specific binding substance is immobilized using a photomask. APTES coated in a region other than the activated region is irradiated with vacuum ultraviolet light to selectively remove APTES, and the first specific binding substance is covalently bound using the amino group in the specific binding substance immobilization region. Can do. However, the method for immobilizing the first specific binding substance is not limited to this, and before joining the substrates, a conventional physical adsorption or a contact printing method is used. Etc. are also possible. After the first specific binding substance is immobilized, the entire flow path is preferably blocked with polyethylene glycol (PEG) or a commercially available blocking agent in order to prevent nonspecific adsorption.

  When the channel is an extended nanochannel, the quantitative device may be formed with only the above-described extended nanochannel as the channel, but it is easy to inject and discharge the sample liquid. In order to achieve this, it is preferable that a microchannel having a larger depth or width is connected to the extended nanochannel (see the following example, Patent Document 1). The size of the microchannel is not particularly limited, but is usually about 1 μm to 1000 μm in depth and about 1 μm to 1000 μm in width.

In the method of the present invention, a sample is circulated through the flow path of the quantitative device, and a test substance in the sample is bound to the first specific binding substance. When the channel is an extended nanochannel and the above-described microchannel is present, the sample liquid is injected into the microchannel, and the sample is circulated through the extended nanochannel connected to the microchannel. At this time, the following formula [I]
5 ≤ (2Dτ) ^1/2 / d [I]
(Where D is the diffusion coefficient of the test substance (m ² / s), τ is the average time (s) that the molecules of the test substance stay on the specific binding substance immobilization region, and d is the depth of the flow path. (m))
It is preferable to distribute the sample at a flow rate that satisfies the above. The diffusion coefficient of D (m ² / s) itself is well known to those skilled in the art and is a value depending on the temperature used by the device. D of the test substance can be measured by a conventional well-known diffusion coefficient measurement using a concentration gradient. Further, as described in the literature (Small, 8 (8), 1237-1242 (2012)), direct measurement may be performed in an extended nanospace or a microspace. When a test substance contained in a specific sample is quantified with a specific quantification device, D and d are constants, so the variable is only τ, and τ is the average flow rate of the sample flowing through the flow path and a specific value. Since it can be calculated immediately from the length of the binding substance immobilization region, when quantifying a test substance contained in a specific sample with a specific quantitative device, the above formula [I] can be used to determine the flow rate and flow rate of the sample appropriately. This can be achieved by selecting The flow rate and flow rate of the sample can be easily adjusted by injecting the sample using a metering pump commonly used in the field of microchannel chips. The reaction temperature in this step is a temperature suitable for the reaction between the test substance and the first specific binding substance. For example, in the case of an antigen-antibody reaction or in the case of a reaction between a receptor and a ligand, 37 ° C., and in the case of reaction between nucleic acid complementary strands, it is a temperature suitable for achieving specific hybridization (eg, 50 ° C. to 60 ° C.).

The above formula [I] was derived as follows. That is, as a result of intensive studies, it was considered that the capture of the target molecule on the surface depends on the number of collisions between the target molecule and the wall surface per unit time. In this device, it is considered how much the molecule can collide with the specific binding substance when passing through the specific binding substance immobilization region. At this time, from the conventional Brownian motion theory, the average movement distance of the target molecule per transit time τ is given by (2Dτ) ^1/2 . That is, the ratio (2Dτ) ^1/2 / d between the average moving distance and the channel depth is considered to be a factor that determines the number of collisions. Conventionally, it has been unknown how many collisions the target molecule can be reliably captured. However, as described below, it was found that the capture efficiency depends on this factor.

The value on the right side of the above formula [I] is preferably 10 or more, and more preferably 30 or more. If this value is 30 or more, almost 100% of the molecules of the test substance bind to the first specific binding substance. In addition, when the value on the right side of the above formula [I] is 5, it has been confirmed that the capture rate is several percent. With this level, the test substance can be quantified. In Example 1 below, D is 40 μm ² / s, τ is 1.8 seconds, d is 200 nm, and when the unit is corrected as described above, the right side of the formula [I] is 60. In Example 2 below, D is 40 μm ² / s, τ is 10 seconds, and d is 800 nm. When the unit is corrected as described above, the right side of equation [I] is 35. The upper limit of the value on the right side is not particularly limited, but is usually 1000 or less, preferably 100 or less, from the viewpoint of measuring quickly and measuring a trace amount of sample. The concentration of the test substance in the sample is not particularly limited as long as the test substance can be quantified, but in a preferred embodiment, the test substance can be quantified even at a low concentration of about 0.1 pM to 10 pM.

  Next, after washing the specific binding substance-immobilized region with a buffer solution or the like, a labeled second specific binding substance that specifically binds to the test substance is circulated through the flow path, thereby the first specific binding. The test substance bound to the substance is bound to the second specific binding substance. Here, the second specific binding substance is a substance that specifically binds to the test substance in a state where the test substance is bound to the first specific binding substance, and when the test substance is an antigen, An antibody or antigen-binding fragment thereof that reacts with the antigen and antigen-antibody, and when the test substance is an antibody, an antigen or antibody that reacts with the antibody by antigen-antibody (if not labeled, another labeled antibody) When the test substance is a nucleic acid, a nucleic acid complementary to a single-stranded region that is not hybridized with the first specific binding substance (nucleic acid) or an antibody that recognizes these diffusions (label) If it is not, a further labeled antibody is reacted).

  The flow rate conditions for reacting the second specific binding substance are preferably equal to or lower than the conditions for binding to the first specific binding substance. The reaction temperature is a temperature suitable for the reaction between the test substance and the second specific binding substance, as in the reaction between the test substance and the first specific binding substance.

  As the label, a known label conventionally used for sandwich immunoassay can be used, and examples thereof include an enzyme label, a fluorescent label, and a chemiluminescent label. Of these, enzyme labels and fluorescent labels are preferred because they can be easily quantified with high sensitivity (see Examples below). In particular, when an enzyme that reacts with the substrate and develops color is used as a label, the colored substance can be quantified using a differential interference thermal lens microscope, and the test substance can be quantified in countable units. Therefore, the measurement sensitivity can be remarkably increased, which is preferable. As an enzyme that reacts with a substrate and develops color, an enzyme that has been conventionally used in ELISA, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), which develops color by an enzymatic reaction, can be used. The enzyme labeling method itself is well known. Among these, when HRP is used as the labeling enzyme and 3,3 ', 5,5'-tetramethylbenzidine (TMB) is used as the substrate, the turnover number of the enzyme (number of molecules per second) It is preferable from the experiment that it is possible to perform a highly sensitive quantification at the count level.

  Next, after washing the specific binding substance-immobilized region with a buffer solution or the like, the label bound to the test substance via the second specific binding substance is quantified. The quantification of the label itself can be performed by a well-known method in the immunoassay field. When an enzyme that reacts with the substrate and develops color is used as a label, a substrate solution of the labeling enzyme is circulated and the enzyme reacts with the substrate to cause color development. At this time, the enzyme reaction is preferably performed in a state where the flow of the sample is stopped from the viewpoint of measurement sensitivity and reproducibility. The stop time is appropriately set so as to satisfy the above formula [I], but is usually about 15 seconds to 4 minutes, and preferably about 30 seconds to 2 minutes. Other conditions such as the reaction temperature may be the same as in the well-known ELISA.

  Next, the colored substance produced from the substrate by the enzymatic reaction is quantified with a differential interference thermal lens microscope (DIC-TLM). DIC-TLM itself is known (Reference: Analytical Chemistry, 81, 9802-9806 (2009)). In quantification with a differential interference thermal lens microscope in a minute space such as an extended nanochannel, it was found that when measurement was performed in the specific binding substance immobilization region, the signal value changed greatly, and quantification was difficult. Therefore, it is preferable to carry out in the region downstream of the specific binding substance immobilization region from the viewpoint of reproducibility of quantification.

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

Example 1
1.1. Antibody patterning method Low temperature bonding was used to prevent functional molecules from being damaged by heat during the thermal bonding process. This method enables glass-glass bonding at 25 to 100 [deg.] C. by activation of oxygen plasma surface containing fluorine. However, this oxygen plasma surface activation process damages surface modifying molecules such as functional groups and antibodies. Here, in order to pattern the functional group for immobilizing the antibody and activate the glass surface to be bonded, vacuum ultraviolet (VUV) light having a strong oxidizing effect by high photon energy (λ = 172 nm) was used.

  FIG. 1 shows a schematic diagram of an antibody patterning operation in an extended nanochannel. In order to introduce a functional group for immobilizing the antibody, the entire surface of the quartz glass substrate (first substrate) was modified with aminopropyltriethoxysilane (APTES) in the gas phase to form a uniform APTES layer. When VUV light is irradiated through a chromium photomask, reactive oxygen species generated from oxygen gas molecules that absorb high energy VUV light oxidize APTES oxidatively. Since this glass surface becomes superhydrophilic and is activated after the decomposition of APTES, it is possible to achieve a strong glass-glass bonding that has a significant meaning in nanofluid control using a high-pressure flow system. In order to design and adjust the size and position of the antibody immobilization region, a partially modified APTES layer was formed by masking a portion of the substrate area from VUV light irradiation using a photomask. An advantage of using VUV light is that patterning and surface activation can be achieved simultaneously.

  Next, the substrate on which APTES was patterned (first substrate) was brought into contact with an upper glass substrate (second substrate) including microchannels and nanochannels, and this substrate was activated by fluorine-containing oxygen plasma. Thereafter, the APTES pattern and the nanochannel were arranged to be orthogonal. The width, depth, and length of each nanochannel were 3.3 μm, 200 nm, and 2 mm, respectively, and 50 nanochannels were prepared in parallel to obtain sufficient analytical data. The substrates were bonded by pressing at a relatively low temperature of 100 ° C. with a force of 5000 N for 2 hours. Trimethoxysilane-poly (which has been shown to significantly reduce protein adsorption on other modified surfaces when physisorbed to reduce the possibility of nonspecific protein adsorption to the nanochannel surface. The surface was chemically modified with (ethylene glycol) (PEG). After bonding, the exposed silica region and the APTES region on the channel surface were modified with silanized PEG. As detailed in Section 2.3 below, this modification did not significantly affect the surface density of the antibody, but it did not completely shield the APTES region from the PEG modification, so the APTES region was partially modified. It was. The capture antibody was chemically immobilized by crosslinking the amino group of the APTES molecule with the antibody using glutaraldehyde. The remaining reactive groups were blocked with ethanolamine. All experimental details regarding substrate bonding and chemical modification for antibody patterning are described in the experimental section.

1.2. Evaluation of pressure resistance performance for nanofluid control An immunoassay device for nanofluid requires pressure-driven fluid control to introduce and capture target molecules by adjusting liquid volume and liquid exchange. This makes it possible to properly separate the immunochemical reactant from other molecules. A schematic diagram of the experimental apparatus for this purpose is shown in FIG. As described above, the liquid flow was controlled by a pressure-driven flow system. The flow of liquid contained in the vial was introduced by air pressure adjusted by a pressure control device connected to a compressor. A capillary made of polyetheretherketone (PEEK) was connected to the inlet and outlet of the microchannel (width 500 μm × depth 6 μm). In order to quickly exchange the solution of the microchannel and the nanochannel, both ends of the microchannel were connected to the outside through a through hole. This solution was introduced into the nanochannel by applying pressure to one inlet of the microchannel. Next, it was examined whether or not a device fabricated using the proposed method would enable pressure-driven fluid control without leakage.

  The fabricated device (FIG. 3 (a)) showed uniform bonding without voids or stripes. The pressure resistance was evaluated by examining the degree of surface activation achieved by VUV light irradiation, and its effect on glass-glass bonding energy was evaluated by measuring the contact angle of water. As shown in FIG. 3B, since the APTES molecule is decomposed by the active oxygen species generated by the VUV light, the contact angle of water on the substrate decreases as the irradiation time t of the VUV light increases. The APTES surface was slightly hydrophobic initially, similar to the previously reported results, and showed a contact angle of 70 ° or less. By t = 10 minutes, the surface became superhydrophilic with a contact angle of less than 5 ° and the surface was fully activated. This degree of surface activation strongly influenced the glass-glass bonding energy.

The measurement results of the bonding energy are shown in FIG. The bonding energy was measured using a crack opening test, which is a standard method widely used for evaluating bonding strength. In addition to the measurement of the bonding energy, leakage was evaluated by observation after introduction of the fluorescein solution. These test results indicated that the more the surface was activated, the higher the bonding energy. At t = 0 minutes (ie, the bond between the APTES modified glass surface and the plasma excited glass surface), the bond energy was 0.16 J / m ² and leakage was observed at pressures below 50 kPa. At t = 12 minutes, the joining energy was 0.89 J / m ² , and no leakage was observed even at a pressure exceeding 2 MPa. The strong bond obtained by irradiation is due to the high silanol group density on the hydrophilic active surface. Since the pressure required to flow a fluid at a speed of fL / second through a nanochannel having a typical size (width and depth: 10 ² nm, length: 1 mm or less) is on the order of several hundred kPa, It was concluded that the device is resistant to leakage in pressure driven flow and can be used in immunoassay experiments.

1.3. Verification of femtoliter reaction space
In order to construct a femtoliter reaction space, it is necessary to design the device surface precisely. Due to the large surface area / volume ratio of the extended nanochannels, non-specific adsorption of target molecules can lead to significant sample loss and consequently prevent quantitative analysis. In general, proteins adsorb irreversibly on the exposed silica surface, resulting in significant non-specific adsorption of capture antibody and target antigen. Therefore, in order to reduce non-specific adsorption, a method of directly coupling silanized PEG to a silanol group is selected. In order to verify that a molecular capture region (ie, an antibody immobilization region) (specific binding substance immobilization region) has been formed as designed, the immobilized antibody is indirectly bound by binding a fluorescently labeled antigen under an antigen-excess condition. Visualized. For this purpose, anti-mouse IgG (antibody) and Dylight 488-labeled mouse IgG (antigen) were used. Prior to visualization, the capillary, microchannel and nanochannel surfaces were blocked with 2% BSA in PBS for 30 minutes. After the reaction using the 68 nM antigen solution and the washing with the reaction buffer, the molecular supplement region (specific binding substance immobilization region) was observed. As shown in FIG. 4A, clear contrast was observed in both the fluorescence intensity image and the line profile. Since the measurement width 130 μm was almost the same as the design width 135 μm of the photomask used in the VUV light irradiation process, the antibody-immobilized surface can be adjusted sufficiently. The non-uniformity of the APTES pattern caused by refraction of VUV light caused a slight decrease in pattern width and a step change in intensity at both ends of the pattern. Nanochannel blockage and leakage were not observed. The above results indicated that the molecular capture region could be constructed at a preset location and was successful with an immunochemical reaction field volume of 86 fL. The reaction field volume reaction volume of microchannel antibody microbeads immobilized filled (10 0 ^nL) 5 orders of magnitude smaller than.

  In order to measure the effectiveness of the PEG modification of the extended nanochannel, the average fluorescence intensity of the molecular capture region and other regions was measured in both the PEG-modified channel and the non-PEG-modified channel. The result of subtracting the background is shown in FIG. Because the PEG molecule significantly rejected the protein, the fluorescence intensity of the PEG-modified non-supplemented region was significantly reduced to an almost electrical background level that was a hundred times lower than that of exposed silica. The slight difference in strength between the PEG-modified and PEG-unmodified supplemental regions is due to the attachment of PEG to unreacted silanol groups in the APTES molecule. A small amount of PEG in APTES can reduce the binding between the capture antibody and the target antigen. From the above results, the PEG modification of the nanochannel was extremely effective.

1.4. Efficient immunochemical reaction in extended nanochannels In contrast to the size of reaction spaces (μm to mm scale) in conventional immunoassay formats such as microfluidic devices or 96-well plates, extended nanochannels (nm scale) ) Is much smaller than the diffusion distance over a few seconds. Under such conditions, due to the short diffusion distance, the introduced antigen necessarily interacts with the immobilized antibody, so that the reaction becomes very efficient and can be captured without losing the target molecule. This is a characteristic characteristic of molecular capture in the extended nanochannel, which makes the extended nanochannel ideally suited for quantifying very small amounts of analyte.

  In order to examine the reaction quantitatively, the amount of surface-bound antigen change over time was evaluated by measuring the change in fluorescence intensity when antigen solutions of various concentrations were introduced under controlled flow rate under continuous flow. did. Prior to introduction of the antigen, the surfaces of the capillary, microchannel, and nanochannel were blocked with 2% BSA in PBS for 30 minutes. Antigen solutions were prepared by three-step dilution from 68 nM using 10 mM PBS containing 2% BSA and 0.05% Tween 20, and the antigen concentrations examined were 0, 2.5, 7.6, 23 and 68 nM. The flow rate experimentally determined by measuring the average rate (68 μm / sec) of a 1 mg / mL fluorescein buffer solution was 45 fL / sec. This average flow velocity was consistent with the theoretical value calculated from the pressure loss assumed to be Hagen-Poiseuille flow. After measuring the fluorescence intensity generated by the passage of each antigen solution, the bound antigen was removed by reducing the pH to 2.4 using glycine / HCl buffer, and the regenerated surface was used again. FIG. 5A shows a plot of changes in fluorescence intensity of the entire immobilized region at intervals of 3 seconds for each concentration of antigen. t = 0 is the reaction starting point, and is the time when the introduced antigen reaches the molecular supplement region. This was calculated from the flow velocity and distance (1 mm or less) between the nanochannel inlet and the molecular supplement region. The intensity increase was plotted by subtracting the intensity at t = 0. The plotted values represent the average of 5 nanochannels and error bars represent ± 2α. The change in fluorescence intensity clearly corresponded to the concentration of the introduced antigen. At the highest concentration of antigen (68 nM), all binding sites were saturated in less than 80 seconds. The intensity of the area without immobilized antibody was also measured, but no increase was observed. The decrease in fluorescence intensity caused by photobleaching was estimated from the slight decrease in intensity at the plateau. Since the total exposure time during one measurement was only 5 seconds, the intensity was 3 digits smaller and the influence of photobleaching could be ignored.

  The concentration dependence of the antigen is examined by examining the initial antigen binding rate, that is, the capture rate. The supplement rate was calculated as follows. To correct the diurnal variation in surface density of the supplemental antibody binding site, the strength of the solution from 0 to 68 nM was determined using the plateau value at 68 nM, the concentration at which almost all of the capture antibody was bound to the fluorescently labeled antigen molecule. Normalized. It was estimated that the available binding sites did not change during the measurement of a series of diluted samples over several hours. The slope determined from the linear fit of the normalized number between 0-18 seconds using the least squares method was defined as the supplement rate. To measure the specificity of the reaction, a 10-fold dilution of bovine serum with Dylight488 labeled mouse IgG was also evaluated. FIG. 5B shows a plot of the capture rate for the first 18 seconds after the start of supplementation against the absolute number and concentration of the introduced molecules. There was a strong relationship between the capture rate and the number of antigen molecules introduced. Since the number of available binding sites [antibodies] decreased rapidly during the first 18 seconds of the reaction, the capture rate was lower than expected assuming a linear increase when the antigen concentration was high. The reaction was specific for the immobilized antibody since the same concentration dependence was shown as for bovine serum. If antigen molecules bind nonspecifically to the immobilized antibody, the capture rate in serum samples (containing many impurities) should be lower than the capture rate in PBS. The amount of sample passing through the channel in the first 18 seconds was 810 fL, and the detection limit based on the absolute number of molecules was 3 zmol.

  In order to confirm the efficiency of the antibody-antigen reaction, the molecular capture rate defined as the number of surface-bound molecules relative to the number of introduced molecules was evaluated and expressed as a percentage. The number of molecules introduced was calculated from the concentration, flow rate and flow duration. Although surface-bound molecules were difficult to assess, their number was estimated from the fluorescence intensity measured by the calibration process. As shown in FIG. 6 (a), a calibration curve of fluorescence intensity was prepared by measuring the intensity of the same kind of fluorescently labeled antigen solution filling the nanochannel. The size of the detection area was the same as the detection area used in the experiment summarized in FIG. 5, and the entire fluorescence intensity was derived over the entire detection area. Since the confocal length of the excitation light (2.2 μm) was longer than the depth of the nanochannel (200 nm), all the molecules in the nanochannel can be detected, so the solution concentration is an arbitrary value. Can calculate the number of molecules in the detection region. Using the obtained calibration curve (FIG. 6B), the intensity was converted to the number of molecules.

  FIG. 6 (c) is a plot of the difference in the number of introduced and supplemented molecules (error bar: ± 2α) within the range of several thousand molecules at an antigen concentration of 7.6 nM. The slope of the line indicating the number of introduced molecules and the slope of the line indicating the number of supplemental molecules are almost the same. FIG. 6D shows a plot of the supplementary efficiency at each point. Large errors were observed for the other points, but very high values (near 100%) were obtained, for example, the supplementary efficiency was 95 ± 20% at t = 27 seconds. The relatively large error was considered to be caused by a low SB ratio (calculated as 0.16 from the intensity value). The weak signal intensity from the surface bound antigen was greatly affected by the solvent related background signal, glass substrate and optical scattering. Quantification of nonspecific adsorption of antigen to the APTES layer in the supplemental region was difficult, but to minimize the possibility of nonspecific adsorption of antigen during the antibody antigen reaction, 0.2% BSA and 0. Since 05% Tween 20 was added to the antigen solution, the effect is considered to be negligible. The surface was blocked by capping with ethanolamine, PEG modification, and physical adsorption of BSA. The surface blocking method used in the experiments is commonly used in immunoassays to minimize nonspecific adsorption.

To account for these data, the depth of the nanochannel was compared to the diffusion distance of the target antigen while passing through the molecular capture region. The movement time t required for the molecules to pass through the molecular supplementary region was 1.8 seconds as calculated from the flow rate and length of the supplemental region. The diffusion distance L (μm) when t was 1.8 seconds was calculated as 12 μm from the formula L = (2Dt) ^1/2 (wherein the diffusion coefficient of IgG (D) = 40 μm ² / s). The calculated diffusion distance was much (one order of magnitude) greater than the depth of 200 nm. Thus, the introduced molecule will definitely interact with the immobilized antibody, resulting in a high capture efficiency. Although the size of the channel was not optimized, it is possible to further reduce the size of the channel in order to reduce the amount of sample required without reducing the capture efficiency. However, the pressure required to create a sufficient flow increases significantly (up to MPa) and increases the risk of clogging the nanochannel between modification and assay.

  Table 1 summarizes the results of comparing the performance of the device described in this study with a conventional immunoassay using antibodies immobilized on microbeads packed in microchannels. In this study, the reaction volume was reduced by 5 orders of magnitude (to 86 fL) and the sample volume was reduced by 6 orders of magnitude (to 810 fL). In addition, the sensitivity (based on the absolute number of molecules detectable) was increased by 5 orders of magnitude without the need for enzyme amplification (up to 3 zmol). Thus, molecular supplementation was demonstrated using a much smaller sample volume than a single cell volume picoliter. In future work, the sensitivity to detection of pM concentration should be improved to analyze several target molecules within a single cell. Since detectable amounts of fluorogenic or chromogenic substrates are produced from fewer target molecules by chemical amplification using enzymatic reactions, one effective method for improving sensitivity is an enzyme-linked immunosorbent assay. (ELISA).

2. Experimental Part Raw materials and chemicals: APTES was purchased from Tokyo Chemical Industry (Tokyo, Japan). Glutaraldehyde (25%) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Trimethoxysilane-PEG (Silane-PEG, MW = 5 kDa) was purchased from NANOCS (New York, NY, USA). Monoclonal anti-mouse IgG2a (ab131231) and mouse IgG2a isotype control (clone 20102) antibodies were purchased from Abcam (Tokyo, Japan) and R & D Systems (Minneapolis, Minnesota, USA), respectively. Mouse IgG2a labeling with the fluorescent dye Dylight 488 was performed using a Dylight 488 microscale antibody labeling kit purchased from Thermo Fisher Scientific (Waltham, Mass., USA). Glutaraldehyde (2.5%) in 10 mM borate buffer (pH 7.0) was used for antibody immobilization. The reaction buffer was 10 mM PBS (pH 7.4) containing 2% bovine serum albumin and 0.05% Tween 20, and the buffer used to regenerate the antibody-immobilized surface was pH 2. 4 glycine / HCl. Bovine serum was purchased from Kojin Bio Inc. (Saitama Prefecture, Japan). All solutions were filtered through a 0.22 μm syringe filter to prevent blockage of the channel.

  Fabrication of the flow path: As described separately, the expanded nano-flow path was subjected to a VIOSIL-SQ fused silica glass substrate (thickness 0.7 mm × width 70 mm × length 30 mm, Shin-Etsu Chemical Co., Ltd., Japan) by electron beam lithography and plasma etching. ) Produced above. The expanded nanochannel was measured to be 3.3 μm wide, 200 nm deep and 2 mm long by scanning electron / atomic force microscope. A total of 50 nanochannels were made in parallel. Both ends of the nanochannel were connected to a microchannel having a width of 500 μm and a depth of 6 μm, which was produced using a conventional UV photolithography technique.

Chemical modification for antibody patterning: A fused silica glass substrate was modified with APTES in the gas phase. Prior to modification, the substrate was immersed in a piranha solution (sulfuric acid: hydrogen peroxide = 2: 1) for 10 minutes. The substrate was thoroughly rinsed, sonicated in ultrapure water, and dried with an air stream. This clean silica substrate was placed in a separable flask with a vial containing 200 μL of APTES solution, then the separable flask was sealed and evacuated using a vacuum pump. The flask was heated in a 120 ° C. oil bath. After 2 hours of reaction, the substrate was removed from the flask and sonicated in anhydrous toluene for 5 minutes. Thereafter, the substrate was rinsed with ethanol and then with ultrapure water and dried under a stream of air. The substrate modified with APTES was placed in a self-made stainless steel container and covered with a chrome mask (the entire mask was 30 mm × 30 mm, and the chrome area was 135 μm × 3 mm at the center of the mask). A VUV light lamp housing (E500-172-120-A2, Excimer, Japan) was placed on this container, and a light source was placed within several mm from the substrate in order to minimize the attenuation of light intensity. This substrate was irradiated with VUV light at an intensity of 5 mW / cm ² for 0 to 15 minutes. As already reported, after irradiation, the substrate patterned with APTES was rinsed with ultrapure water and dried under air flow. This substrate was bonded to another silica substrate including a microchannel whose surface was activated by fluorine-enhanced plasma and an extended nanochannel. Briefly, after washing with a piranha solution, a clean silica substrate having a flow path was placed in a plasma vessel and treated with fluorine-containing oxygen plasma (60 Pa O ₂ , 250 W power) for 40 seconds. The substrate was removed from the plasma container and contacted with the APTES patterned substrate. In order to increase the bonding strength of the substrate, this device was pressurized at 5,000 N and 100 ° C. for 2 hours using a bonding machine (Bondtec Corporation, Japan). The bonded device was held at room temperature (25 ° C.) for 24 hours. All reagents for antibody immobilization were introduced into the channel by a pressure driven flow of 200 kPa. A water / ethanol (5/95) solution of 0.1 wt% trimethoxysilane-PEG was passed through the channel for 1 hour, and then the channel was washed with ethanol and water for 10 minutes. To chemically cross-link APTES and antibody through the amino group of the protein, 2.5% glutaraldehyde in 10 mM borate buffer was passed through the flow channel for 2 hours, then 1 in 25 μg / mL capture antibody in PBS. I passed the time. A 0.5 M ethanolamine solution (pH 8.3) containing 0.5 M NaCl was passed through the channel for 10 minutes to block the remaining reactive groups.

  Measurement of contact angle: The contact angle of pure water on the surface of APTES irradiated with VUV was measured using a contact angle meter (DM-500, Kyowa Interface Science, Japan).

  Fluorescence observation of immunochemical reaction: Immobilized antibody was visualized using fluorescently labeled antigen to confirm antibody activity. Prior to introduction of the target antigen solution, the flow path was filled with reaction buffer. Next, the target antigen solution was introduced into the nanochannel from one of the microchannels. Changing to the regeneration buffer was accomplished by changing the vial. A fluorescence micrograph of the nanochannel was obtained using an inverted fluorescence microscope (IX71, Olympus, Japan) equipped with a charge coupled device (CCD) camera (C9100-13, Hamamatsu Photonics, Japan). The fluorescent signal resulting from excitation induced using a mercury lamp was collected using a 40 × objective lens with a numerical aperture of 0.60. For observation over time, irradiation of excitation light at an exposure time of 100 ms was minimized by synchronizing the CCD camera and the open / close shutter in the excitation light path. The obtained micrograph was analyzed using Aquacosmos software (Hamamatsu Photonics Co., Ltd.). The total intensity in the detection window at an arbitrary location in the micrograph was determined. The motor-driven stage made it possible to observe the set position with a spatial resolution of 0.1 μm.

Example 2 Sandwich immunoassay using DIC-TLM A device similar to that in Example 1 was produced in the same manner as in Example 1. The extended nanochannel portion of the device is schematically shown in FIG. The height of the extended nanochannel was 800 nm, the width was 2 μm, and the length of the specific substance binding region selectively coated with APTES was 3 mm. The first specific binding substance immobilized on APTES was an anti-mouse IgG polyclonal antibody. After immobilizing the anti-mouse IgG polyclonal antibody, blocking was performed by treatment with 2% BSA solution in PBS for 30 minutes.

  The following operations were all performed at 20 ° C. The sample solution was injected in the same manner as in Example 1. The sample solution is a solution containing mouse IgG antibody as an antigen in PBS. The antigen concentration is 0 (no antigen), 0.2 pM, 0.4 pM, 0.8 pM or 1.7 pM, and the volume is 16 pL. (95 seconds at a flow rate of 166 fL / sec, pressure 25 kPa). At this concentration and volume, the number of antigen molecules contained in the sample solution is 0, 2, 4, 8, and 16, respectively. In the same manner as in Example 1, the expanded nanochannel was washed with a buffer solution (400 kPa, 30 seconds), and then a solution of HRP-labeled anti-mouse IgG2a goat polyclonal antibody (Abcam) in PBS (concentration 100 pM) at 25 kPa at 30 kPa. Circulated for seconds. After washing the extended nanochannel in the same manner as above, a substrate solution (1.7 mM TMB (trade name SureBlue TMB-1 (manufactured by KLM, USA) and 0.02% hydrogen peroxide in the buffer) was added to the extended nanoflow. In this state, the flow of the liquid was stopped for 60 seconds, and the enzyme reaction was carried out.After 60 seconds, the buffer solution was recirculated and the downstream of the specific binding substance immobilization region. The blue pigment | dye which is an enzyme reaction product was measured by DCI-TLM in the position 1.5 mm downstream from the edge (refer FIG. 7).

  The DIC-TLM used is a known one having two laser oscillation devices and other optical devices. The excitation beam was a diode-pumped solid-state laser (Ignis660 (trade name), manufactured by Laser Quantum) with a wavelength of 660 nm, the output was 205 mW, and its intensity was adjusted by a mechanical chopper with a modulation frequency of 3.30 kHz. The probe beam was an emission line with a wavelength of 532 nm of a YAG laser. Both beams were linearly polarized by a Glan-Thomson prism, and the plane of polarization was rotated by a half-wave plate. The two beams were coaxial with a dichroic mirror. The probe beam was divided into two orthogonally polarized beams by a DIC prism. On the other hand, the excitation beam was not split by the first DIC prism by controlling the polarization. The split probe beam and excitation beam were both focused by the objective lens (40x, N.A. 0.75) and passed through the sample. Only the probe beam was interfered by the lower DIC prism and polarizing filter. Both the interference probe beam and the excitation beam were collected by a condenser lens and passed through a filter. Only the probe beam intensity was monitored with a photodiode. A pre-amplified signal from the photodiode was synchronously amplified using a lock-in amplifier (5610B (trade name), NF, Yokohama). The time constant of the lock-in amplifier was 1 second and data was acquired every 0.2 seconds. The magnitude of the signal was recorded on a personal computer.

  In the absence of blue dye molecules that are the product of the enzyme reaction, the intensity of the probe beam is zero due to interference. When the dye molecules absorb the excitation beam and generate heat, the refractive index of the solvent changes locally, causing a phase change only for one split probe beam. As a result, a phase difference occurs between the split probe beams, and this phase difference generates a new polarization component. Only this component is detected as a signal. The signal is proportional to the absorbance of the sample.

  The results are shown in FIG. As shown in FIG. 8, the peak height of the signal changes according to the number of molecules of the antigen (target molecule) in the sample. As a result, it has been clarified that this method can quantitate antigen molecules in a sample at a count level. Conventional analytical devices are used for quantification of concentration and require about 1 million molecules. However, using the method and device of the present invention, a significant performance improvement has been found that it can be quantified at countable levels.

Claims (9)

A first substrate, a second substrate bonded to the first substrate, and a flow formed on a bonding surface of the first and second substrates and communicating with the outside of the first and second substrates And a specific binding substance immobilization region in which a first specific binding substance that specifically binds to a test substance is immobilized, and is a partial area of the flow path. 10 μm to 10 μm, and the length of the specific binding substance immobilization region is 1 μm to 10 cm, and the sample in the sample is circulated through the flow path of the quantification device for the test substance in the sample. Binding a substance to the first specific binding substance;
After washing the specific binding substance-immobilized region, a labeled second specific binding substance that specifically binds to the test substance is circulated through the flow path and bound to the first specific binding substance. Binding the test substance and the second specific binding substance;
A method for quantifying a test substance in a sample, comprising washing the specific binding substance-immobilized region and then quantifying a label bound to the test substance. The flow rate of the sample is expressed by the following formula [I]
5 ≤ (2Dτ) ^1/2 / d [I]
(Where D is the diffusion coefficient of the test substance (m ² / s), τ is the average time (s) that the molecules of the test substance stay on the specific binding substance immobilization region, and d is the depth of the flow path. (m))
A method for quantifying a test substance in a sample, the flow rate satisfying   The method according to claim 2, wherein the flow rate of the sample is a flow rate at which the value on the right side of the formula [I] is 30 or more.   The method according to claim 1, wherein the channel is an extended nanochannel having a height of 10 nm to 1000 nm.   The method according to any one of claims 1 to 4, wherein the first specific binding substance is an antibody, an antigen-binding fragment thereof, or an antigen, and the test substance is an antigen or an antibody.   The label is an enzyme label, and the enzyme is an enzyme that reacts with the substrate to develop a color, and further includes a step of causing the label enzyme and the substrate to react with the substrate to develop a color. 6. The method according to any one of claims 1 to 5, which is carried out by quantifying the substance that has developed color as a result of differential interference thermal lens microscopy.   The method according to claim 5 or 6, wherein the enzyme reaction is performed in a state where the flow of the sample is stopped.   The method according to any one of claims 1 to 7, wherein the quantitative determination by the differential interference thermal lens microscope is performed in a region downstream of the specific binding substance immobilization region.   A first substrate, a second substrate bonded to the first substrate, and a flow formed on a bonding surface of the first and second substrates and communicating with the outside of the first and second substrates And a specific binding substance immobilization region in which a first specific binding substance that specifically binds to a test substance is immobilized, and is a partial area of the flow path. A quantification device for a test substance in a sample used in the quantification method according to any one of claims 1 to 8, wherein the quantification method according to any one of claims 1 to 8, wherein the specific binding substance immobilization region has a length of 10 nm to 10 µm. .