Selectively bonding substance immobilization carrier

Abstract

To stabilize measurement results by restricting variation in detection signals between reaction zones, in a selectively bonding substance immobilization carrier having a plurality of reaction zones on the same carrier.SOLUTION: In a selectively bonding substance immobilization carrier, a residual amount of an N-hydroxysuccinic imide ester on a carrier surface, generated at the stage of fixing a selectively bonding substance, is restricted. In particular, when a carrier surface composed from a polymethyl methacrylate ester (PMMA) is analyzed according to a time-of-flight secondary ion mass analysis (TOF-SIMS), an intensity ratio of a peak C4H4 NO2-(m / z 98.24) relative to a peak of C4H5O2- (m / z 85.04) is 0.02 or less, or an intensity ratio of a peak C4H4 NO3- (m / z 114.02) relative to a peak of C4H5O2- (m / z 85.04) is 0.005 or less in the carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a carrier having immobilized thereon a substance that selectively binds to a substance to be measured. [Background technology]

[0002] A selective binding substance-immobilized carrier is a carrier having immobilized on its surface a substance such as a nucleic acid or a protein (referred to as a "selective binding substance" in this specification) that selectively binds to a substance to be measured. The substance to be measured can be detected by detecting the selectively bound substance through fluorescence or the like, and molecular identification and diagnosis can be performed from the intensity change or pattern.

[0003] The method of immobilizing a selective binding substance on a carrier mainly involves chemically binding the substance to the carrier surface. For example, a carrier having a carboxy group formed on its surface is used, and the carboxy group on the carrier is condensed with the amino group of the selective binding substance to form an amide bond, thereby directly immobilizing the selective binding substance on the carrier surface.

[0004] On the other hand, in addition to the method of directly immobilizing a selective binding substance on the carrier surface, a method of immobilizing the substance via a linker molecule is also known. For example, Patent Document 1 describes a method of immobilizing DNA on the carrier surface using amino acids and peptides as linkers, and by immobilizing the DNA via a linker, it is possible to obtain an effect of improving the detection signal when the substance to be measured is reacted.

[0005] In addition, in order to improve the measurement efficiency, selective binding substance immobilized carriers have been developed that can simultaneously measure multiple samples containing the target substance by providing multiple reaction areas on the same carrier. For example, Patent Document 2 describes selective binding substance immobilized carriers having 12 and 24 reaction areas on the same carrier. When providing multiple reaction areas on the same carrier, it is important to suppress the variation in detection signals between reaction areas so that there is no difference in the measurement results depending on the position of the reaction area on the carrier. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2006-208012 A [Patent Document 2] International Publication No. 2015 / 147004 Summary of the Invention [Problem to be solved by the invention]

[0007] As shown in Comparative Example 1 described later, the present inventors immobilized DNA on a carrier having 24 reaction regions via a linker made of a peptide by the method described in Patent Document 1 in order to improve the detection signal. The obtained DNA-immobilized carrier was evaluated for the variation between reaction regions of the detection signal obtained by hybridization reaction with a sample containing a microRNA, which is the substance to be measured, using the coefficient of variation (CV) as an index. As a result, it was revealed that the CV exceeded 5%, indicating a large variation. In other words, it was revealed that when a selective binding substance is immobilized on a carrier having multiple reaction regions, the detection signal obtained for the substance to be measured varies between reaction regions, resulting in differences in the measurement results. [Means for solving the problem]

[0008] As a result of intensive research, the present inventors have clarified by time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis that the cause of the variation in detection signal between reaction regions is N-hydroxysuccinimide ester (hereinafter, NHS ester) remaining on the carrier surface. They have also found that the variation in detection signal between reaction regions can be suppressed by suppressing the amount of NHS ester remaining on the carrier surface, and have completed the present invention.

[0009] That is, the present invention is constituted by the following aspects (1) to (4). (1) A carrier having a selective binding substance immobilized on an immobilization region on its surface, the carrier having a plurality of reaction regions on the same carrier, the carrier being made of polymethacrylic acid methyl ester, the selective binding substance being immobilized on the immobilization region by a method including a step of forming an N-hydroxysuccinimide ester, and in anion analysis by time-of-flight secondary ion mass spectrometry (TOF-SIMS) on the surface of the carrier in a region where the selective binding substance is not immobilized, C4H5O2 - C4H4NO2 for the peak at (m / z 85.04) - (m / z 98.24) peak intensity ratio is 0.02 or less or C4H5O2 - C4H4NO3 for the peak at (m / z 85.04) - A support characterized in that the intensity ratio of the peak at (m / z 114.02) is 0.005 or less. (2) The carrier according to claim 1, wherein the selective binding substance is immobilized on the carrier via an amino acid, a peptide or a derivative thereof. (3) The carrier according to claim 1 or 2, wherein the selective binding substance is a nucleic acid. (4) The support according to any one of claims 1 to 3, wherein the detection target is microRNA. Effect of the Invention

[0010] By detecting a substance to be measured contained in a specimen using the DNA-immobilized carrier of the present invention, it becomes possible to perform measurements with reduced variation in detection signals between reaction regions. [Brief description of the drawings]

[0011] [Figure 1] (a) is a top view showing an example of a selective binding substance-immobilized carrier with 24 reaction regions on the same carrier, and (b) is a cross-sectional view taken along the plane A in (a). Each reaction region has a concave structure, and the selective binding substance is immobilized on the bottom surface of the concave portion. [Diagram 2](a) is a top view showing an example of a selective binding substance-immobilized support with 24 reaction regions on the same support, and (b) is a cross-sectional view taken along the plane indicated by B in (a). Each reaction region has a concave-convex structure, with multiple convex portions on the bottom surface of the concave portions, and the selective binding substance is immobilized on the top surface of the convex portions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to a carrier having a selective binding substance immobilized on an immobilization region on its surface, the carrier having a plurality of reaction regions on the same carrier, the carrier being made of polymethacrylic acid methyl ester, the selective binding substance being immobilized on the immobilization region by a method including a step of forming an N-hydroxysuccinimide ester (NHS ester), and in anion analysis by time-of-flight secondary ion mass spectrometry (TOF-SIMS) on the surface of the carrier in a region where the selective binding substance is not immobilized, C4H5O2 - C4H4NO2 for the peak at (m / z 85.04) - (m / z 98.24) peak intensity ratio is 0.02 or less or C4H5O2 - C4H4NO3 for the peak at (m / z 85.04) - The carrier is characterized in that the intensity ratio of the peak at m / z 114.02 is 0.005 or less.

[0013] The carrier of the present invention is produced through a process in which a selective binding substance is immobilized via an intermediate containing an NHS ester.

[0014] In a selective binding substance-immobilized support having multiple reaction regions on the same support, the variation in detection signals between reaction regions can be improved by reducing the amount of residual NHS ester that is generated as an intermediate in the process of immobilizing the selective binding substance onto the support.

[0015] The carrier of the present invention has a selective binding substance immobilized on a partial region of its surface, and has a plurality of reaction regions on the same carrier for reacting the immobilized selective binding substance with a sample to be measured. The number of reaction regions on the same carrier may be two or more, but five or more is preferable, at which the effect of the present invention, that is, suppression of variation in detection signals between reaction regions, is more significantly exhibited, more preferably 12 or more, and even more preferably 24 or more.

[0016] The reaction regions on the same carrier are preferably separated by a partition to prevent mixing of samples to be measured. For example, as shown in FIG. 1, a reaction chamber having a concave structure provided on the carrier and having a selective binding substance immobilized on the bottom surface of the concave is considered as one embodiment of the reaction region. In addition, in order to improve the detection sensitivity during detection, a reaction region having a concave-convex structure as disclosed in JP-A-2004-264289 can also be used. When a reaction region having a concave-convex structure is used, a structure in which a plurality of convex portions are present on the bottom surface of the concave portion and a selective binding substance is immobilized on the upper surface of the convex portions can be considered as shown in FIG. 2.

[0017] In the present invention, the carrier on which the selective binding substance is immobilized is made of polymethylmethacrylate (PMMA), which has excellent processability and optical properties. The carrier may contain additives other than PMMA. For example, carbon black may be added to make the carrier black, which can reduce background noise during fluorescence detection.

[0018] The selective binding substance in the present invention means a substance that can selectively bind to a substance to be measured directly or indirectly, and representative examples thereof include nucleic acids, proteins, sugars, and other antigenic compounds. Examples of nucleic acids include DNA, RNA, and PNA. A single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part of the base sequence, and thus corresponds to a selective binding substance. Examples of proteins include antibodies, antigen-binding fragments of antibodies such as Fab fragments and F(ab')2 fragments, and various antigens. An antibody or its antigen-binding fragment selectively binds to a corresponding antigen, and an antigen selectively binds to a corresponding antibody, and thus corresponds to a selective binding substance. Examples of sugars include polysaccharides, and various antigens. Substances having antigenicity other than proteins and sugars can also be immobilized. The selective binding substance used in the present invention may be commercially available or may be obtained from living cells, etc. A particularly preferred selective binding substance is nucleic acid. Among nucleic acids, oligonucleic acids having a length of 10 to 100 bases are preferred because they can be easily artificially synthesized using a synthesizer and can be easily immobilized on a carrier surface by introducing a reactive functional group into the end. From the viewpoint of hybridization stability, a length of 20 to 100 bases is more preferred. Examples of reactive functional groups to be introduced into the end of a nucleic acid include an amino group, a hydroxyl group, a thiol group, and a carboxyl group, and an amino group is preferred.

[0019] The selective binding substance is immobilized on the surface of the carrier by a method including a step of forming an NHS ester, and can be immobilized, for example, by a method consisting of the following steps (1) to (4).

[0020] Step (1) generating a carboxy group on the surface of the support; Step (2) converting the carboxy group into an NHS ester; Step (3) reacting the NHS ester with a linker; Step (4) is the step of immobilizing a selective binding substance via the linker.

[0021] In step (1), methods for generating carboxyl groups on the surface of the support include treatment with acid or alkali, ultrasonic treatment in warm water, and oxygen plasma treatment. Since PMMA, the base material of the support, has methyl esters in its side chains, carboxyl groups that serve as reaction sites can be easily generated by acid or alkali hydrolysis.

[0022] In step (2), the carboxyl group on the carrier surface can be converted to an NHS ester by a condensation reaction using a condensing agent in the presence of N-hydroxysuccinimide. Usable condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), with EDC being preferred.

[0023] In step (3), the molecule used as the linker is preferably a bifunctional molecule having a functional group used for immobilization on the carrier and a functional group used for binding to the selective binding substance. Examples of the bifunctional molecule include amino acids, peptides, and derivatives thereof, and peptides and their derivatives are more preferred in terms of signal enhancement effect. The degree of polymerization of the peptide is preferably 2 to 100, more preferably 2 to 10.

[0024] In step (3), the method for reacting the linker with the NHS ester may be a reaction with a functional group having NHS ester reactivity (e.g., an amino group, a thiol group, or a hydroxyl group) present in the linker molecule, and an example of such a method is to contact the support with a neutral or weakly alkaline solution containing a linker having an amino group.

[0025] In step (4), the selective binding substance can be immobilized via the linker by reacting a functional group present in the linker molecule with a functional group in the selective binding substance, for example, by a condensation reaction of a carboxy group in the linker molecule with an amino group in the selective binding substance. In the condensation reaction, the carboxy group in the linker molecule may be converted to an NHS ester, as in the case of converting the carboxy group on the surface of the PMMA molecule, and the resulting NHS ester may be bonded to a selective binding substance having an amino group.

[0026] In addition, steps (2) and (3) may be repeated. For example, when an amino acid is used as a linker, steps (2) and (3) can be repeated to produce a polymer of amino acids, i.e., a peptide, on the support. Steps (2) and (3) are preferably repeated 2 to 10 times.

[0027] Among these steps, a portion of the NHS ester converted in step (2) does not react with the linker molecule in step (3) and remains on the surface of the carrier, resulting in variation in the detection signal between reaction regions. Therefore, by reducing the amount of remaining NHS ester, it is possible to improve the variation.

[0028] Methods for reducing the amount of remaining NHS ester include changing the reaction conditions in steps (2) and (3) and changing the type and purity of the reagents used. For example, the amount of remaining NHS ester can be reduced by using a condensation agent with a low melting point, which is an index of purity, for the condensation agent used in step (2).

[0029] The amount of NHS ester remaining on the support surface was determined by time-of-flight secondary ion mass spectrometry (TOF-SIMS) using the secondary ion C4H4NO2 - (m / z 98.24) and C4H4NO3 - The secondary ion C4H5O2 from PMMA for the peak at m / z 114.02 -It can be expressed as the intensity ratio of the peak at m / z 85.04.

[0030] The amount of remaining NHS ester required in the present invention is determined by the TOF-SIMS analysis of the area on the surface of the selective binding immobilization carrier where the selective binding substance is not immobilized. - C4H4NO2 for the peak at (m / z 85.04) - (m / z 98.24) peak intensity ratio is 0.02 or less, or C4H5O2 - C4H4NO3 for the peak at (m / z 85.04) - The intensity ratio of the peak at (m / z 114.02) is 0.005 or less.

[0031] The substance to be measured using the carrier of the present invention is a substance that selectively binds to the selective binding substance of the present invention. Examples include nucleic acids, proteins, polysaccharides, etc., and nucleic acids are preferred, and among nucleic acids, RNA is more preferred, and microRNA is even more preferred.

[0032] Examples of specimens containing the substance to be measured include body fluids such as blood, serum, plasma, urine, and saliva, as well as dilutions and extracts thereof, with serum and plasma extracts being preferred. EXAMPLES

[0033] Example 1 (1) Preparation of the carrier A mold for injection molding was prepared using the known LIGA (Lithographic Galvanoformung Abfromung) process, and a carrier made of polymethyl methacrylate (PMMA) and having a shape as described below was obtained by injection molding.

[0034] The carrier has a concave-convex structure with an external shape of 76 mm in length, 26 mm in width, and 1 mm in thickness. The carrier has 24 concave portions, each measuring 6.48 mm in length, 6.90 mm in width, and 0.12 mm in depth, arranged in 3 columns and 8 rows. Within these concave portions, there are 576 convex portions, each measuring 0.1 mm in diameter and 0.12 mm in height, arranged in 24 columns and 24 rows.

[0035] (2) Formation of carboxyl group The support shown in (1) was immersed in a 10N sodium hydroxide solution at 70°C for 15 hours. It was then washed with pure water, a 0.1N HCl solution, and pure water in that order. In this way, the side chains of PMMA on the support surface were hydrolyzed to generate carboxyl groups.

[0036] (3) Conversion reaction to NHS ester The support obtained in (2) was immersed in 2-morpholinoethanesulfonic acid monohydrate (manufactured by Fujifilm Wako Pure Chemical Industries, hereinafter referred to as MES) buffer (adjusted to pH 6.0 with 0.1N sodium hydroxide aqueous solution, 50 mM) in which N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, hereinafter referred to as MES) was dissolved to a final concentration of 10 mM and 1-ethyl-3-(3-dimethylaminopropylcarbodiimide (manufactured by Sigma-Aldrich, BioXtra grade, lot number: BCCB8365, hereinafter referred to as EDC) to a final concentration of 10 mM, and stirred with a micro stirrer at room temperature for 1 hour, followed by washing with pure water.

[0037] (4) Addition reaction of amino acids to NHS esters The carrier obtained in (3) was immersed in borate buffer (adjusted to pH 8.3 with 1N sodium hydroxide solution, 50 mM) in which glycine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved to a final concentration of 10 mM, and after stirring with a microstirrer at room temperature for 1 hour, it was washed with pure water. Furthermore, the operations of (3) and (4) were repeated three times to obtain a carrier bound with four glycine molecules.

[0038] (5) Immobilization of probe DNA In order to detect hsa-miR-149-3p (sequence number 1), hsa-miR-2861 (sequence number 2), and hsa-miR-4463 (sequence number 3), DNA consisting of the base sequences of the following sequence numbers 4 to 6, which are complementary to each other, were synthesized.

[0039] (SEQ ID NO: 4) 5'-GCACAGCCCCCGTCCCTCCCT-3' (5'-end aminated) (SEQ ID NO: 5) 5'-CCGCCCACCGCCAGGCCCC-3' (5'-end aminated) (SEQ ID NO: 6) 5'-GGCCCCACCCCAGTCTC-3' (5'-end aminated) These DNAs were dissolved in pure water at a concentration of 0.27 mmol / μL to prepare a stock solution. The stock solution was diluted 10-fold with phosphate buffered saline (manufactured by Fujifilm Wako Pure Chemical Industries, hereinafter referred to as PBS) in which EDC (Sigma-Aldrich BioXtra grade, lot number: BCCB8365) was dissolved to a final concentration of 10 mM to prepare a spotting solution. 40 μL of each spotting solution was taken out and spotted on the upper surface of the convex part of the carrier prepared above using a spotting robot (Japan Laser Electronics Co., Ltd., GTMAS Stamp-2). The spotted carrier was placed in a sealed plastic container and incubated for 20 hours at 37° C. and 100% humidity. Finally, the carrier was washed with pure water and centrifuged and dried with a spin dryer.

[0040] (6) Preparation of sample RNA Blood was collected from one healthy individual, and serum was prepared. From the prepared serum, RNA contained in the serum sample (hereinafter referred to as sample RNA) was extracted using the "3D-Gene" RNA extraction reagent from liquid sample kit (Toray Industries, Inc.). The obtained sample RNA was labeled using the "3D-Gene" miRNA labeling kit (Toray Industries, Inc.).

[0041] (7) Evaluation of hybridization signals 10 uL of the labeled RNA solution prepared in (6) above was poured into each reaction area (recess) of the DNA-immobilized carrier prepared in (5) above. The DNA-immobilized carrier was sealed to close the reaction area, and then placed in a stirring device "Bioshaker 5000" installed in an oven temperature-controlled at 37°C, and stirred at 5000 rpm for 16 hours. After stirring, the carrier was washed with pure water and dried by centrifugation in a spin dryer, and the hybridization signal was measured using a high-resolution fluorescence detection device (Toray, 3D GeneScanner).

[0042] The fluorescence intensity at the site where SEQ ID NOs: 1 to 3 were immobilized was calculated, and the variation in the fluorescence intensity of each of the 24 reaction regions present on the same carrier was calculated as CV (average intensity / standard deviation).

[0043] (8) Measurement of carrier by TOF-SIMS The region of the DNA immobilized carrier prepared in (5) above where the probe DNA was not immobilized was subjected to TOF-SIMS analysis under the following conditions to obtain a secondary ion mass spectrum and a secondary ion image. In the obtained mass spectrum, the secondary ion peak of N-hydroxysuccinimide (C4H4NO2 - , C4H4NO3 - ) and secondary ion peaks from the resin (C4H5O2 - ) peak intensity ratio (C4H4NO2 - / C4H5O2 - , C4H4NO3 - / C4H5O2 - The results are shown in Table 1.

[0044] (TOF-SIMS conditions) Equipment: ION-TOF TOF.SIMS5 Secondary ion polarity: negative Mass range (m / z): 0~1500 Raster size: 300μm×300μm Number of scans: 24 scans Number of pixels (1 side): 256 pixels Measured vacuum (before sample introduction): 4×10-7 Pa or less Primary ion species: Bi3 2+ Primary ion acceleration voltage: 25 kV Pulse width: 15.2ns Bunching: Yes (high mass resolution measurement) Static neutralization: yes Rear acceleration: 9.5kV (9) Differential Scanning Calorimetry (DSC) of EDC The melting peak temperature of the EDC (lot number: BCCB8365) used in (3) was determined by differential scanning calorimetry (hereinafter, DSC) under the following conditions, and was found to be 118.5° C. The results are shown in Table 1. (DSC conditions) Equipment: TA Instruments Q100 Data processing: TA Instruments "Universal Analysis 2000" Atmosphere: Nitrogen flow (50mL / min) Temperature and heat calibration: High purity indium (Tm=156.61℃, ΔHm=28.71 J / g) Temperature range: 50~180℃ Heating rate: 10℃ / min Sample size: approx. 1 mg Sample container: Standard aluminum container.

[0045] Comparative Example 1 The DNA immobilized carrier was prepared, evaluated, and analyzed in the same manner as in Example 1, except that the EDC used in Example 1(3) (conversion reaction to NHS ester) was "lot number: BCCF5030" instead of "lot number: BCCB8365," and the lot of EDC used in (5) (immobilization of probe DNA) was also used.

[0046] The peak melting temperature of EDC (lot number: BCCF5030) was determined in the same manner as in Example 1(9) and was found to be 119.5°C.

[0047] Comparative Example 2 The DNA immobilized carrier was prepared, evaluated, and analyzed in the same manner as in Example 1, except that the EDC used in Example 1(3) (conversion reaction to NHS ester) was "lot number: BCCD9983" instead of "lot number: BCCB8365," and the lot of EDC used in (5) (immobilization of probe DNA) was also used.

[0048] The peak melting temperature of EDC (lot number: BCCF9983) was determined in the same manner as in Example 1(9) and was found to be 119.2°C. result

[0049] [Table 1] As a result of "(7) Evaluation of hybridization signals," in Comparative Examples 1 and 2, the CV of fluorescence intensity between each reaction region exceeded 5%, whereas in Example 1, the CV between each reaction region was 5% or less.

[0050] The results of "(8) Measurement of the carrier by TOF-SIMS" show that C4H4NO2 - / C4H5O2 - , C4H4NO3 - / C4H5O2 - The peak intensity ratios of both were high in Comparative Examples 1 and 2, but low in Example 1. From this, it is considered that the amount of NHS ester remaining on the support was high in Comparative Examples 1 and 2, but low in Example 1.

[0051] As a result of "(9) Differential scanning calorimetry (DSC) of EDC," the melting peak temperatures of the EDC used in Comparative Examples 1 and 2 were 119.5°C and 119.2°C, respectively, while the melting peak temperature of the EDC used in Example 1 was 118.5°C.

[0052] Considering these facts, it is presumed that when the support is treated with a condensing agent with a relatively low melting peak temperature, the amount of NHS ester remaining on the support is kept low, thereby improving the CV of the fluorescence intensity between the reaction regions.

[0053] The reason why the variation in detection signal between reaction regions becomes large when NHS ester remains on the carrier surface is thought to be that some of the carboxyl groups on the carrier surface and in the linker, which are necessary for the immobilization reaction of the probe DNA, remain converted to NHS ester, inhibiting the immobilization reaction of the probe DNA and causing the amount of immobilization to vary between reaction regions. [Explanation of symbols]

[0054] 1. Carrier 2. Recess (reaction area) 3. Selective binding substances 4 Convex

Claims

1. A carrier having a selective binding substance immobilized on its surface, wherein a plurality of reaction regions are present on the same carrier, the carrier is made of polymethyl methacrylate, and the selective binding substance is immobilized on the immobilization region by a method including a step of forming N-hydroxysuccinimide ester. In the anion analysis of time-of-flight secondary ion mass spectrometry (TOF-SIMS) on the surface of the region of the carrier where the selective binding substance is not immobilized, C ４ H ５ O ２ － (The intensity ratio of the peak of m / z 85.04) to C ４ H ４ NO ２ － (The intensity ratio of the peak of m / z 98.24) is 0.02 or less or C ４ H ５ O ２ － (The intensity ratio of the peak of m / z 85.04) to C ４ H ４ NO ３ － (The intensity ratio of the peak of m / z 114.02) is 0.005 or less. A carrier characterized by this.

2. The carrier according to claim 1, wherein a selectively binding substance is immobilized on the carrier via an amino acid, a peptide, or a derivative thereof.

3. The carrier according to claim 1 or 2, wherein the selectively binding substance is a nucleic acid.

4. The carrier according to claim 1 or 2, wherein the target for detection is microRNA.

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