JP2007248206A - Quantitative method of functional group on surface of base material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for quantifying the number of functional groups present on the surface of a glass base material optimum to a biochip. <P>SOLUTION: This quantitative method of the functional groups on the surface of the base material is composed of the following processes A-E: a process A: the process for forming a semi-hermetically closed space with an average thickness of 500 μm or below and a micro-volume wherein the area of the base material (11) is 100-10,000 mm<SP>2</SP>on the base material (11), a process B: the process for filling the space with a liquid containing a substance reacting with the functional groups on the base material, a process C: the process for quantitatively reacting the functional groups on the base material with the substance, a process D: the process for recovering the liquid after reaction and a process E: the process for quantifying the substance in the recovered liquid to calculate difference. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for quantifying functional groups on the surface of a substrate, and more specifically to a method for quantifying functional groups on the surface of a biochip substrate.

  The surface of a glass substrate used for biochips or the like needs to be subjected to various chemical modifications in order to exhibit the effect. However, conventionally, quantitative analysis of functional groups added by chemical modification of the glass substrate surface has not been performed. This is partly because such a measurement is very difficult. Furthermore, as a second factor, if the change in physical properties of the entire substrate surface due to chemical modification of the substrate surface can be confirmed, the substrate material can be obtained even if the molecular level of the substrate surface cannot be grasped quantitatively. There is no major problem with respect to the surface characteristics of the above, and it can be mentioned that it was sufficient as a base material for biochips. For confirmation of the change in physical properties of the entire substrate surface, for example, an evaluation test such as a contact angle of water or a change in transmittance and antifouling performance is used (Non-Patent Document 1).

  However, biochips that have been actively developed in recent years are required to be able to comprehensively and quantitatively measure genes with low expression concentrations called low expression (Patent Document 1). It is important to quantitatively grasp the number of functional groups present on the surface of the base material. However, it has been extremely difficult to directly compare the physicochemical properties of the substrate surface with the performance as a biochip.

  Therefore, various attempts have been made to obtain a correlation with the performance of the biochip by measuring the functional group amount on the surface of the glass substrate. However, conventionally, it was only possible to qualitatively measure the amount of functional groups on the surface of the glass substrate, and therefore, it was not possible to accurately control the amount of functional groups on the glass substrate optimal for biochips and the like. . FT-IR, ESCA, fluorescence image scanning, etc. were used as qualitative analysis methods for the amount of functional groups on the surface. Although these methods can qualitatively determine the amount of functional groups, they are quantitative. It was difficult to grasp the amount of functional groups.

JP 2006-29953 A Technical Information Association "" Wettability "and Control-Surface Modification / Interface Control, Evaluation, Contact Angle-" January 2000, Chapter 3 "Measurement and Contact Angle of Wetness"

  An object of the present invention is to provide a technique capable of quantitatively evaluating a functional group on a substrate surface, which has been difficult with the prior art, even with a small amount of liquid.

The present invention provides a method for quantifying a functional group on a substrate surface, which comprises the following steps A to E.
Step A: A step of forming a semi-enclosed space of a minute volume having an average thickness of 500 μm or less on the substrate and an area of the substrate of 100 square mm to 10,000 square mm.
Step B: A step of filling the space with a liquid containing a substance that reacts with the functional group on the substrate.
Step C: a step of quantitatively reacting the functional group on the substrate with the substance.
Step D: A step of recovering the liquid after the reaction.
Step E: A step of quantifying the substance in the recovered liquid and obtaining a difference.

  Furthermore, the present invention relates to a method for quantifying the functional group on the surface of the substrate, wherein the functional group to be detected is an amino group, a quantification method in which the substance that reacts with the amino group is a substance containing an isothiocyanate group and a phenyl group, and an isothiocyanate. The functional group on the substrate surface is characterized in that high-performance liquid chromatography is used for the method for quantifying the functional group on the surface of the substrate, wherein the substance containing a group and a phenyl group is phenylene isothiocyanate, and the method for detecting the substance that reacts with an amino group And a method for quantifying the functional group on the substrate surface, wherein the solvent in the liquid is dimethylformaldehyde or N-methylpyrrolidone, and the functional group on the substrate surface further containing a non-volatile tertiary amine in the liquid. Substrate surface that does not react with amino groups and can be quantified by high-performance liquid chromatography as an internal standard. Quantitative method of functional group, quantification method of functional group on substrate surface whose internal standard substance is dimethylaniline, quantification method of substrate surface whose substrate to be quantified is a substrate surface-treated with aminosilane, and There is also provided a method for quantifying the surface of a substrate, wherein the substrate is a biochip substrate, and the substrate to be quantified is a substrate surface-treated with aminosilane.

  By using the method for quantifying functional groups on the substrate surface of the present invention, it is possible to quantitatively measure the functional groups on the substrate surface, and the group having an optimal functional group amount as a glass substrate used for biochips and the like. It becomes possible to provide the material.

In the step of forming a minute volume space in which the average thickness is 500 μm or less on the substrate in step A and the area of the substrate is 100 to 10,000 square mm ² , the thickness may be 500 μm or less. The thinner the thickness, the more the opportunity for the substance that reacts with the functional group on the base material in Step B and the functional group on the base material to approach within the diffusion distance. By reducing the volume of the space, the concentration of the liquid containing the substance that reacts with the functional group on the base material in the process B can be increased, and an error due to adsorption or the like until it is quantified in the process E Is preferably 100 μm or less. Furthermore, 50 μm or less is more preferable. If the thickness of the space with a very small volume is less than 5 μm, it becomes difficult to recover the liquid in Step D, and sampling of high performance liquid chromatography (hereinafter referred to as HPLC) is also difficult. Therefore, the thickness is preferably 5 μm or more. It is. Furthermore, 10 μm or more is more preferable.

  The space of the minute volume may be a space that holds the liquid during the reaction time of the functional group and does not volatilize the liquid when the space is filled with the liquid that reacts with the functional group. Such a space is obtained by, for example, stacking a cover glass having convex portions having an average thickness of 500 μm or less on both edges on a base material such as a slide glass as shown in FIG. Is possible.

  Any method may be used for filling the space containing a substance that reacts with the functional group on the base material (hereinafter referred to as a functional group-reactive substance) into the space in Step B. However, a normal means such as a syringe or a syringe may be used. May be used.

  In Step C, since the functional group on the base material and the functional group reactive substance are quantitatively reacted, it is necessary that the functional group on the base material and the functional group reactive substance sufficiently react with each other. Therefore, the time required for the reaction to proceed sufficiently is required before the recovery of step D. This time depends on the functional group on the substrate and the functional group reactive substance.

  In the step of recovering the liquid after the reaction in the step D, the functional group-reactive substance in the liquid obtained by recovering a small volume of the liquid in the step E is quantified, and an amount enough to obtain the difference may be recovered. . There are various methods of recovery, but for convenience, when the space of the minute volume is composed of a base material and a cover glass, it is possible to open the cover glass and recover with a syringe or the like. The amount to be recovered may be an amount that can be detected by an apparatus that can determine the difference in the step of quantifying the functional group reactive substance in the liquid in the following step E and determining the difference.

  In the step of quantifying the substance in the liquid recovered in the step E and obtaining the difference from the quantification result of the reactive substance before the reaction, any apparatus capable of obtaining the difference may be used, such as HPLC or gas chromatography ( GC) etc. can be used.

  The functional group to be detected of the base material does not need to be a specific functional group. However, in the biochip, DNA and protein are easily immobilized on the surface of the base material, and DNA and protein are more firmly fixed. In many cases, an amino group can be preferably used as the functional group because it is often used as a reaction point for constructing a functional group for conversion into a substrate surface.

  When the functional group is an amino group, the substance that reacts with the amino group can be detected with at least a functional group that is known to react specifically with the amino group, and a test device using optical detection such as HPLC. Thus, it is preferable to use a substance (hereinafter referred to as an amino group reactive substance) containing a site having light (ultraviolet ray) absorption characteristics or fluorescence characteristics such as a phenyl group. Specifically, NBD-F and NBD-Cl having a nitroaryl halide group, DMEQ-COCl and Sulforhodamine 101 Acid Chloride having an acid chloride group, 5-FAM and 6-TAMRA having a succinimide moiety, and fluorescein isothiocyanate having an isothiocyanate group In addition, phenylene isothiocyanate, other combinations of orthophthalaldehyde and N-acetylcysteine, ninhydrin, and the like can be used, but are not limited to the above substances.

  Among them, the use of phenylene isothiocyanate (hereinafter referred to as PITC) is a minimum configuration of a functional group that specifically reacts with an amino group and a benzene ring that absorbs ultraviolet rays. Even if it is present on the surface of the equipment, the volume of the amino group reactant itself can avoid saturating the quantitative results according to the present invention, and PITC is well known for its properties in amino acid labeling and protein analysis. It is preferable in that it has a track record in similar fields. In this case, it takes about 1 to 8 hours for the reaction in Step C to proceed. It takes 4 to 8 hours at room temperature for the amino group on the substrate to completely react with PITC, but if the reaction time is excessively extended, error factors such as PITC autolysis and DMF volatilization may occur. In order to expand, practically, the amino group and PITC reaction proceed sufficiently, and a reaction time of about 3 hours is suitable for improving the quantitativeness in measurement.

  As a method for detecting a substance that reacts with an amino group, GC or HPLC can be used because it is only necessary to measure a small amount of sample and to be able to separate and quantify each compound. Although GC is superior in terms of detection sensitivity, the use of HPLC has a track record in terms of quantification, and PITC can be quantified more specifically by detecting light absorption at a wavelength of 254 nm.

  The solvent in the liquid may be any solvent that uniformly dissolves the amino group reactant and has little interaction with the amino group and the amino group reactant. Specifically, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, methanol, ethanol, dimethylformaldehyde, N-methylpyrrolidone, or the like can be used, but is not limited thereto. Among them, dimethylformaldehyde or N-methylpyrrolidone is low in volatility, so that an accurate operation in the above steps B and D is easy, the solubility of the amino group reactant is high, and the wettability with respect to the equipment surface is appropriate. Furthermore, especially when PITC is used as an amino group reactant, it is preferable because anhydrous grade solvents are readily available for the purpose of minimizing decomposition other than bonding with amino groups on the surface of PITC equipment. Used.

  Furthermore, it is possible to further include a tertiary amine in the liquid. When a tertiary amine is further contained in the liquid, it is expected to improve the reactivity with the amino group of the amino group reactant.

  Further, when the substituent of the tertiary amine is an alkyl group, the light absorption is small, so that it is difficult to hinder the analysis in the HPLC analysis in the above step E. As such a substance, for example, triethylamine can be preferably used. The amount used is preferably 0.01 to 1% by volume in the solution when phenylene isothiocyanate is used in the solution. The effect is not seen at 0.01% by volume or less, and the use of more than 1% by volume causes a problem of promoting the self-decomposition of PITC.

When the functional group to be detected of the substrate to be quantified is an amino group, it is desirable to use the substrate surface-treated with a silane coupling agent (aminosilane) having an amino group. As the usable aminosilane, R has an organic functional group and X represents a hydrolyzable group that reacts with an inorganic material, and has an RSiX ₃ chemical structure. At least one of the ends of the organic functional group R is amino. X is preferably a chlorine, alkoxy group or the like. Examples of the alkoxy group include methoxy group, ethoxy group, propoxy group, butoxy group, pentyloxy group, hexyloxy group, and the like.

  Examples of aminosilanes include, but are not limited to, N- (2-aminoethyl) -3aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, and 3-aminopropyltrimethoxysilane. . Among these, 3-aminopropyltrimethoxysilane is particularly preferable because it has a simple structure and is easy to handle.

  As the glass used for the substrate, any kind of glass can be used as long as the silane coupling agent can be bonded to the surface. Glasses such as synthetic quartz, borosilicate glass, and soda lime glass are preferable in terms of availability and achievements in biochip applications, but they are made of metal, metal oxide, or metal nitride, or these May be a substrate whose surface is coated by sputtering or vapor deposition. Since the amount of functional groups such as amino groups on the surface of the substrate is quantified by the quantification method of the present invention, it is possible to produce a high-quality biochip substrate according to the use of the biochip.

  Further, the liquid of the present invention may contain a substance that does not react with an amino group, has little adsorption to the substrate surface, and can be quantified by high performance liquid chromatography as an internal standard substance. This is because when the internal standard substance is contained in the liquid, it may be possible to improve the quantitativeness of the amino group on the substrate. As the internal standard substance, for example, substances such as toluene, pyridine, acetaminophen, N-phenylacetamine, dimethylaniline can be used, but are not limited thereto.

A soda-lime glass substrate having a size of 25 mm × 76 mm × thickness 1 mm was sonicated in a 10% NaCl aqueous solution for 10 minutes and rinsed three times with pure water to obtain Sample 0. Further, this substrate was immersed in a solution of 3-aminopropyltrimethoxysilane (hereinafter referred to as aminosilane) 1% (v / v) in H ₂ O: MeOH = 1: 1 and left overnight at room temperature. Thereafter, the sample was rinsed three times with MeOH, dried, and baked in an oven at 120 ° C. for 2 hours to obtain Sample 1.

  A synthetic quartz glass substrate having a size of 25 mm × 76 mm × thickness 1 mm was sonicated in 1% hydrochloric acid for 10 minutes and rinsed three times with pure water. The substrate was immersed in a 1% (v / v) MeOH / H 2 O solution of 3-aminopropyltrimethoxysilane and left overnight at room temperature. Thereafter, it was rinsed three times with MeOH, dried, and baked in an oven at 120 ° C. for 2 hours to obtain Sample 2.

A high performance liquid chromatograph (hereinafter referred to as HPLC) used for measurement of this sample was prepared under the following conditions. (1) Detection wavelength: 254 nm, (2) Column: Cadenza CD-C18 manufactured by Imtakt, (3) Flow rate: 1.0 mL / min, (4) Solvent: H ₂ O: Acetonitrile = 4: 6. Under these conditions, a dimethylformamide solution containing 0.1% triethylamine (v / v) (hereinafter referred to as DMF solution) was prepared, and PITC was diluted 125,000 times with DMF solution to obtain Test Solution 1. 100 μL of the test solution 1 contains approximately 6.75 × 10 ⁻⁹ mol (hereinafter referred to as test solution concentration) of PITC.

  “Gap cover glass (trade name)” made by Matsunami Glass Co., Ltd., having a cover glass with an area of 10 square cm and a spacer having a thickness of 20 μm is placed on samples 0, 1 and 2 (hereinafter referred to as each sample). The volume of the space 1 sandwiched between the cover glass was set to 20 μL. Fill the space 1 with 20 μL of the test solution 1 slowly using capillary action while using the micropipette for 20 μL and bringing the tip of the pipette tip into contact with the flat end of the cover glass (hereinafter referred to as the filled volume). This time was set to time 0, and the whole of each sample and the cover glass was individually put into a sealed container made of polypropylene.

  Since the test liquid 1 is decomposed by natural standing after preparation, PITC is decomposed, so that the error due to this is subtracted by the following procedure to ensure quantitative accuracy. That is, a part of the test liquid 1 is quickly divided into 4 to 5 HPLC sample bottles (hereinafter referred to as “no-operation test liquid”) by the same volume (20 μL) as the space 1 after preparation, and subjected to HPLC analysis as quickly as possible. Further, HPLC analysis is also performed when an appropriate time (1 hour, 2 hours, etc.) has elapsed from time 0, such as immediately before, immediately after, or immediately before or after a test solution collected from the space 1 of each sample described later. After the completion of all the HPLC analyses, the PITC peak area of the non-operating test solution is approximated by an exponential function with the elapsed time from the time 0 to the HPLC analysis time taken as a predicted curve. For the PITC peak area of each sample, the difference from the expected area at the analysis time (elapsed time from time 0) of each sample on the prediction curve is calculated, and the number obtained by dividing this difference by the predicted area is calculated for each sample. The net amount by which the amino group of aminosilane and PITC reacted on the surface (hereinafter referred to as net reduction rate).

When 3 hours passed from time 0, test solution 1 was collected from space 1 of each sample by the following method. That is, when tweezers are applied to one side of the cover glass and lifted, and the side opposite to the lifted side remains in contact with the glass substrate, the test liquid 1 in the space 1 is transferred to the glass substrate by surface tension. As a result, the tip of a micropipette for 20 μL was applied and carefully sucked. The sucked test liquid 1 was transferred to an HPLC sample bottle and subjected to HPLC analysis to obtain the peak area of PITC, and the net reduction rate was evaluated by the above calculation method. Further, by the following calculation, how much aminosilane covers the surface area of the glass substrate (hereinafter referred to as amino group density) was evaluated. That is, from the molecular model of aminosilane, it is assumed that the alkyl chain site of aminosilane exists perpendicular to the plane of the glass substrate surface, and it is the most when viewed from above perpendicular to the plane of the glass substrate surface. The area of a square having a wide width of 0.5 nm and having this as one side was defined as the area where one aminosilane molecule covers the glass substrate surface in this quantitative method. When squares assuming the state described on the left were formed on a glass substrate surface having a theoretical density of 10 cm ^{2 in} a single layer, the number of squares was estimated to be 6.64 × 10 ⁻⁹ mol.

  The amino group density of each sample was determined by the formula: amino group density = test solution concentration × input volume × net decrease rate / fine amino group density. The results are shown in Table 1. In sample 0, the amino group density is 1.99%, and even the base material that should not have amino groups is reduced in this manner by the quantitative operation. However, sample 1 and sample 2 treated with aminosilane clearly show significantly greater amino group density compared to sample 0, so that this reaction method and instrumentation allowed the chemically coated glass substrate to be chemically treated. It can be said that PITC could be reacted with an amino group that was effective.

Sample Amino group density (mol / 10 cm ² ) Amino group density (%)
Sample 0 1.32 × 10 ⁻¹⁰ 1.99
Sample 1 6.63 × 10 ⁻¹⁰ 9.98
Sample 2 9.78 × 10 ⁻¹⁰ 14.7

  If the method for quantifying functional groups on the surface of a substrate material of the present invention is used, the amount of functional groups required for a biochip or the like can be quantified, so that a high-quality substrate can be provided.

The figure which shows an example of the formation method of the space in this invention

Explanation of symbols

11: Slide glass 12: Gap cover glass 13: Spacer attached to the gap cover glass

Claims (9)

A method for quantifying a functional group on a substrate surface, comprising the following steps A to E.
Process A: The process of forming the space of the trace volume whose average thickness is 500 micrometers or less on a base material, and the area of this base material is 100-10000 mm < ² >.
Step B: Filling the space with a liquid containing a functional group-reactive substance that reacts with a functional group on the substrate.
Step C: a step of quantitatively reacting the functional group on the substrate with the functional group-reactive substance.
Step D: A step of recovering the liquid after the reaction.
Step E: A step of quantifying the reactive substance in the recovered liquid and obtaining a difference.   The method according to claim 1, wherein the functional group to be detected is an amino group.   The method according to claim 2, wherein the substance that reacts with an amino group contains an isothiocyanate group and a phenyl group.   The method according to claim 3, wherein the substance containing an isothiocyanate group and a phenyl group is phenylene isothiocyanate.   5. The method according to claim 3, wherein high-performance liquid chromatography is used as a method for detecting a substance that reacts with an amino group.   6. The method according to claim 2, wherein the solvent in the liquid is dimethylformaldehyde or N-methylpyrrolidone.   The quantification method according to claim 2, further comprising a tertiary amine in the liquid.   The quantification method according to any one of claims 2 to 7, wherein the substrate to be quantified is a substrate surface-treated with aminosilane. The quantitative determination method according to claim 8, wherein the base material is a biochip substrate.

Images