JP2012141140A - Probe immobilization method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a probe immobilization method in which probe modification is restricted.SOLUTION: A probe immobilization method for immobilizing a probe 12 onto a substrate 11 comprises the steps of: preparing the substrate 11 and the probe 12; and processing the substrate 11 to fix the probe 12 onto the substrate 11 without chemical reaction. This allows the probe 12 to be adsorbed and fixed to the substrate 11 in a natural reaction without any chemical reaction upon fixing the probe 12, thereby making it possible to restrict the probe 12 from being modified with change in pH and temperature in a chemical reaction.

Description

  The present invention relates to a method for immobilizing probes to be immobilized on an array substrate used for DNA arrays, protein arrays, sugar chain arrays and the like.

  Immobilizing probes such as nucleic acids, peptides, antibodies, and lectins is considered useful for the construction of array chips used in various diagnostic chips, array arrays used in DNA arrays, protein arrays, sugar chain arrays, etc. Yes.

  Conventionally, a silane coupling reaction is known as a technique for immobilizing a probe such as an antibody on a substrate surface. This can be achieved, for example, by bringing a solid phase carrier into contact with a silane coupling agent having an acid anhydride functional group, such as 3- (triethoxysilyl) propyl succinic anhydride. Immobilizing the probe by the silane coupling method, including holding the phase carrier in a temperature range of 0 ° C. to 60 ° C. and performing the binding treatment of the physiologically active substance to the acid anhydride functional group. be able to.

  As a prior art document related to the invention of this application, for example, Patent Document 1 is known.

JP 2009-229319 A

  However, in the conventional method for immobilizing a probe, there are cases where the function of the probe cannot be fully utilized due to a chemical reaction.

  That is, the procedure for the silane coupling reaction is complicated, and it is necessary to replace the solvent holding the probe with a reagent for silane coupling. The probe is affected by changes in pH, salt concentration, temperature, and surfactant. May be denatured. In this way, the functional group is added to the substrate by a chemical reaction, and thus the process becomes complicated and troublesome, and there is a problem in that it causes variations for each immobilized substrate lot.

  Furthermore, when the silane coupling reaction is used, it is difficult to control the direction in which the probe is immobilized on the substrate by the above reaction method because the probe surface that binds to the substrate cannot be specified. That is, the silane coupling method often used for a chemical reaction cannot specify the probe surface to be bonded to the substrate and has a long linker between the probe and the substrate, so that the orientation of the probe becomes difficult.

  Therefore, an object of the present invention is to suppress the denaturation of the probe.

  In order to achieve the above object, the probe immobilization method of the present invention has a step of adsorbing and immobilizing a probe on a substrate by treating the substrate spontaneously or physically without any chemical reaction.

  By physically treating the substrate of the present invention, the probe can be adsorbed and fixed spontaneously to the substrate, so that denaturation of the probe with changes in pH or temperature in a chemical reaction can be suppressed. It becomes. Furthermore, since no chemical reaction is involved, the number of processes can be reduced, and variations in each immobilized substrate lot during measurement can be reduced. Furthermore, the distance between the probe and the substrate can be made extremely short, and the probe can be oriented.

Sectional schematic diagram of the immobilized substrate of the probe in Example 1 (A), (b) Sectional drawing for demonstrating the fixing method of the probe in Example 1 (A), (b) Sectional drawing for demonstrating the fixing method of the probe in Example 1 Sectional schematic diagram of the immobilized substrate of the probe in Example 1

Example 1
Hereinafter, the method for immobilizing the probe in this embodiment will be described.

  FIG. 1 is a schematic cross-sectional view of an immobilization substrate immobilized in the probe immobilization method of this embodiment.

  The probe 12 is immobilized on the immobilized substrate 10 by covalently bonding the surface molecule on the surface of the substrate 11 and the aldehyde group of the probe 12.

  Next, a probe immobilization method in the present embodiment will be described with reference to FIGS.

First, the substrate 11 and the probe 12 for immobilization are prepared, and the prepared substrate 11 is subjected to plasma treatment to add radicalized functional groups (substrate functional groups 13) to the substrate 11. For example, as shown in FIG. 2 (a), a substrate 11 made of silicon, glass, quartz, silicon oxide, silicon oxynitride, or a mixture of any of these is formed by oxygen plasma. When treated, a number of radicalized hydroxyl groups (—OH) (substrate functional groups 13) are formed. Alternatively, as shown in FIG. 3A, the substrate 11 made of silicon nitride, silicon oxynitride, or a mixture thereof is radicalized when treated with laughing gas plasma, NOx plasma, or mixed plasma of nitrogen and hydrogen. Many amine groups (—NH ₂ ) (functional groups 13 of the substrate) are formed. Alternatively, a hydroxyl group (—OH) radicalized by oxygen plasma treatment may be formed on the substrate 11, and then a radical —ON group formed by nitrogen plasma treatment may be formed. Note that plasma is a gas and molecular molecules accelerated by a DC voltage, a high-frequency voltage applied between the electrodes, or an electric field generated by a microwave while maintaining a constant reduced pressure of an inert gas gas or a reactive gas in the chamber. And can be generated using impact ionization. The number and density of the functional groups 13 of the substrate formed by such plasma treatment can be strictly controlled by the power / gas partial pressure and the degree of vacuum at the time of plasma generation.

  The functional group 13 of the radicalized substrate easily reacts with the functional group 14 of the prepared probe 12 to form a covalent bond as shown in FIG. 2B or 3B. By doing so, the probe 12 is adsorbed and fixed to the substrate 11. The functional group 14 of the probe here is an aldehyde group, thiol group, carboxyl group, activated carboxyl group, disulfide group, activated disulfide group, alkyl group, alkene group, alkyne group, aldehyde group, ketone group, azide group. , Amines or their derivatives.

  The effect by the probe immobilization method in a present Example is shown.

  The probe immobilization method in the present invention does not need to be subjected to chemical treatment such as chemical treatment because the probe does not need to be substituted with a specific reactive group as in the conventional silane coupling method. That is, the functional group 13 of the probe such as an aldehyde group present in the probe 12 is generated by generating a functional group 13 of the substrate that is radicalized by plasma processing the substrate 11 without processing the probe itself by a chemical reaction by chemical processing. 14 can be adsorbed and fixed to the substrate 11 as it is. Therefore, when the probe 12 is immobilized, it is not necessary to replace the solution mixed with the probe 12 with a solution necessary for chemical reaction, and the probe 12 is immobilized regardless of the pH or temperature. Can be As a result, denaturation of the probe 12 due to substitution with a solution containing an undesirable pH, salt concentration, temperature or surfactant for the probe 12 can be avoided. Furthermore, since there is no chemical reaction as in the prior art, it is possible to reduce the number of processes and to reduce the variation of each 10 lots of immobilized substrates during measurement. Furthermore, since it is not necessary to have a long linker between the probe 12 and the substrate 11, the orientation control of the probe 12 becomes easy.

Note that oxygen gas, laughing gas, and NOx have been described as the gases used for plasma, but any other gas such as nitrogen, hydrogen, ammonia, C ₂ F ₆ , CF ₄ , or the air can be used for plasma. Alternatively, any mixed gas thereof may be used.

  Note that a resin substrate such as ABS resin, polycarbonate, polystyrene, polyethylene, polypropylene, vinyl chloride, COP (cycloolefin polymer), COC (cyclic olefin copolymer) may be treated with the plasma gas as the substrate 11. Similar effects can be obtained.

  In addition, when sugar is used as the probe 12, there is no need to add a functional group to the probe 12, so that the number of steps can be further reduced. In this case, the aldehyde group can be exposed by reducing the end of the sugar that is the probe 12 to a reducing sugar. Then, the reducing sugar is plasma-treated and immobilized on the substrate 11 having the radicalized functional group 13, whereby the probe 12, which is a sugar containing the reducing sugar at the terminal, can be adsorbed and immobilized.

  In the step of using the saccharide as the probe 12 as a reducing saccharide, an enzymatic reaction can be used, so that chemical treatment such as chemical treatment is not necessary, and denaturation of the probe can be suppressed.

  As shown in FIG. 4, when the probe 12 has no functional group, the probe can be fixed to the radicalized substrate 11 by adding an epitope 15 having a functional group to the probe 12. For example, when the probe 12 is the protein 12a, a reducing sugar obtained by reducing the end of the sugar 15a as the epitope 15 is added to the lysine residue or the serine residue of the protein 12a, which is the probe 12, by glycosyltransferase, and the reducing sugar aldehyde The probe 12 can be adsorbed and immobilized on the substrate 11 via the group. By using a reducing sugar as the linker, the linker can be shortened, and the orientation control of the probe 12 can be facilitated as compared with the conventional case.

  The present invention is useful as a method for immobilizing probes to be immobilized on an array substrate used for DNA arrays, protein arrays, sugar chain arrays, and the like.

DESCRIPTION OF SYMBOLS 10 Immobilization substrate 11 Substrate 12 Probe 12a Protein 13 Substrate functional group 14 Probe functional group 15 Epitope 15a Sugar

Claims (10)

A probe immobilization method for immobilizing a probe on a substrate,
Preparing a substrate and a probe;
A probe immobilization method in which a probe is immobilized on a substrate by processing the substrate without a chemical reaction. Plasma treating the substrate after the step of preparing the substrate and the probe;
The method for immobilizing a probe according to claim 1, further comprising immobilizing the probe on the plasma-treated substrate. The plasma treatment is performed by plasma treatment using oxygen, nitrogen, hydrogen, laughing gas, NOx gas, ammonia, C ₂ F ₆ , CF _4, air, or a mixed gas thereof. Immobilization method of the probe. 3. The probe immobilization method according to claim 2, wherein the substrate is made of any one of silicon, glass, quartz, silicon oxide, silicon nitride, silicon oxynitride, or a mixture thereof. 3. The probe immobilization method according to claim 2, wherein the substrate is made of any one of ABS, polycarbonate, polystyrene, polyethylene, polypropylene, vinyl chloride, COP, COC, or a mixture thereof. Preparing a probe having an aldehyde group in the step of preparing the probe;
In the step of fixing the probe to the plasma-treated substrate,
The probe immobilization method according to claim 2, further comprising a step of immobilizing the plasma-treated substrate and the aldehyde group of the probe having the aldehyde group. Prepare probes with reactive groups consisting of thiols, carboxyls, activated carboxyls, disulfides, activated disulfides, alkyls, alkenes, alkynes, aldehydes, ketones, azides or their derivatives and amines instead of the aldehyde groups. The probe immobilization method according to claim 6. Preparing a sugar as the probe;
Reducing the sugar end to a reducing sugar;
The method for immobilizing a probe according to claim 2, further comprising a step of immobilizing the reducing sugar on a plasma-treated substrate. Preparing sugar,
Reducing the sugar end to a reducing sugar;
Adding a reducing sugar to the probe;
The method for immobilizing a probe according to claim 2, further comprising a step of immobilizing the reducing sugar on a plasma-treated substrate. The step of adding a reducing sugar to the probe has a step of adding a reducing sugar to a predetermined lysine residue or serine residue of the probe by glycosyltransferase, and the probe is attached to the plasma-treated substrate via the reducing sugar. The probe immobilization method according to claim 9, wherein the probe is immobilized.

Images