JP3815621B2 - Biochip manufacturing method - Google Patents

Description

  The present invention relates to a method for producing a biochip in which non-specific adsorption of biomolecules is suppressed by blocking a hydrophilic polymer.

  In recent years, biochips used for biomolecule interaction analysis, profiling of expressed molecules, or diagnosis have attracted attention. The operation is facilitated by immobilizing the biomolecule on the substrate, and in some cases, the interaction of a very large number of substances can be analyzed.

  When observing the interaction, nonspecific adsorption becomes a problem. In other words, it refers to the case where the target substance adsorbs nonspecifically to molecules that do not interact. As a result, a false positive determination is given, which is not preferable. In order to suppress nonspecific adsorption, biochips in which hydrophilic polymers such as dextran and polyethylene glycol are immobilized on the surface have been developed. It is known that hydrophilic polymers have an effect of suppressing nonspecific adsorption of biomolecules.

  A biosensor that suppresses nonspecific adsorption by forming a gel matrix of a hydrophilic polymer on the biosensor surface is shown. In this method, functional groups are introduced into the gel matrix, and biomolecules are immobilized by covalent bonds using the functional groups. Specifically, the carboxyl group of carboxymethyl dextran was activated with water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the formed succinimide group and the biomolecule were successfully immobilized (Non-patent Document). 1). However, since the formed succinimide group does not react 100%, it is necessary to block the remaining succinimide. Here, succinimide groups are blocked using ethanolamine, and a technique using a hydrophilic polymer as a blocking material is not shown.

  A means for reacting a hydrophilic polymer on a substrate after immobilizing a biomolecule is known (Patent Document 1). However, here, the maleimide group on the chip is reacted with the thiol group of the target molecule to immobilize the target molecule on the chip, but the subsequent step of blocking the maleimide group is not included. For this reason, the maleimide group remains as it is, and there is a concern of causing nonspecific adsorption problems. Further, since a hydrophilic polymer capable of reacting with the immobilized biomolecule is used, the biomolecule may be modified and denatured.

  The present invention provides a means for efficiently blocking a functional group used for immobilization of a target molecule without inhibiting the target molecule and suppressing nonspecific adsorption.

Anal. Biochem. 198, 268, 1991 US Pat. No. 6,127,129

  An object of the present invention is to obtain a biochip that suppresses nonspecific adsorption of biomolecules. In particular, the object is to obtain a chip that can accurately evaluate the interaction even when used for analyzing the interaction with the protein.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. After reacting the functional group (A) on the chip with the functional group (B) of the target molecule to immobilize the target molecule on the chip, the functional group (A) remaining on the chip surface is converted to the functional group ( 1. Biochip preparation method in which a functional group (A) is covalently blocked by reacting a hydrophilic polymer having C) 2. The method according to 1, wherein a hydrophilic polymer having a functional group (C) only at the terminal is used. 3. The method according to any one of 1 to 2, wherein the hydrophilic polymer has a molecular weight of 400 or more. 4. The method according to any one of 1 to 3, wherein the hydrophilic polymer is polyethylene glycol. 5. The method according to any one of 1 to 4, wherein the chip surface is gold. The method according to any one of 1 to 5, wherein the substrate of the chip is a transparent substrate

  By adopting the method for producing a biochip of the present invention, a biochip in which nonspecific adsorption is suppressed can be easily obtained.

  The biochip production method of the present invention is not used for the immobilization reaction with respect to the method of immobilizing the target molecule on the chip by reacting the functional group (A) on the chip with the functional group (B) of the target molecule. And a functional group (A) remaining on the chip is reacted with a hydrophilic polymer having the functional group (C) to covalently block the biochip. Therefore, the blocking method by physical adsorption or ion adsorption is not included in the present invention.

The functional group (A) can react with the functional group (B) of the target molecule to be immobilized, and can also react with the functional group (C) of the hydrophilic polymer.
Functional group (A), functional group (B), functional group (C) is not particularly limited, carboxyl group, amino group, succinimide group, sulfated succinimide group, maleimide group, epoxy group, thiol group, Examples include an aldehyde group, a vinyl group, a tosyl group, a tresyl group, an acetic anhydride group, an isocyanate group, an isothiocyanate group, an acyl azide group, a chlorinated sulfuric acid group, and an imide ester group. Here, the functional group (A) and the functional group (B), and the functional group (A) and the functional group (C) are a reactive pair. Functional group (A) and functional group (B), functional group (A) and functional group (C) are basically different functional groups, but functional group (B) and functional group (C) are the same. May be different functional groups.

  The means for introducing the functional group (A) onto the chip is not particularly limited. Examples thereof include a self-assembled surface method for aligning molecules on the substrate surface, a method of introducing using a reaction reagent, and a means for coating a substance having a functional group on a chip. Also included are means for introducing the functional group (A) using a cross-linking agent starting from the functional group introduced on the surface.

The position of the functional group (B) in the target molecule is not particularly limited, and may be any of a terminal portion, a side surface portion, and an unspecified number of locations in the target molecule.
The functional group (C) in the hydrophilic polymer is preferably present at the polymer end, and more preferably at one end. If the functional group (C) is present in the main chain of the polymer or a side chain that is branched from the main chain, there is a risk of steric hindrance to the immobilized target molecule, which is not preferable. Further, when a large number of functional groups (C) are present in the main chain or side chain, the functional groups (C) still remain even if they are blocked by reacting with the functional groups (A). The possibility that the functional group (C) also causes nonspecific adsorption cannot be denied. Therefore, it is preferable that the functional group (C) exists only at the terminal. More preferably, a hydrophilic polymer having a functional group (C) only at one end and a low activity methoxy group or hydroxyl group at the other end is preferable.

  The molecular weight of the hydrophilic polymer is preferably 400 or more, and more preferably 1,000 or more. This is because the higher the molecular weight / repeating unit, the stronger the effect of suppressing nonspecific adsorption. However, a molecular weight of 50,000 or more is not preferable because it may cause steric hindrance to the immobilized target molecule and adversely affect it.

The hydrophilic polymer refers to a compound having a repeating unit that is soluble in water or swells in water, and may be a synthetic product or a natural product.
Specifically, as the hydrophilic polymer, for example, polyethylene glycol, polyvinyl alcohol, poly (meth) acrylic acid, poly (meth) acrylate, poly (meth) acrylamide, polyethyleneimine, polyvinylpyrrolidone, carboxylic acid Or the salt, the sulfonic acid, the monomer containing the salt, or polyester, polyurethane, carboxymethylcellulose which copolymerized hydrophilic parts, such as polyethyleneglycol, and polysaccharides, such as chitosan, carrageenan, and glucomannan, are mentioned.

  However, since the hydrophilic polymer in the present invention is intended to suppress nonspecific adsorption, a compound having an ionic functional group may not be preferable. Among these, those having no reactive moiety such as OH groups, carboxylic acids and salts thereof, amines, and imines such as polyethylene glycol, poly (meth) acrylamide, and polyvinylpyrrolidone are preferable, and polyethylene glycol is most preferable. Polyethylene glycol (sometimes referred to as polyoxyethylene) is highly hydrophilic and has a high effect of suppressing non-specific adsorption because there is no reactive functional group.

  The chip according to the present invention can be applied to various applications, but it is particularly effective for optical detection methods such as surface plasmon resonance (SPR), sum frequency generation (SFG), localized plasmon resonance (LPR), and ellipsometry. Demonstrate. In a general chip or array, detection by a labeling means using a fluorescent substance or a radioisotope is used as a method for detecting an interaction. In this case, it is possible to detect only whether or not the label substance is finally bound, and even if a substance not related to the interaction is adsorbed non-specifically, it is not erroneously detected. Therefore, if the interacting target substance is not non-specifically adsorbed to the negative control, it can be determined that the measurement is accurate.

  However, in the label-free optical detection method, any substance adsorbed on the chip is detected as a signal. That is, it is difficult to distinguish nonspecific adsorption of a substance that is not a measurement target from specific adsorption. Therefore, since a means for suppressing the non-specific adsorption more severely is required, the blocking method by the covalent bond of the present invention is very effective.

  In the ELISA method and the interaction analysis method using a label substance, physical adsorption such as bovine serum albumin or casein is generally selected as a blocking method. Although the method of physical adsorption is easy, it is not stable and desorbs from the chip surface over time. In the above optical detection method, even desorption of a blocking agent is detected, so that blocking by a covalent bond is necessary.

  In SPR, SFG, LPR, and ellipsometry, a metal substrate is used. In the present invention, the material of the substrate is preferably gold which is very stable in acid, alkali, organic solvent and the like. In fact, gold is a material frequently used in the above optical detection method. Further, the material supporting gold is preferably transparent, and more preferably transparent glass. This is because transparent glass is not only easily available, but also very suitable for measuring SPR and LPR.

  As a method of forming the metal substrate, a method of coating a thin metal layer is preferable. The method for coating the metal is not particularly limited, but generally, a vapor deposition method, a sputtering method, an ion coating method, or the like is selected. In order to provide an optical detection method, it is necessary to control the thickness of the thin metal layer at the nano level. The thickness of the thin metal layer is not particularly limited, but is generally selected in the range of 30 nm to 80 nm. In order to suppress peeling of the thin metal layer, a chromium layer or a titanium layer of 0.5 nm to 10 nm may be coated on the substrate in advance.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

[Example]
(Double-stranded DNA immobilization)
4 armPEG (SUNBRIGHT PTE-100SH manufactured by NOF Corporation) whose terminal functional group is a thiol group was dissolved in a mixed solution of 7 ml of ethanol: water = 6: 1 at a concentration of 1 mM. The molecular weight of 4armPEG is 10,000, which is a molecule in which four PEG chains having approximately the same length from the center are present, and is very hydrophilic. In addition, all four ends of PEG are thiol groups, and particularly exhibit metal binding properties to gold.

  A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thickness and gold was vapor-deposited at 45 nm was immersed in the 4 armPEG thiol solution for 3 hours to bond 4 armPEG thiol to the entire gold substrate.

  A photomask shown in FIG. 1 was placed on this slide, and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO INC.) For 2 hours to remove 4 armPEG thiol in the UV irradiation section. The photomask has 96 0.5 mm square holes, and the hole spacing is 1 mm. UV light is transmitted through the holed portion of the photomask, and the slide is irradiated and patterned. In the part that was not irradiated, 4armPEG remains and functions as a reference part as a background part of the chip.

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. A heterobifunctional polyethylene glycol (NHS-PEG-MAL, manufactured by Nektar) having a succinimide (NHS) group and a maleimide (MAL) group at the end of a molecular weight of 3,400 is phosphate buffer (20 mM phosphate, 150 mM NaCl, It was dissolved in pH 7.2) at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours. Since the amino group of 8-AOT and the NHS group of NHS-PEG-MAL reacted and the MAL group remained unreacted, a maleimide group could be introduced to the surface via PEG.

Using an automatic spotter device (MultiSPRinter Spotter: manufactured by Toyobo Co., Ltd.), two types of DNA were annealed on the obtained surface and spotting was performed. A spot for immobilizing two types of double-stranded DNA (dsDNA) and a spot for immobilizing nothing (Blank) were prepared. The DNA sequence used is shown in Table 1. The sequence of the DNA on the immobilization side is designed so that the target sequence is inserted from the 5 ′ thiol end via a thymine 15-base spacer. The complementary DNA sequence has a sequence of only a portion complementary to the target sequence of the immobilized DNA, and does not have a thiol and thymine 15-base spacer. Here, GATAreg is a sequence that GATA-1 can recognize and bind to, and the GATA sequence is contained in the center from the 5 ′ side. On the other hand, the GATAmut sequence is a sequence in which AGTA, G, and A are replaced instead of GATA, and GATA-1 is generally a sequence that cannot be recognized.
The maleimide group formed on the surface of the chip reacts with the thiol group of the immobilized DNA, and the DNA can be covalently immobilized on the surface.

  The DNA annealing conditions were as follows: Prepare a solution in a 5 SSC solution (75 mM sodium citrate, 750 mM NaCl, pH 7.0) so that the 5 ′ thiol-terminated DNA was 25 μM and its complementary DNA was 100 μM. For 5 minutes, rapidly cooled to 0 ° C for 15 minutes, and then incubated at 37 ° C for 3 hours. The annealed dsDNA was spotted with about 10 nl and reacted for 15 hours to immobilize the dsDNA on the surface.

(Blocking of unreacted maleimide groups)
After washing the dsDNA-immobilized surface with a phosphate buffer, in order to block unreacted maleimide groups, one end functional group is a thiol group and the other functional group is a methoxy group (Japan Oil and fat SUNBRIGHT MESH-50H) was dissolved in a phosphate buffer (20 mM phosphoric acid, 150 mM NaCl, pH 7.2) at a concentration of 10 mg / ml, and 250 μl was poured onto the chip and allowed to react for one hour. The molecular weight of the PEG thiol used here is 5,000, and the hydrophilicity is very high, and an effect of suppressing nonspecific adsorption can be expected.

(Measurement by SPR imaging method)
The dsDNA-immobilized chip thus obtained is set in an SPR imaging device (MultiSPRinter: manufactured by Toyobo Co., Ltd.), for measuring transcription factors of 20 mM Hepes, 300 mM NaCl, 0.2 mM ZnCl ₂ , 0.005% Tween 20, pH 7.9. Buffer was flowed into the flow cell.
After confirming that the signal from the SPR was stabilized, the transcription factor GATA-1 was prepared in the above-mentioned transcription factor measurement buffer at a concentration of 10 μM and injected into the cell in the SPR device. When injecting GATA-1, polyIdC was added to the buffer at a concentration of 100 μg / ml to suppress non-specific adsorption to nucleic acids. The cell was injected into the cell at a rate of 100 μl / min for 10 minutes, and a buffer solution containing no transcription factor was allowed to flow, and changes in the SPR signal of binding and dissociation were observed. In addition to GATAreg and GATAmut, signal changes were observed in Blank and Background.

(Observation results and discussion)
A graph of signal change is shown in FIG. Here, it was observed that GATA-1 was bound to the GATAreg sequence and hardly bound to GATAmut. The Blank part (blocking the maleimide group with PEG thiol) and the Background part (4armPEG) in this graph showed no signal change, indicating that GATA-1 was hardly bound.
In this way, it was possible to suppress nonspecific binding of GATA-1 at the Blank portion where the maleimide group was blocked with PEG thiol. As a result, it can be considered that the binding data of GATAreg and GATAmut does not contain a signal due to nonspecific adsorption, and it can be said that the reliability of the data is increased.
Thus, by blocking the surface functional groups covalently with a hydrophilic polymer, it was possible to easily obtain a biochip with suppressed nonspecific adsorption.

[Comparative example]
(Double-stranded DNA immobilization)
It carried out similarly to the Example.
(Blocking of unreacted maleimide groups)
Here, the blocking operation was not performed, and the process proceeded directly to the next step (measurement by SPR imaging method).
It carried out similarly to the Example.

(Observation results and discussion)
A graph of signal change is shown in FIG. Here, GATA-1 binds to the GATAreg sequence, and the tendency for the amount to bind to GATAmut to be small is the same as in the example. However, an increase in signal is observed in the blank part (maleimide group) in this graph. , Indicating that nonspecific adsorption of GATA-1 occurred.
However, the background component (4armPEG) showed no signal change, indicating that GATA-1 was hardly bound, and nonspecific adsorption to Blank was due to the maleimide group remaining on the surface. Can be considered. The signal due to the binding of GATA-1 to GATAmut tends to be larger than that in Examples, but this is not due to the binding to the GATAmut sequence but can be considered to be due to the maleimide group remaining on the surface.
Therefore, non-specific adsorption may occur due to functional groups remaining without reacting, which may lead to erroneous results. From this result, it can be said that the reliability of the data is low.

  The biochip produced by the method of the present invention suppresses nonspecific adsorption and enables accurate measurement. In addition, the creation method is easy and it is important to contribute to the industry.

Photomask used in Examples and Comparative Examples Changes in SPR signal in Examples SPR signal change in the comparative example

Claims (6)

  After the functional group (A) on the chip reacts with the functional group (B) of the target molecule to immobilize the target molecule on the chip, the functional group (A) remaining on the chip surface is converted to the functional group ( A biochip manufacturing method in which a hydrophilic polymer having C) is reacted to covalently block the functional group (A)   The method according to claim 1, wherein a hydrophilic polymer having a functional group (C) only at the terminal is used.   The method according to claim 1, wherein the hydrophilic polymer has a molecular weight of 400 or more.   The method according to claim 1, wherein the hydrophilic polymer is polyethylene glycol.   5. The method according to claim 1, wherein the chip surface is gold.   6. The method according to claim 1, wherein the substrate of the chip is a transparent substrate.

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