JP4099706B2 - Biochip for surface plasmon resonance measurement - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biochip capable of evaluating the interaction between substances by surface plasmon resonance. In particular, the present invention relates to a biochip in which a difference from the background becomes clear when surface plasmon resonance is measured by immobilizing a biomolecule on a biochip using a functional group such as an amino group in the biomolecule. .
[0002]
[Prior art]
In recent years, attempts have been made to evaluate interactions between biomolecules as a means of examining functions of biomolecules such as DNA and proteins or clarifying expressed genes and proteins. One of the methods is an interaction analysis method using a surface plasmon resonance (SPR) method. SPR is a method in which reflected light is irradiated by irradiating a metal thin film on which an analyte is immobilized, and the interaction with a sample is measured by a change in resonance angle.
[0003]
The advantage of SPR is that labeling substances such as fluorescence and radioisotopes are not required for interaction analysis, and real-time measurement is possible. It may be very cumbersome and difficult to introduce the label substance into the substance, and the original function and activity of the substance may be lost by the label operation.
In order to observe the interaction by SPR, it is necessary to immobilize molecules on the surface of the metal thin film. Examples of a method for immobilizing a biomolecule on a solid phase include a means for immobilizing by a covalent bond or an ionic bond starting from a functional group introduced on the surface.
[0004]
Most conventional techniques measure a single point on the substrate, but as described in the literature, it is possible to detect a change in SPR at a localized portion of the substrate surface by the SPR imaging method applying the SPR technique. (For example, Non-Patent Document 1). That is, if a plurality of substances are immobilized at different locations on the substrate, the interaction analysis of the plurality of substances can be performed simultaneously.
[0005]
Most chips used for SPR introduce a functional group into the entire substrate, and there is no distinction between the immobilization site and the background portion when the biomolecule is not immobilized. Therefore, when applied to the SPR imaging method, it may not be possible to ignore the nonspecific adsorption of the sample to the functional group present in the background part. In addition, a technique for spotting a biomolecule solution at a specific position by a minute amount is essential, and an expensive spotting device is required.
[0006]
US patents disclose means for preparing an array in which a biomolecule is immobilized at an immobilization site and a hydrophilic polymer having no functional group or binding molecule as a starting point is present in the background portion. Reference 1). In the present invention, when a biomolecule or biomolecule assembly is immobilized on the surface, a reversible hydrophobic protecting group is immobilized on the background portion, and spotting is easy. However, after immobilizing a biomolecule or biomolecule assembly on the surface, it is necessary to remove the hydrophobic protecting group using a basic organic solvent and immobilize the hydrophilic polymer, which is very complicated and There is a concern that the basic organic solvent adversely affects the biomolecule or the biomolecule assembly. In addition, there is a concern that it may adversely affect the biomolecule or biomolecule assembly immobilized when the hydrophilic polymer is immobilized. Specifically, the hydrophilic polymer binds to the immobilized biomolecule, and the function of the biomolecule is impaired.
[0007]
Therefore, there is a demand for a biochip capable of immobilizing a biomolecule or a biomolecule assembly at the immobilization site while maintaining the activity, with little nonspecific adsorption in the background portion.
The present invention is a biochip in which a substance that suppresses nonspecific adsorption is immobilized on the background portion, and a substance having a negatively charged functional group that is a starting point for immobilization is immobilized on the immobilization site. Makes it possible to obtain
[0008]
[Non-Patent Document 1]
Anal. Chem. 1997, 69, 1449-1456.
[Patent Document 1]
US Pat. No. 6,127,129 [0009]
[Problems to be solved by the invention]
An object of the present invention is to obtain a biochip for SPR measurement that has almost no nonspecific adsorption in the background portion and can be immobilized while maintaining the activity of the biomolecule or biomolecule assembly at the binding site.
[0010]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
[0011]
1. A biochip for measuring surface plasmon resonance (SPR) having a portion for immobilizing a biomolecule or a biomolecule assembly (an immobilization site) and a background portion other than the immobilization site, and having a molecular weight of 2000 to 20000 A pH 7.4 phosphate buffer solution in which L-lysine was dissolved at a concentration of 1 mg / ml was contacted with the chip surface at 30 ° C., and 15 minutes later, obtained by poly-L-lysine binding to the immobilization site. SPR measurement biochip having a signal ratio of 3 or more due to non-specific adsorption to the background portion other than the immobilized site and signal due to SPR
2. The biochip according to 1, wherein the functional group introduced into the immobilization site is a carboxyl group or a nitrilotriacetic acid (NTA) group.
3. The biochip according to 1 or 2, wherein a nonionic substance is immobilized on the background part.
4). 4. The biochip according to any one of 1 to 3, wherein a transparent glass or transparent plastic on which gold is deposited is used as a substrate.
[0015]
5. The biochip according to any one of 1 to 4, wherein the surface plasmon resonance is SPR imaging.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below. The present invention is a biochip for SPR measurement, in which a biomolecule or a biomolecule assembly is immobilized on the surface, and is effective for interaction analysis, screening of expressed genes, proteins and the like. In the present invention, the immobilization site of the biomolecule or biomolecule assembly is clearly distinguished from the background part other than the immobilization site, and the immobilization site preferably has a negative charge as a starting point for immobilization. A substance having a functional group is immobilized. A biomolecule or biomolecule assembly can be immobilized on the surface by a covalent bond, an ionic bond or the like via a negatively charged functional group.
[0017]
The biochip of the present invention is suitable for a method of analyzing a substrate surface such as SPR imaging, and can immobilize a plurality of substances at a plurality of immobilization sites and simultaneously analyze the interaction. The substrate is preferably a transparent plastic or glass so that it can be analyzed by SPR, and a metal thin film is formed on the surface thereof. The metal thin film is particularly preferably a surface on which gold is deposited.
[0018]
Examples of the functional group having a negative charge include a carboxyl group and an NTA group, and the introduction method is not particularly limited. For example, an alkanethiol substance having a negative charge at the terminal is allowed to act directly on the gold deposition surface, A method of introducing a substance having a negative charge directly through a gold-sulfur bond is conceivable.
[0019]
As another method, an alkanethiol substance having a positive charge at the terminal is allowed to act directly on the gold vapor deposition surface, a positive charge is introduced by a gold-sulfur bond, and then a compound having a plurality of negative charge sites is ionized. A method may also be considered in which a negatively charged substance is indirectly introduced to the surface by binding.
Still further, a thiol substance having a reactive group is allowed to act directly on the gold vapor deposition surface, and the reactive group is introduced by a gold-sulfur bond, and then has a group capable of reacting with the reactive group and has a negative charge. A method of introducing a negatively charged substance on the surface by reacting a compound having the same may be considered. In this case, the reactive group of the reactive group of the thiol substance and the compound having a negative charge includes a hydroxyl group, an amino group, a carboxylic acid group, and a thiol group. There are a glycidyl group, an acid anhydride group, an isocyanate group, a vinyl group, and the like, which are used in appropriate combination.
[0020]
The method of binding a biomolecule or a biomolecule assembly with a negatively charged functional group such as a carboxyl group or NTA group on the biochip surface thus introduced is not particularly limited, but an amine coupling method Alternatively, a biomolecule or a biomolecule assembly can be introduced onto the surface by a nickel chelate histidine tag method or a crosslinking agent. As an example of introducing by a crosslinking agent, for example, the surface carboxyl group can be activated with carbodiimide and reacted with an amino group in the biomolecule, or the biomolecule can be immobilized using an epoxy-based crosslinking agent or the like.
[0021]
The background is preferably immobilized with a nonionic substance in order to suppress nonspecific adsorption. For example, a method of directly immobilizing an alkanethiol having a hydroxyl group or a hydrophilic polymer such as polyethylene glycol on the gold surface is mentioned. Another example is a method in which an alkanethiol having a functional group at the terminal is introduced and a nonionic substance is indirectly immobilized on the surface using the functional group.
[0022]
Examples of means for separating the immobilization site and the background part include a patterning technique by light irradiation and a stamp technique. For example, in the method of patterning by irradiating light, gold-sulfur bonds can be oxidized and washed away by ultraviolet rays, and alkanethiol can be newly bonded to the gold surface.
[0023]
The biochip is set in an SPR device and observed in a buffer solution. Since the SPR resonance angle varies depending on the refractive index of the buffer solution, the measurement is performed with the same buffer solution. At the stage where the buffer solution is introduced, the shooting angle is set. The measurement is preferably carried out at an angle 0.5 to 1 degree smaller than the incident angle (SPR resonance angle) at which the biochip causes SPR resonance and the reflected light intensity is minimized. When a poly-L-lysine having a molecular weight of 2000 or more and 20000 or less dissolved in a phosphate buffer solution of pH 7.4 at a concentration of 1 mg / ml is brought into contact with the chip surface at 30 ° C., the biochip of the present invention Poly-L-lysine binds to the immobilization site by the binding, and the SPR resonance angle shifts to the wide-angle side, so that the reflected light intensity increases. As the phosphate buffer here, 10 mM phosphate and 150 mM sodium chloride are used. Since a nonionic substance is immobilized on the background part, adsorption of a polymer having a positive charge is small.
[0024]
An SPR imaging apparatus using a white light source capable of analyzing the slide surface is used as the SPR apparatus. The reflected image from the slide is captured over time by SPR imaging taken by a CCD camera. The difference between the captured image and the image before contacting the polymer can be obtained by arithmetic processing. A phosphate buffer solution having a molecular weight of 2000 or more and 20000 or less dissolved in poly-L-lysine at a concentration of 1 mg / ml is brought into contact with the chip surface at 30 ° C. for 5 minutes, and then the phosphate buffer solution. In the case of washing for 5 minutes, the signal increase by SPR obtained by poly-L-lysine binding to the immobilization site is compared with the signal increase by nonspecific adsorption to the background part other than the immobilization site. The comparative control is an image before contacting with poly-L-lysine. By this operation, the signal change after contacting with poly-L-lysine and after washing for 5 minutes and after washing for 5 minutes can be compared. When the signal ratio of the signal due to SPR obtained by poly-L-lysine binding to the immobilization site and the signal due to non-specific adsorption to the background part other than the immobilization site is 2 or more, the biomolecule is attached to the immobilization site. Alternatively, it is preferable because it is easy to introduce a biomolecule assembly. The signal ratio is preferably 3 or more. In addition, when obtaining a signal ratio, it sets so that the fixed site | part and background part to be compared may not leave | separate 1 mm or more on a board | substrate. As a method for analyzing an image with a computer, for example, image processing software V ++ (Digital Optics) can be used to capture signal changes in real time.
[0025]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
[0026]
[Example]
After depositing 1 nm of chromium on a transparent glass substrate made of SF10 having a thickness of 1 mm and 18 mm × 18 mm, gold was deposited by 45 nm. The thickness of the deposition was monitored with a crystal oscillator. A substrate with gold deposited on the surface is immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 16 hours to form a self-organized surface of 8-AOT. It was.
[0027]
Molecular weight 2000 succinimide group-terminated polyethylene glycol (mPEG-SPA, manufactured by Sheawater Polymers) was dissolved in phosphate buffer at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours to immobilize PEG on the surface. . A photomask having a pattern of chromium drawn on quartz was placed on the substrate, and patterned by irradiation with a 1000 W xenon arc lamp manufactured by Oriel for one hour. The photomask pattern has 100 squares of 500 μm × 500 μm arranged. After irradiation, it was washed with Milli-Q water and ethanol, then immersed in 1 mM ethanol solution of 7-Carboxy-1-Heptanethiol (7-CHT, manufactured by Dojindo Laboratories) for 16 hours, and 7-CHT was irradiated on the irradiated part. A self-assembled surface was formed. The biochip thus prepared has a carboxyl group at the immobilization site, and nonionic PEG is immobilized at the background portion.
[0028]
This biochip was set in an SPR imaging device (SPRIimager: manufactured by GWC Instruments) and observed at an angle of 1 degree smaller than the SPR resonance angle of the immobilized site. Observation was carried out at 10 mM phosphate buffer, 150 mM NaCl, pH 7.4, 30 ° C. 5 minutes after contacting with a phosphate buffer of pH 7.4 in which poly-L-lysine (manufactured by Sigma) having a molecular weight of 4000-15000 of 1 mg / ml is contacted, the above-mentioned buffer is flowed for 5 minutes. The photograph after this was continuously taken every 5 seconds, and the result of analyzing the signal changes in the immobilized site and the background is shown in FIG. The time-dependent change in reflected light intensity at 6 points was obtained, and a signal ratio was obtained at 3 points. The result was 5.51, 5.56, 4.06, and the average was 5.04, which was more than 2.
The phosphate buffer solution at pH 7.4 uses 10 mM phosphate and 150 mM sodium chloride, and the biochip is contacted with this solution at 30 ° C. for 5 minutes, and then the same pH 7.4 phosphate buffer solution. Washed off at 30 ° C. for 5 minutes.
[0029]
A flow cell was used for contact and washing with the biochip, and the flow cell made of POM was used as shown in FIG. Specifically, a silicon rubber O-ring with a wire diameter of 1.5 mm and an inner diameter of 15.5 mm is set in the groove of the flow cell, and a biochip is stacked on the flow cell so that the gold vapor deposition surface faces the flow cell. The chip was fixed so that the distance between the chips was about 200 μm. A tube was connected to the injection part and the discharge part of the flow cell, and a poly-L-glutamic acid solution and a buffer solution were introduced with a buffer solution pump. The introduction flow rate was 200 μl / min in all cases.
The measurement temperature was 25 ° C.
[0030]
Moreover, the result of having taken the difference of the image before poly-L-lysine contact and after buffer inflow is shown in FIG. 2 (schematic diagram). A darker portion indicates that the reflected light intensity is increased.
FIG. 3 shows a graph in which the density of this image difference is observed with a dotted line. Also from this figure, it can be seen that the signal ratio of the signal of the background part and the signal of the immobilization site is 2 or more.
[0031]
[Comparative example]
A gold-deposited substrate similar to the example was immersed in a 1 mM ethanol solution of 7-CHT for 16 hours to form a 7-CHT self-assembled surface on the entire surface, and carboxyl groups were introduced into the entire substrate. When this surface was brought into contact with 1-mg / ml poly-L-lysine (manufactured by Sigma) having a molecular weight of 4000-15000, the poly-L-lysine binds to the entire substrate, so the signal ratio was 1.00. The biochip in this case has no distinction between the immobilization site and the background part.
[0032]
【The invention's effect】
The biochip for SPR of the present invention hardly adsorbs biomolecules or biomolecule aggregates to the background part, adsorbs to the binding site while maintaining the activity efficiently, and measures SPR. Sometimes the difference from the background becomes clear.
[Brief description of the drawings]
FIG. 1 is a diagram showing the results of analyzing signal changes in the immobilization site and background obtained by SPR, obtained in Example. FIG. 2 before contact with poly-L-lysine and after inflow of buffer in Example. Fig. 3 is a graph showing the difference between images. Fig. 3 is a graph observing the density difference of the image in Fig. 2 with a dotted line. Fig. 4 is a top view and a cross section of the flow cell used in the example Figure

Claims (10)

Biological molecule or organism molecules having a hydrophilic polymer to the background portion other than a functional group and immobilization sites with negative charged biological molecule or moiety to immobilize biomolecules assembly (fixed sites) A biochip for measuring surface plasmon resonance (SPR) for immobilizing an assembly ,
After contacting the biochip with a pH 7.4 phosphate buffer solution in which poly-L-lysine having a molecular weight of 2000 or more and 20000 or less is dissolved at a concentration of 1 mg / ml at 30 ° C. for 5 minutes. When washed with phosphate buffer for 5 minutes,
A biochip for SPR measurement, wherein a signal ratio of SPR signal obtained by poly-L-lysine binding to an immobilization site and signal due to non-specific adsorption to a background part other than the immobilization site is 2 or more . The biochip according to claim 1, wherein an alkanethiol substance having a functional group having a positive charge at its end is immobilized at an immobilization site. The biochip according to claim 1 or 2 , wherein the negatively charged functional group is a carboxyl group or a nitrilotriacetic acid (NTA) group . The biochip according to any one of claims 1 to 3, wherein the hydrophilic polymer is polyethylene glycol. The biochip according to any one of claims 1 to 4 , wherein a transparent glass or transparent plastic on which gold is deposited is used as a substrate. The biochip according to any one of claims 1 to 5 , wherein the surface plasmon resonance is SPR imaging .
Io chip . A method for producing a biochip for surface plasmon resonance (SPR) measurement for immobilizing a biomolecule or a biomolecule assembly, comprising the following steps (1) to (3).
(1) First step of immobilizing hydrophilic polymer on gold deposition surface on substrate
(2) Second step of patterning the substrate obtained by (1)
(3) Third step of introducing a negatively charged functional group to the gold deposition surface exposed by patterning 8. The method for producing a biochip according to claim 7, wherein an alkanethiol substance having a negatively charged functional group at its end is immobilized at an immobilization site. The method for producing a biochip according to claim 7 or 8, wherein the negatively charged functional group is a carboxyl group or an NTA group. The method for producing a biochip according to any one of claims 7 to 9, wherein the hydrophilic polymer is polyethylene glycol.

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