JP2004125624A - Biochip for measuring surface plasmon resonance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a biochip for measuring SPR for immobilizing a biomolecular or a biological molecule aggregate to a combined site, while maintaining activity without hardly non-specific adsorption in the background section. <P>SOLUTION: The biochip for measuring surface plasmon resonance (SPR) comprises a part (immobilization site) for immobilizing a biomolecular or a biological molecule aggregate, and a background section other than the immobilization site. A phosphoric acid buffer solution of pH 7.4, where poly-L-lysine, whose molar weight is 2,000 or higher and 20,000 or lower is dissolved at a concentration of 1 mg/ml, is made to be in contact with a chip surface at 30°C for five minutes, before cleaning for five minutes in the phosphoric acid buffer solution. The ratio of the signal by SPR obtained by poly-L-lysine combined with the immobilization site to the signal obtained by non-specific adsorption to the background section other than the immobilization site is 2 or larger. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biochip capable of evaluating an interaction between substances by surface plasmon resonance. In particular, by immobilizing a biomolecule to a biochip using a functional group such as an amino group in the biomolecule, the biochip becomes clear from the background when surface plasmon resonance is measured. .
[0002]
[Prior art]
In recent years, attempts have been made to examine the functions of biomolecules such as DNA and proteins, or to evaluate the interaction between biomolecules as a means for 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 of irradiating a metal thin film on which an analyte is immobilized with light, monitoring reflected light, and measuring the interaction with a sample by a change in resonance angle.
[0003]
The advantages of SPR are that no label substance such as fluorescence or radioisotope is required for interaction analysis, and that measurement can be performed in real time. It may be very complicated and difficult to introduce a label substance into a substance, and the label may lose its original function and activity due to label operation.
To observe the interaction by SPR, it is necessary to immobilize the molecule 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 a biomolecule by a covalent bond or an ionic bond starting from a functional group introduced on the surface.
[0004]
Most of the conventional techniques measure one point on a substrate, but it is possible to detect an SPR change in a localized portion of the substrate surface by an SPR imaging method applying the SPR technique as described in the literature. (For example, Non-Patent Document 1). That is, if a plurality of substances are immobilized at different places on the substrate, the interaction analysis of the plurality of substances can be simultaneously performed.
[0005]
Most of the chips used for SPR also have a functional group introduced into the entire substrate, and there is no distinction between an immobilized site and a background portion in a state where a biomolecule is not immobilized. Therefore, when applied to the SPR imaging method, non-specific adsorption of the sample to the functional group present in the background may not be ignored. In addition, a technique of spotting a biomolecule solution at a specific position in a very small amount is also indispensable, and an expensive spotting device is required.
[0006]
The U.S. patent discloses means for immobilizing a biomolecule on an immobilization site and producing an array in which a hydrophilic polymer having no functional group or binding molecule as a starting point in a background portion is immobilized. Reference 1). According to the present invention, when a biomolecule or a biomolecule aggregate is immobilized on a surface, a reversible hydrophobic protecting group is immobilized on a background portion, and spotting is easy. However, after the biomolecules or biomolecule aggregates are immobilized on the surface, it is necessary to remove the hydrophobic protecting groups using a basic organic solvent, and immobilize the hydrophilic polymer. There is a concern that the basic organic solvent may adversely affect the biomolecules or biomolecule aggregates. In addition, there is a concern that the immobilization of the hydrophilic polymer may adversely affect the immobilized biomolecules or biomolecule aggregates. Specifically, the point is that 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 which has almost no non-specific adsorption in the background portion and can immobilize a biomolecule or biomolecule aggregate at an immobilization site while maintaining its activity.
The present invention provides a biochip in which a substance that suppresses nonspecific adsorption is immobilized on a background portion, and a substance having a negatively charged functional group serving as a starting point for immobilization is immobilized on the immobilization site. It is 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 provide a biochip for SPR measurement which has almost no non-specific adsorption in a background portion and can be immobilized on a binding site while maintaining the activity of a biomolecule or a biomolecule aggregate.
[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 surface plasmon resonance (SPR) measurement having a portion for immobilizing a biomolecule or a biomolecule assembly (immobilization site) and a background portion other than the immobilization site, wherein the polychip has a molecular weight of 2,000 to 20,000. A phosphate buffer of pH 7.4, in which L-lysine is dissolved at a concentration of 1 mg / ml, is brought into contact with the chip surface at 30 ° C., 15 minutes later, and obtained by poly-L-lysine binding to the immobilization site. A biochip for SPR measurement, wherein the signal ratio between the signal obtained by SPR and the signal obtained by non-specific adsorption to a background portion other than the immobilization site is 3 or more.
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. 3. The biochip according to 1 or 2, wherein a nonionic substance is immobilized on a background portion.
4. 4. The biochip according to any one of 1 to 3, wherein a transparent glass or a transparent plastic on which gold is deposited is used as a substrate.
[0015]
5. The biochip according to any one of claims 1 to 4, wherein the surface plasmon resonance is SPR imaging.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail. The present invention is a biochip for SPR measurement, in which a biomolecule or an assembly of biomolecules is immobilized on a surface, and is effective for interaction analysis and screening of expressed genes and proteins. In the present invention, the immobilized site of the biomolecule or biomolecular assembly and the background portion other than the immobilized site are clearly distinguished, and the immobilized site preferably has a negative charge as a starting point for immobilization. A substance having a functional group is immobilized. The biomolecule or biomolecule assembly can be immobilized on the surface through a negatively charged functional group by a covalent bond, an ionic bond, or the like.
[0017]
The biochip of the present invention is suitable for a method for analyzing the surface of a substrate, such as SPR imaging, and can immobilize a plurality of substances on a plurality of immobilization sites and simultaneously analyze an interaction. For analysis by SPR, the substrate is preferably made of transparent plastic or glass, 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 a method for introducing the functional group is not particularly limited. For example, an alkanethiol substance having a negative charge at a terminal is allowed to directly act on a gold deposition surface, A method in which a substance having a negative charge is directly introduced by a gold-sulfur bond can be considered.
[0019]
As another method, a alkanethiol substance having a positive charge at the terminal is allowed to act directly on the gold 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 of binding by binding and indirectly introducing a negatively charged substance to the surface is also conceivable.
Still further, a thiol substance having a reactive group is allowed to act directly on the gold deposition surface to introduce a reactive group by a gold-sulfur bond, and then has a group capable of reacting with the reactive group and generates a negative charge. A method of reacting a compound having the compound and introducing a negatively charged substance to the surface is also conceivable. In this case, the reactive group of the thiol substance and the reactive group of the compound having a negative charge include 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, and these are used in an appropriate combination.
[0020]
The method of binding the negatively charged functional group such as a carboxyl group or NTA group on the surface of the biochip thus introduced to a biomolecule or a biomolecule aggregate is not particularly limited. Biomolecules or biomolecule aggregates can be introduced to the surface by using a histidine tag method, a nickel chelate histidine tag method or a crosslinking agent. Examples of the introduction with a cross-linking agent include, for example, activating a carboxyl group on the surface with carbodiimide and reacting with an amino group in the biomolecule, or immobilizing the biomolecule using an epoxy-based cross-linking agent.
[0021]
It is preferable that a nonionic substance is immobilized on the background in order to suppress nonspecific adsorption. For example, there is a method in which an alkanethiol having a hydroxyl group or a hydrophilic polymer such as polyethylene glycol at the terminal is directly immobilized on the gold surface. 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]
Means for separating the immobilization site from the background portion include a patterning technique by light irradiation, a stamp technique, and the like. For example, in the method of patterning by irradiating light, a gold-sulfur bond is oxidized and washed and removed by ultraviolet light, and alkanethiol can be newly bonded to the gold surface.
[0023]
The biochip is set on an SPR device and observed in a buffer. Since the SPR resonance angle changes depending on the refractive index of the buffer, the measurement is performed with the same buffer. At the stage when the buffer solution is introduced, the angle at which the image is captured is set. It is preferable that the measurement is performed 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 intensity of reflected light is minimized. When poly-L-lysine having a molecular weight of 2,000 or more and 20,000 or less dissolved in a phosphate buffer having a concentration of 1 mg / ml and a pH of 7.4 at 30 ° C. is brought into contact with the biochip of the present invention at 30 ° C., ion The binding binds poly-L-lysine to the immobilization site and shifts the SPR resonance angle to the wide-angle side, so that the intensity of reflected light increases. The phosphate buffer used here uses 10 mM phosphoric acid and 150 mM sodium chloride. Since the nonionic substance is immobilized on the background portion, the adsorption of the polymer having a positive charge is small.
[0024]
As the SPR device, an SPR imaging device using a white light source capable of analyzing a slide surface is used. The image reflected from the slide is captured over time by SPR imaging captured by a CCD camera. The difference between the captured image and the image before contacting the polymer can be obtained by arithmetic processing. After contacting the chip surface with a pH 7.4 phosphate buffer in which poly-L-lysine having a molecular weight of 2,000 or more and 20,000 or less is dissolved at a concentration of 1 mg / ml at 30 ° C. for 5 minutes, the phosphate buffer After 5 minutes of washing with, the signal increase due to SPR obtained by poly-L-lysine binding to the immobilization site is compared with the signal increase due to non-specific adsorption to a background portion other than the immobilization site. The control is an image before contact with poly-L-lysine. By this operation, it is possible to compare the signal change before contacting with poly-L-lysine and after washing for 5 minutes after contacting for 5 minutes. If 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 area other than the immobilization site is 2 or more, the biomolecule will be added 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. When obtaining the signal ratio, the immobilized site and the background portion to be compared are set so as not to be separated from each other by 1 mm or more on the substrate. As a method of analyzing an image by a computer, for example, a signal change can be captured in real time using image processing software V ++ (Digital Optics).
[0025]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to 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 to a thickness of 45 nm. The thickness of the evaporation was monitored with a crystal oscillator. The substrate on which gold was deposited was 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-assembled surface of 8-AOT. Was.
[0027]
Succinimide group-terminated polyethylene glycol having a molecular weight of 2,000 (mPEG-SPA, manufactured by Sheawater Polymers) was dissolved in a phosphate buffer at 10 mg / ml, reacted with 8-AOT on the gold surface for 2 hours, and PEG was immobilized on the surface. . A photomask in which a pattern was drawn with chromium on quartz was placed on the substrate, and irradiation was performed with a 1000 W xenon arc lamp manufactured by Oriel for one hour to perform patterning. The pattern of the photomask is one in which 100 squares of 500 μm × 500 μm are arranged. After irradiation, the plate was washed with Milli-Q water and ethanol, immersed in a 1 mM ethanol solution of 7-Carboxy-1-Heptanethiol (7-CHT, manufactured by Dojindo Laboratories) for 16 hours, and the irradiated portion was irradiated with 7-CHT. Formed a self-assembled surface. The biochip thus produced has a carboxyl group at the immobilization site, and nonionic PEG is immobilized on the background.
[0028]
The biochip was set on an SPR imaging device (SPRImager: manufactured by GWC Instruments) and observed at an angle where the incident angle was 1 degree smaller than the SPR resonance angle of the immobilized site. The observation was performed at 30 mM at 10 mM phosphate buffer, 150 mM NaCl, pH 7.4. Before and after contact with a phosphate buffer of pH 7.4 in which 1 mg / ml of poly-L-lysine having a molecular weight of 4000-15000 (manufactured by Sigma) is dissolved, the buffer is allowed to flow for 5 minutes and then for 5 minutes. The photograph after this was taken continuously every 5 seconds, and the result of analyzing the change in the signal of the immobilized site and the background is shown in FIG. The change over time in the reflected light intensity at six points was determined, and the signal ratio was obtained at three points. The results were 5.51, 5.56, 4.06, averaged 5.04, and exceeded 2.
The phosphate buffer of pH 7.4 uses 10 mM phosphoric acid and 150 mM sodium chloride. Contact with the biochip is performed at 30 ° C. for 5 minutes in the solution, and then the same phosphate buffer of pH 7.4 is used. At 30 ° C. for 5 minutes.
[0029]
A flow cell was used for contact and washing with the biochip, and the flow cell used was made of POM and shown in FIG. Specifically, an O-ring made of silicon rubber having a wire diameter of 1.5 mm and an inner diameter of 15.5 mm was set in the groove of the flow cell, and a biochip was stacked thereon such that the gold vapor-deposited surface faced the flow cell. The chip was fixed so that the interval between the chips was about 200 μm. The tubes were connected to the inlet and outlet of the flow cell, and a poly-L-glutamic acid solution and a buffer were introduced by a buffer pump. The introduction flow rate was 200 μl / min.
The measurement was performed at a temperature of 25 ° C.
[0030]
FIG. 2 (schematic diagram) shows the result of the difference between the image before contact with poly-L-lysine and the image after inflow of the buffer solution. The darker part indicates that the reflected light intensity is higher.
FIG. 3 shows a graph in which the shade of the difference between the images is observed by a dotted line. From this figure, it can be seen that the signal ratio of the signal of the background portion to the signal of the immobilization site is 2 or more.
[0031]
[Comparative example]
The same gold-deposited substrate as in the example was immersed in a 1 mM ethanol solution of 7-CHT for 16 hours to form a self-assembled surface of 7-CHT on the entire surface, and a carboxyl group was introduced into the entire substrate. When 1 mg / ml of poly-L-lysine having a molecular weight of 4000-15000 (manufactured by Sigma) was brought into contact with this surface in the same manner as in the example, the poly-L-lysine was bonded to the entire substrate. 1.00. In this case, the biochip has no distinction between an immobilized site and a background site.
[0032]
【The invention's effect】
In the biochip for SPR of the present invention, a biomolecule or a biomolecule aggregate hardly non-specifically adsorbs to a background portion, and adsorbs to a binding site while maintaining activity efficiently, and SPR was measured. At this time, the difference from the background becomes apparent.
[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the results of analysis of signal changes in an immobilization site and a background obtained by SPR obtained in an example. FIG. 2 Before and after contact with poly-L-lysine and in a buffer solution in the example. FIG. 3 is a graph showing the results of image differences. FIG. 3 is a graph in which the shade of the image difference in FIG. 2 is observed with a dotted line. FIG. 4 is an upper surface and a cross section of the flow cell used in the example (a cross section of a dotted line in the top view). Figure

Claims (5)

A biochip for surface plasmon resonance (SPR) measurement having a portion for immobilizing a biomolecule or a biomolecule assembly (immobilization site) and a background portion other than the immobilization site, and a poly-chip having a molecular weight of 2,000 to 20,000. After contacting the chip surface with a phosphate buffer of pH 7.4 in which L-lysine is dissolved at a concentration of 1 mg / ml for 5 minutes at 30 ° C. and then washing with the phosphate buffer for 5 minutes, immobilization is performed. SPR measurement biochip in which the signal ratio of a signal due to SPR obtained by poly-L-lysine binding to the immobilized site and a signal due to non-specific adsorption to a background portion other than the immobilized site is 2 or more The biochip according to claim 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 claim 1, wherein a nonionic substance is immobilized on the background portion. The biochip according to claim 1, wherein a transparent glass or a transparent plastic on which gold is deposited is used as a substrate. The biochip according to any one of claims 1 to 4, wherein the surface plasmon resonance is SPR imaging.

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