JP4099707B2 - 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 that makes the difference from the background clear when surface plasmon resonance is measured by immobilizing the biomolecule on the biochip using a functional group such as COOH 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.
[0004]
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.
[0005]
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.
[0006]
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.
[0007]
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.
[0008]
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.
[0009]
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 positively charged functional group that is a starting point for immobilization is immobilized on the immobilization site. Makes it possible to obtain
[0010]
[Non-Patent Document 1]
Anal. Chem. 1997, 69, 1449-1456.
[Patent Document 1]
US Pat. No. 6,127,129 [0011]
[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.
[0012]
[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.
[0013]
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 Fixation is obtained when a phosphate buffer solution having a pH of 7.4 in which L-glutamic acid is dissolved at a concentration of 1 mg / ml is brought into contact with the chip surface at 30 ° C. for 10 minutes and then washed with the phosphate buffer solution for 10 minutes. SPR measurement bio, characterized in that the signal ratio between the signal by SPR obtained by poly-L-glutamic acid binding to the immobilized site and the signal by nonspecific adsorption to the background part other than the immobilized site is 2 or more Chip.
[0014]
2. 2. The biochip according to 1, wherein the functional group introduced into the immobilization site is an amino group.
[0015]
3. 3. The biochip according to 1 or 2, wherein a nonionic substance is immobilized on the background part.
[0016]
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.
[0017]
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 biochip having the characteristics of the present invention, the immobilization site of the biomolecule or the biomolecule assembly is clearly distinguished from the background part other than the immobilization site, and the immobilization site is preferably for immobilization. It is preferable that a substance having a positively charged functional group as a starting point is immobilized. A biomolecule or biomolecule assembly can be immobilized on the surface by a covalent bond, an ionic bond, or the like starting from a functional group having a positive charge. In this case, when a heterogeneous cross-linking agent is used for a functional group having a positive charge, a biomolecule or a biomolecule assembly can be more effectively introduced to the surface.
[0019]
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.
[0020]
Examples of the functional group having a positive charge include an amino group and are not particularly limited. However, as an example of a method for introducing a functional group, an alkanethiol substance having a positive charge at the terminal is allowed to act directly on the gold deposition surface. A method of introducing a substance having a positive charge directly through a gold-sulfur bond is conceivable.
As another method, an alkanethiol substance having a negative charge at the terminal is allowed to act directly on the gold deposition surface, a negative charge is introduced by a gold-sulfur bond, and then a compound having a plurality of positive charge sites is ionized. A method may also be considered in which a positively 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 a positive charge is possessed by a group that can react with the reactive group. 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 compound having a positive charge with the reactive group of the thiol substance 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.
[0021]
A functional group having a positive charge such as an amino group on the surface of the biochip immobilized in this way can be effectively bound to a biomolecule or a biomolecule assembly. A method for binding a biomolecule or a biomolecule assembly is not particularly limited. For example, the COOH group of the biomolecule is activated and immobilized on the surface, or a cross-linking agent having a succinimide group is used. Biomolecules can be immobilized on the surface.
[0022]
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.
[0023]
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.
[0024]
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-glutamic acid 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 As a result of the binding, poly-L-glutamic acid binds to the immobilization site, 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 portion, adsorption of a negatively charged polymer is small.
[0025]
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 in which poly-L-glutamic acid is dissolved at a concentration of 1 mg / ml is brought into contact with the chip surface for 10 minutes at 30 ° C. In the case of washing for 10 minutes, the signal increase due to SPR obtained by poly-L-glutamic acid binding to the immobilized site is compared with the signal increase due to non-specific adsorption to the background part other than the immobilized site. The comparative control is an image before contacting with poly-L-glutamic acid. By this operation, it is possible to compare a change in signal before contacting with poly-L-glutamic acid and after contacting for 10 minutes and washing off for 10 minutes. In addition, although the flow cell as shown in the embodiment is used for the measurement, the flow cell having the shape shown in the embodiment may not be used as long as the relationship between the capacity in the flow cell and the flow rate is almost the same as that in the embodiment. .
If the signal ratio of the signal due to SPR obtained by poly-L-glutamic acid that binds 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 at the immobilization site Alternatively, it is preferable because it is easy to introduce a biomolecule assembly. The signal ratio is more 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.
[0026]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
[0027]
[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. The substrate on which gold was deposited was immersed in a 1 mM ethanol solution of 7-Carboxy-1-Heptanethiol (7-CHT, manufactured by Dojindo Laboratories) for 16 hours to form a 7-CHT self-assembled surface. 0.2M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Nacalai Tesque), 0.05M N-hydroxysuccinimide (manufactured by Nacalai Tesque), amino group-terminated polyethylene glycol molecular weight 5000 (mPEG- Amine, 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. The photomask which drew the pattern with the chromium on the crystal | crystallization was set | placed on the board | substrate, and it patterned by irradiating with the 1000W high pressure mercury lamp made from Ushio Electric for 1 hour.
[0028]
The photomask pattern has 100 squares of 500 μm × 500 μm arranged. After irradiation, the plate was washed with milliQ water and ethanol, then immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 16 hours, and the portion irradiated with light was exposed to 8 -A self-assembled surface of AOT was formed. The biochip thus produced has an amino group at the immobilization site, and nonionic PEG is immobilized in the background portion.
[0029]
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. 10 minutes after contact with poly-L-glutamic acid (manufactured by Sigma) having a molecular weight of 3000-15000 with a molecular weight of 3000-15000, photographs after flowing the above buffer solution for 10 minutes continuously every 5 seconds. FIG. 1 shows the result of photographing and analyzing the signal changes in the immobilization site and the background.
[0030]
The change in reflected light intensity with time was obtained at a total of 6 points including 3 immobilization sites and 3 background portions, and signal ratios at three locations were obtained. The results were 5.20, 3.93, 19.2 and the average was 9.46, which was above 2.
[0031]
The phosphate buffer solution at pH 7.4 uses 10 mM phosphate and 150 mM sodium chloride, and the biochip is brought into contact with this solution at 30 ° C. for 10 minutes, and then the same pH 7.4 phosphate buffer solution. And washed off at 30 ° C. for 10 minutes. 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.
[0032]
In addition, FIG. 2 (schematic diagram) shows the results of taking the difference between the images before the contact with poly-L-glutamic acid and after the inflow of the buffer solution. 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.
[0033]
[Comparative example]
A gold-deposited substrate similar to the example was immersed in a 1 mM ethanol solution of 8-AOT for 16 hours to form a self-organized surface of 8-AOT on the entire surface, and amino groups were introduced into the entire substrate. When this surface was contacted with poly-L-glutamic acid (manufactured by Sigma) having a molecular weight of 3000-15000 of 1 mg / ml in the same manner as in the example, poly-L-glutamic acid was bound 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.
[0034]
【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 the signal changes in the immobilization site and background obtained by SPR, obtained in Example. FIG. 2 before contact with poly-L-glutamic acid and after influx 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 an upper surface and a cross section of the flow cell used in the example (cross section of a dotted line in the top view). 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 positively charged biomolecules or portions for immobilizing biomolecules assembly (fixed sites) A biochip for measuring surface plasmon resonance (SPR) for immobilizing an assembly ,
After the biochip was brought into contact with the chip surface at 30 ° C. for 10 minutes with a phosphate buffer solution of pH 7.4 in which poly-L-glutamic acid having a molecular weight of 2000 or more and 20000 or less was dissolved at a concentration of 1 mg / ml In the case of washing with phosphate buffer for 10 minutes,
A biochip for SPR measurement, wherein a signal ratio of SPR signal obtained by poly-L-glutamic acid 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 functional group having a positive charge is an amino 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 vapor-deposited is used as a substrate. The biochip according to any one of claims 1 to 5, the surface plasmon resonance is characterized by a SPR imaging. 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 functional group having a positive charge at the end of the gold vapor deposition surface exposed by patterning 8. The method for producing a biochip according to claim 7, wherein an alkanethiol substance having a functional group having a positive charge at its end is immobilized at an immobilization site. 9. The method for producing a biochip according to claim 7, wherein the functional group having a positive charge is an amino 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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