JP2005069979A - Biochip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a biochip having no fear of contamination in an array forming stage, enabling the alignment of the array formed on the biochip and easy to mount, attach and detach. <P>SOLUTION: The biochip comprises a transparent substrate coated with a metal and the thickness of the transparent substrate is 1.20-4.00 mm. This biochip is preferably constituted so as to flow a sample-containing liquid onto the surface to which molecules are fixed of the transparent substrate and can be suitably used for measuring a surface plasmon. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biochip that can be easily set in an interaction analyzer.

  In recent years, biochips used for biomolecule interaction analysis, gene expression, profiling of expressed molecules, or diagnosis have attracted attention. Operation is facilitated by immobilizing biomolecules on the substrate, and a lot of information can be obtained at once.

  Among these, optical analysis methods such as surface plasmon resonance (SPR) are methods that can observe interactions in real time without labeling, and have been established as molecular analysis means. In the SPR method, molecules are immobilized on a biochip composed of a metal substrate, and a solution containing a substance to be analyzed (analyte) is exposed to the substrate surface. The change in refractive index near the surface due to the generated interaction can be grasped by the shift amount of the SPR angle or the change amount of the reflected light intensity.

  Since this is a technique for detecting light and can be measured in real time, the Kinetics value of the intermolecular interaction can be obtained. In order to obtain the binding / dissociation rate constant, it is necessary to eliminate the concentration gradient on the substrate surface. The most effective method of reducing the concentration gradient is to flow a liquid containing an analyte over the substrate surface. The concentration gradient is destroyed by the shear rate due to the flow of the liquid.

  There are many situations where it is difficult to prepare a large amount of an analyte-containing solution at a measurable concentration. Therefore, a flow path capable of flowing a liquid in the smallest possible amount on the biochip substrate surface is produced.

  As a method of forming the flow channel, a method of securing a basin with an O-ring by grooving a plastic provided with a liquid inlet / outlet hole, a silicon-based polymer such as PDMS is poured into a silicon mold having a flow channel shape, Examples include a method of solidifying and adhering to the substrate surface, a method of creating a thin gasket, and creating a small gap on the substrate. In any case, it is not easy to attach and detach in a state where the substrate is aligned, and some device is required.

  As seen in Patent Document 1, a conventional substrate in which a plastic plate is attached to a substrate has been invented and used. By manipulating the plastic plate, the substrate can be easily attached and detached.

  However, it is common sense that a composite substrate with plastic immobilizes molecules on the surface of the substrate with the substrate set in an SPR device. That is, the reaction solution and the immobilized substance are also introduced into the apparatus and flowed onto the substrate to immobilize the substance on the surface. However, this method cannot measure an array in which a plurality of substances are immobilized on the surface. In order to observe a plurality of interactions using an array, a method should be adopted in which an array is prepared outside the SPR device, set on the device, and the interaction is observed.

When the molecule is immobilized outside the SPR device and then set on the SPR device, there is a risk that the adhesive between the substrate and the plastic elutes and adheres to the substrate surface. Therefore, great care must be taken in the operation of cleaning and drying the chip. Also, the immobilization process is performed on the substrate alone. The method of bonding with plastic is also complicated.
Therefore, there is a need for an inexpensive biochip that can be easily attached to and detached from the SPR device and does not cause contamination.

Japanese National Publication No. 04-501462

  An object of the present invention is to obtain a biochip that can be aligned with an array formed on a biochip without any fear of contamination at the stage of forming the array, and can be easily attached and detached.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. 1. A biochip comprising a transparent substrate coated with a metal, and the thickness of the transparent substrate is in the range of 1.20 mm to 4.00 mm. 2. The biochip according to 1, wherein a liquid containing a sample can flow on the surface of the surface of the transparent substrate on which the molecules are immobilized. The biochip according to any one of 1 to 2, which is used for surface plasmon resonance measurement

  There was no risk of contamination at the stage of forming the array, the alignment of the array formed on the biochip was possible, and a biochip that was easy to attach and detach could be obtained.

  The biochip of the present invention comprises a transparent substrate coated with a metal. As the metal to be coated, gold, silver, chromium, titanium, aluminum or the like is preferably used alone or in combination. In particular, gold is particularly preferable because it is stable and a molecule can be immobilized by introducing a functional group to the surface by a gold-sulfur bond. When gold alone is directly coated on the substrate, the gold is easily peeled off, and therefore it may be treated with chromium or titanium before coating with gold.

  Although the thickness of coating is not specifically limited, Usually, it coats in the range of 20-200 nm. The coating method is not particularly limited, and vapor deposition, sputtering, ion coating method and the like are selected. Examples of the material for the transparent substrate include transparent plastics such as polycarbonate, polyolefin, polyethylene terephthalate, and polymethyl methacrylate, and glass. Glass is preferable because it is inexpensive and easily available, and has very little distortion.

  The transparent substrate is preferably composed of a single material. If it is composed of a plurality of materials, there is a risk of peeling at the interface. This is because if an adhesive is used to prevent peeling, the adhesive is eluted and there is a risk of causing contamination.

  The biochip of the present invention is preferably used for surface plasmon resonance (SPR). SPR is a commonly used analysis method and is preferable because it is label-free and allows real-time detection. SPR imaging in which a chip surface is irradiated with a polarized light beam and a reflected image is taken is more preferable because a plurality of interactions can be simultaneously analyzed by an array formed on a biochip.

  An optical system for observing SPR is preferable because the Kretschmann arrangement is easy and the sensitivity is high. In the Kretschmann arrangement, a method of using the biochip by bringing the non-metal surface of the biochip into contact with the prism is preferable.

  The thickness of the transparent substrate in the present invention is preferably 1.20 mm or more and 4.00 mm or less. It is preferable that the depth of the dent in the portion where the biochip is mounted is 0.70 mm or more because the chip is accommodated and positioning can be performed. Particularly when the flow path is formed of an O-ring, the O-ring has a round cross section, so that the O-ring is compressed when the chip is pressed from above, and the flow path has an appropriate space above the chip. Is formed. Naturally, the depth of the groove in which the O-ring is mounted is smaller than the wire diameter of the O-ring, and the O-ring protrudes above the surrounding surface. In order to mount the chip without compressing the O-ring, it is necessary to provide a chip mounting portion having a dent deeper than the ledge of the O-ring. Actually, it is preferable to provide a chip mounting portion that is 0.50 mm or more deeper than the protrusion of the O-ring, and more preferably 1.00 mm or more.

  Since the wire diameter of an O-ring that can be easily obtained is 1.00 to 3.00 mm, the dent of the chip mounting portion is preferably 1 mm or more. Since it is preferable to adapt an O-ring having a wire diameter of 1.50 to 2.00 mm in standard products that are widely used, the dent of the chip mounting portion is more preferably 1.25 mm or more, and 1.75 mm or more. Further preferred.

1
In order to bring the prism into contact with the non-metal surface of the chip, the chip thickness is preferably 0.20 mm or more thicker than the depth of the recess in which the chip is mounted. This is because, when the chip thickness is smaller than the dent, a prism having one side larger than one side of the chip cannot be used, so that the use range is limited. A method in which a contact liquid having almost the same refractive index as that of the chip / prism is inserted between the chip and the prism and the measurement is performed as optically the same is easy and inexpensive in the Kretschmann arrangement. When bubbles are generated in the contact liquid when the contact liquid is added, the prism can be easily removed by sliding left and right. When the chip thickness is smaller than the dent, the range in which the prism is slid is limited, which is not preferable.

Therefore, the thickness of the chip is preferably 1.20 mm or more, more preferably 1.45 mm or more, and further preferably 1.95 mm or more.
However, if the tip is too thick, it will be difficult to grasp the tip with tweezers. Therefore, the thickness of the chip is preferably 4.00 mm or less, and more preferably 3.00 mm or less.

  The shape of the biochip is preferably rectangular for positioning and storage, and more preferably square. In this case, the corner may be rounded.

The area of the entire surface for immobilizing the molecules of the transparent substrate is preferably 25 mm ² or more. If it is less than 25 mm ² , an array having a large number of spots cannot be formed on the chip, which is not preferable.

  It is preferable that a liquid containing a sample can flow on the surface of the surface on which the molecules of the transparent substrate are immobilized. This is because, when measuring the interaction, in order to destroy the concentration gradient on the surface of the substrate, it is preferable to have a flow because it is suitable for Kinetics measurement. In addition, since the liquid can be flowed, it is preferable that the interaction of various substances can be observed by switching the liquid to be flowed.

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

[Example]
3 nm chromium was vapor-deposited on an 18 × 18 mm, 2 mm thick SF10 glass slide, and further 45 nm gold was vapor-deposited to obtain a biochip. This biochip was mounted on a PEEK chip holding jig shown in FIG. A cross-sectional view taken along the dotted line in FIG. 1 is shown in FIG. The depth of the dent of the chip mounting portion (1) in FIGS. 1 and 2 is 1.80 mm, and the biochip is 0.20 mm thicker. The depth of the O-ring mounting groove (2) in FIGS. 1 and 2 is 1.10 mm, and the O-ring wire diameter is 1.50 mm. The protrusion of the O-ring with respect to the surrounding surface is 0.40 mm. The depth of the chip mounting portion (1) was sufficiently deep, and the chip could be easily mounted.

In the part surrounded by the O-ring in (2), there are two 0.3φ holes, so that the liquid containing the sample can flow in and out, and on the surface of the surface that immobilizes the chip molecules The liquid containing the sample can be flowed.
It was possible to set this chip together with a chip holding jig on a surface plasmon resonance imaging apparatus (MultiSPRinter: manufactured by Toyobo Co., Ltd.) and use it for observation of interaction.

[Comparative example]
3 nm chromium was vapor-deposited on an 18 × 18 mm, 1 mm thick SF10 slide glass, and further 45 nm gold was vapor-deposited to obtain a biochip. When this chip is mounted on the chip holding jig of the embodiment, it is difficult to mount the prism.

  Therefore, a chip holding jig having a cross section shown in FIG. 3 was manufactured. The depth of the dent of the chip mounting portion is 0.80 mm, and the biochip is 0.20 mm thicker. The depth of the O-ring mounting groove in FIG. 3 is 1.10 mm, and the O-ring wire diameter is 1.50 mm. The protrusion of the O-ring with respect to the surrounding surface is 0.40 mm. Since the dent of the chip mounting portion is 0.80 mm with respect to the O-ring bulge of 0.40 mm, there is only a 0.40 mm difference. In such a jig, it is difficult to mount the chip, and it is difficult to perform positioning.

  According to the present invention, there is no fear of contamination at the stage of forming an array, and the alignment of the array formed on the biochip is possible, and a biochip that can be easily attached and detached can be obtained. The present invention enables quick measurement and contributes to the industry.

Chip holding jig used in Examples and Comparative Examples. (1) is a tip mounting portion, and (2) is an O-ring mounting groove. Sectional drawing of the jig | tool for chip | tip holding used in the Example. The chip mounting part depth is 1.5mm. Sectional drawing of the jig | tool for chip | tip holding used in the comparative example. The chip mounting part depth is 0.5mm.

Claims (3)

  A biochip comprising a transparent substrate coated with a metal, and the thickness of the transparent substrate is in the range of 1.20 mm to 4.00 mm   The biochip according to claim 1, wherein a liquid containing a sample can flow on the surface of the surface of the transparent substrate on which the molecules are immobilized.   The biochip according to claim 1, wherein the biochip is for surface plasmon resonance measurement.