JP4281108B2 - Biochip - Google Patents

Description

  The present invention relates to a metal substrate biochip having a marker that facilitates discrimination of spot positions.

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 large number of substances can be analyzed simultaneously.
A general biochip is a DNA chip. A DNA chip is obtained by immobilizing DNA molecules on a solid substrate, and is used for observation of DNA-DNA interaction. Recently, the degree of accumulation of molecules has become very high, and the amount of information obtained from a single measurement has also increased. However, labeling methods such as fluorescence, chemiluminescence, and radioisotopes are necessary for detection of binding. Therefore, it is not suitable for the interaction of biomolecules such as proteins and sugars that are difficult to label.

As a label-free interaction analysis method, an optical detection method such as a surface plasmon resonance (SPR) method, an ellipsometry method, or a sum frequency generation method has attracted attention. Among them, the SPR imaging method using the SPR method is attracting attention because it can observe the interaction in a two-dimensional region. That is, this is a method in which the entire surface of the chip is irradiated with a polarized light beam and the reflected light is photographed with a CCD camera. If an array is produced on a metal substrate chip, interaction at multiple points by the SPR imaging method becomes possible (Patent Document 1).

However, the optical system of the SPR imaging method is complicated, and the CCD camera takes an image from the back surface of the chip, so that the pattern formed on the chip surface is reversed and displayed on the CCD camera. Therefore, it is difficult to specify the location where the sample is fixed. In order to solve this problem, a method of fixing to the surface with a predetermined pattern has been taken (Non-Patent Documents 1 and 2). Thus, when it is immobilized on the surface with a pattern, it becomes easy to determine which molecules interacted. However, in order to form a pattern, it is necessary to fix one sample to a plurality of spots, and there is a problem that the throughput of measurement is limited.
The present invention realizes a biochip suitable for multipoint interaction analysis by providing a marker for specifying the spot position on the chip.

US Pat. No. 6,127,129 Nelson et. al. Anal. Chem. 73 (2001) 1-7 Smith et. al. J. et al. Am. Chem. Soc. 124 (2002) 6810-6811

  The subject of this invention is obtaining the biochip which consists of a metal substrate which can discriminate | determine the position of a spot.

As a result of intensive studies, the present inventors have found that the above problem can be solved by using a biochip using the means described below.
1. A marker having an array portion formed on a metal substrate and patterned with a monolayer describing the spot location of the array portion, and having an average molecular weight of molecules coated on the marker portion And the ratio of the average molecular weight of the molecules coated on the marker portion and the peripheral portion of the array portion is 0.5 or less , thereby changing the thickness of the chip surface layer having a different refractive index. A biochip for measurement of surface plasmon resonance imaging, characterized in that it can be identified .
2. The metal substrate is a transparent substrate coated with a gold thin film. The biochip as described in.
3. 1. The molecule coated on the marker portion and the peripheral portion of the array portion is PEG thiol. Or 2. The biochip as described in.
4). 2. The molecular weight of PEG thiol is 5000. The biochip as described in.
5. 1. The marker is a distinguishable character and / or number. ~ 4. The biochip according to any one of the above.
6). 1. The molecule as described in 1. above, wherein the molecule coated on the marker moiety is 7-carboxy-1-heptanethiol. ~ 5. The biochip according to any one of the above.

The biochip obtained by the present invention can easily know the position of the spot by the marker in the interaction on the metal substrate by the optical detection method.

  The biochip of the present invention has an array portion formed on a metal substrate. The metal substrate is preferably a transparent substrate coated with a gold thin film. This is because it is suitable for optical detection methods such as SPR, ellipsometry, and sum frequency generation method. In addition, it is preferable to use alkanethiols or disulfides having a functional group at the terminal because a self-assembled surface can be formed on the gold surface by a gold-sulfur bond and the functional group can be introduced to the surface. The functional group is not particularly limited. For example, a substance having an amino group after the carboxyl group is introduced to the gold surface by using an alkanethiol having a carboxyl group and activated by water-soluble carbodiimide and N-hydroxysuccinimide. Can be immobilized on the surface.

The method for coating the gold thin layer is not particularly limited, and examples thereof include a vapor deposition method, a sputtering method, and an ion coating method. In general, chromium or titanium is coated under a thickness of 1 to 10 nm under the gold in order to suppress gold peeling. Although the thickness of gold is not limited, it is generally coated with 30 to 100 nm.
The material of the transparent substrate is not particularly limited, and examples thereof include plastics such as glass, polycarbonate, and polyolefin. The array portion on the metal substrate refers to a portion in which molecules are regularly arranged and immobilized on the substrate or can be immobilized.

  As an optical detection method, the SPR method sensitive to a change in the refractive index of the surface is preferable. The SPR method is a label-free and real-time measurement method, and has a track record as an interaction analysis method. Among these, the SPR imaging method is more preferable because it can observe the interaction of a two-dimensional region. If an array is made, the interaction at multiple points can be measured.

  The biochip of the present invention is characterized in that it is provided with a marker that indicates the spot location of the array portion. By providing the marker, it is possible to easily determine where the substance immobilized on the array is. In particular, when the SPR imaging method is used as an optical detection method, it is common to photograph with a CCD camera from the back of the chip. When taking a picture from the back, the pattern on the chip is reversed so that the location is difficult to distinguish. The presence of the marker makes it possible to easily identify the spot location.

There may be one marker or many markers, and the marker may have any shape. However, in order to easily distinguish the rows and columns of the array, the shape of the marker is preferably a distinguishable character and / or number. For example, when a sample to be immobilized is prepared on a commercially available 96-hole plate or 384-hole plate, it is conceivable to reflect the letters and numbers of the rows and columns described on the plate as they are. In the case of a 96-hole plate, there may be a case where alphabetical characters from A to H are written on the vertical axis and numbers from 1 to 12 are written on the horizontal axis.
The array preferably has an 8 × 12 spot portion, a 12 × 16 spot portion, or a 16 × 24 spot portion in order to correspond to a commercially available 96-hole plate or 386-hole plate.

  The method for introducing the marker is not limited to the method as long as it is a method that can be detected by a detection system. Examples thereof include a method of attaching a polymer, an organic substance, an inorganic substance or the like on a metal surface of a marker part or a part other than the marker or between a substrate and a metal layer, or attaching a substance different from other parts.

  As a method of attaching a polymer, an organic substance, an inorganic substance, etc. to a marker portion or a portion other than a marker, masking and vapor deposition, masking and coating, printing by letterpress or ink jet method, etc., after coating the entire surface Examples include the method of removing unnecessary parts by decomposing or altering them by light, laser, radiation irradiation, etc., removing unnecessary parts, and attaching other substances after removing unnecessary parts. Can do. When making it adhere on a metal surface, a marker can be provided by the said method on the board | substrate which provided the metal layer. When making it adhere between board | substrate-metal layers, a metal layer can be provided after making a marker adhere on a board | substrate by the said method.

  There are no problems as long as the polymer or organic substance can be attached to a metal or a substrate, and if it is attached to the metal, it is not a problem as long as it is not lost when the measurement liquid is flowed. Organic materials and these compositions are exemplified, but organic materials and polymers containing sulfur such as thiol, sulfild, disulfide, etc. in the molecule that can be firmly bonded to gold are preferably used.

Moreover, it is preferable that the difference in height on the chip surface between the marker and another part is 3 μm or less. More preferably, it is 1 micrometer or less, More preferably, it is 100 nm or less, Most preferably, it is 50 nm or less. Furthermore, the marker is preferably introduced as a monomolecular layer. If the chip surface has irregularities exceeding 3 μm, the flow of the surface deteriorates when a solution containing a substance to be analyzed is exposed to the chip surface, which is not preferable for evaluating the kinetics of the interaction. This is because it is preferable. The unevenness at the molecular level can be sufficiently discriminated by an optical detection method such as the SPR imaging method.
The surface irregularities are determined by measuring the scale of the irregularities, such as a stylus type or non-contact surface roughness meter, interference microscope, tunnel microscope, calculation from SPR angle, cut and observed with a cross section, etc. I can do it.

  Furthermore, it is preferable that the marker part is formed at the same time when the spot part is formed. If either the marker portion or the spot portion is formed in advance, the spot pattern may be limited or a positional shift may occur. Note that the term “simultaneous” here does not matter if it is not completely simultaneous in time, as long as it is performed continuously without moving the same process or substrate. As a method of simultaneously forming the marker part and the spot part, masking is performed when the metal layer is provided, and the marker part and the spot part are provided by performing vapor deposition of metal, etc. When the spot part is provided by spotting on the metal layer, And a method of providing a marker part when the surface of the metal surface corresponding to the background part is surface-treated to provide the spot part.

  Specifically, when providing a spot portion by spotting on a metal layer, for example, spotting is performed on the metal with an automatic spotter or the like, and the same or different substance is used therein. Provide a marker. Examples of the spotting and marker include a method using a pin or a pen, a method using an ink jet, and the like. Further, a marker may be engraved on the substrate using a laser or the like after the spotting process.

In the case of providing the marker portion when the surface of the metal surface corresponding to the background portion is surface-treated to provide the spot portion, for example, the entire surface of the metal surface is coated with a hydrophilic compound corresponding to the background portion, and then the spot portion is applied. The hydrophilic compound or the like is removed, and in this case, a method of removing the hydrophilic compound in the marker portion can be mentioned.
The most preferable example of this method is a method of oxidizing alkanethiol by known photolithography (described in Patent Document 1). That is, it is a means for irradiating the spot portion for immobilizing molecules with ultraviolet rays to distinguish the peripheral portion from the spot portion. In this method, not only the spot portion but also the marker portion can be patterned, and also can be patterned with a monomolecular layer.

However, in order to identify unevenness at the molecular level by the SPR imaging method, the molecular weights of the immobilized molecules must be different. This is because the difference cannot be identified unless the thicknesses having different refractive indexes are changed in the surface layer of the chip.
Specifically, the ratio of the average molecular weight of the molecules bonded to the marker portion to the average molecular weight of the molecules bonded to the surrounding portion is preferably 0.5 or less or 2 or more. When the molecule to be bonded is a polymer having a molecular weight distribution, the weight average molecular weight by GPC is selected as the average molecular weight.

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

[Example]
(Step 1)
PEG thiol (SUNBRIGHT MESH-50H manufactured by NOF Corporation), whose alkyl chain has 2 carbon atoms and the terminal functional group is a thiol group, is dissolved in 7 ml of ethanol: water = 6: 1 mixed solution at a concentration of 1 mM. It was. PEG thiol has a molecular weight of 5000 and is very hydrophilic. Moreover, the terminal of PEG is a methoxy group and has almost no reactivity.

  A gold-deposited slide in which 3 nm of chromium was deposited on an SF10 glass slide of 18 mm square and 1 mm thickness and gold was deposited to 45 nm was immersed in the PEG thiol solution for 3 hours, and PEG thiol was bonded to the entire gold substrate in a monomolecular layer. .

(Step 2)
The photomask shown in FIG. 1 (the chrome surface is shown above) was placed on this slide, and irradiated with a 500 W ultra-high pressure mercury lamp (USHIO) for 2 hours to remove PEG thiol in the irradiated area. The photomask is provided with an array portion and a marker portion. There are 96 spots in the array portion, and markers are present reflecting the 96-well plate. That is, the vertical axis is 8 rows from A to H, and the horizontal axis is 12 columns from 1 to 12. In this case, the photomask is placed on the gold surface with the chrome face down. Accordingly, the gold surface is patterned in the left-right direction by irradiation with ultraviolet rays. In the SPR imaging method, images are taken from the back of the chip, and as a result, letters and numbers are projected in the correct direction.

(Step 3)
Next, the slide was immersed in a 1 mM solution of 7-carboxy-1-heptanethiol (7-CHT: Dojindo Laboratories) having a molecular weight of 176 for 1 hour, and 7-CHT was immobilized on the immobilization part as a single molecule.
In the biochip thus obtained, 7-CHT is immobilized on the marker part and the array part, PEG thiol is immobilized on the surrounding part, and the molecular weight ratio is 0.035. The biochip is patterned with a single molecule.
This biochip was set in an SPR imaging device (manufactured by Toyobo Co., Ltd.), and Milli-Q water was allowed to flow. The SPR image at that time is shown in FIG. Thus, letters can be clearly read on the vertical axis and numbers on the horizontal axis.

The SPR angle of this character part and the background part was measured. The SPR angle is an angle at which the reflected light intensity is minimized. The SPR angle of the character part was 53.80 °, the SPR angle of the background part was 54.06 °, and the displacement of the SPR angle was 0.26 °.
Based on the N-phase Fresnel equation by Hansen (J. Opt. Soc. Am. 1968, 58, 380-390), the layer thickness was calculated for 830 nm light. The calculation was performed assuming that the refractive index of SF10 glass was 1.710, gold n = 0.155, k = 5.1634, thickness 45 nm, PEG layer refractive index 1.40, and water refractive index 1.33. A displacement of .26 ° corresponded to a thickness difference of 11 nm. Since it is 50 nm or less, it is estimated that the influence on the flow on the chip is very small.
A sample prepared in a 96-well plate can be fixed to the biochip thus obtained, and the spot position can be easily referred to.

Photomask used in the examples Example SPR imaging image

Claims (6)

A marker having an array portion formed on a metal substrate, patterned with a monolayer describing the spot location of the array portion, and having an average molecular weight of molecules coated on the marker portion And the ratio of the average molecular weight of the molecules coated on the marker part and the peripheral part of the array part is 0.5 or less , so that the thickness having different refractive index in the surface layer of the chip is changed, and the difference A biochip for measurement of surface plasmon resonance imaging, characterized in that it can be identified . The biochip according to claim 1, wherein the metal substrate is a transparent substrate coated with a gold thin film. The biochip according to claim 1 or 2, wherein the molecules coated on the marker portion and the peripheral portion of the array portion are PEG thiols. The biochip according to claim 3, wherein the molecular weight of PEG thiol is 5000. The biochip according to any one of claims 1 to 4, wherein the marker is a distinguishable character and / or number. The biochip according to any one of claims 1 to 5, wherein the molecule coated on the marker portion is 7-carboxy-1-heptanethiol.

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