Patent Application
20160216254
The present invention relates to a substrate for biochips, to which substrate biologically relevant substances such as proteins, nucleic acids, peptide derivatives, saccharides and derivatives thereof, natural products and small molecule compounds are immobilized as probes; and to a biochip comprising the same.
Biochips such as protein chips, peptide chips and DNA chips are widely used for diagnosis and research of various diseases. The biochips, which have been widely used, are usually those obtained by immobilizing biologically relevant substances such as proteins, peptides and DNAs on a glass substrate such as a slide glass (for example, Patent Document 1, Patent Document 2 and the like).
In recent years, higher densification of biochips is demanded. In cases where biochips are highly densified, each reaction spot becomes small, and therefore the amount of the biologically relevant substances (which are expensive in many cases) immobilized on each spot can be reduced. Further, if comprehensive analysis of genes or proteins can be conducted on a sheet of biochip, the procedures are simple and costs can be reduced, which is preferred.
Although the present inventors made an effort to prepare biochips with high density, it was proved that in cases where the density of reaction spots is increased, various solutions of different biologically relevant substances used in forming each reaction spot are mixed with each other, and a biologically relevant substance(s) immobilized to each reaction spot is/are then likely to be contaminated (contamination).
An object of the present invention is to provide a substrate for biochips, in which contamination of a biologically relevant substance(s) to be immobilized does not occur even when reaction spots of a substrate for biochips are highly densified; and a biochip comprising the same.
The present inventors intensively studied to find that contamination of a biologically relevant substance(s) can be prevented by providing a plurality of grooves, which surround each of reaction spots individually and independently, on the substrate, thereby completing the present invention.
That is, the present invention provides a substrate for biochips, the substrate comprising a substrate and a plurality of reaction spots disposed on the substrate, wherein a plurality of grooves which surround each of said reaction spots individually and independently are provided on the substrate. Also, the present invention provides use of the substrate according to the above-described present invention as a substrate of biochips. Further, the present invention provides a biochip in which a biologically relevant substance(s) is/are immobilized on each of the reaction spots of the substrate for biochips, according to the above-described present invention.
By the present invention, a substrate for biochips, in which reaction spots are highly densified and even so contamination of a biologically relevant substance(s) to be immobilized does not occur; and a biochip comprising the same are provided for the first time. According to the substrate for biochips of the present invention, even when the reaction spots are highly densified, contamination of a biologically relevant substance(s) does not occur, and therefore the accurate analysis is possible. Also, according to the present invention, the reaction spots can be highly densified, and as a result, the size of each spot can be reduced. Therefore, the amount of a biologically relevant substance(s), which is/are expensive, to be immobilized on each spot can be reduced.
A preferable specific example of the present invention will be explained based on
Materials constituting the substrate 10 may be the same as those constituting known substrates for biochips, and examples of the materials include glass such as slide glass; plastics such as acrylic resin, polystyrene and polyethylene terephthalate; metals such as aluminum and stainless steel; carbon such as amorphous carbon and diamond-like carbon. Among these, carbon has excellent properties that autofluorescence is not induced, a biologically relevant substance(s) can be immobilized easily, processing of the substrate is easy, and high flatness and surface precision can be attained. Therefore, a substrate whose surface at least is composed of carbon is preferred. In order to promote the accuracy of measurement when used as a biochip, the surface of the substrate is preferably as flat as possible and may be polished as required. The surface roughness Ra is preferably 2 nm or less, and more preferably about 1 nm. Even though the substrate body is made of glass, metal, plastics or the like, the above-described excellent effects can be attained as long as the surface of the substrate is composed of carbon, and the substrate has flat surface.
For accurate analysis, it is preferable to bind a biologically relevant substance(s) covalently to the substrate more strongly to attain immobilization. For this reason, the surface of the reaction spot 14 preferably has a functional group(s) through a covalent bond, to covalently bind to a biologically relevant substance(s). Preferable examples of the functional group(s) to covalently bind to a biologically relevant substance(s) include amino groups and carboxyl groups, but the functional group(s) is/are not restricted thereto. Methods for binding amino groups or carboxyl groups covalently to the surface of the substrate composed of carbon are known (the above-described Patent Document 1 and Patent Document 2), and the amino groups and the carboxyl groups can be bound covalently to the surface of the substrate by these known methods. Preferably, an amino group-containing polymer or a carboxyl group-containing polymer is bound covalently to the surface of the substrate (Patent Document 2).
A plurality of reaction spots are present on the substrate, and the number of the reaction spots is usually not less than 300, preferably not less than 1000, more preferably about 10000 to 40000 per substrate having a size of 25 mm×75 mm slide glass.
On the substrate for biochips of the present invention, the groove 12, which surrounds each of the reaction spots individually and independently, is provided. The groove 12 surrounds each of the reaction spots individually. Also, each groove 12 surrounds each of the reaction spot 14 independently, that is, the grooves each surrounding a different reaction spot do not intersect or come into contact with one another. The width of the groove is not restricted, but it is usually about 10 μm to 100 preferably about 20 μm to 50 μm; and the depth of the groove is about 0.5 μm to 50 μm, preferably about 1 μm to 20 μm. The planar shape of the groove 12 (the two-dimensional shape when the substrate is viewed from the top) is not restricted, but it may be a circle, square, rectangle, polygon, or the like. Preferably, the groove 12 may be formed easily by direct writing with a laserbeam. The size of the groove may be any size as long as the groove surrounds each reaction spot.
A biologically relevant substance(s) is/are immobilized to the reaction spots. The biologically relevant substance(s) may be any substance which is used as a probe in biochips, and examples thereof include any optional polypeptide (including natural or synthetic protein and oligopeptide), nucleic acid (including DNA and RNA, and artificial nucleic acid), saccharide, lipid, complex thereof (glycoprotein and the like), and derivative thereof (modified protein, nucleic acid and the like). In cases where a functional group(s) is/are added to the surface of the reaction spots, these biologically relevant substances are bound covalently to the functional groups by well-known methods.
When each of the biologically relevant substances is immobilized to each reaction spot of the substrate for biochips of the present invention, a solution containing a biologically relevant substance(s) (usually a solution containing a biologically relevant substance(s), surfactant, reagents and the like in aqueous buffer solution) is spotted (dropped).
The thus obtained biochip may be used in exactly the same manner as in the conventional biochips.
The present invention will now be described more concretely by way of Examples. However, the present invention is not restricted to the Examples below.
Using, as a substrate material, an amorphous carbon plate (25.0×75.0 mm, tolerance ±0.1 mm, plate thickness 1.000 mm, tolerance ±0.025 mm) which was polished such that the surface roughness Ra was 1 nm, a 15-minute ultraviolet irradiation (18.5 mW/cm2, 254 nm) was performed by an ultraviolet irradiation apparatus (SEN LIGHTS Co., Ltd., Photo Surface Processor PL16-110).
The reaction spots had a diameter of 1.5 mm, and 1536 reaction spots were formed. Around each reaction spot, grooves each surrounding the reaction spot were formed respectively by direct writing with a laserbeam (Apparatus: MDV9600A, produced by KEYENCE CORPORATION, Output 200 kW). Each groove had a diameter of about 2 mm, a depth of about 10 μm and a width of about 40 μm.
Amino groups were bound covalently on the surface of the reaction spots as follows:
1. The substrate was subjected to scrub washing and drying with a spin dryer.
2. The substrate was irradiated with UV having a wavelength of 185 nm/235 nm in the air for 5 minutes.
3. The substrate was coated with a solution of polyallylamine (PAA) in ethanol with a Baker applicator
4. The coated substrate was incubated at high humidity of 95% Rh for 15 minutes.
5. A 15-minute vacuum drying was performed under reduced pressure of not more than 0.095 MPa.
6. A 3-minute UV irradiation was performed under reduced pressure of not more than 0.095 MPa to immobilize PAA.
7. The substrate was washed by performing a 5-minute immersion in pure water twice, and dried with a spin dryer for 40 seconds.
Next, mKate protein was immobilized to each reaction spot by a covalent bond. This procedure was carried out concretely in the following way. The substrate, which was grooved and modified with amino groups, was immersed in 0.2 mM Sulfo-SMPB (Thermo scientific) solution which was a cross-linker. After adding maleimide groups to each reaction spot, the substrate was immersed in 50 mM solution of GSH having a thiol group to bind GSH to the maleimide group. The mKate protein was synthesized in the form having FLAG and GST tags added in the N-terminal side, by using an extract of wheat germ. A solution of FLAG-GST-mKate protein was then spotted to the spot, to which GSH was bound, using automatic pipetter, thereby allowing GSH and GST to react to bind each other. When the mKate protein solution was spotted to each reaction spot, as schematically shown in
The above-described binding of the mKate protein was confirmed by reacting Anti-FLAG-HyLight-647 and measuring fluorescence intensity. As a result, independent fluorescences of each spot could be detected due to the effect of the groove, and the binding of the mKate protein on the whole surface in a reaction spot could be confirmed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013-185020 | Sep 2013 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2014/068444 | 7/10/2014 | WO | 00 |
