A biochip enables the following steps to be carried out simultaneously in a large amount: fixing on a support (substrate) bio molecules such as DNA, protein, sugar chains or the like, or cells; contacting the thus fixed bio molecules, cells or the like (hereinafter referred to as ‘probes’) with bio molecules or any other compounds (hereinafter referred to as ‘targets’); and detecting the thus resulting characteristic interactions. Among such biochips, a DNA chip is prepared by fixing several thousands or several tens of thousands kinds of DNA fragments and synthetic oligonucleotide (hereinafter, they will be referred to as ‘DNA probes’) respectively at predetermined positions on a glass substrate, a silicon substrate or the like that is square, several centimeters on the side. Such a DNA chip is used, for example, for measuring simultaneously a number of genes being expressed. It can be used also in genetic screening for detecting the presence of a particular gene, for example.
The genetic screening using a DNA chip can be performed in the following steps, for example.    1. Messenger RNA (mRNA) is extracted from cells or blood of a specimen.    2. Complementary DNA (cDNA) is reverse-transcribed and replicated from the mRNA, fragmented and bonded to fluorochrome for labeling, thereby preparing a target.    3. The thus labeled cDNA (target) is contacted with the DNA chip so as to bond to the DNA probe on the substrate. The cDNA as the target is bonded to DNA probe complementary to the cDNA.    4. The DNA chip is washed in order to remove the target not being bonded to the DNA probe.    5. Color development of the fluorochrome is observed with a fluorescence microscope so as to detect the position and amount of the targets on the DNA chip substrate.
Processes for manufacturing a DNA chip is classified roughly into two types. The first type utilizes a photolithography method that is used in the semiconductor manufacturing technique. According to this process, four kinds of mononucleotides as constituent units of DNA are bonded chemically in a predetermined order respectively at predetermined positions on the substrate so as to form oligonucleotide (see Patent Document 1, for example). As used herein, a word “oligonucleotide” denotes a polymer of several or several tens of nucleotides, and it is formed for example by fragmenting a polynucleotide with a high polymerization degree (such as a natural nucleic acid), or its molecular weight is smaller than that of the polynucleotide.
The second type is a process for manufacturing a DNA chip, where oligonucleotide to be fixed is dissolved in a liquid previously, and the liquid is micro-dripped on predetermined positions of a substrate and fixed thereto. Methods for fixing the oligonucleotide onto the substrate include, for example, a process using chemical bonding, a process using physical adsorption and the like, and an ink jet method can be applied to the micro-dripping (see Patent Document 2, for example). In the ink jet method, droplets are discharged from a number of holes toward a substrate, where each of the holes formed on a nozzle plate has a diameter of several tens of micrometers, thereby placing the liquid on predetermined positions of the substrate. This method is used commonly as an ink jet printing method.
According to a process for manufacturing a DNA chip by an ink jet method, a DNA chip can be manufactured easily at a low cost in comparison with the first method. Therefore, the ink jet method is expected to contribute to the process for manufacturing DNA chips in the future. However, the process can cause problems from the following viewpoints:                (1) accuracy in placing droplets on predetermined positions of a substrate; and        (2) spreading and bleeding of a solution discharged on a substrate by the ink jet method.        
The accuracy in placement as raised in (1) above is an essential factor for the quality of the DNA chip, and the accuracy can be improved by developing an ink jet discharger that allows highly-accurate printing. Some commercially-available ink jet printers can place droplets having a diameter of several tens of micrometers on a substrate with a positional accuracy of ±30 μm. Reduction in size and the positional accuracy of the droplets are considered to improve remarkably by future remodeling of the apparatuses.
Spreading and bleeding of a solution on a substrate as mentioned in (2) above will restrict the density in placing the DNA probe regions on the substrate. Namely, for raising the density of the DNA probe regions, the respective DNA probe solutions must be placed on the substrate at a narrow spacing. The solutions will overlap each other due to spreading or bleeding of the solutions. When the spacing between the respective solutions is widened for avoiding the overlapping of the solutions, the probe density will decrease.
FIGS. 5A-5F show schematically that a DNA probe solution discharged by an ink jet method is spreading on a substrate. The same reference numerals in FIGS. 5A-5F are assigned to the same elements. FIG. 5A-5C show a case where a DNA probe solution discharged toward the substrate by an ink jet method does not spread on the substrate. Specifically, FIG. 5A shows that a DNA probe solution 51 is discharged by an ink jet method toward a substrate 53 in a direction indicated by an arrow 52. FIG. 5B shows a DNA probe solution 54 on the substrate 53, which was observed just after contacting the DNA probe solution 54 with the substrate 53. FIG. 5C shows a DNA probe solution 55 on the substrate 53, which was observed after a lapse of time from the contact. As shown in FIGS. 5A-5C, substantially there is no difference between a contact area of the solution 54 observed just after contacting the solution 54 with the substrate 53 (FIG. 5B) and a contact area of the solution 55 on the substrate 53 over time (FIG. 5C), and this indicates that the DNA probe solution will not spread on the substrate 53. In contrast, FIGS. 5D-5F show schematically a case where a DNA probe solution spreads on a substrate. Specifically, FIG. 5D shows that the DNA probe solution 51 is discharged by an ink jet method toward the substrate 53 in a direction indicated by the arrow 52. FIG. 5E shows a DNA probe solution 56 on the substrate 53, which was observed just after contacting the DNA probe solution 54 with the substrate 53. FIG. 5F shows a DNA probe solution 57 on the substrate 53 after a lapse of time from the contact. As shown in FIGS. 5D-5F, the contact area of the solution 56 just after contacting the solution 56 with the substrate 53 (FIG. 5E) increases over time. Namely, the solution 57 spreads on the substrate 53 (FIG. 5F).
FIGS. 6A-6C are plan schematic views showing a DNA chip substrate before fixing DNA probe solutions and after discharging the DNA probe solutions by an ink jet method. The same reference numerals in FIGS. 6A-6C are assigned to the same elements. FIG. 6A shows a DNA chip substrate 61 before fixing DNA probe solutions onto fixation regions 62. FIG. 6B shows the DNA chip substrate 61 for a case where DNA probe solutions 63 are discharged but not spreading on the substrate 61. FIG. 6C shows a DNA chip substrate 61 for a case where DNA probe solutions 64 are discharged to spread on the substrate 61. When the DNA probe solutions do not spread on the substrate, the solutions 63 are placed on predetermined regions 62 as shown in FIG. 6B. When the DNA probe solutions spread, the solutions 64 at adjacent regions will be mixed with each other as shown in FIG. 6C. For preventing the overlapping of the DNA probe solutions, spacing between the respective solutions (spacing between the DNA probe regions) must be increased. However, this will lower the density of the DNA probe regions on the substrate, and the number of spots of the DNA probe solutions allowed to be placed on a DNA chip will be restricted.
The tendency of spreading and bleeding of the DNA probe solutions provided by the ink jet method on a substrate often is effected from the method of fixing the DNA probes to the substrate. As mentioned above, examples of the method for fixing the DNA probes onto a substrate include chemical bonding and physical adsorption, and both the chemical bonding and physical adsorption use polar groups in DNA probes. Therefore, either polar groups to react chemically with the polar groups of the DNA probes or polar groups to form ionic bonds or hydrogen bonds with the polar groups of the DNA probes will exist on the substrate to fix the DNA probes. A substrate surface having polar groups has a high surface energy and favorable wettability with respect to liquids. Therefore, a DNA chip substrate generally has a high surface energy and thus the DNA probe solution discharged by an ink jet method easily will spread or bleed on the substrate.
In a method disclosed for solving the problems of spreading and bleeding of the DNA probe solution on a substrate, regions for fixing the DNA probes are made to be hydrophilic with the surrounding regions being hydrophobic in order to prevent the DNA probe solution from spreading from the fixing regions (see Patent Document: D3). Accordingly, DNA chips can be manufactured in this method by: preparing a substrate whose surface is water-repellent in an untreated condition and becomes hydrophilic by a treatment with light beams or heat; forming a pattern of water-repelling regions and hydrophilic regions on the substrate surface by irradiating with light through a metal mask for example; and then discharging solutions containing DNA probes on the hydrophilic regions by an ink jet method.
Patent Document D1: U.S. Pat. No. 5,405,783
Patent Document D2: JP 2001-66305 A
Patent Document D3: JP 2003-28864 A