A liquid discharging device includes a plurality of liquid discharge sections. Each liquid discharge section includes a reservoir, a nozzle that discharges a solution supplied from the reservoir, and discharge energy generating means that generates energy to discharge the solution from the nozzle. The number of the liquid discharge sections corresponds to the number of probe types to be formed. The nozzles are two-dimensionally arranged. Using this liquid discharging device, probe liquids are discharged from the corresponding reservoirs onto a solid-phase substrate to form a predetermined two-dimensional probe array of high-purity probes on the substrate. This process exhibits high reproducibility and processability, and the resulting probe array has high array density.

1. Field of the Invention
The present invention relates to a method for making a probe support on a solid-phase substrate and an apparatus used for the method. This apparatus includes a liquid discharging device for two-dimensionally arranging and fixing the probe array on the solid-phase substrate.
2. Description of the Related Art
In base sequence analysis of gene DNAs and gene examination of simultaneous multiple items with high reliability, DNAs having objective base sequences must be identified using a plurality of probes. DNA microchips have lately attracted attention as means for providing the plurality of probes which are used in the identification. Furthermore, high-throughput screening of pharmaceuticals and combinatorial chemistry requires methodical screening in which many objective proteins and drugs (for example, 96, 384, or 1,536) are arranged. Technologies developed for these purposes include methods for arranging many drugs, automatic screening processes, dedicated apparatuses, and software for controlling a series of screening operations and for statistically processing the results.
In a parallel screening operation, a probe array is basically used to detect the interactions and reactions on the probes under the same conditions in which the probe array includes many known probes which select substances to be evaluated. In general, the interactions and reactions on the probes which are used are preliminarily determined; hence, probes loaded in one probe array belong to the same type or category in a large classification, for example, a group of DNA probes having different base sequences. That is, substances used in a group of probes are, for example, DNAs, proteins, and synthetic chemical substances (drugs). In many cases, the probe array used includes plural probe groups. However, some screening operations allow the use of an array of probes including DNAs having the same base sequence, proteins having the same amino acid sequence, and the same chemical substance, the array having many points of these constituents. These arrays are mainly used in drug screening and the like.
In a probe array including different groups of probes, each group generally includes a group of DNAs having different base sequences, a group of proteins having different amino acid sequences, and a group of different chemical substances, and these constituents are arranged into an array according to a predetermined order on a substrate. In particular, a DNA probe array is used in base sequence analysis of gene DNAs and highly reliable gene examination of simultaneous multiple items.
One of the requirements for a probe array including different groups of probes is to arrange as many types of probe as possible, for example, DNA probes having many types of base sequence on one substrate, in other words, to arrange these probes as densely as possible.
U.S. Pat. No. 5,424,186 discloses a method for fixing plural types of probe into an array on a substrate. The array of DNA probes having different base sequences is prepared on the substrate by a successive extension reaction of DNAs on a solid-phase substrate using a photodegradable protective group and a photolithographic process. According to this method, the resulting DNA probe array can contain 10,000 or more types of DNA having different sequences per 1 cm2. In this DNA synthesis by the successive extension reaction, the photolithographic process is repeated for each of the four bases (A, T, C, and G) using dedicated photomasks to selectively extend these bases at predetermined positions in the array. Plural types of DNA having required base sequences are thereby synthesized on the substrate in a predetermined array. Thus, the production costs and time required increase with the length of the DNA chains. Furthermore, the synthetic yield of nucleotide synthesis in each extension step is not 100%; hence, the proportion of DNAs having base sequence defects is large. In addition, the use of the photodegradable protective group results in a lower synthetic yield rate compared with the use of a general acidolyzable protective group; hence, the proportion of DNAs having designed base sequences will not be necessarily high in the final array product.
Since the product is directly synthesized in the solid-phase substrate, DNAs having base sequence defects cannot be removed from the DNAs having designed base sequences. Moreover, the base sequences of the synthetic DNAs in the final array on the substrate cannot be identified. This is the most significant and essential problem in this method. If a defective product without extension of a predetermined base in some extension steps is yielded due to an erroneous procedure, screening using this product will produce erroneous results. However, there is no way to prevent such results.
Another method is proposed in which DNAs for probes are preliminarily synthesized, purified, and subjected to confirmation of the lengths of the bases, if necessary, and each DNA is applied onto a substrate by a device such as a microdispenser to make a probe array. PCT publication WO95/3350 discloses a method for supplying DNAs on a membrane by a capillary. This method principally enables the formation of an array of about 1,000 DNAs per square centimeter. In this method for making the probe array, a solution of each probe is applied at predetermined positions on the substrate by one capillary dispenser and this procedure is repeated. If a dedicated capillary is prepared for each probe, no problems will arise. However, if a small number of capillaries are used to repeat the same procedure, the capillaries must be thoroughly washed before changing the type of probe in order to prevent contamination. Moreover, positions to be applied must be controlled for every procedure. Accordingly, this method is not necessarily suitable for making an array containing many types of probe and having high density. Since the probe solution is applied to the substrate by tapping the tip of the capillary thereon, reproducibility and reliability are not perfect.
For 96-well microplates and 384-well microplates used in high-throughput screening of drugs, a microdispencer device for supplying different drug solutions to individual wells is commercially available from, for example, Robbins Scientific (Trade name: HYDRA). In this device, microsyringes are two-dimensionally arranged and the minimum discharge volume thereof is 100 nl. When this device is applied to the formation of an array, the array density is limited by the minimum discharge volume, inhibiting the formation of a higher density array.
In other known methods, a solution of a substance required for solid-phase synthesis of a DNA is applied onto a substrate by an inkjet process for each extension step. For example, European Examined Patent Publication No. EP 0 703 825B1 discloses a method of solid-phase synthesis of plural types of DNA having predetermined base sequences. In this method, nucleotide monomers and activators, which are used in solid-phase synthesis of DNAs, are supplied through individual piezo-jet nozzles. The application using this inkjet process is highly reliable due to high reproducibility in the discharge volume compared with the application of the solution using the above capillary and is suitable for making higher-density probe arrays. Since this method also involves successive extension reactions of DNAs on the substrate, the method has the same problems described in U.S. Pat. No. 5,424,186 above: for example, the base sequences of DNAs synthesized on the substrate cannot be confirmed. This method is free from problems inherent in photolithography using a dedicated mask for every extension step, but has a problem in that predetermined probes may be fixed to individual points. In addition, this patent only discloses a method using a small number of piezo-jet nozzles which are separately provided. Thus, this method using these nozzles is not necessarily suitable for making high-density probe arrays, like the above method using the capillary.
U.S. Pat. No. 5,658,802 describes that "the invention provides apparatus and methods for making arrays of functionalized binding sites on a support surface. The invention further provides apparatus and methods for sequencing oligonucleotides and for identifying the amino acid sequence of peptides that bond to biologically active macromolecules, by specifically binding biologically active macromolecules to arrays of peptides or peptide mimetics."
U.S. Pat. No. 5,847,105 described that "a method and apparatus are provided for preparing a substrate upon which is located microdrop-sized loci at which chemical compounds are synthesized or diagnostic tests are conducted. The loci are formed by applying microdrops from a dispenser from which a microdrop is pulse fed onto the surface of the substrate."
U.S. Pat. No. 5,658,802 describes that "arrays of electro-mechanical dispensers are used to form extremely small drops of fluid and locate them precisely on substrate surfaces in miniature arrays. The printed arrays may consist of DNA, immunoassay reagents or the like. A positioning support such as an X-Y table moves the dispensing devices and substrate surfaces relative to each other to locate the drops on the substrates in predetermined patterns. Arrays of probes as dense as one thousand per square centimeter with center-to-center spacing as small as twenty-five micrometers are formed".
Japanese Unexamined Patent No. 11-187900 discloses a method for forming spots containing probes on a solid phase in which a probe solution is applied onto the solid phase through a thermal inkjet head. A known inkjet head for general printers is used as a device for discharging the solution in this method. However, the structure of this head is not always suitable for making probe arrays.
Inkjet heads for printing are developed for printing characters and images. Thus, monochrome (generally black) printing requires one color (black) ink, and color printing generally requires three primary color inks of yellow (Y), cyan (C) and magenta (M). Although some color printing processes use concentrated and diluted inks of black or Y, M, and C, if necessary, the number of types of ink is at most ten.
A conventional head for inkjet printing, which discharges large volumes of inks onto paper, is provided with containers (reservoirs) for storing sufficient ink, nozzles for discharging the inks, and ink channels for conducting the inks to the nozzles.
In contrast, it is preferred that the liquid discharging device used in the production of probe arrays discharge as many types of solution as possible. In a liquid discharging device having plural nozzles, it is preferred that the number of reservoirs provided be equal to the number of these nozzles.
Since large volumes of solutions are not consumed in the production of probe arrays by the liquid discharging to device compared with printing on paper by general purpose inkjet heads, the volume of the reservoirs of the liquid discharging device may be relatively small.
In general purpose inkjet printing heads, a predetermined ink must be discharged onto a predetermined position of the paper to form characters and images. Thus, these heads have a structure which allows each nozzle to be independently selected at any one time. This structure results in a complicated head configuration.
In contrast, the liquid discharging device used in the production of probe arrays does not necessarily require a structure for independently selecting each nozzle at any one time.
As described above, in the conventional general purpose inkjet printing heads having a head structure which allows each nozzle to be independently selected at any one time, power transistors and logic circuits, which are necessary for discharging the inks through predetermined nozzles, may be provided outside or inside the head.
Types of jet for discharging the liquid are classified into a thermal jet type for discharging the liquid by thermal energy from a heater and a piezo-jet type for discharging the liquid by deformation of a piezoelectric element by a voltage applied thereto. The thermal jet type has a simple structure compared with the piezo-jet type and is suitable for miniaturization of heads and formation of multinozzle heads. Thus, the thermal jet type is suitable for production of probe arrays using a liquid discharging device having many nozzles.
Printing heads for the current thermal jet type are provided with many nozzles, for example, 128 or 256 for each color for the purpose of improving the printing rate. Since the number of signals for determining ink discharge from the nozzles increases with an increase in the number of nozzles, the number of contact points between the head and the exterior increases. In order to overcome this problem, the head is provided with power transistors for driving heaters, a shift register for transmitting data including characters and images to be printed, a decoder, and an AND circuit, a NAND circuit, and the like to reduce the number of contact points.
The head provided with the power transistors and logic circuits is generally composed of silicon, with devices such as MOS transistors and bipolar transistors being formed therein
As described above, semiconductor devices are generally formed in the printing head in a conventional method. This method requires a complicated manufacturing process which causes increased costs and a decreased head yield. The decrease in yield is particularly noticeable as the number of nozzles increases, that is, as the number of circuits in the head increases.
As described above, a liquid discharging device for making probe arrays requires many nozzles, for example, 1,000 or more nozzles. If the configuration of a conventional inkjet head is applied without modification to this liquid discharging device, an increase in cost and a decrease in yield are inevitable.
In the case of manufacturing a probe array including an arrangement of different solutions which is formed by discharging different solutions through plural nozzles, the probe array can be formed by one-shot discharge through each nozzle in most cases, and the positions for discharging the solutions can be preliminarily determined. Thus, a complicated discharge control is not always necessary in the production of the probe array by this method, unlike printing on paper. Accordingly, an inexpensive liquid discharging device having a simplified structure and a high yield is desired.

Method for making probe support and apparatus used for the method