DESCRIPTION PROBE SOLUTION, PROBE CARRIER, AND METHOD OF MANUFACTURING PROBE CARRIER TECHNICAL FIELD The present invention relates to a method of manufacturing a probe carrier having a probe capable of specifically binding to a target substance, a method of determining the positions of spots on a probe carrier and detecting a probe array and a target substance, a method of specifying a nucleotide sequence of a single-stranded nucleic acid in a sample and a method of determining the quantity of a target substance in a sample.

BACKGROUND ART For rapidly and accurately determining the nucleotide sequence of a nucleic acid, detecting a target nucleic acid in a sample, or identifying bacterial species, for example, a method has been proposed which employs a probe, a substance specifically binds to a target nucleic acid having a specific nucleotide sequence, to form a probe array having a plurality of types of probes arranged in the form of an array on a solid support. According to this method, the specific binding of a test sample to

a plurality of types of probes can be evaluated using this probe array substrate. The probe array, also called probe carrier. The probe array comprise a substrate, such as a glass substrate, a plastic substrate or a membrane, and spots of probes which are formed of several tens to thousands of types of DNA fragments, arranged and immobilized densely in a form of array.

Recently, studies have been vigorously made on use of such a probe array to detect and quantify a target substance. For example, the specification of U. S. Patent No. 5, 405, 783 describes a method of photolithographically preparing a probe array on a solid carrier by use of a DNA successive extension reaction. WO 95/35505 pamphlet describes a method of preparing a probe array by supplying DNA onto a membrane from capillaries. The specification of European Patent No. 0703825 describes a method of preparing a probe array by synthesizing a plurality of types of DNA on a solid phase by supplying DNA through a piezo jet nozzle. Japanese Patent Application Laid-Open No. H11-187900 describes a method of preparing a probe array by supplying a liquid containing a probe through an ink jet head to apply liquid drops to a solid phase.

In any one of these methods, it is important to suppress and reduce variation in size and shape

between spots, to arrange spots at the same intervals, and keep the spots clean while preventing contamination with dust and other.

Control of the size, shape and position of each spot (so as to be arranged at predetermined positions) is important to prepare a probe array with probes arranged more densely. Development of a method of preparing such a probe array with good yield has been desired. After manufacturing a probe array, it is also very important to check whether the size, shape and position of each spot are maintained within predetermined ranges. This check, in order to put a probe array on the market, must be performed from a quality-guarantee point of view.

For example, one method for preparing a probe array is described in Example 1 of Japanese Patent Application Laid-Open No. H11-187900, where a DNA probe array is manufactured by washing a glass substrate with ultrasonic waves and an alkali, treating the surface of the glass substrate with a coupling agent containing a silane compound having an amino group, and then with N- (maleimidecaproyloxy) succinimide, and spotting a transparent liquid containing a single-stranded DNA probe having a nucleotide sequence of 5'ACTGGCCGTCGTTTTACA3'on the glass substrate by an inkjet method.

After the probe array was prepared, whether the size, shape and position of spots are within predetermined range of values are checked and evaluated by taking image of each of the spots of the probe array by means of an optical microscope or a laser microscope and analyzing the image by using image analysis software.

However, the method described in Japanese Patent Application Laid-Open No. Hll-187900 has a problem. A good-contrast image is not acquired at times since transparent liquid is spotted on a transparent glass substrate. In addition, long spotting time of a liquid may result in vaporization of the liquid. In such a case, it is often difficult to evaluate the size, shape and position of the spots.

As described above, when the images of spots cannot be analyzed and the positions of the spots are not determined by drying of the probe solution during or after the probe array preparation step, it is not easy to confirm whether the size, shape and position of spots were maintained within predetermined values.

Japanese Patent Application Laid-Open No. 2002- 69349 describes color materials having a copper phthalocyanine structure, such as C. I. AcidBlue 249, C. I. Direct Blue 86, C. I. Direct Blue 199, and C. I.

Direct Blue 307 as a color material for ink for inkjet printing. Other than these, there are

water-soluble xanthene dyes, monoazo dyes, diazo dyes, triazo dyes, and tetraazo dyes.

DISCLOSURE OF THE INVENTION The present invention has been made to solve the aforementioned problems and directed to facilitating acquisition of spot images with good contrast by forming spots of a probe solution containing an indicator (such as a dye, pigment, polymer, inorganic particles, inorganic-compound particles, inorganic complex and inorganic-organic complex), thereby attaining evaluation of the size, shape and position of the spots by image analysis.

Furthermore, since the size, shape and position of spots can be easily observed even after a probe solution is dried, quality control can be made.

Moreover, evaluation time can be reduced, so that the yields of production and evaluation test of a probe carrier can be improved.

To attain the aforementioned objects, the present inventors prepared a probe carrier having probes capable of specifically binding to a target substance, such as a probe array, adding a substance such as a dye, pigment, polymer, inorganic particles, inorganic-compound particles, inorganic complex and inorganic-organic complex to a probe solution to be used in manufacturing a probe array, and spotting the

probe solution onto a probe carrier. In this way, visualization of spot-image and observation of the size, shape and position of spots even after the probe solution was dried become possible.

The present invention relates to a method of preparing a probe carrier having a plurality of independent spots thereon by spotting a plurality of probe solutions onto predetermined positions on the carrier, where each probe solution contains a probe capable of specifically binding to a target substance in a liquid solvent, and each of the probe solutions further contains an indicator optically detectable when contained in a spot.

Further in the method of the present invention, spotting may be performed by an inkjet method.

Alternatively, spotting may be performed by a pin method. The probe may be a nucleic acid.

In the present invention, it is preferable that the indicator be at least one selected from the group consisting of dyes, pigments, polymer substances, inorganic particles, inorganic compound particles, inorganic complexes and inorganic-organic complexes.

The present invention also relates to a method of obtaining at least one piece of information selected from size, shape and position of each of a plurality of spots on a probe carrier, the probe carrier having the spots each of which contains a

probe capable of specifically binding to a target substance and which are arranged independently on the probe carrier, the method comprising the steps of: introducing a detectable indicator into the spots ; optically detecting the indicator by a detection unit; and obtaining a piece of information based on the optical data obtained by the detection unit.

The present invention also relates to a method of identifying a probe contained in each of a plurality of spots on a probe carrier, the probe carrier having the spots each of which contains a probe capable of specifically binding to a target substance and which are arranged independently on the probe carrier, the method comprising the steps of: introducing into the spots, an optically detectable indicator for allowing the identification of the probe contained in each of the spots; optically detecting the indicator contained in each of the spots by a detection unit; and identifying the probe contained in each of the spots based on optical information obtained by the detection unit.

The present invention also relates to an apparatus for obtaining information concerning at least one item selected from the group consisting of

size, shape and position of a predetermined spot on a probe carrier, the probe carrier having the spots each of which contains a probe capable of specifically binding to a target substance and which are arranged independently on the probe carrier, the device comprising: a support unit for supporting the probe carrier having spots each of which contains an optically detectable indicator; and a unit for optically detecting the indicator contained in the spots to obtain information concerning the spots.

The present invention also relates to an apparatus device for identifying a probe contained in a predetermined spot on a probe carrier, the probe carrier having a plurality of spots each of which contains a probe capable of specifically binding to a target substance and which are arranged independently on the probe carrier, the device comprising: a support unit for supporting the probe carrier having spots each of which contains a probe and an optically detectable indicator for allowing the identification of the probe; and a unit for optically detecting the indicator contained in each of the spots and for obtaining information for identifying the probe.

The present invention also relates to a probe solution comprising a liquid medium, a probe capable of specifically binding to a target substance, and at least one optically detectable indicator selected from the group consisting of dyes, pigments, polymers, inorganic particles, inorganic compound particles, inorganic complexes and inorganic-organic complexes, and a liquid solvent.

The present invention also relates to a probe carrier that comprises a carrier and a plurality of spots formed independently on the carrier, characterized in that each of the spots contains a probe capable of specifically binding to a target substance and at least one optically detectable indicator selected from the group consisting of dyes, pigments, polymers, inorganic particles, inorganic compound particles, inorganic complexes and inorganic-organic complexes.

According to the present invention, by spotting a probe solution containing an indicator (dyes, pigments, polymer substances, inorganic particles, inorganic compound particles, inorganic complexes and inorganic-organic complexes), it is possible to acquire images of spots with good contrast and, therefore, possible to evaluate the sizes, shapes, and positions of spots by image analysis.

Furthermore, the size, shape and position of

each spot can be easily observed even after the spotted probe solution was dried. Therefore, the guarantee of quality is facilitated.

Moreover, time for evaluating the aforementioned items can be reduced. Therefore, it is possible to produce a probe carrier in a more stable yield.

Other features and advantages of the present invention will be apparent from the following description taken in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the figures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

FIG. 1 is a flow chart showing a process for spot evaluation and probe identification ; and FIG. 2 is a view showing a structure of a system for spot evaluation and probe identification where a denotes optical detection system, b denotes a carrier having spots thereon, and c denotes a stage for arranging carriers having spots thereon.

BEST MODE FOR CARRYING OUR THE INVENTION Preferred embodiments of the present invention will now be described in detail in accordance with the accompanying drawings.

An indicator to be contained in a probe solution is a substance that can be optically detected when it is spotted on a carrier together with a probe. To obtain quantitative information about the spots or to quantify probe amounts, the indicator is preferably nonvolatile and that allows quantitative optical detection. The term "nonvolatile"used herein refers to a property: never vaporizing and drying together with the solvent of a probe solution when the probe solution is spotted and dried on a carrier to form a probe carrier.

More specifically, an indicator is preferably not gasified or sublimed during the drying step in the probe carrier production. The temperature of the drying step is preferably 150°C or less, more preferably 110°C or less in consideration of maintaining the properties of a probe.

Examples of an indicator include dyes, pigments, polymer substances, inorganic particles, inorganic- compound particles, inorganic complexes and inorganic-organic complexes. A probe-array prepared by using a probe solution containing an indicator is subjected to a reaction called hybridization reaction

with a fluorescent-labeled target substance, and then fluorescence is observed or fluorescence intensity is measured. Therefore, the hybridization reaction or detection of fluorescence should not be interfered by the indicator. Thus, the indicator should be contained in such a concentration that facilitates visual and microscopic observation of the spots formed on a substrate and facilitates image acquisition of spots with a good contrast, but not inhibiting the binding reaction between a target substance and a probe or the detection of the fluorescence.

Examples of dye indicators include color materials having a copper phthalocyanine structure such as C. I. Acid Blue 249, C. I. Direct Blue 86, C.

I. Direct Blue 199, and C. I. Direct Blue 307 as described in JP-A-2002-69349 ; and also water-soluble dyes such as xanthene dyes, monoazo dyes, diazo dyes, triazo dyes, and tetraazo dyes color materials. A concentration of a dye in a probe solution is preferably from 0.005 to 5 % by mass and more preferably from 0.005 to 0.5% by mass.

Examples of pigment indicators include carbon black such as No. 2300, No. 900, MCF88, No. 40, No.

52, MA7, MA8, No. 2200B (the products mentioned supra are manufactured by Mitsubishi Kasei), RAVEN1255 (manufactured by Columbia), REGAL 400R, REGAL 660R,

MOGULL (the products mentioned supra are manufactured by Cabot), Color Black FW1, Color Black FW18, Color Black S170, Color Black 150, Printex 35, Printex U (the products mentioned supra are made by Degussa), C.

I. Pigment Yellow 2, C. I. Pigment Yellow 3, C. I.

Pigment Yellow 13, C. I. Pigment Yellow 16, and C. I.

Pigment Yellow 83.

The concentration of a pigment indicator in a probe solution is preferably from 0.01 to 10% by mass, and more preferably, from 0.01 to 1% by mass.

Examples of polymer indicators include polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), paogen, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), dextran, and pluran. Of them, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) are preferable since they are of general use and stable with probes.

The concentration of a polymer indicator in a probe solution is preferably from 0.001% to 0. 5% by mass and more preferably from 0.01 to 0. 2% by mass.

A polymer substance is preferably dissolved or stably dispersed in the solvent of the probe solution because it is mixed in a probe solution and used to make spots. A polymer substance is not particularly limited, but it is a water-soluble polymer when water is used as the solvent of a probe solution, or a polymer stably soluble in an organic solvent when the

organic solvent is used as the solvent of the probe solution. In a case of a water-soluble polymer, the polymer preferably has a polymerization degree as low as about 500 to 600 in consideration of solubility in water. Furthermore, the polymer preferably has a hydrolysis degree of 87 to 89. When an organic solvent is used for the probe solution, the type of polymer may vary depending upon the type of organic solvent. Examples of a polymer stably dispersed in a probe solution include polymer beads and a polymer micelle. In consideration of dispersion stability, spot-formability and detectability, a small-sized polymer substance is preferably used. For example, polymer beads having an average particle-diameter of 3 Fm or less are used, and more preferably, polymer beads having an average particle-diameter of 1 Km or less are used. In the case of polymer micelle, an average particle diameter of 10 nm or more is preferable.

To detect a polymer substance, detection by using a metal microscope or an ellipsometer may be mentioned. The analysis method used in practice may vary depending upon the type of polymer substance.

When a sufficient amount of a polymer material is dissolved in a solvent, detection can be made by measuring differential interference, reflection light, and transmission light by using a metal-microscope,

but when a small amount of a polymer material is dissolved in a solvent, microscopic measurement is difficult, and an ellipsometer is preferably used.

The ellipsometer method has difficulty in identification of probes, but it is suitable for determination of the size, shape and position of spots. When polymer beads are used as a polymer substance dispersed in a probe solution, detection can be properly made by a metal microscope. To be more specific, specific data on the size, shape and position of spots can be obtained based on the measurement of differential interference, reflection light and transmission light. Furthermore, when a polymer substance is a polymeric micelle, detection is properly made by a metal microscope. More specifically, specific data on the size, shape and position of spots can be obtained by using a transmission light having a wavelength of 200 to 400 nm in measurement depending upon the state of a polymeric micelle after drying.

Further, various substances such as inorganic particles, inorganic compound particles, inorganic complexes and inorganic-organic complexes can be used for the indicator. When a probe solution is applied by an ink jet system, the particles of an indicator are desirably small enough to be ejected and have no adverse effect upon ejection.

The concentration of an indicator such as inorganic particles, inorganic compound particles, an inorganic complex and an inorganic-organic complex in the probe solution is preferably from 0.01 to 5 % by mass and more preferably 0. 1 to 2 % by mass.

As an example of inorganic particles, metal particles may be mentioned. Examples of metal include Au, Ag, Cu, Ni, Co, Cr, Al, Ta, Pt, Pd, Zn and Sn. Examples of inorganic compound particles include metal oxide particles such as TiO2, A1203 and ZrO.

Examples of inorganic complexes and inorganic- organic complexes include metal complexes and organic metal complexes. Examples of metals used in such metal complexes include Au, Ag, Cu, Ni, Co, Cr, Al, Ta, Pt, Pd, Zn and Sn.

An average particle diameter of inorganic compound particles preferably falls within the range of 1 nm to 100 nm in consideration of the stability of a probe solution and spots. Inorganic particles, inorganic compound particles, inorganic complexes, and inorganic-organic complexes are appropriately detected by a metal microscope. In the case of inorganic complexes and inorganic-organic complexes, specific absorption or light reflection property differs depending upon types of complexes. Therefore, specific data on the size, shape and position of

spots can be obtained by measuring reflection light and transmission light. In the case of inorganic particles and inorganic compound particles, the size, shape and position of spots can be identified by the determination of reflection light scattered by particles. In this manner, the sizes, shapes, and positions of spots can be determined and probes can be identified.

The ratio of an indicator to a probe contained in a probe solution is preferably 2000: 1 to 20 : 1.

When a pin method is used, the limitation of inorganic particles, inorganic-compound particles, inorganic complex and inorganic-organic complex is not strict, since they are not ejected. When an ink- jet method is used, although various inorganic particles, inorganic-compound particles, inorganic complexes and inorganic-organic complexes may be used, they must have small sizes enough to be ejected and good ejection properties.

The indicators mentioned above may be used singly or in combination thereof in a probe solution.

The indicator according to the present invention is not necessary to be adsorbed or immobilized on a substrate surface. When a hybridization reaction is performed with a target substance, the indicator may not be present on the substrate.

When an indicator is present on a substrate during hybridization, it is more preferable that the indicator is contained in the range in which hybridization would not be affected. Alternatively, a step of removing an indicator by washing may be provided before the hybridization reaction.

As a probe, it is preferable to use at least one selected from the group consisting of DNA, RNA, cDNA, PNA, oligonucleotides, polynucleotides, other nucleic acids, oligopeptides, polypeptides, proteins, enzymes, enzyme substrates, antibodies, epitopes, antigens, hormones, hormonal receptors, ligands, ligand receptors, oligosaccharides, and polysaccharides.

A probe solution is prepared, for example, as follows. First, an aqueous solution containing 7.5% by mass of glycerin, 7. 5% by mass of urea, 7. 5% by mass of thiodiglycol, and 1% by mass of acetylene alcohol (trade name: acetylenol EH, manufactured by Kawaken Fine Chemicals Co., Ltd.) is prepared, and separately a TE solution (10 mM Tirs-HCl (pH8)/1 mM an aqueous EDTA solution) containing HS-(CH2) 6-O-PO2- 0-ssDNA in an amount of about 400 mg/ml is prepared.

Then the aqueous solution is added to the TE solution to a final concentration of a single-stranded DNA of 8 J. M. The surface tension of this probe solution falls within the range of 30 to 50 dyne/cm and its

viscosity is 1.8 cps (measured by an E type viscometer, manufactured Tokyo Keiki, Inc.) Examples of a method of forming spots of such a probe solution on a probe carrier include a pin method and an inkjet method; however may not limited to these and any method may be used as long as a method makes it possible to form spots of a probe solution on a probe carrier. In the case of an inkjet method, both a piezo system liquid ejection system and a so-called bubble jet system may be used.

In the bubble jet system, heat energy is used as ejection energy, and a solution is ejected from an ejection port by the pressure of a bubble generated in the solution.

The diameter of a spot can be represented by a diameter of a circle that has an equivalent area to that occupied by the spot. The diameter of a spot may be from 1 to 500 Rm and more preferably from 1 to 200 Fm.

An indicator contained in a probe solution can be visually detected or optically detected by using a detection unit such as a camera, optical microscope, and laser microscope as detection data. The detection data was subjected to image analysis, by which spots can be evaluated (for size, shape, and position) and the probe contained in a spot (which probe is contained which spot is determined) can be

identified. Preferably, different types of indicators are used in different amounts between spots. In this manner, identification of the spots and identification of the probes can be easily made.

Since the indicator does not vaporize even if the probe solution is dried, determination of spots and identification of probes can be performed either immediately after spotting or after drying of the spots.

The present invention will be explained in more detail below.

FIG. 1 is a simple flowchart showing procedures including preparing a probe carrier, evaluating spots (for size, shape and position) formed on a substrate, and identifying probes in spots (determining which probe is contained in which spot).

FIG. 2 shows an example of a structure of a system for performing the steps (4) and (5) of the flowchart shown in FIG. 1.

The drawings will be explained below.

In FIG. 1, a carrier is provided in step (1), a probe solution containing an indicator is prepared in step (2), and the probe solution is spotted on a carrier surface in step (3). The carrier having spots formed thereon is subjected to detection by an optical detection system in step (4) to acquire information about spots (as images etc. ). The image

data was subjected to image analysis. in step (5). In this way, the size, shape and position of spots are evaluated and the probe contained in each spot is identified. The term"size of a spot"means the area of a spot formed on a carrier surface.

In FIG. 2, a plurality of carriers having spots formed thereon are arranged on a stage (c). Images of spots are acquired by an optical detection system (a). A drive control section (d) controls the optical detection system (a) and the stage (c) can be driven independently. The spot images thus acquired are accumulatedly stored in an image acquisition section (e). The acquired image is analyzed by an image analysis section (f) with respect to spot area, spot shape (circulality) and spot position. The analytical results are stored in a spot data section, (g). The contrast ratios and color extraction results of spot images analyzed are also stored as the results.

When different indicators are added to different probe solutions and the images of the resultant spots are analyzed with respect to contrast ratio and color extraction, the obtained analysis data are stored (registered) in a probe identification section h, in advance, as reference values. The types of probes contained in spots can be identified by comparing the results stored in the

section (g) with the reference values.

Now, the present invention will be explained in more detail below. Examples are not limited to those illustrated herein.

Examples Example 1 Detection of target substance by probe array prepared using probe solution containing blue dye (1) A surface-treated glass substrate and a single-stranded DNA having a nucleotide sequence of 5'ACTGGCCGTCGTTTTACA3' (SEQ ID NO : 1) were prepared as follows.

(1-1) Washing of Substrate A glass plate of 1 inch x 1 inch was placed in a rack and immersed in an ultrasonic washing detergent overnight. After ultrasonic washing in the detergent for 20 minutes, the detergent was removed by rinsing with water. After rinsing with distilled water, ultrasonic treatment was performed in a container containing distilled water for 20 minutes.

The glass plate was immersed for 10 minutes in a 1 N sodium hydroxide solution preheated to 80°C. Then, the plate was washed with water and distilled water to prepare a glass plate for a probe array.

(1-2) Surface Treatment

An 1 wt% aqueous solution of a silane coupling agent (Product name: KBM603; Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an amino group (N-ß-(aminoethyl)-Y- aminopropyltrimethoxysilane) was stirred at room temperature for 2 hours to hydrolyze methoxy groups of the above silane compound. Then, the substrate was immersed in this solution at room temperature (25°C) for 20 minutes, drawn up from the solution, and dried by blowing nitrogen gas to both sides of the glass plate. Then, the glass plate was baked for 1 hour in an oven heated to 120°C to complete silane coupling treatment to introduce an amino group on the surface of the substrate. Then, 2.7 mg of N- (6- maleimidocaproyloxy) succinimide (Dojin Co. , Ltd.) (abbreviated as EMCS hereinafter) was weighed and dissolved in a mixture of DMSO/ethanol (1: 1) to a final concentration of 0.3 mg/ml to prepare an EMCS solution. The glass plate subjected to silane coupling treatment was immersed in the EMCS solution at room temperature for 2 hours for the reaction of the amino groups carried on the surface of the glass plate by silane coupling treatment and the carboxyl groups of the EMCS solution. In this condition, the glass plate obtained maleimido groups derived from EMCS on its surface. The glass plate drawn up from

the EMCS solution was washed successively with a mixed solvent of dimethylsulfoxide and ethanol and with ethanol and then dried under a nitrogen gas atmosphere.

(1-3) Synthesis of DNA Probe A single-stranded (ss) nucleic acid of SEQ ID NO: 1 was synthesized using an automatic DNA synthesizer. A thiol (-SH) group was introduced at the terminus of the ss DNA of SEQ ID NO : 1 using Thiol-Modifier (Glen Research Co., Ltd.) during synthesis by the automatic DNA synthesizer.

Following ordinary deprotection, DNA was recovered, purified with high performance liquid chromatography, and used in the following experiments.

SEQ ID No. 1 'HS- (CHz) 6-0-P02-0-ACTGGCCGTCGTTTTACA ' (2) Four types of solutions were prepared by adding cyan ink (trade name: BCI-5C, manufactured by Canon) to an aqueous solution to concentrations of 0. 01%, 0.1%, 1% and 10%, respectively. The aqueous solution contained 7. 5wt% glycerol, 7. 5% urea, 7. 5% thiodiglycol, and lwt% acetylene glycol (ACETYLENOL, Kawaken). To each of these 4 types of solutions, a single-stranded DNA of the step (1) was dissolved to a final concentration of 8 FM, to make probe solutions.

(3) Each of the probe solutions prepared in the step (2) was loaded in a bubble-jet head of BTJ850 (trade name, manufactured by Canon Inc. ) so as to eject the solution. Sixteen matrixes were prepared each having 144 spots (12 rows x 12 columns) arranged at intervals of about 130 pm on the glass substrate prepared in the step (1). Of the 16 matrixes, each solution was used to form 3 matrixes. The remaining 4 matrixes were formed as a control with a probe solution containing no cyan ink (0%).

(4) After spots were formed, an image was taken by using a CCD color camera serving as an optical data acquisition unit for obtaining data on spots.

As a result, with the matrixes formed with the above four probe solutions, good images were successfully obtained with better contrast than the control matrixes, enabling evaluation of the size, shape and position of spots based on image analysis.

The sizes of spots were evaluated based on a value of a spot area obtained by using an image analysis software as follows. Average of the spot areas of matrixes formed with one of the probe solutions was determined for respective solutions and then variation (35 value) was determined. A value of variation/average was calculated for matrixes formed with the same probe solution. The results are shown in Table 1. The evaluation standard is 0.25 or less.

Table 1 Evaluation results of spot size (variation/average) Concentration oo 0. 10 10 10% of cyan ink Variation/0. 18 0.17 0.18 0.19 0.17 average

In Table 1, the denotation"0%"means that no cyan ink is contained. From Table 1, results with all solutions satisfied the standard. The formation of good spots was confirmed and evaluation was easy.

The shapes of spots were evaluated based on a radius ratio obtained by the image analysis software, as follows. Average of the radius ratios of spots formed with a probe solutions and variation (3a value) were determined for each solution. A value of variation/average was calculated for matrixes formed with the same probe solution. The results are shown in Table 2. The evaluation standard was 0.25 or less.

Table 2 Evaluation results of spot shape (variation/average) Concentration -of cyan ink average average In Table 2, the denotation"0%"means that no cyan ink is contained. From Table 2, results with all solutions satisfied the standard. Formation of

good spots was confirmed and evaluation was easy.

The position of a spot was evaluated based on the X-Y coordinate of a gravity-point, which was obtained by the image analysis software, as follows.

The X-Y coordinate of the gravity point of each spot was determined. Then, the coordinate-conversion was performed by making a correction of 0 based on the least-squares method. The converted coordination was defined as (XN, YN). After the coordinate conversion is performed, the X-Y coordinate (Xg, Yg) of a gravity point with respect to each matrix is obtained. Based on this, an ideal lattice coordinate (Xr, Yr) was obtained. If the difference between the real coordinate (XN, YN) and the desirable lattice coordinate (Xr, Yr) was obtained, deviation of the position of a spot from the ideal lattice coordinate can be obtained. Deviation data of 144 spots were obtained from a single matrix. Each of the deviation amounts of spots was expressed by a combination of the X direction deviation and the Y-direction deviation for each solution, and an average of deviation amounts of all spots formed with one solution was obtained. The evaluation results are shown in Table 4 with reference symbols defined in Table 3. The evaluation standard was less than 13.0 Fm.

Table 3 Relationship between deviation amount and symbol Symbol @ O A X Deviation 0-4. 9 5. 0-9. 9 10. 0-12. 9 13. 0 or amount [m] more

Table 4 Evaluation results of spot position Concentration of 0% 0. 01% 0. 1% 1% 10% cyan ink Deviation amount in the X direction [pm] Deviation amount in the Y direction [pm] In Table 3, the denotation"0%"means that no cyan ink is contained. From Table 3, the results with all aqueous solutions satisfied the standard of evaluation. The formation of spots in good positions was confirmed and evaluation was performed easily.

Furthermore, after a probe solution was dried by drying a substrate having spots formed thereon, observation was made by a CCD color camera. As a result, the positions of spots were easily confirmed.

As is the same as in Table 3, all aqueous solutions satisfied the standard.

(5) The following experiment was performed to check the effect. of cyan ink on hybridization reaction. Using each solution, probe carriers having probe spots thereon were prepared as above, and three

of them were stored in an 1M NaCl/50 mM phosphate buffer solution (pH 7.0) and three of them were dried.

Then the samples were soaked in a 2% aqueous bovine serum albumin solution, allowed to stand for 2 hours, and subjected to a blocking reaction. Thereafter, a hybridization reaction was performed in an aqueous solution containing the target substance: a single- stranded DNA (5'TGTAAAACGACGGCCAGT3') complementary to that of the step (1) (SEQ ID NO: 2), at 45°C for 2 hours. The resultant samples were scanned for fluorescence by a DNA microarray scanner (trade name: GenePix 4000B, manufactured by Axon Instruments.

Inc. ). The results of the probe carriers stored in the buffer solution are shown in Table 5 and the results of the dried probe carriers are shown in Table 6. Average values and 36 values shown in the tables were normalized with the value of the control formed with a probe solution containing no cyan ink (0%).. 36 value/average values were obtained by calculation using actual measurement values.

Table 5 Concentration of cyan ink in probe solution and fluorescent intensity of probe carrier (stored in Buffer solution) Cyan ink concentration 0. 01% 0. 1% 1% 10% (mass %) Average value 1.00 1.15 0. 96 0. 17 0. 00 36 value 1 0.83 0.54 0.06 0. 02 36 value/average value 0. 32 0. 23 0. 18 0.11 2. 16

In Table 5, the denotation"0%"means that no cyan ink is contained. From Table 5, the binding reaction between a target substance and a probe capable of bind specifically to the target substance was not substantially inhibited when the concentration of cyan ink in a probe solution was 0.01 to 0. 1%. Therefore, it was confirmed that sufficient fluorescent intensity was observed at the fluorescent detection time. At the concentration of 1%, it was found that the binding reaction between the target substance and a probe capable of specifically binding the target substance was more or less inhibited, however, fluorescent intensity was much higher than that of the background.

Table 6 Concentration of cyan ink in probe solution and fluorescent intensity of dried probe carrier Cyan ink concentration 0% 0. 01% 0. 1% 1% 10% - (mass %) Average value 1. 00 1.16 G. 86 0.07 0.00 3o value 1.00 0.94 0.60 0.04 0. 00. ja value/average value 0. 29 0. 23 0. 20 0. 14 0. 30 In Table 6, the denotation"0%"means that no cyan ink is contained. From Table 6, the binding reaction between a target substance and a probe capable of binding specifically to the target

substance was not substantially inhibited when the concentration of cyan ink in a probe solution was 0. 01 to 0. 1%. Therefore, it was confirmed that sufficient fluorescent intensity was observed at the fluorescent detection time. At the concentration of 1%, the binding reaction between the target substance and a probe capable of specifically binding the target substance was more or less inhibited; however, fluorescent intensity was much higher than that of the background.

From the foregoing, it was clear that spot images can be easily obtained with good contrast by forming spots of the probe solution. As a result, the size, shape and position of spots were successfully evaluated by image analysis. After the probe solution was dried, the positions of spots formed on a substrate were successfully and easily observed. Further, hybridization and fluorescence detection are not hindered by the presence of a coloring material in a certain range.

Example 2 Detection of target substance by probe array prepared by using probe solution containing black dye (1) A surface-treated glass substrate and a single-stranded DNA having a nucleotide sequence of 5'ACTGGCCGTCGTTTTACA3' (SEQ ID NO : 1) were prepared in the same manner as in Example 1.

(2) Four types of solutions were prepared by adding black ink (trade name: BCI-5BK, manufactured by Canon Inc. ) to an aqueous solution to concentrations of 0. 01%, 0. 1%, 1% and 10%, respectively. The aqueous solution contained 7. 5wt% glycerol, 7.5% urea, 7. 5% thiodiglycol, and lwtG acetylene glycol (ACETYLENOL, Kawaken). To each of these 4 types of solutions, a single-stranded DNA of the step (1) was dissolved to a final concentration of 8 M, to make probe solutions.

(3) Each of the probe solutions prepared in the step (2) was loaded in a bubble-jet head of BTJ850 (trade name, manufactured by Canon Inc. ) so as to eject the solution. Sixteen matrixes were prepared each having 144 spots (12 rows x 12 columns) arranged at an interval of about 130 m on the glass substrate prepared in the step (1).

(4) After spots were formed, an image was taken by using a CCD color camera serving as an optical data acquisition unit for obtaining data on spots.

As a result, in the four cases, good images were successfully obtained with better contrast than conventional ones, attaining evaluation of the size, shape and position of spots based on image analysis.

Furthermore, after a probe solution was dried by drying a substrate having spots formed thereon, observation was made by a CCD color camera. As a

result, the positions of spots were easily confirmed < and the positions were successfully evaluated. Not only immediately after the spots were formed but also after the probe solution of the spots were dried, the size, shape and position of spots of all the solutions satisfied the standard, as is the same as in Example 1. Therefore, evaluation was successfully performed.

(5) Probe carriers stored in a buffer solution and those dried were prepared in the same manner as in Example 1. Then the samples were soaked in a 2% aqueous bovine serum albumin solution, allowed to stand for 2 hours, and subjected to a blocking reaction. Thereafter, a hybridization reaction was performed in an aqueous solution containing the target substance: a single-stranded DNA (5'TGTAAAACGACGGCCAGT3') (SEQ ID NO : 2), complementary to SEQ ID NO : 1, at 45°C for 2 hours.

The resultant samples were scanned for fluorescence by a DNA microarray scanner (trade name: GenePix 4000B, manufactured by Axon Instruments. Inc. ). The results of the probe carriers stored in the buffer solution are shown in Table 7 and the results of the dried probe carriers are shown in Table 8. Average values and 36 values shown in the tables were normalized with the value of the control formed with a probe solution containing no black ink (0%). 3s

value/average values were obtained by calculation using actual measurement values.

Table 7 Concentration of black ink in probe solution and fluorescent intensity of probe carrier stored in buffer solution Black ink 0% 0. 01% 0. 1% 1% 10% concentration (mass %) Average value 1.00 0.99 0. 92 0. 67 0.01 3a value 1. 00 0. 87 0. 72 0.41 0.06 3s value/average value 0.24 0.21 0.19 0.14 1.30 In Table 7, the denotation"0%"means that no black ink is contained. From Table 7, the binding reaction between a target substance and a probe capable of binding specifically to the target substance was not substantially inhibited within the concentration of black ink in a probe solution of 0.01 to 1%. Therefore, it was confirmed that sufficient fluorescent intensity is observed at the fluorescent detection time. At the concentration of 1%, it was found that the binding reaction between the target substance and the specific probe was not much inhibited and fluorescent intensity was much higher than that of the background.

Table 8 Concentration of black ink in probe solution and fluorescent intensity of dried probe carrier Black ink 0% 0. 01% 0. 1% 1% 10% concentration (mass %) Average value 1. 00 0.91 0.60 0.06 0.00 3o value 1.00 0.97 0. 64 0. 05 0.00 3o value/average value 0.20 0. 21 0. 22 0. 17 0. 07

In Table 8, the denotation"0%"means that no black ink is contained. From Table 8, the binding reaction between a target substance and a probe capable of binding specifically to the target substance was not substantially inhibited within the concentration of black ink in a probe solution of 0.01 to 0. 1%. Therefore, it was confirmed that sufficient fluorescent intensity is observed at the fluorescent detection time. At the concentration of 1%, the binding reaction between the target substance and a probe capable of specifically binding the target substance was more or less inhibited; however, fluorescent intensity was much higher than that of the background.

From the foregoing, it was clear that spot images can be easily obtained with good contrast by forming spots of the probe solution. As a result, the size, shape and position of spots were successfully evaluated by image analysis. After the probe solution was dried, the positions of spots

formed on a substrate were successfully and easily observed. Further, hybridization and fluorescence detection are not hindered by the presence of a coloring material in a certain range.

In Examples mentioned above, the same indicator was used in all probe solutions; however the indicators used in probe solutions may be different from each other. In this case, if a plurality of indicators different in absorption wavelength are used, probe solutions can be determined based on color difference The present invention is not limited to the above embodiments and various changes and modifications can be made within the spirit and scope of the present invention. Therefore to apprise the public of the scope of the present invention, the following claims are made.

This application claims priority from Japanese Patent Application No. 2003-324648 filed September 17, 2003, which is hereby incorporated by reference herein.