CN1781020A

CN1781020A - Biochip and biochip kit, and method of producing the same and method of using the same

Info

Publication number: CN1781020A
Application number: CN 200480011176
Authority: CN
Inventors: 奥村胜弥; 三原诚; 吉冈睦彦
Original assignee: JSR Corp; Octec Inc
Current assignee: JSR Corp; Octec Inc
Priority date: 2003-04-25
Filing date: 2004-04-23
Publication date: 2006-05-31

Abstract

The invention provides a biological chip, a biological chip reagent case, and the production process and the use process, target substance in analyte and probe reaction in high efficiency for short time, quantitative determination which can be conducted in high B/F separating efficiency and high sensitiveness, a process which uses the biological chip reagent case, the reaction between the target substance in analyte and the probe, and separating classifying process and determination and identification process of the target substance in the analyte and the like. The biological chip of the invention has a well whose bottom is provided with a filter, wherein the filter comprises straight pores with equal aperture which are arranged in equal pore gap. Dispersion which is dispersed with probe carrier particles is held in the well, and analyte is plunged into the well to react with the probe carrier particles. Solvent such as analyte solvent and the like can be guided into the well through the filter, or be exhausted from the well.

Description

Biochip and biochip kit, and manufacture method and using method

Technical field

The present invention relates to a kind of biochip, have well, the filter that is made of the straight pore with equal equal aperture is set in its bottom; And the biochip kit that constitutes by this biochip and container; And the target material in the interactional analyte substance of probe carrier particulate and reaction between the probe or interaction method; B/F separates the method for target material from analyte; The method of the target material being separated classification from analyte; And the reaction of target material in the detected material and bioprobe, interactional detection authentication method.

Background technology

Double-stranded DNA, though for example by the heating as single stranded DNA the time, also because its structure be complementary, so have the character that mutually combines, utilize this character to establish the Northern hybrid method.Therefore, will have the dna fragmentationization of suitable length particular sequence with restriction enzyme etc. in the past, and maybe synthetic oligonucleotide be used as probe, retrieval has and the dna fragmentation of its complementary series and oligonucleotide etc., should retrieve as standard method.

But even when handling small amounts of analyte, also there is the problem that can not handle at short notice in Northern hybrid method complicated operation.Therefore, developed the DNA chip, as the convenient disposal method of using the Northern hybrid method, fixed above-mentioned oligonucleotide etc. to high-density on substrates such as microslide, the method that can analyze with the short time now, just is widely used.

As the representative example of this DNA chip, fixing oligonucleotide on planar substrates such as silicon.This chip is, on substrate, fix after 1 base, make this base and the combination respectively of other bases, can generate and be elongated to length with 20～30 bases, under rayed or having under the condition that acid exists, make the oligonucleotide of base sequence directly synthetic on chip, thereby make the DNA chip with regulation.For example open the DNA chip of having put down in writing the synthetic oligonucleotide of on substrates such as silicon In Situ etc. in the clear 63-27000 communique the spy.

Perhaps, the nucleic acid such as oligonucleotide that can make cDNA or pre-synthetic suitable length on substrate in conjunction with and make, in the method, the nucleic acid of preparing to separate is separated from particulate carrier, it is carried out point sample with sample applicator at assigned position, that is: use Blang (Brown) method.

But in former approach, owing to synthetic alkali basic sequence on chip, if in a part of base sequence resultant fault takes place, then causing its entire chip is inferior goods.Its result, the yield rate of chip becomes y ^4npHere, y is a positive compound probability in base synthesis procedure, and n is the base number, and p is a number of probes.Therefore, when number of probes increased, yield rate descended by geometric series.Here, standby probe is set, and the probe that replaces bad probe is set, so substrate area becomes big.

In addition, in latter's method, the precision of sample applicator etc. is depended in the point sample amount of DNA and point sample position, is difficult to obtain good repeatability.

And, in these microarraies or DNA chip, may carry the probes such as nucleic acid of tens thousand of projects of a relevant analyte, also the sundry item inspection can be arranged in the time of tens thousand of project, probe is fixed on the substrate, the reaction of analyte and probe becomes the reaction of analyte in fixing probe and the liquid, is called as solid-liquid reaction that is:.Usually, the reaction efficiency of solid-liquid reaction is poor, and promptly the hybridization reaction with analyte needs a few hours.

And, in order to detect nucleic acid accurately with detecting target base sequence, need be in narrow space high-level efficiency fixedly have nucleic acid (hereinafter referred to as " probe ") with this nucleic acid complementary series.The quantity that can be fixed on the above-mentioned probe on this type of substrate determines according to the occupied area of each probe and the size of DNA chip self, surpass physically some above fixedly be impossible.Therefore, as the DNA chip that is using now, in making its this class chip that immerses analyte and reaction because there is the considerably less situation of amount of the analyte that may use, so chip from as little better.

But, if reduce the size dimension of chip, then because area that can stationary probe also diminishes thereupon, and narrow space and cause detection signal to die down, thereby detection sensitivity descends.

Be improvement the problems referred to above points, recently, make and sell the chip (for example, rolling up p.69-78 (calendar year 2001)) that on 3D glue, is fixed with above-mentioned probe with reference to Analyticai Clinica Acta the 444th.In said chip, probe is fixed on the 3D glue by solid, because probe density is very high, the intensity of detection signal also uprises, but noise signal strength also uprises thereupon, so S/N is difficult to significantly improve accuracy of detection than not improving.Here, as the factor that noise signal strength rises, think to be difficult to remove the product that the non-specific responding of chip by being fixed with above-mentioned probe on 3D glue and analyte forms.

In addition, also developed a kind of chip: after tying up hollow line, on above-mentioned hollow line inwall, make the probe combination, form the three dimensional stress probe, make probe density rising (opening the 2000-181074 communique) thus with reference to the spy.

And, in recent years also developed a kind of biochip: can use porous matter aluminum oxide substrate with perpendicular magnetic anisotropy pore, stationary probe on the inwall of this upright opening, improve probe density with three-dimensional structure, and utilize release (the Blow down) that make the hole vertically become sky to construct, after making analyte and probe reaction, clean and remove non-target material (with reference to the flat 9-504864 communique of special table (the international communique that discloses No. 01/12846)).

Like this in above-mentioned any one method that the pore inwall of inwall that makes probe and hollow line or porous matter aluminium oxide combines, for probe stationary on fixation wall, need be when analyte and probe reaction, analyte is near to or in contact with the probe that has been fixed.But the size of this probe is compared extremely small with the reaction compartment volume, observe with the rule of reaction compartment, probe only with from fixation wall a little projection come out, analyte is extremely low near to or in contact with the probability of probe.Therefore, in said method, need long stirring can produce reaction.

And on the inwall of three-dimensional structure during stationary probe, if be provided with the degree of depth in hole very dark, it is big that the surface tension of hole inwall becomes, and makes the input of detection bodies and cleaning fluid etc. discharge the difficulty that becomes like this.

As mentioned above, also practicable a kind of method: replace to the fixation wall stationary probe, allow particulate carry probe, in the solution that contains this probe carrier particulate, the target material in probe carrier particulate and the analyte is reacted.In this method, the probe carrier particulate in solution when three dimensions disperses because probe carrier particulate and analyte can move mutually, have reactive very high characteristics.For example, known detection authentication method: make the target goods and materials in the analyte and the probe specific reaction on emulsion particle surface, separate, resolve special efficacy ground and the content of the target material after probe reaction combines by B/F (Bound form/Free form).

P.5037-5048 in (1986), put down in writing a kind of method at ucleic Acid Research the 14th volume: make after the nucleic acid probe of the target material of detection bodies and carrying particulate hybridizes in solution, unreacted analyte is removed in centrifuging.This method is widely used now.But, when using centrifuging like this, need disengaging time, and separating property is not so high problem.

Open in clear 63-27000 communique, No. 2975603 communique of special permission the Jap.P. spy, put down in writing a kind of method: the particulate as the carrying probe uses magnetic particle, fixes this magnetic particle by magnet, and cleans and remove unreacted analyte.This method also is widely used now.But magnetic particle generally needs size in order to bring into play magnetic characteristic be more than 1 micron, owing to contain ferrite (ferrite) the metal magnetic composition of etc.ing and cause proportion very big, easy sedimentation and cohesion, also should be noted that keeping quality and operability.

Open in the clear 63-27000 communique the spy, put down in writing a kind of method: make after the nucleic acid probe of the target nucleic acid of analyte and carrying particulate hybridize in solution, filter cleaning, measure the signal of the reaction that is concentrated on the comfortable filter paper with filter paper.This method also is widely used.But in the method, the amount of the emulsion particle that uses as particulate is very many, not only need filtration time, and filter paper stops up easily.

And any one method of putting down in writing in the above-mentioned document, use the probe carrier particulate all can not carry out for the analyte of a kind checking (Mutiplex Assay) simultaneously or multinomially separating classification simultaneously by the multinomial order of a plurality of probes.

As using the probe carrier particulate to carry out the method that multinomial order is checked simultaneously, added the identification marking particulate that is used to discern a plurality of probes, proposed to use the method for this particulate.For example at United States Patent (USP) the 5th; 736; No. 330 communique, spy open in clear 62-81566 communique, the special fair 7-54324 communique; put down in writing a kind of method: use a plurality of particulates or the different a plurality of particulates of mean particle dia through fluorescent color; particulate carries different probes respectively; with the special kind of setting probe such as color and particle diameter, with the reaction of flow cytometer (Flow cytometer) check and analysis thing and probe carrier particulate.This method also is widely used now.

But, in the method, filter probe carrier particulate with analyte response with 96 orifice plates of subsidiary filter, or with methods such as centrifugings through the B/F after separating, inject flow cytometer and detect.At this moment, analyte was once processed with centrifugal separating tube with the plate or the centrifuging of subsidiary filter, afterwards in order to hang in the flow cytometer, and when increasing operation, might by plate, pipe produce pollute and analyte adhere to the impairment that causes.

And in the method with color identification, the kind of the color that is used to discern is limited, utilize in certain wavelength coverage and the combination of light intensity can be carried out wide about 100 kinds of beam split, therefore, about 100 kinds color and combination of strength, that is: the limit of checking simultaneously being arranged is 100 kinds of probes.

Other are opened in the flat 11-243997 communique the spy, have put down in writing the method by particle size and shape recognition particulate thereof.And, open in the 2000-346842 communique the spy, put down in writing a kind of method: particulate with one dimension or two-dimensional arrangements, in order to distinguish kind different probe particulate, is used the slight grain that varies in size as separating the next door.

In above-mentioned each document, put down in writing, the identification particulate carries out detecting by flow cytometer in order to discern particulate in any one method that multinomial order checks simultaneously.But, by in the method for flow cytometer, particulate is flowed in the tubule at this, owing to transparent part irradiating laser, discern particulate continuously to this tubule, there is the cost problem of detection time.And, in order to shorten detection time, and do not spend time of a particulate of identification, might reduce accuracy of detection.And the tubule of flow cytometer costs an arm and a leg, and the expense that the tubule of each analyte is replaced in exchange also is a problem.Therefore, it is necessary cleaning the tubule of replacing each analyte, needs the time in order to clean more fully.

In biochip, can be the DNA chip that is fixed with dna fragmentation or oligonucleotide as its probe; Other are fixed with protein such as antigen and antibody or in addition with the protein-chip of these protein specific reactions or interactional chemical substance etc., and the problems referred to above exist in these protein-chips etc. equally.

These DNA chips and protein-chip be by being fixed on the various probes on the substrate, can be applied to the functional analysis of the gene that biology has; In order to confirm the status of patient of diseases such as various infection diseases, and the clinical examination of carrying out; The many types of parsing of gene; Be called as (Tailor-made) treatment of suiting the medicine to the illness, selected according to the treatment medicine of patient's gene order; The chemical substance or the screening of albumen or the toxicity screening of chemical substance that are used for the pharmaceuticals exploitation; Other various screenings etc.

But in order to be applied to above-mentioned various uses, detection sensitivity is insufficient in the biochip that is now using.For example, in order to find disease at incunabulum, be necessary that incunabulum in disease is in the stage, that detection is moved to the extracellular from cell, the mark of disease origin of trace extremely, but the mark that detects this denier with the detection sensitivity that has biochip now is not enough, needs more highly sensitive biochip.

And, in order to obtain the correct information of diseased state that relevant patient takes a disease, make Error Diagnostics reduce to minimum, be inadequate with single probe, use a plurality of different probes to carry out multinomial order and check simultaneously.Therefore, expectation can be carried out the biochip that multinomial order is checked simultaneously with high detection sensitivity.

Summary of the invention

The present invention researches and develops in order to solve above-mentioned prior art problems, its objective is:

Providing a kind of can target material and probe in chien shih analyte in short-term react expeditiously; B/F separation efficiency height; Detect the biochip and the manufacture method thereof of evaluation with high sensitivity.

In addition, the purpose of this invention is to provide a kind of biochip kit, between a plurality of biochips that form by well (well) or between a plurality of biochips that constitute by well and a plurality of container that constitutes by well, can directly between well, carry out receiving of liquid.

And, the purpose of this invention is to provide following method:

Use this biochip, from an analyte, separate the classification separation stage division of more than one target material at least;

The separation stage division of the above target material of separating at least one concurrently simultaneously from most analytes;

The test method of the above target material of separating at least one from an analyte;

The test method of the above target material of separating at least one concurrently simultaneously from most analytes.

(i) feature of biochip of the present invention is, has the well that the bottom is provided with filter, and described filter comprises the straight pore with equal equal aperture that forms by impartial span.

(ii) the feature of biochip of the present invention is, in the biochip described in (i), the thickness of above-mentioned filter is 1～10 μ m.

(iii) the feature of biochip of the present invention is, at (i) or in the biochip (ii), the open area ratio of above-mentioned filter is 15～60%.

(iv) the feature of biochip of the present invention is, in (i)～(iii) any described biochip, the Facing material of above-mentioned filter is silicon dioxide, titania or aluminium oxide.

(v) the feature of biochip of the present invention is, in (i)～(iv) any described biochip, has a plurality of above-mentioned well that is integral with each other.

(vi) the feature of biochip of the present invention is, in (i)～(iv) any described biochip, has single above-mentioned well.

(vii) the feature of biochip of the present invention is, in that (i)～(in vi) any described biochip, the upside of the filter in above-mentioned well or downside are provided with the reinforcement flank.

(viii) the feature of biochip of the present invention is, in that (in the biochip vii), above-mentioned reinforcement is a shape that is formed with a plurality of through holes with flank.

(ix) feature of biochip of the present invention is, (vii) or (in the biochip viii), above-mentioned reinforcement directly combines with above-mentioned filter with flank.

(x) feature of biochip of the present invention is, (vii) or (in the biochip viii), above-mentioned reinforcement flank extends to form with identical materials continuously with above-mentioned filter.

(xi) feature of biochip of the present invention is, in (i)～(x) any described biochip, in the bottom of above-mentioned well, in its circumferential edges Rack, the atresia portion that does not form the filter pore is set.

(xii) feature of biochip of the present invention is, in (i)～(xi) any described biochip, when the bottom is provided with first filter, in the mode that well is clipped in the middle, in the opposite side of first filter second filter is set.

(xiii) feature of biochip of the present invention is, in (i)～(xii) any described biochip, accommodates in above-mentioned well and has the dispersion liquid that has disperseed the probe carrier particulate.

(xiv) feature of biochip of the present invention is, the ratio in the particle diameter of above-mentioned particulate and the aperture of filter is particle diameter/aperture=1.1～2.5, and particle diameter＜span＜particle diameter * 10.

(xv) feature of biochip of the present invention is, the particle diameter of above-mentioned particulate, the aperture of filter and span satisfy the relation of particle diameter＞aperture+span/2.

(xvi) feature of biochip of the present invention is, in the biochip described in (xiii), accommodate in above-mentioned well and have the dispersion liquid that has disperseed the probe carrier particulate, this probe carrier particulate has at least a means of identification that is used to provide the probe identifying information.

(xvii) feature of biochip of the present invention is, in the biochip described in (xvi), above-mentioned means of identification is that selection is at least a from the color of probe carrier particulate, shape, particle diameter and gene order.

(xviii) feature of biochip of the present invention is, at (xvi) or in the biochip (xvii), the a plurality of probe carrier particulates identical at the probe identifying information of whole above-mentioned means of identification are housed in the same well, and identical for the above-mentioned probe identifying information that is accommodated in a plurality of probe carrier particulates in each well.

(xix) feature of biochip of the present invention is, at (xvi) or in the biochip (xvii), the a plurality of probe carrier particulates identical at the probe identifying information of whole above-mentioned means of identification are housed in the same well, and for the above-mentioned probe identifying information difference that is accommodated in a plurality of probe carrier particulates in each well.

(xx) feature of biochip of the present invention is, at (xvi) or in the biochip (xvii), the a plurality of probe carrier particulates different at the probe identifying information of at least a above-mentioned means of identification are housed in the same well, and for a plurality of probe carrier particulates that are accommodated in each well, the formation of the above-mentioned probe identifying information in its whole means of identification is identical.

(xxi) feature of biochip of the present invention is, at (xvi) or in the biochip (xvii), the a plurality of probe carrier particulates different at the probe identifying information of at least a above-mentioned means of identification are housed in the same well, and for a plurality of probe carrier particulates that are incorporated in each well, the formation difference of the above-mentioned probe identifying information in its whole means of identification.

(xxii) feature of biochip kit of the present invention is, has container, and this container or accommodate a plurality of wells that are integral with each other or single well in (i)～(xxi) any described biochip perhaps is connected with above-mentioned well.

(xxiii) feature of biochip kit of the present invention is, in the biochip kit described in (xxii), said vesse and above-mentioned well form as one.

(xxiv) feature of biochip kit of the present invention is, in the biochip kit described in (xxii), said vesse and above-mentioned well form independently of each other.

(xxv) feature of biochip kit of the present invention is, in (xxii)～(xxiv) any described biochip kit, said vesse has and the corresponding well of the well of above-mentioned biochip.

(xxvi) feature of biochip kit of the present invention is, in the biochip kit described in (xxv), at the bottom formation through hole of said vesse.

(xxvii) feature of biochip kit of the present invention is, at (xxv) or in the biochip kit (xxvi), above-mentioned biochip is connected with said vesse, so that corresponding well interconnects.

(xxviii) feature of biochip kit of the present invention is, in (xxii)～(xxvii) any described biochip kit, said vesse is to use the plate that is formed with through hole and does not form at least a in the plate of through hole, forms by stacked a plurality of above-mentioned plates.

(xxix) feature of biochip kit of the present invention is, (i)～(xxi) any described a plurality of biochips is connected each other, so that corresponding well interconnects.

(xxx) feature of biochip kit of the present invention is, in (xxii)～(xxix) any described biochip kit, the par that is arranged on the well sidepiece upper end of the par of well sidepiece lower end of above-mentioned biochip and said vesse that is arranged on separation or above-mentioned biochip directly is connected, and well is interconnected.

(xxxi) feature of biochip kit of the present invention is, in (xxii)～(xxix) any described biochip kit, well sidepiece lower end at above-mentioned biochip is provided with, with the recess of the location usefulness of the set protuberance tabling in the well sidepiece of said vesse that separates or above-mentioned biochip upper end; Or with the location protuberance of the set recess tabling in the well sidepiece of said vesse that separates or above-mentioned biochip upper end.

(xxxii) feature of the manufacture method of biochip of the present invention is, when making (i)～(xxi) any described biochip, for form with the material of different component at least by the two-layer plate that constitutes, carry out etching by etched pattern, be carved into two layers border and form wellhole from a lateral erosion, carry out etching by the etching pattern simultaneously, etch into two layers border and form the filter hole, obtain the biochip of well and filter combination thus from opposite side.

(xxxiii) feature of the manufacture method of biochip of the present invention is, when making (i)～(xxi) any described biochip, makes filter, rib and well respectively by the etching silicon wafer, and is then that it is stacked.

(xxxiv) feature of the method for operating of biochip kit of the present invention is, in the solution in (xxii)～(xxxi) that the well that is housed in container and biochip forms independently in this container of any described biochip kit, by the aboveground of biochip moved down, the solution in this container is contacted with probe carrier particulate and/or solution in the well.

(xxxv) feature of the method for operating of biochip kit of the present invention is, the interface that is housed in the solution in the container of any described biochip kit in (xxii)～(xxxi) by making moves up and down, and the solution in this container is contacted with probe carrier particulate and/or solution in the well.

(xxxvi) feature of the method for operating of biochip kit of the present invention is, any described biochip kit in (xxii)～(xxxi) is had be used between the container that connects biochip and the chip or produce pressure reduction each other at the well of this chip, produce contact, liquid the moving between well of interior liquid of well and probe carrier particulate thus, or the two produces simultaneously.

(xxxvii) feature of the method for operating of biochip kit of the present invention is, any described biochip kit in (xxii)～(xxxi) is had be used between the container that connects biochip and the chip or the well of this chip produces pressure reduction each other, produce contact, liquid the moving between well of interior liquid of well and probe carrier particulate thus, or the two produces simultaneously.

(xxxviii) feature of the method for operating of biochip kit of the present invention is, in (xxxiv)～(xxxvii) any described method, by the solution in the said vesse is contacted with probe carrier particulate and/or solution in the well, the content in the biochip is mixed, spreads, reacts, separates or cleans.

(xxxix) feature of the method for operating of biochip kit of the present invention is, in (xxxiv)～(xxxviii) any described method, drops into identical analyte in each well of above-mentioned biochip.

(xl) feature of the method for operating of biochip kit of the present invention is, in (xxxiv)～(xxxviii) any described method, drops into the analyte that differs from one another in each well of above-mentioned biochip.

(xli) feature of the reaction method of target material in the analyte of the present invention and probe is may further comprise the steps:

Specific particulate is placed in the well of biochip in (xxii)～(xxxi) any described kit, forms (xviii)～(xxii) any described chip;

In the well of above-mentioned biochip, drop into the solution that contains analyte, make the above-mentioned particulate in this analyte and all wells form the possibility state of contact; And

In the above-mentioned solution that the biochip container is accommodated, make and abovegroundly move down or the interface that is housed in the above-mentioned solution in the biochip container is moved up and down, perhaps make solution circulation inside and outside the well, thereby make target material and probe reaction in the analyte by applying pressure reduction.

(xlii) method that B/F separates the target material from analyte of the present invention is characterized in that, may further comprise the steps:

Specific particle is placed in the well of biochip of (xxii)～(xxxi) any described kit, forms (xviii)～(xxii) any described chip;

In the well of above-mentioned biochip, drop into the solution that contains analyte, make the above-mentioned particulate in this analyte and all wells form the possibility state of contact;

Make the interfacial level of above-mentioned solution be reduced to the lower surface of the filter that is lower than bottom, thereby the analyte of the probe reaction that will be not be carried with above-mentioned particulate is removed in each well; And

In the well of biochip, drop into cleaning fluid, by said vesse cleaning fluid is circulated in the well of this biochip, cleaning fluid is discharged in input inside and outside the well of this biochip, and discharges cleaning fluid from well, thereby cleans the material beyond the target material of removing the probe combination.

(xliii) the analyte of the present invention separation stage division of material that hits is characterized in that, may further comprise the steps:

Specific particle is placed on (xxx) or (xxxi) described in kit in the well of biochip in, form (xviii)～(xxi) any described chip;

Make the interfacial level of above-mentioned solution be reduced to the lower surface of the filter that is lower than bottom, the analyte of the probe reaction that do not carried with above-mentioned particulate is removed in each well;

In the well of biochip, drop into cleaning fluid, by said vesse cleaning fluid is circulated in the well of this biochip, cleaning fluid is discharged in input inside and outside the well of this biochip, and discharges cleaning fluid from well, thereby cleans the material beyond the target material of removing the probe combination; And

Make the recess, protuberance or the partes glabra that are provided with in the well sidepiece lower end of above-mentioned biochip chimeric with corresponding protuberance, recess or the partes glabra that has in container upper end with it, in the well of biochip, drop into separating agent solution then, the target material in the separate analytes from above-mentioned particulate thus, and move in the well of said vesse.

(xliv) the interactional detection authentication method of target material in the analyte of the present invention and probe is characterized in that, may further comprise the steps:

Specific particle is placed in the well of the biochip in (xxii)～(xxxi) any described kit, forms (xviii)～(xxi) any described chip;

In the well of biochip, drop into cleaning fluid, by said vesse cleaning fluid is circulated in the well of this biochip, cleaning fluid is discharged in input inside and outside the well of this biochip, and discharges cleaning fluid from well, thereby cleans the material beyond the target material of removing the probe combination;

Particulate in the well is positioned in the pore of filter; And

Reaction or interaction between the target material of detection evaluation probe that above-mentioned particulate carried and analyte.

(xlv) the interactional detection authentication method of target material in the analyte of the present invention and probe, it is characterized in that, in (xliv) described method, for each detection of particulates two aspect information, be the probe identifying information of particulate, and reaction between the target material of probe that above-mentioned particulate carried and analyte or interactional information.

(xlvi) the interactional detection authentication method of target material in the analyte of the present invention and probe, it is characterized in that, in (xliv) described method, for the particulate in each well, discern the probe identifying information that is carried on the above-mentioned particulate, the interaction of the target material in relevant probe of instrumentation and the analyte then, with the interactional status information based on each particulate, calculating with the well is the interaction information of unit.

According to the present invention,

Providing a kind of can target material and probe in chien shih analyte in short-term react expeditiously;

B/F separation efficiency height;

Can carry out the biochip and the manufacture method thereof of highly sensitive detection by quantitative.

And, according to the present invention, provide a kind of biochip kit, between a plurality of biochips that constitute by well or between a plurality of biochips that constitute by well and a plurality of container that constitutes by well, can directly between well, carry out receiving of liquid.

And, according to the present invention, provide and use this biochip,

The separation stage division of the target material from an analyte more than the separating at least one;

The separation stage division of the target material more than the separating at least one side by side from most analytes;

The test method of separating at least one and more than one target material side by side from most analytes.

Description of drawings

The sectional view of bottom one side of the well that Fig. 1 has for the biochip in one embodiment of the present of invention;

Fig. 2 is the mode sectional drawing of the pore example of filter;

Fig. 3 is provided with the mode sectional drawing of reinforcement with bottom one side of the well of flank;

Fig. 4 is provided with the state vertical view of reinforcement with flank for the filter surface;

Fig. 5 is provided with the electron micrograph of reinforcement with the bottom of flank for the filter surface;

Fig. 6 is provided with the mode sectional drawing of reinforcement with the bottom of flank;

Fig. 7 is provided with the mode sectional drawing of reinforcement with the bottom of flank;

Fig. 8 is for being provided with the bottom mode sectional drawing of positioning combination with the well of recess in reinforcement with flank;

Fig. 9 is for being provided with the bottom mode sectional drawing of positioning combination with the well of recess in reinforcement with flank;

Figure 10 is the upper base surface at well, begins to be provided with Rack the bottom mode sectional drawing of the well of atresia portion from its circumferential edges;

Figure 11 is for making the key diagram of the method for rib or well with the light modeling;

Figure 12 is the upper surface figure and the A-A ' sectional view of the biochip in the embodiments of the invention;

Figure 13 is the upper surface figure and the A-A ' sectional view of biochip in the embodiments of the invention;

Figure 14 is for having the sectional view of the biochip of first filter and second filter simultaneously;

Figure 15 is the sectional view that the manufacture method of the biochip kit that has connected biochip and container is described;

Figure 16 is on the plate of the bottom of the container that forms biochip kit or cap, has used the example sectional view of level and smooth transparent plate as optical detection;

Figure 17 forms the example sectional view of the biochip kit of container for using plate;

Figure 18 is the example sectional view with the biochip kit of chip multistage setting;

Figure 19 is a key diagram of making the method for the chip that is made of a plurality of wells;

Figure 20 is the key diagram of the method for operating of the biochip that the present invention relates to;

Figure 21 is the chip profile figure of an operation of the separation stage division of the target material in the analyte that the present invention relates to of explanation and detection method;

Figure 22 is the chip profile figure of an operation of the separation stage division of the target material in the analyte that the present invention relates to of explanation and detection method;

Figure 23 is the chip profile figure of an operation of the separation stage division of the target material in the analyte that the present invention relates to of explanation;

Figure 24 is provided with the sectional view of the biochip kit of trickle through hole for the shaft bottom face of container;

Figure 25 is the chip profile figure of an operation of the detection method of the target material in the analyte that the present invention relates to of explanation;

Figure 26 is for taking in the example key diagram of the biochip of the present invention of probe carrier particulate in the well;

Figure 27 is the method key diagram that drops into analyte in the well of biochip of the present invention;

Figure 28 is for being positioned the probe carrier particulate electron micrograph on the filter hole;

Figure 29 is the electron micrograph of being taken pictures in the bottom of each well of chip;

Figure 30 flows in the filter of chip of embodiment 3 the cow's serum dilution, when the lower cover of container is discharged the cow's serum dilution down, and the chart of the summation of expression discharge rate and the relation of efflux time;

Figure 31 is illustrated in eight kinds of combinations different with span of the aperture of chip, with 18gf/cm ²Pressure reduction carry out the chart of the test findings identical with embodiment 3.

Embodiment

Below, with reference to accompanying drawing the present invention is carried out specific description.Fig. 1 is the bottom sectional view of the well that biochip has in the embodiments of the invention.

As shown in the figure, this well 16 is provided with the filter 18 that is formed with pore 19 in its bottom.The formation of this filter 18 is, when the liquid such as medium that make analyte or dissolving or be dispersed with analyte pass through, in well 16, accommodate, do not discharge with the interactional probe carrier particulate of analyte to the outside.

In the present invention, on filter 18, be formed with straight pore by impartial span with equal equal aperture.And " impartial aperture " described in this instructions its meaning is that aperture error is smaller or equal to 20% of CV (Coefficient of Variation) value, preferably smaller or equal to 10%.Aperture error can be made according to the aftermentioned method smaller or equal to 20% pore of CV value, can make the difference in size in the probe carrier particulate that is housed in the well 16 and aperture very small.

There is the residual quantity about 10 times in aperture for the membrane filter that used etc. in the past, and when for example using the filter in 0.2 μ m aperture, in fact the particle diameter that can be filtered is about 5 μ m.By comparison, when using above-mentioned filter with equal equal aperture,, can make the difference in particle diameter and hole very little, therefore can make the aperture bigger, in the filterableness that improves filter, can also reduce filter pressure because the aperture of filter pore is strict impartial.

In the above-mentioned filter with equal equal aperture, the aperture of its pore has no particular limits, and is generally 0.01 μ m～100 μ m, preferred 0.1 μ m～50 μ m, more preferably 0.5 μ m～15 μ m.

And the meaning of so-called " impartial span " is that the error of span is smaller or equal to 15% of CV (Cofficient of Variation) value.Have no particular limits for span, but because span when very narrow, the intensity of filter weakens, and when span is very big, open area ratio descends,, and be preferably 0.5 μ m～10 μ m so common span is 2 times smaller or equal to the aperture.And so-called " span " is the bee-line of the not perforate part between each hole of adjacency.

And in the present invention, the meaning of so-called " directly " pore is that pore does not form branch on the path.For example, the open centre that forms on a side filter surface begins to do vertical line to opposite side filter surface; Begin when vertical line is done on an above-mentioned side filter surface from the lip-deep open centre of this opposite side filter, even two vertical lines depart from, but this point of the pressure loss is so not serious again, therefore can adopt this class pore.And, also can adopt to come down to this two vertical line and do not depart from, and the different pore in aperture of the aperture of filter first side (upper surface side) and second side (lower face side).As above-mentioned pore shape can be the shape shown in (a)～(d) of Fig. 2 for example.There is no particular limitation for the section shape of the pore vertical with connecting direction, can be in cylinder, rectangular pyramid, the polygonal pyramid any one, but from making the minimum viewpoint of meniscus (meniscus), preferred obtuse angle shape or circle.But the volume of well is more than or equal to ormal weight, and during for example more than or equal to 0.1 microlitre, meniscus is just no problem, adopts quadrangular or rectangular pyramid etc. also no problem.

Form the length minimum of the pore of filter with above-mentioned straight pore, thereby, reducing with the contact area of porous wall, it is minimum that the pressure resistance that filtration is produced reaches.

And, even being deposited in first side of filter, the probe carrier particulate causes when stopping up, particulate can not enter the inside of filter and rest on filter surface (forming the so-called model that entirely shuts), brush away (flushing) by second side blow, particulate is disperseed to first side from the filter surface from filter.Therefore, the obstruction that can remove filter once more makes it to recycle, and prolongs the life-span of filter.

As mentioned above, owing on filter, form straight pore with equal equal aperture with impartial span, the probe carrier particulate is arranged on the opening of first side, wash away liquid such as analyte from second side as required, particulate is disperseed to first side, thereby can easily carry out the reaction and the detection of analyte and probe carrier particulate.

And, do not allow to discharge the probe carrier particulate to second side of filter, catch under the situation of particulate at needs 100%, not because of factors such as consideration particle size, so that the aperture of filter is greater than the minimum diameter of particulate, and the problem that transmission coefficient and filtration efficiency are reduced just can be caught 100% particulate.

The common filter paper and the aperture of membrane filter, span are not impartial, and hole self is three-dimensional complex structure, have a hole thinner than filter surface in that filter is inner.Therefore, a part particles contained in the filtrate is caught by the hole of filter inside, the obstruction of inaccessible model in the middle of generation is called as, for removing this obstruction, from second side send into particulate that liquid will be present in filter in the filter when first side-draw goes out, insufficient by the effect of washing away, can not all particulates be taken out from filter in first side, be enclosed in the filter, the particulate that is used in detection reduces.And the particulate capturing efficiency of filter has ratio,, selects the filter of " becoming the maximum diameter that the minimum grain size＞filter of the size distribution of old object directly distributes " for reaching 100% capturing efficiency, but transmission coefficient and filtration efficiency reduce thus.

In the present invention, preferred 1～10 μ m of the thickness of filter, more preferably 2～7 μ m.When the thickness of filter during greater than 10 μ m, it is big that the filtration resistance during filter liquide becomes; When the thickness less than 1 μ m of filter, the physical strength deficiency of filter.

In the present invention, the open area ratio of filter is preferred 15～60%, and more preferably 20～50%.When open area ratio less than 15%, filtration efficiency reduces; When open area ratio greater than 60% the time, the physical strength deficiency of filter.

Filter can be made of the material of the various forms with pore.Can be specifically: by the mould stamping parts, weave cotton cloth, on filter, bore a hole and the filter of formation with laser or neutron ray; Make resin or metallic film filter injured and that form by the tension force reaming; The filter that base material perforation is formed by photoetching; By filter of resin forming etc.

As mentioned above, have the filter of the pore that forms by equal equal aperture, with impartial span, for example can to the organic or inorganic filter painting erosion resistant agent of regulation, make the hole of blank regulation form filter by the photoetching process manufacturing by pattern etching.In order to ensure the aperture of thickness with specify mechanical intensity and regulation, need carry out the etching of high orientation (aspect), can also implement by the anisotropy etching.

And, also can make by the method for expansion alloy.For example, be that the stainless steel foil of 30 μ m forms staggered otch with mould to thickness, its expansion is formed the through hole of rhombus.According to this method, the ultimate range that can obtain diamond hole is that 30 μ m, open area ratio are about 60% filter.Then, the expansion alloy filter that obtains is carried out punching press with convex mold again handle, form the concave surface of regulation, obtain integrally formed a plurality of wells thus.Perhaps, make the gang of wells of up/down perforations such as cellular or ball-shaped in addition with resin or metal, at coating of the bottom surface of gang of wells and dry moldable resin solution, by warmed-up filter of hot tie-in and well, connect above-mentioned expansion alloy filter and gang of wells, thereby form well.

And, as described below, can be by well sidepiece and the integrated method of filter being made with resin.

As the particularly preferred manufacture method of biochip can be: with the plate of the different various material formations of forming, for example for aluminium/aluminium oxide, metal silicon/silicon dioxide or Titanium/titania etc., by carry out pattern etching respectively from both sides, form filter and well.Specifically, for example, by for metal oxide layers such as aluminium oxide, silicon dioxide, titania, be etched to the border of this metal oxide layer and metal level and form filter, then for metal levels such as aluminium, metallic silicon, Titaniums, be etched to the border of this metal level and metal oxide layer, make the biochip that well and filter combine.

According to this method, by using the multilayer material of formation one in advance such as metal and metal oxide, can making filter and well are integrally combined, can not take place from the problems such as interface leakage between filter and the well.

And, owing to can use transparent materials such as aluminium oxide, silicon dioxide, titania, when detecting, can observe directly the motion of particulate by the filter layer by light as the filter layer.

As the material that forms filter,,, specifically, when preferred solution is water system, select hydrophilic material preferably with the high material of solution compatibility that is housed in the well reducing aspect such as filtration resistance; When solution is oiliness, select oil loving material.

Can use as hydrophilic organic material, for example the multipolymer of the various acrylic acid esters such as multipolymer of tygon vinylite, crosslinked polyethylene alcohol resin, polyureas acetate resin, polyamide, poly-imines, cellulose acetate resin, triacetyl cellulose, nitrocellulose resin, epoxy resin, 2-methyl-prop rare acyl hydroxyethyl Phosphorylcholine and methyl-prop diluted acid ester etc.

Can use as oil loving organic material, for example tygon, polypropylene, polystyrene, plexiglass, liquid crystal polymer, polycarbonate, polyamide, poly-imines, polyethylene terephthalate, polyethylene naphthalenedicarboxylate phenolic ester, cycloolefin, polymethylpentene, polyene propyl ester, polysulfones, polyethersulfone etc.

And, can use as inorganic material, for example metals such as iron, nickel, copper, zinc, aluminium, silicon, titanium, tantalum, magnesium, molybdenum, tungsten, rhodium, palladium, silver, gold, platinum, stainless steel, brass, red brass, bronze, phosphor bronze, aluminium copper, almag, Aludur, alumin(i)um zinc alloy, iron-nickel alloy; Metal oxide such as silicon dioxide, aluminium oxide, titania, zirconia, oxidation are smooth; Metal nitrides such as SiN, TiN, TaN; Metal carbide such as SiC, WC; Adamas, graphite, diamond-like coal material with carbon elements such as (Diamond Like Carbon:DLC); Glass such as soda-lime glass, pyrex, Pyrex glass (trade mark), quartz glass etc.

And, also can on the surface of lipophilicity material, implement plasma treatment, corona treatment or ion processing etc., form hydroxyl, carboxyl etc.And, also can form hydrophilic metal or metal oxide from the teeth outwards by electroplating to wait; Perhaps also can plate the hydrophilic material such as multipolymer, polyethyleneglycol derivative of for example 2-methyl-prop rare acyl hydroxyethyl Phosphorylcholine and methyl-prop diluted acid ester; Perhaps also can be coated with after the epihydric alcohol methylpropenoic acid ester, make epoxy ring-opening.

Particularly, the preferred surface material is the material as filter of aluminium oxide, silicon dioxide, titania in above-mentioned.Because their possess hydrophilic properties, the non-specific adsorption of biological analyte is very low, and the analyte of water system and cleaning fluid immerse in the filter easily.And, not only on the surface but also by also use identical material in inside, because filter is transparent, when using optical detecting method, can easily detect reaction, the interaction of identifying microparticle probes and analyte mark by transparent filter as detection method.

When being the material formation filter of aluminium oxide, silicon dioxide, titania, also can use aluminium, metallic silicon or Titanium to form the chip that constitutes by filter and well, carry out oxidation afterwards and form aluminium oxide, silicon dioxide, titania with Facing material; Perhaps can also prepare the plate that constitutes by aluminium/aluminium oxide, metal silicon/silicon dioxide, Titanium/titania, use this plate to form well and filter.

＜well portion 〉

There is no particular limitation for the quantity of a well that biochip had, can set arbitrarily according to the kind of the particulate that has probe that holds or the number of particulate, can use the chip that constitutes by single well or the chip that constitutes by a plurality of wells that form one in any one.And, even the kind of probe carrier particulate is a kind of, can when holding the most particulate that is used for separation and purification etc., a plurality of wells be set, in each well, disperse to hold identical probe particulate.

Shown in the (a) and (b) of Fig. 1, well can be formed by the material different with filter, and shown in Fig. 1 (c), can use also that identical materials is integrally formed in fact with filter.And the material of the material of filter and well is to form with different material in the (a) and (b) of Fig. 1, but also can form respectively after filter and the well with same material, again with they combinations.

Shown in Fig. 1 (b), (c), well does not form the part of filter in the filter layer, be connected with filter, and this is preferably implemented on the good face of physical strength.

There is no particular limitation for the diameter of well portion and height, can set arbitrarily according to the number of required particulate and the volume of reaction.When the diameter of well became big, the area on filter surface also became greatly, may cause that filter is crooked or destroyed because of the general pressure that applies to filter, but by following rib is set, can avoid this bending or destruction.

And in having the chip of a plurality of wells, the diameter of these wells is unnecessary identical, considers the quantity and the reaction volume of the particulate that holds, and the well of different-diameter can be set.

There is no particular limitation for the shape of the wellhole that is made of the well sidepiece, for making minimum preferred obtuse angle of meniscus or circle.

＜reinforcement flank 〉

Above-mentionedly be provided with in the well of filter in the bottom, because the thin thickness of filter, when the aperture of well open area ratio big or filter was big, the undercapacity of bottom waited filter damaged easily or damage in use.Here, in these cases, preferably the intensity servicing unit that the rib shape is set in upper surface one side or lower surface one side of filter replenishes and strengthens bottom.

Fig. 3 is provided with the bottom model lateral sectional view of above-mentioned reinforcement with the well of flank.In (a) of Fig. 3, has the flank 23 of projecting strip part in the lower surface one side setting of filter 18.In (b) of Fig. 3, flank 23 is set in upper surface one side of filter 18.

Fig. 4 is for being provided with the state upper surface synoptic diagram of reinforcement with flank 23 at filter 18 surperficial upsides.As shown in the figure, it is shaped as the integrated that is formed with a plurality of through holes 29, preferably is connected with rib between through hole 29.Not being defined as circle, rectangle, this class polygon of hexagon etc. with the cross section of the perforation direction approximate vertical of through hole 29, is to reduce obtuse angle polygons such as meniscus phenomenon circular or hexagon.In Fig. 5, the actual reinforcement that on filter, is provided with of the expression electron micrograph of flank.

Reinforcement uses the rib height of flank according to the floorage of well and the material of rib, preferred 10 μ m～2000 μ m, more preferably 100 μ m～1000 μ m.

By above-mentioned reinforcement flank is set, improved the physical strength of filter, use big also passable of chip area.And, even the very thin patience that also can obtain mechanical pressure of filter owing to can use the shallow filter of filter hole depth, can further reduce filter pressure simultaneously.

Reinforcement can form respectively and combine with filter shown in Fig. 6 (a) with flank; Perhaps shown in Fig. 6 (b), (c), also can rib or well material be smashed and fill up and with addition process etc. in conjunction with the pore of filter 18.The sidewall 20 of well can be connected filters 18 and with the bottom of its perforation as the part of reinforcement with flank 23.

When the reinforcement that forms is in addition combined with filter with flank or well sidewall, preferably do not use coupling agent and directly combination.Directly, accumulate the method for formation etc. with flank or well sidewall by stacked filter and reinforcement, also can implement following method in conjunction with being.

In the method, because not directly combination, can eliminate when filtering the problem that reinforcement is entered the interface with the interface peel or the filtering object liquid of flank or well sidewall and filter by coupling agent etc.Especially when the chip that opening diameter is little, well quantity is many of use well, the combination of well sidewall and filter become the difficulty and with geometric growth, be easy to generate liquid at the interface and leak, but by do not use coupling agent with well and filter directly in conjunction with the time, can remove liquid and leak problem.And, be provided with reinforcement with flank as the physical strength supplementary material of filter, be connected with filter when insufficient, physical strength just can not be improved.

But, do not use coupling agent like this and directly combination, make filter and rib integrated, thereby improve physical strength with full intensity.

And, shown in Fig. 7 (a) and (b), can use with the filter same material and form the reinforcement flank continuously, owing to be integrally formed, then have desirable intensity.Here so-called same material can be identical with former material, for example with a part of oxidation of metallic silicon and obtain the silica/silicon materials of monox; With a part of carbonization of metallic silicon or nitrogenize and obtain silicon/SiC, silicon/SiN material of SiC or SiN etc.; With the above-mentioned same material that is considered as.The sidewall of well also can similarly form with filter continuously.As its manufacture method, when making filter in advance, do not make the perforation of well or flank, then when forming well or rib, position in this part, form well or rib, adopt on the identical materials cutting or etching and produce filter and reinforcement with the method for flank or well sidewall etc., undertaken by the aftermentioned method.

And on the part of the sidewall 20 of flank 23 or well, the pore of filter 18 can not exist as required yet.Specifically, shown in Fig. 1 (b), (c), when making filter 18, be pre-formed the part that does not have pore, the sidewall 20 of flank 23 or well be positioned on this part that does not have pore make; Perhaps when making flank 23 and filter 18, can implement the hole fill method simultaneously by addition process.

In the well shown in the (a) and (b) of the (a) and (b) of Fig. 8 and Fig. 9, lower surface or upper surface at filter are provided with above-mentioned reinforcement flank 23, use the lower end position of the well sidepiece 20 in the flank 23 simultaneously in this reinforcement, the recess 27 of the location usefulness that the protuberance that forms and be provided with is chimeric on another container.The concrete shape of recess 27 can be to form suitable shape corresponding to the protuberance that is provided with on another container.Promptly, at the recess 27 of aboveground formation is that solution in will the well of regulation is when moving and replacing well to the regulation of other containers, has ditch as the cloudy type part of container connector, because of chimeric, can make the solution in the well outside the established part of container, not leak and move with the positive type part of container.The location is not particularly limited with the shape of the ditch of recess, as long as cloudy type part can be provided with positive type protuberance partly chimeric with container.

And the location that well is not set is with recess or protuberance, also can not form recess 27 and form level and smooth bottom surface shown in Fig. 8 (c), Fig. 9 (c) and carry out combination.This even surface is level and smooth more, and the combination between the face is just firm more.For example, use commercially available semiconductor silicon wafer, when especially using super level and smooth silicon wafer, can obtain extremely firm faying face.

By being set, above-mentioned location moves with the liquid that the well of recess produces, have a plurality of wells that contain mobile solution respectively and having under the situation of container of corresponding a plurality of wells, can be effectively between corresponding well the while shift solution in whole corresponding well concurrently.And, also go for moving to the liquid that the chip with corresponding well carries out from container with a plurality of wells, perhaps the liquid that carries out between a plurality of chips moves.

Rib with above-mentioned ditch can be made with normally used rib in the same manner by following the whole bag of tricks.There is no particular limitation for the width of well sidepiece and shape, and the width of well sidepiece is preferably greater than and equals 100 μ m, more preferably greater than equaling 20 μ m smaller or equal to 10mm, most preferably more than or equal to 300 μ m smaller or equal to 5mm.When the width less than 100 μ m of well sidepiece, make the location become difficult with the protuberance of recess and container is chimeric; Perhaps when in conjunction with even surface, because deviations diminishes bonded area.When the width of well sidepiece surpassed 10mm, the occupied part of well became too small in the regulation area, and the row yielding of chip is reduced.

The recess that above-mentioned positioning combination is used with whether be that reinforcement is irrelevant with the part of flank 23, and be formed at the lower end of the sidepiece 20 of well.The recess that the above-mentioned on the other hand lower end at well forms also can be protuberance or partes glabra, at this moment, with cell therefor or chip on should form the recess or the partes glabra of chimeric usefulness.

In well shown in Figure 10, in the upper base surface of well, in the width of its circumferential edges regulation, atresia portion 28 is set, do not form the pore 19 of filter 18 in this atresia portion 28.

As the Rack of this atresia portion 28, begin to be preferably 5 μ m～50 μ m from the circumferential edges of bottom, more preferably 10 μ m～30 μ m.Especially, shown in Figure 10 (b), the surface of preferred atresia portion 28 is what tilt.If this atresia portion not, when the mural margin of rib or well was provided with pore, particulate was trapped on the mural margin, or also had phenomenon generation such as accumulations.By atresia portion is set, the physical strength on the connecting portion between well and filter improves, and the possibility of peeling off reduces.And, can prevent that solution from the laminar flow that moves to central part from the mural margin of well or rib taking place, produce particulate and be detained or be deposited in phenomenon on the mural margin of well or rib.And, when following optical detection, there is not particulate in order to make at mural margin, can reflect the detection that prevents to hinder particulate on the mural margin by light from the borehole wall.

Well with this atresia portion can be made by following method: make the filter that is provided with virgin coil in specific zone in advance, make well or reinforcement navigate to virgin coil and combination with flank; Or by addition process on virgin coil stacked well or reinforcement with flank etc.And shown in Figure 10 (b), the structure of the surface tilt of atresia portion 28 can carry out wet etching in the short transverse of well after being pre-formed filter portion, forms by the residual etching remnants of defeated troops.

Below, the various manufacture methods of above-mentioned well are described.

First method is by electroplating the method for stacked filter etc.In the method; prepare in advance and carried out the substrate that electric conductivity is handled,, the part of not electroplating is protected with resist thereon with figures of stamping such as resists; by electric current is flow through between substrate and the electroplating solution, only the established part at substrate forms metal or ionic polymers material.

At first, prepared to carry out the substrate that electric conductivity is handled, thereon painting erosion resistant agent.This resist is formed on conductive board after the film, prepare photomask and carry out exposure imaging, on conductive sheet, form the post against corrosion (post) of the reverse pattern of filter with UV light.Depend on the exploring degree of resist by the boundary of said method pattern,, can make pattern errors 5 μ m, asperratio and be 2 post against corrosion for example by using THB-110N (trade name, JSR Corp.'s system).

Then, between these posts, make according to electrochemical plating to exchange or DC current flows through metal or ionic resin is electrically filled.Can use gold, nickel, copper, iron, iron-nickel alloy etc. as the metal material that is filled according to electrochemical plating.Can use nickel sulfamic acid bath (mixed solution of 60% sulfaminic acid solution 700g/l, nickelous bromide 5g/l, boric acid 35g/l is bathed 50 ℃ of temperature) etc. as the electroplate liquid that is used for the nickel electroforming; Can use copper sulphate bath (mixed solution of copper sulphate 200g/l, sulfuric acid 60g/l, chlorion 30mg/l is bathed 30 ℃ of temperature) etc. as copper electroplating liquid; Can use sulfaminic acid iron bath (mixed solution of sulfaminic acid iron 400g/l, sulfaminic acid amine 30g/l, formalin 100mg/l is bathed 46 ℃ of temperature) etc. as ferroelectric plating bath.For example, when using nickel sulfamic acid to bathe, by voltage 6V, current density 3A/dm ²Direct current about 10 to 20, can obtain the electronplate that thickness is 5 μ m.

And, can use epoxy resin, acryl resin, polyimide resin etc. as the resin material that is filled by electrochemical plating.Can use the acryl resins such as PowerTop U of PAINT that the Ekote of Shimizu K. K. company for example makes, Japanese Co., Ltd. as these resins.

No matter metal, resin can add various adjuvants,, the chemical property of the film of formation is changed for example by adding metal oxide microparticle, fluoroplast particulates etc. such as silicon dioxide, titania, aluminium oxide.

Then, when the resin electroforming, by being heated to about 200 ℃ at last, when making resin cure (cure), resist is removed in calcining.When using electrochemical plating, for example remove resist by membrane removal device THB-S1 (trade name, JSR Corp.'s system).

Make as mentioned above after the filter, can use the method identical, re-use electrochemical plating and make well or reinforcement flank with the method that forms filter.That is, on filter, form diaphragm same as described abovely, carry out photoetching then, carry out material by electrochemical plating and fill.In this case, be generally the height that the makes well thickness more than or equal to filter, claimed film is thickening also, therefore preferably the stacked dry film photoresist of diaphragm is used.Usually, the ratio that once forms live width and thickness is very difficult greater than 2 electronplate, in these cases, could form the well of specified altitude repeatedly for several times by the operation of photoetching and electrochemical plating formation electronplate.

In second method, make filter, well, reinforcement with at least a in the flank by engraving method.Etched object is, be not defined as metal, metal oxide, organism etc. for the material that can form filter, but particularly preferably be the high material of physical strength, specifically, engineering plastics such as liquid crystal polymer, polycarbonate, polyamide, poly-imines, polyethylene terephthalate, polyethylene naphthalenedicarboxylate phenolic ester, polyene propyl ester, polysulfones, polyethersulfone for example; Metals such as iron, nickel, copper, zinc, aluminium, silicon, titanium, tantalum, magnesium, molybdenum, tungsten, rhodium, palladium, silver, gold, platinum, stainless steel, brass, red brass, bronze, phosphor bronze, aluminium copper, almag, Aludur, aluminium zinc magnesium alloy, iron-nickel alloy; Metal oxide such as silicon dioxide, aluminium oxide, titania, zirconia, oxidation are smooth; Metal nitrides such as SiN, TiN, TaN; Metal carbide such as SiC, WC; Adamas, graphite, diamond-like coal material with carbon elements such as (Diamond LikeCarbon:DLC); Glass such as soda-lime glass, pyrex, Pyrex glass (trade mark), quartz glass etc.

Can carry out etching according to usual way.But the excessive etching of asperratio may the aperture take place or shape is unequal, and in fact the asperratio that obtains by common chemical etching is smaller or equal to 5, preferably smaller or equal to 3.But, form obconic filter because of in the scope of this asperratio, initiatively using, also can obtain the good filter of filterableness.

And when making well or rib shape by etching, preferred asperratio is more than or equal to 5, can carry out asperratio more than or equal to 5 etching according to the anisotropy of material etching.For example, the very big dependent character of crystallization of utilizing monocrystal materials such as silicon to show for KOH aqueous solution or ethylene diamine pyrocatechol (EDP), 4 methyl hydrogen amine oxide etching solutions such as (TMAH).Use under the situation of silicon, (111) etching speed of face is quite slow with respect to other crystal planes, use the silicon wafer of (110) face, and the direction of the opening edge that makes mask material and (111) face is consistent carries out chemical etching, can carries out asperratio and be about 100 etching as the surface.These methods are recorded in accurate engineering meeting, write: " nanoscale process technology ", Nikkan Kogyo Shimbun (1993).

And, as required, can use isoionic reactive ion etching (RIE).For example be used in SF ₆In contain the gas of the chlorine elemental gas of fluorine Lyons system, carry out vertical different in nature etching with substrate.These methods are recorded in " ' 02 up-to-date semiconductor processing techniques ", Press Joumal society (2001).

Especially, the preferred plate that constitutes of the various material of being made up of difference for example for aluminium/aluminium oxide, metal silicon/silicon dioxide or Titanium/titania etc., carries out pattern etching from both sides respectively and forms the method for filter and well.Specifically, for example for metal oxide layers such as aluminium oxide, silicon dioxide, titania, photoetching is to the border of this metal oxide layer and metal level and form filter, then for metal levels such as aluminium, metallic silicon, Titaniums, photoetching is to the border of this metal level and metal oxide layer, makes well and filter in conjunction with biochip by this method.

According to this method, can filter and well be integrally combined by using integrally formed plied timbers such as pre-prepd metal and metal oxide, do not exist from problems such as the generation leakages of the interface between filter and the well.

And, owing to can use transparent materials such as aluminium oxide, silicon dioxide, titania, thereby when utilizing light to detect, can observe directly the motion of particulate by the filter layer as one deck of filter.

Can use as above-mentioned plied timber, for example surface of aluminum plate is oxidized to specific thickness and obtain the aluminium/alumina plate of aluminium oxide, the Titanium of oxidizing metallic titanium using/titania plate, the silicon wafer that has oxide film or SOI (Silicone On Insulator) wafer etc. similarly.

And, can pass through etching method etching silicon wafer, form filter, rib and well respectively, the flatness of utilizing silicon wafer is stacked and obtain chip with it.In this case, the thickness that is used to make the silicon wafer of filter, rib and well is set respectively aptly, and engraving method also can use dry etching and wet etching for each filter, rib and well respectively.

In the method, and compare with the method for rib or well, needn't open hole, open after filter, rib and the aboveground through hole of setting diameter respectively, because only with they stacked formations, so make easy with slope with same material etching filter.And, carry out combination according to the flatness of silicon, owing to do not use coupling agent and combination interface, do not exist the liquid that produces from the interface to leak so can make, and the high chip of peel strength.

The stacked filter of location interval, rib and well for regulation preferably set in advance the positioning combination mark thereon, position combination according to this mark.And, in solution when stacked, in order to prevent and produce peeling off of lamination surface, preferably step up lamination surface from the lateral deviation of plywood.

In third party's method, make filter by the anodic oxidation of metal.Can use aluminium, silicon etc. as the object metal.Below be that example is to concrete example explanation with aluminium.At first, prepare aluminium sheet.According to circumstances aluminium sheet preferably is pre-formed the concave surface of side about than the about 100 μ m of the depth as shallow of well.For example the degree of depth of well is the thickness of 5mm, filter when being 20 μ m, and preparing aluminium plate thickness is 5.0mm and the aluminium sheet with 4.9mm concave surface.In concave surface inside, be purpose with the physical strength of keeping in the anodic oxidation step, fill the filling material of montanic acid wax, Tissuemat E waxes such as (Clariant Co., Ltd. systems) etc., can form the back at filter and remove by heat fusing.

Then, on an opposite side of these concave surfaces, be formed for offering the pilot hole in filter hole.The error of this pilot hole is smaller or equal to 1 μ m, preferably smaller or equal to 0.5 μ m; Its degree of depth is preferably greater than and equals 0.1 μ m.This pilot hole also can use the resist photoetching to obtain except that by the method for using the mould punching press.

Then, when being applied voltage in oxalic acid or sulfuric acid solution, above-mentioned aluminium carries out anodic oxidation.Its concrete grammar is recorded in the spy and opens the 2003-11099 communique.Make after the filter by anodic oxidation, connect, splash into strong acid or their acid mixture or iron chloride such as phosphoric acid, hydrofluorite, nitric acid to the concave surface of aluminium well in order to make the bottom surface and the filter hole that on aluminium, are formed with concave surface.When for example using iron chloride, can carry out 50 ℃, 20 minutes etching, the shaft bottom face is connected to the surface in filter hole.Said method is recorded in applied physics the 69th volume No. 5 (2000), spy and opens 2000-258650 communique etc.According to this anodised method, can make the aperture and be that 5～450nm, asperratio are about 100, size is the filter about 5～10mm.

In cubic method, make filter with the nano print (nanoprint) of resin.At first, prepare to be used for forming the substrate of filter by nano print.Substrate can be level and smooth plate, also can be the plate that has the concave surface of the bottom about the about 100 μ m of residual thickness in order to form wellhole in advance.This concave surface also can be filled wax same as described abovely.

The mould that pre-prepd nano print is used.There is no particular limitation for the manufacture method of mould, but smaller or equal to the manufacture method of the mould in the hole of tens μ m qualification is arranged to being provided with the aperture, usually according to the method for the anisotropic etching or the X-ray lithography of the crystal anisotropy that utilizes silicon, promptly make by MEMS (Micro Electro Mechanical System) technology.Perhaps can use the mould of making based on according to the MEMS technology, carry out metal plating and the reverse mould that obtains.Above-mentioned pre-prepd casting mold is installed to pressue device.Can use the nano print device of the nano print device of NPHT-1 that Tectoria Co., Ltd. makes, Hitachi Co., Ltd, bright prosperous machine worker Co., Ltd. etc. as this nano print device.

As the good resin of resin preferred flow by the nano print moulding, liquid crystal copolymer or crystallinity nylon resin etc. are more suitable.Perhaps can also in to the pressurization of oligomer such as epoxy resin or urethane acrylate, carry out photo-hardening or thermmohardening.Epoxy resin or urethane acrylate are liquid resin, in addition can also use the membranaceous material of pre-tire (pre-preg).

The resin of residual skin shape according to the not demoulding of filter of above-mentioned nano print extrusion forming and in the bottom surface, like this moulding failure seldom, and the demoulding easily.And consider the difficulty or ease of the demoulding and the damage of mould, the preferred asperratio in the hole of formation is smaller or equal to 10, is more preferably less than to equal 5.

And, on purpose skin portion is stayed as well portion during moulding, in addition can be by hollowing out or etching makes this skin portion form well.Under the situation of film residue, after substrate is peeled off the gum residue thin slice, can offer through hole to the filter part specifically to released part boring or photoetching as the well utilization.Perhaps can also carry out machine drilling to a part, remove last tens～hundreds of μ m with etchings such as acid or alkali.

In the 5th method, make rib or well with light chisel.At first, make filter by any one method in the nano print of above-mentioned electroforming, etching, anodic metal oxide, resin.Perhaps can also use filter by pressure processing, expanded metal manufactured.These filters as substrate, or are fixed on filter on other substrates, form well or rib thereon.

Specifically, shown in Figure 11 (a), filter 18 is fixed on the substrate 71, and be set to light chisel trough 72, the lifter 73 of supporting substrate 71 is descended, inject 1 layer of UV hardening resin to the shunting of filter 18 1 sides, make flank 23 (the perhaps well portion) sclerosis of the resin bed of 1 layer of part by the UV rayed.Carry out repeatedly lifter 73 move and the operation of UV irradiation after the stacked specified layer of having hardened, the duplexer that will contain substrate 71 takes out from the UV resin liquid, cleans unhardened part, removes substrate 71, and sulfuration and obtain having rib or well filter.

In utilizing the method for this light chisel, make filter 18 be positioned at the below, when forming the flank 23 of closed form or well by light chisel above it, very difficult discharge is the unhardened resin of portion within it, owing to inner resin because of the surface tension gathering that makes progress, and might influence the shape of rib or well.Therefore, shown in Figure 11 (b), preferably make filter 18 be positioned at the top, thereunder growing up by light chisel forms the method for flank 23 or well.The light chisel device that is used to implement this method is the SLP-4000 that the commercially available DENKEN of Co., Ltd. makes, and the manufacturing of handling moulding is entrusted.And existing light chisel mainly carries out the sclerosis of ray hardening resin by laser radiation, and irradiation mode is consistent in the above-mentioned situation, can carry out rayed by photomask and come stacked UV resin, can shorten the moulding time.

In the 6th method, well or rib combine with filter by hot tie-in.For example, add the hot filter part, well or rib are carried out the hot tie-in combination under non-heated state.Filter is partly made by temperature conductivity good metal or pottery, and rib is processed by the thermoplasticity forming materials.The end face of filter is combined with heating material, utilize high-temperature electric conduction to add hot filter and hot tie-in well and rib.

Perhaps, utilize the heating of electromagnetic induction heating or dielectric.Employed here well or rib are that the material that heats with athermic low dielectric constant by electromagnetic induction heating or dielectric is that thermoplastic resin is made.Here the dielectric constant of institute's materials used is preferably smaller or equal to 3.5.Can use as concrete resin material, for example tygon, polypropylene, polystyrene, plexiglass, liquid crystal polymer, polycarbonate, polyamide, polyethylene terephthalate, polyethylene naphthalenedicarboxylate phenolic ester, cycloolefin, polymethylpentene, polyene propyl ester, polysulfones, polyethersulfone etc.

With the well that makes above-mentioned molding resin material become to be scheduled to someway or the shape of rib.Well preferably is loaded in a plurality of wells of filter and is all made integratedly, can use as its method, is shaped by jet moulding, press molding or special-shaped extrusion and makes a plurality of continuous pipes with empty well hole, pipe is become the method for ring section; Offer the method in hole etc. onboard with methods such as brills.

And employed here filter material is preferably by the heated material of dielectric heating means, and preferred dielectric constant is more than or equal to 3.8.Specifically, can use for example metals such as gold, silver, nickel, copper, iron, aluminium, titanium, silicon, stainless steel, tantalum, molybdenum; Metal oxides such as silicon dioxide, aluminium oxide, titania; Metal nitrides such as SiN; Glass such as soda-lime glass, pyrex, Pyrex glass (trade mark), quartz glass etc.Perhaps, also can use the resin material potpourri that is mixed with inorganic fillings.

Clamping filter etc. between well and rib, in well and the rib any one and filter crossover are fixed with stationary fixture, or clamp and pressurize and carry out electromagnetic induction heating or dielectric heating with resin roller etc., filter is partly generated heat, well and rib thermosol are received on the filter by its heating.Frequency as electromagnetic induction or dielectric heating can be used 30kHz to 300MHz, preferred 1～100MHz.

In the 7th method, well is attached to filter with coupling agent.In advance coupling agent is coated on the side end face of well, is pasted to filter again, add thermal as required.Perhaps,, waxes such as Clariant Japan LICOWAX LP are coated aboveground, added hot filter again with following method combination, and the coating cooling.

In the method for all directions, make well or rib by the insert molding of filter.In insert molding, ready-made filter is inserted the mould mould, can make filter and resin one-body molded by carrying out resin forming.

In the 9th method, filter and well adopt magnetic material by the magnetic force combination respectively.For example, any one of filter or well used kicker magnet, and corresponding well or filter are made of magnet, by forming the well that has filter with the magnet combination.Can use nickel, iron etc. as kicker magnet; Can use ferrite, samarium-cobalt magnet, neodymium series magnet, aluminium cobalt series magnet etc. as magnet.

Filter is under the situation of magnet, for example on the surface of level and smooth copper, nickel, chromium etc. and golden connectivity good metal plate, carries out deposition of gold with the thickness of tens dusts, formulate pattern then, that is, after the coating photoresists, at the residual resist of part that forms the filter pore.Do not have the electrolytic iron hydrochlorate then and electroplate, form thickness and be after the ferrous acid salt deposit of 1～20 μ m, peel off resist, peel off the ferrous acid salt deposit from the deposition of gold layer at last with anticorrosive additive stripping liquid controlling.This ferrous acid salt deposit is electroplated and can be used the magnetism will Vol22 of association with for example Japan, No9, and 1998 1225-1232, Japan uses the magnetism will Vol24 of association, No4-2, the enforcement that is as the criterion of the condition of 2000 515-517.

Above-mentioned with ferrite as magnet, for example above-mentioned electroformed nickel of passing through is electroplated and is made well, according to both magnetic force and in conjunction with making the well that has filter.

And, rib is under the situation of magnet, for example can use laser radiation is that the magnetic powder forms laminar plasticity magnet sheet (for example the agX of M Co., Ltd. makes) etc. with resin adhesive mixing ferrite, samarium system or neodymium, offer thickness and be after the hole about 0.3mm～1mm, combine by magnet with above-mentioned ferronickel hydrochlorate filter, and form the well that has filter.

In the tenth method, make well and filter combination by mechanically chimeric.For example, before the aboveground resist of peeling off the electrochemical plating manufacturing by above-mentioned nickel, go back painting erosion resistant agent and formulate pattern, the taper resist ditch than the only little 50 μ m of the width～100 μ m of well is set in the upper surface center of the borehole wall.In this ditch, electroplate with soft metals such as tin or gold or resin and to fill ditch.Then, peel off resist, be formed on the aboveground protruding structure well of nickel matter with height and the width veteranellinae shape protuberances such as tin, gold or resin about equally with stripper.Simultaneously, also use resist in same position, form matrix shape, with the height of above-mentioned convex with width equates and the nickel harder than protuberance, polyimide resin etc. as cloudy type part in filter one side.Then, both are mechanically chimeric with pressurization such as roller, thus be combined into the well that has filter.And, also can at the borehole wall upper surface concave shape be set on the contrary, and convex form be set in filter one side.

In biochip of the present invention, also can be when the bottom of well is provided with first filter, side (by the opposition side of well) is provided with the structure of second filter thereon.Second filter obtain by said method in the bottom is provided with the well of filter, accommodate the probe carrier particulate; And second filter can by the plane between, the even surface chimeric with coupling agent, magnetic force, machinery in conjunction with, mechanically clamp in conjunction with method and be provided with by said method gained filter; The biochip that therefore can be had filter up and down.

And the biochip that is formed with filter in the bottom of each well can pass through for example following method manufacturing.

(1) shown in Figure 19 (a), it is wavy that filter 18 is formed, and is connected with the suitable film of its protruding top 32 and recessed top 34 usefulness formation sidewall 20 or with filter, and forms each well 16,16 ... method

(2) shown in Figure 19 (b), will be shaped to the shape that is interval with recess in accordance with regulations with stamping die by the filter that metal or resin are made, with this recess as well 16,16 ... method; Perhaps also form the method for well with enclosure space in conjunction with other filter or film with methods such as cementing agent, magnet or machinery are chimeric to a side

(3) shown in Figure 19 (c), at well 16,16 ... diapire 22, wear the method that through hole forms filter 18 with laser, pressurization, photoetching etc.; Perhaps also with filter with cementing agent, magnet or the chimeric method that is combined in aboveground portion of machinery

(4) shown in Figure 19 (d), do not have diapire 22 and form well by sidewall 20, connect the method for filter 18 in its bottom; Perhaps also with filter with cementing agent, magnet or mechanical chimeric combination and cover the method for aboveground portion

(5) to the method with laser beam perforation, machinery pressurization or photoetching such as metal, metal oxide, metal/metal oxide or resin.

Biochip of the present invention has integrally formed a plurality of wells or single well; In this well, accommodate the dispersion liquid that is dispersed with the probe carrier particulate.The particulate that well is accommodated is the particle diameter bigger than filter pore, make the particulate of chip be statically placed in the hole of filter and when carrying out optical detection, the aperture of the particle diameter of probe carrier particulate and filter pore is particle diameter/aperture=1.1～2.5, and particle diameter＜span＜particle diameter * 10, the more preferably relation of particle diameter＜span＜particle diameter * 4.Here, so-called " span " is the bee-line of the part of Kong Buwei sky between each hole of adjacency.

Particle diameter/aperture was less than 1.1 o'clock, and particulate enters pore, and particulate can not increase from the probability that pore breaks away from.Particle diameter/aperture was greater than 2.5 o'clock, and the solution that will contain particulate filters by filter, and microparticle residue is on filter the time, and particulate does not drop on the hole, and randomly or the particulate probability that is arranged on the filter that overlaps each other uprise.Can not break away from above-mentioned relation preferable particle size/aperture=1.15～2.0, more preferably particle diameter/aperture=1.2～1.6 in order to prevent particulate from entering pore.

And when chip was used for separation and purification, the particle diameter of setting particulate, the aperture and the span of filter satisfied the relation of particle diameter＞aperture+span/2.When satisfying this and concerning, the solution that will contain particulate filters by filter, when making particulate rest on the filter, for particulate is not rested on particulate is rested at least one span, on at least more than or equal to 50% interval, there is the filter hole of not blocked, consequently can prevents filtration resistance by particulate.

Whole particle numbers that well is accommodated are according to purposes and difference, and being used to separate under the situation of classification is 100 times～1/100 times of filter pore number, preferably 10 of filter pore number times～1/10 times scope; Under the situation that detection is identified in each probe the absolute number of particulate be 1～1000, preferred 1～500, more preferably 1～200.When the particle number in well is a lot, makes filtration resistance become big, strainability when filtering and reduce, perhaps filter is destroyed.Even to be 1000 also be fully to significant particle number when detection is identified, the more also nonsensical filterableness difficulty that becomes that also makes.

The absolute number of the probe carrier particulate of accommodating to each well is can be with following method control.

That is, can one by one control particulate with flow cytometry on one side when dropping into particulate solution, with CCD camera etc. practically count each particulate and with its input on one side.Perhaps, can measure the particle concentration of particulate solution in advance, the proportion by particle concentration in this solution and particulate calculates particle number, according to this as a result inverse go out the particulate solution amount, and use input such as sample applicator, nearly like in the particle number input unit chamber.And, can also use the particulate of point sample specified quantity several times, to count particle number behind its each point sample and calculate in shortagely, point sample is until zero the method that is similar in shortage.

Here, particle number preferably has approximate homogeneity to a certain degree, its error range preferably the CV value 20% in, more preferably in 10%.

As the particulate of carrying probe,, can use any one of organic fine particles, inorganic particles, organic-inorganic particulate, phase change gel body etc. as long as the diameter particulate bigger than the pore diameter of filter is not particularly limited.

Can use as organic fine particles, specifically, monomer with butadiene-based, polystyrene, divinyl benzene series, acrylic, acrylate system, methacrylate system, acrylamide, benzoguanamine system, nylon system, polyethenol series and fluorine system etc., individually or the raw material monomer that is used in combination more than two kinds, the particulate that obtains by emulsion polymerization or suspension polymerization.Perhaps can also use the methods such as film emulsification of making particulate from plate extrusion resin with the impartial hole of porous matter.These particulates can be arranged particle diameter with grader as required.

And, can also use when polymerization or add magnetic such as ferrite after the polymerization and the material that obtains; Or the material that obtains after with particulate plating iron hydrochlorate of the method for opening flat 10-83902 etc. according to the spy; Or natural cross-linked gel body such as cellulose, starch, agarose, galactose; Synthesizing cross-linked gelinite such as acrylamide.

In the preferred CV value 10% of the size distribution of these particle diameters, more preferably in 5%.The CV value was greater than 10% o'clock, and the probability that particulate enters in the filter hole uprises.

About cinnamic particulate, for example can obtain according to the method that the spy opens the record of clear 57-168163 communique.

And magnetic particle for example can obtain according to the method that the spy opens the record of flat 11-176622 communique.

Particulate as inorganic system can use metal oxide microparticle, metal sulfide particulate, metal particle.

As metal oxide microparticle silicon dioxide microparticle most preferably, can use commercially available various silicon dioxide microparticles.

And, can use commercially available various particulates, for example, IMMUTEX (trade name, JSR), MCI gelinite (trade name, Mitsubishi Chemical), Toypearl (trade name, Tosoh Corporation), Daiso gelinite (trade name, Daiso Co., Ltd.), Shodex (trade name, clear and electrician), Sunsphere (trade name, Asahi Glass) PL-PEGA, (trade name, Polymer Laboratories), PL-CMS (trade name, Polymer Laboratories), PL-PBS (trade name, PolymerLaboratories), PL-DMA (trade name, Polymer Laboratories), AM resin (trade name, Novabiochem), P500 (trade name, Toray Industries, Inc.), Toraypearl (trade name, Toray Industries, Inc.), Techpolymer (trade name, ponding changes into product industry), MP/MR series (trade name, combine and grind chemistry), BELLPEARL (trade name, clock spins), EPOSTER (trade name, Japanese catalyst industry), cellulose powder (Chisso Corp., Asahi Chemical Industry), Sephacell (trade name, Amarsham-Pharmacia society), Sephallose (trade name, Amarsham-Pharmacia society) etc.

Can be to above-mentioned microparticle surfaces by using various functional groups such as amino, carboxyl, carbodiimide, epoxy radicals, tosyl, N-succinimido, dimaleoyl imino, mercapto, sulfinyl, hydroxyl, trimethoxy silicyl, triacetic acid itrile group, benzo sulfophenyl, polyethylene imine based, quaternary ammonium group (tri-n-octylmethylammonium), 18 (carbon alkane) base; Or γ-glycidoxypropyltrime,hoxysilane etc. carries out surface modified, and forms the probe binding site.

And, when carrying out above-mentioned surface modified, space during for the probe that makes particulate carrying and analyte response is repelled and is reduced, can be the compounds of the two ends of 10～100 alkylene in conjunction with functional group's structure with having at the carbon number, for example ethylene glycol bisthioglycolate diglycidyl ester derivant, N-k-maleimide undecanoic acid, sulfydryl propyl trimethoxy silicane, cup propadiene derivant, liquid crystal use as spacer.And the oligonucleotide of revising with amino, carboxyl, thiol also can be used as spacer or the spacer binding site of holding concurrently and uses.

As the particulate of carrying probe, under the situation of using organic fine particles, the preferred 0.02 μ m of its particle diameter～120 μ m, more preferably 0.1 μ m～60 μ m.On operability, produce difficult point under the little situation of particle diameter, make the filter of catching these particulates difficulty that becomes, and since the very for a short time analyte that makes in filter hole stop up easily.And under the big situation of particle diameter,, the space repulsion reduces owing to making reaction rate.

And preferred as required particulate is porous matter or hollow form.Therefore, even under the big situation of particle diameter, also can prevent to reduce because of weight causes that the particulate sedimentation makes with the reactivity of analyte.

As the particulate of carrying probe, when using inorganic particles, the preferred 0.1 μ m～0.1mm of its particle diameter, more preferably 1 μ m～0.05mm.On operability, produce difficult point under the little situation of particle diameter, especially catch the particulate difficulty that becomes by the pore of filter.And under the big situation of particle diameter, owing to sedimentation makes reactive the reduction.

And preferred as required particulate is a porous matter.Therefore, even under the big situation of particle diameter, also can prevent to cause particulate sedimentation etc. because of weight.

As the method for the probe carrier particulate being accommodated each well, can use in advance the particulate input corresponding to the method in the unit of each well.Perhaps, also can use the particulate that will have the probe binding site on the surface to drop in each well with sample applicator, then, will be corresponding to the method well of regulation, that diverse probe drops into to the well of correspondence with sample applicator etc.

In the former method, as the solid phase synthesis machine that can use commercially available each company to make to the method for particulate bonding probes, for example oligonucleotide synthesizer, peptide synthesizer, sugar chain synthesizer, according to the various low molecular compound synthesizers of combinatorial chemistry etc.In solid phase method, can use silica dioxide granule or tygon divinylbenzene particulate in order to react with separating as carrier; can be also with these solid-phase synthesized carrier particles and the particulate carrier that is housed in the chip 10, by accommodating to chip 10 point samples with the synthetic probe carrier particle of synthesizer.These synthesizers, especially the oligonucleotide synthesizer is popularized widely and is used, and now, synthesizer for example is set between the lights, just can make the particulate that has solidified oligonucleotide in the second day morning, only its point sample be made the customized chip with any nucleotide in the extremely short time.

Can use as the probe that carries in the particulate, for example the protein of nucleic acid, molecular weight 500～1,000,000, lipid, sugar chain, cell, protein expression cells (protein expression cells), the adaptive son of oligonucleotide (aptamer), virus, enzyme, molecular weight are 50～1,000,000 the lead compound with pharmacologically active, or with regard to chemical substance that maybe may have specific physiological activity of specific physiological activity etc.

Here, protein as molecular weight 500～1,000,000 can be, specifically, antibody, various antigen such as synthetic peptide, memebrane protein, enzyme, transport protein, cell factor, lymphokine, IgA and IgE or material with bioluminescence functions such as luciferin, luciferase, photoprotein, green fluorescent protein matter.

As lipid can be, specifically, phosphatidic acid, phosphatidylinositol, laccol, various gangliosides etc.

The adaptive son of oligonucleotide is, protein, enzyme, stain, amino acid, nucleotide, growth factor, gene expression regulatory factor, cell adhesion molecule, the functional nucleic acid that has binding ability with bion etc.; Specifically, the adaptive son of NS3 proteinase of the adaptive son of fibrin ferment, the adaptive son of elastoser, activated protein C, hepatitis C virus.

As molecular weight is that 50～1,000,000 low molecule lead compound can be, specifically, medical candidate substances such as part, phenylpiperidine derivative, sulfanilamide (SN)/sulfonic acid, steroids, derivatives of prostaglandins such as matrix, coenzyme, regulatory factor, agglutinin, hormone, neurotransmitters, antisense oligonucleotides, ribozyme, the adaptive son of oligonucleotide.

The probe carrier particulate can have: be used to provide at least a means of identification of probe identifying information, this identifying information is used to discern the kind of probe.As this means of identification can be color, shape and the particle diameter of probe carrier particulate.

These means of identification also can a plurality ofly be used in combination, for example the combination of color and particle diameter, CF, color and gene order etc.For with color as means of identification, with dipping particulates and painted such as dyestuffs, pigment, fluorescent dye.About these dyestuffs, pigment, fluorophor, phosphor, have the combination of different absorptions, emission wavelength and color depth thereof by combination, can obtain more probe identifying information.For example, the kind of using color is 2, color depth is 3 concentration, can obtain 9 kinds of identifying informations.Can use IMMUTEX of JSR for example etc. as chromiole.And fluorescent particles can use the Probes as Molecular, the carboxyl modified that the FluoSpheres method luorescent microspheres of Inc. sells, sulfonic acid modification, the modification of acetaldehyde sulfonic acid, amine-modified particle etc.

These color identifying informations detect as follows.At first, to the particulate radiation source that carries out colour code.When colour code is fluorophor, phosphor, need corresponding fluorescence or phosphorescence stimulation wavelength light source.With the CCD camera, the fluorescence of emitting from particulate or phosphorescence etc. are carried out optical detection, the detection information that obtains is carried out statistical treatment then, obtain the probe identifying information with photoelectron amplitron etc.

And, when during as identifying information, in advance particle size being changed according to each probe with particle diameter.As these particle diameters, from detection sensitivity, preferably make the particle diameter difference, more preferably greater than equaling 0.5 μ m more than or equal to 0.3 μ m unit.

Like this, by in well, accommodating a plurality of probe carrier particulates that can discern, in a well, can carry out multinomial detection simultaneously, thereby shorten reaction time and operation steps.And, use has the chip of a plurality of wells, by positional information and a plurality of probe carrier particulate that can discern of combination well, for example the well number is 100 wells, the kind of the probe carrier particulate that can discern is 100, therefore can obtain accommodating the biochip of 100 * 100=10000 kind probe.

Probe carrier particulate with above-mentioned means of identification is accommodated in following each well.

(i) will be contained in the identical well at the identical a plurality of probe carrier particulates of identifying information in whole means of identification, simultaneously for a plurality of probe particulates that are contained in each well, identifying information is identical.That is the identical and kind of color of probe means of identification such as color, particle diameter, the also identical a plurality of particulates of identifying informations such as size of particle diameter, in a well, have been accommodated.Between each well, also accommodate identical particulate.And, under the identical situation of clear and definite in advance identifying information, can ignore the step of probe identifying information and probe identification.

(ii) will be contained in the identical well, simultaneously for a plurality of probe particulates that are contained in each well, identifying information difference at the identical a plurality of probe carrier particulates of identifying information in whole means of identification.That is the identical and kind of color of probe means of identification such as color, particle diameter, the also identical a plurality of particulates of identifying informations such as size of particle diameter, in a well, have been accommodated.But between each well, accommodated the different particulate of identifying information such as color category, particle size of same identification means.

(iii) will be contained in the identical well at the different a plurality of probe carrier particulates of the identifying information at least a means of identification, simultaneously for a plurality of probe particulates that are contained in each well, the formation of the above-mentioned identifying information in its whole means of identification is identical.That is at least a for probe means of identification such as color, particle diameters, a plurality of probe carrier particulates that identifying informations such as color category, particle size are different, in a well, have been accommodated.The formation of this identifying information is identical between each well.

(iv) will be contained in the identical well, simultaneously for a plurality of probe particulates that are contained in each well, the formation difference of the above-mentioned identifying information in its at least a means of identification at the different a plurality of probe carrier particulates of the identifying information at least a means of identification.That is at least a for probe means of identification such as color, particle diameters, a plurality of probe carrier particulates that identifying informations such as color category, particle size are different, in a well, have been accommodated.The formation difference of this identifying information between each well.

Corresponding to the content of test, the formation of selecting the quantity of well aptly and being contained in the probe carrier particulate in the well.For example, can be: (a) of Figure 26 be depicted as the state of accommodating identical probe carrier particulate in the single well; (b) of Figure 26 is depicted as the state of accommodating the different probe carrier particulate of identifying information in the single well; (c) of Figure 26 is depicted as the state of accommodating identical probe carrier particulate in a plurality of wells; (d) of Figure 26 is depicted as when accommodating the different probe carrier particulate of identifying information in a plurality of wells, the identical state of the formation of identifying information between each well; (e) of Figure 26 is depicted as when accommodating the different probe carrier particulate of identifying information in a plurality of wells, the state that the formation of identifying information is different between each well etc.

For example, when the well that contains above-mentioned probe carrier particulate drops into analyte, shown in Figure 27 (a), above-mentioned opening input from well 16, can also as Figure 27 (b) the analyte of accommodating in the container that is shown in 12 and the bottom of each well 16 contact, perhaps be shown in the container 12 that has corresponding to the compartment of each well 16 as (c) of Figure 27, in each compartment, accommodate different analytes, by its bottom with each well 16 is contacted, in each well 16, accommodate different analytes.

＜biochip kit 〉

Biochip kit of the present invention has container, and this container is accommodated the well of biochip and biochip, perhaps is connected with biochip.

There is no particular limitation for the material of container, can use in organic material and the inorganic material any one.Can use as organic material, specifically, tygon, the tygon vinyl acetate resin, the tygon vinyl, polypropylene, polystyrene, polybutadiene, polyacrylonitrile resin, plexiglass, AS resin (vinyl cyanide and cinnamic multipolymer), ABS resin (acrylonitrile and butadiene and cinnamic multipolymer), AAS resin (vinyl cyanide and acrylate and cinnamic multipolymer), polycarbonate, polyamide, polyethylene terephthalate, polyethylene naphthalenedicarboxylate acid esters (polyethylene naphthalate), aliphatic polyester, PLA, polyglycolic acid, fatty polyamide, alicyclic polyamide, cyclenes, polymethylpentene, Polyvinylchloride, polyvinyl acetate, polyarylate, polysulfones, polyethersulfone, polyimide, Triafol T, the cellulose acetate resin, nitrocellulose resin, epoxy resin, teflon, fluorinated ethylene propylene copolymer, tetrafluoroethene perfluor alcoxyl vinyl ether copolymers, polychlorotrifluoroethylene, the vinyl TFE copolymer, polyvinylidene fluoride, poly-ethylene fluoride, silicones etc.

Manufacturing process as these organic materials can be, for example, and casting, shaping without pressure, injection pressure forming, ejection compress moulding, compression molding, transfer shaping, shaping by stock removal, light chisel etc.

Can use as inorganic material, specifically, metals such as nickel, copper, iron, aluminium, titanium, silicon; Metal oxides such as silicon dioxide, aluminium oxide, titania; Glass such as soda-lime glass, pyrex, Pyrex glass (trade mark), quartz glass etc.

These inorganic material can form container shapes by the whole bag of tricks such as moulding or surface treatment processing, for example press molding, plate etching, laser boring, sandblast, to the surface coated of resin or canister etc.

And use to form porose plate or do not form porose plate, its any one or both sides are carried out multilayer laminated, can form the container of regulation.These plates are in order to prevent from the laminated portions leakage liquid, and are necessary level and smooth between the plate face of laminated portions.Therefore, plate is ground level and smooth, or use level and smooth plate such as silicon wafer.During in conjunction with these level and smooth plates, even do not use coupling agent etc. also can obtain very firm combination.

When using silicon wafer, also can use as required to be formed with oxide film, perhaps can carry out oxidation again after the plywood as plate.

The container that uses above-mentioned plate is according to for example following method manufacturing.At first, prepare at container bottom plate that use, that have small air access hole.When using the degree of depth of very dark air access hole, stacked as required many undermost plates.Therefore, the identical level and smooth plate of shape is cut off in having with the vessel water level land of stacked any amount, to form the container hole of prescribed depth.The degree of depth of container hole can at random be controlled by stacked many identical shaped plates.To during with the shape of container, for example to form when having differential hole shape in depth direction control, can obtain any hole shape by stacked difform plate.

On the container cover that is disposed at topmost, can use and offer the vertical hole that liquid or air pressure are come in and gone out that is used for plate, perhaps also dissected valley on the plate face in advance is as the lid of the topmost of plate duplexer.And, for carrying out optical detection, can on the plate of the topmost of duplexer or bottom, use transparent materials such as glass.

Usually, the small degree of depth in aperture is very dark, and promptly the hole of so-called high asperratio is very difficult on making, but can make the container in the hole with any asperratio by said method.

By said method, can make the container that has with the corresponding hole of chip well, also can make the container that has a hole for a plurality of wells.

In order to form the container that has with the corresponding well of well of chip, according to the stacked plate of the degree of depth of container hole with hole shape cross section identical with the well water plane section.

To have a container with the corresponding hole of a plurality of wells in order forming, to cover plate more than or equal to the horizontal section area of a plurality of wells according to the stacked aperture of the degree of depth of container hole.

And, be provided with in the container in hole at container bottom, as mentioned above, the plate that forms container bottom can use to be offered porose plate in advance and makes.By the hole being set at container bottom, for example, when the well of connection biochip and container, can exert pressure to the well of biochip, from the hole of container bottom the air of container is discharged, the interior liquid of well of biochip is moved to container.

The aperture of container bottom is under the big situation in aperture, because the leak of liquid in the container, then need be smaller or equal to the aperture of certain size, its aperture is decided by the degree of depth in hole or the compatibility of hole and liquid, is generally 50 μ m～1mm, preferred 0.1mm～0.5mm.The aperture is during smaller or equal to 50 μ m, stops up and filtration resistance becomes big; The aperture then may produce leak of liquid during more than or equal to 1mm.

Can use biochip and container, the well of the same diameter of mutual correspondence is connected to form biochip kit.In this case, in connecting the continual-connecting-part of well, the area of section of the well of the mobile upstream side by making liquid can make the positioning error minimum of well on the continual-connecting-part greater than the area of section of the well of downstream side.

Biochip can be connected by recessed/protruding, protruding/the recessed or super even surface that is connected on the continual-connecting-part with container.By above-mentioned any one method of attachment, can prevent that liquid from leaking from the joint portion to adjacent well.

And, with pressing above-mentioned same quadrat method between the biochip, can connect by the combination between continual-connecting-part fovea superior/protruding, protruding/recessed or super even surface.

By the connection of biochip and container or the connection between the biochip, the solution that can produce between the mutual well moves, i.e. direct dirty moving between the well of so-called solution.Owing to the mobile devices such as syringe that do not need to be used to move,, move the required time and can reduce solution so follow the solution minimizing that is attached to injector wall and can not produce pollution etc.

The biochip kit that biochip is connected with container can be for example in order to the below manufactured.Here in the method for example, container is to have the plate of through hole and do not form in the plate in hole any one folded formation of a plurality of flaggies by stacked use.

Biochip kit shown in Figure 15 (a) is by following method manufacturing.At first, prepare: be formed with through hole by the etching silicon wafer with biochip 10 have same thickness plate 87a and; The plate 87b of any thickness and; Etching silicon wafer and be formed with the gateway of gas or liquid or the plate 87c of liquid main access.Then, by go up location and plywood 87b ' plate 87a ' in addition at plate 87c ', make and be provided with plywood with the differential hole of secondary.The one-sided chip that biochip 10 will be fitted is inserted in hole to the plate 87a ' of this plywood, the half part biological chip agent box that manufacturing biochip and container coincide.Similarly, stacked 87a, 87b, 87c make plywood, insert the one-sided chip that biochip 10 will be fitted then, make half same part biological chip agent box.

Then, the particulate that combines probe is contained in the above-mentioned half part biological chip agent box by the methods such as open surface point sample from well.Make the half part biological chip agent box of accommodating this particulate as the bottom, second half part biological chip agent box is as top, by make well forward surface that they have to and combination.Therefore, make container and close the biochip kit that chip becomes one.At this moment, make kit positioning combination up and down, can correctly locate according to the positioning combination mark that sets in advance.

And the biochip kit shown in Figure 15 (b) prepares to have the chip of wide outermost upper and lower end face, the upper and lower end face of biochip 10 can be laminated to etching silicon wafer and forming on the end face of plate 87b (87b ') of through hole.

Shown in Figure 16 (a),,, can use for example by that constitute, level and smooth transparent plates 94 such as quartz glasss as optical detection usefulness for the bottom that forms container or the plate of cap.Optical detection is implemented for the reaction of observing the particulate that is filled in filter.According to as the structure shown in the figure, can make from the bottom or cap minimum to the distance of filter, thereby improve the open area ratio of optical system.But in this case, the volume of accommodating the container of liquid diminishes, and may cause the analyte and the cleaning fluid of accommodating inadequate.In this case, shown in Figure 16 (b),, the pipe 89 that is connected with pump 90 is installed, is made the liquid circulation by managing 89 at 88 places, gateway of liquid; Perhaps make liquid only in kit and manage up and down in the definite part of installing port and move up and down, the part of pipe is used as the kit containers impact damper, solve the volume deficiency of container.

Shown in Figure 17 (a),, can enlarge the volume of container by stacked many plate 87b (87b ').And, shown in Figure 17 (b), make the aperture alternation of plate 87b (87b '), thereby inner surface of container is tilted.In this case, on the direction of internal tank, container diameter becomes greatly gradually from gateway 88, inner surface of container is tilted, thereby can make solution import discharge equably from each well of biochip.

Shown in Figure 17 (c), have self-existent well 91 with biochip 10 corresponding wells 16, can be connected the container of its bottom formation corresponding to the hole 92 of this well.The diameter in the hole 92 of this container bottom is according to preferred 100～500 μ m of the height in hole, more preferably 150～300 μ m.The aperture is during greater than 500 μ m, and liquid may flow out from container bottoms; The aperture is during less than 100 μ m, and it is big that the resistance when adding decompression becomes.

In above-mentioned container, be provided with in the biochip kit in footpath, make target substance B/F after separating in the analyte according to the particulate bonding probes, from particulate, separate the target material by any separation method, apply pressure reduction to upper container and following container then, the well 91 of the downward container of target material in the well 16 of biochip 10 is moved.For Xiang Gejing applies pressure reduction equably, upper container, down any one in the container be preferably formed have common in biochip the container of the receiving space of each well.

The volume of container can pass through, and as mentioned above, stacked any the plate with perforate adjusted to any volume.And at least one is a transparent panel in the bottom of container or the cap, during detection with transparent panel as the bottom, make particulate fill the filter hole, carry out optical detection by transparent panel; In addition, can also separate the target material liquid of being caught and move to the well of the container corresponding with the well of biochip by particulate.

Shown in Figure 18 (a), also can connect between the biochip 10 by the well of correspondence.And, shown in Figure 18 (b), between each biochip 10, the container with hole corresponding with these wells 93 can be set.

In each well of each biochip in multistage is disposed at container, when putting into particulate, separate, can improve the B/F separating power by the particulate that has probe of accommodating in the multistage chip being carried out B/F with same probe.And, respectively when first section and second section of multistage chip put into the particulate with different probe, can use so-called Two Step In Vitro Selection with instrument etc.

Figure 12 and Figure 13 be the expression biochip kit of the present invention an embodiment upper surface as and A-A ' sectional view.As Figure 12 and shown in Figure 13, biochip kit 14 has container 12 and biochip 10.

Biochip 10 is by a plurality of

wells

16,16 of cutting apart on sidewall 20 ... constitute.At these

wells

16,16 ... in, for example, the kind of collecting post carrying probe is at the different probe carrier particulate of each well 16.

The diapire 22 of well 16 is formed by filter 18.This filter 18 stops the probe carrier particulate that is contained in well 16, make simultaneously have use that this biochip 10 separates, analyte, the various damping fluid of detection etc.; Cleaning fluid; Separating agent; Labelled reagent; Secondary antibodies; Emulsion; The various solution of protein decomposition enzyme, ionization agent etc. pass through.

Can be to the above-mentioned various solution that the inside of container 12 imports by the whole unit of filter 18 discrepancy.That is, above-mentioned various solution can pass through filter 18, and by and the reception room 26 of above-mentioned various solution well 16 adjacency, that constitute by the gap between chip 10 and the container 12, from filter 18 to well 16 inflow and outflows arbitrarily.

Perhaps, sidewall 20 also can similarly be formed by filter 18.By the filter 18 that on this sidewall, forms, between the well 16,16 of adjacency, can move above-mentioned various solution.In this case, the filter that forms diapire 22 can form with identical material mutually continuously with the filter that forms sidewall 20, also can use the compound filter of the filter formation of unlike material respectively.

Container 12 and chip 10, shown in the (a) and (b) of Figure 12, can both are integrally formed with same material; Perhaps also can make chip 10 and container 12 integrated in container 12 inside by connecting and fixing both.

Perhaps, shown in the (a) and (b) of Figure 13, biochip kit 14 also can be respectively be made of the two of independently container 12 and chip 10, and chip 10 is contained in the inside of container 12.In this case, container 12 need be all not in full accord in various operations, can change its container and solution accepting container according to its unit operation.

In the biochip kit 14 of above-mentioned formation, the probe carrier particulate that is contained in each well 16 is specified the content of probe according to the position of its well, can a plurality of probes are contaminated and be housed on the chip.

Be contained in probe carrier particulate solution and the solution with analyte, various damping fluid, cleaning fluid, separating agent, labelled reagent, secondary antibodies, emulsion, protein decomposition enzyme, ionization agent etc., the well 16,16 that the probe carrier particulate can accommodated in the above-mentioned biochip ... between freely move.By the structure of suitable selection chip 10 or the stirring condition of above-mentioned solution, can make above-mentioned solution to

whole well

16,16 ... circulation, thus the chance that the probe of above-mentioned solution and the carrying of multiple particulate contacts or reacts given.

Whole wells

16,16 ... preferably by being formed with the filter of diapire 22 or sidewall 20, directly abut against the solution reception room 26 that the gap by chip 10 and container 12 constitutes.

This solution reception room 26 is, for example shown in the (a) and (b) of Figure 12, by the gap between the upper base surface of the bottom lower surface of chip 10 and container 12 constitute outside; Can also be shown in the (a) and (b) of Figure 13, by respectively independently the gap between chip 10 and the container 12 constitute.

According to above-mentioned formation, by stirring and adding decompression as required, make above-mentioned solution pass through one deck filter 18 from the solution reception room 26 that it is commonly accommodated, arrive a plurality of

wells

16,16 of having accommodated the particulate that carries the variety classes probe respectively side by side ... thereby, contact side by side, react.

And the unreacted analyte that does not react in specific well 16 is pressed onto also trial contact in the well 16 that reaches other by stirring or adding and subtracting again, reacts.Like this at each well 16,16 ... between, attempt making unreacted above-mentioned solution to contact, react one by one with the probe carrier particulate.

Then, because solution reception room 26 and each well 16,16 ... only pass through one deck filter 18 and adjacency, pressure loss minimum.And, because when arriving solution reception room 26, carry out side by side, thereby can make contact, reaction time the shortest with the reaction of probe carrier particulate in each well 16.

And pore aperture and its degree of depth by with its filter 18 of suitable condition enactment can drop to the pressure loss very low.Since just in the spatial movement that does not have filter 18,

whole wells

16,16 that above-mentioned solution can contain the probe carrier particulate ... and freely move between the solution reception room 26 that constitutes by the common chamber of above-mentioned solution.

Perhaps, can form such structure: first filter is set at bottom time, at the opposition side of first filter second filter is set, to clamp well.This is for example shown in (a) of Figure 14.Shown in figure,, upside filter 18 ' is set as second filter on the top of this biochip.This upside filter 18 ' is housed in the probe carrier particulate in the well 16 and installs afterwards.As its installation method can use that above-mentioned coupling agent, magnetic force, machinery are chimeric, the combination of even surface, machinery pressurization etc.And shown in Figure 14 (b), in filter 18 ' and the well 16 with bottom surface filter 18 any one is installed respectively on upper container 81 and following container 82 in advance, connect after upper container 81 and the following container 82, also can use machinery pressurization installation methods such as coupling agent, magnet, clamping.In the method, according to the setting of mechanical pressure/reset, upside filter 18 ' and bottom surface filter 18 at random can be cut off.Perhaps, shown in Figure 14 (c), have the well 16 of bottom surface filter 18, install on the contrary up and down on itself and the following container 82 upper container 81 setting, and any one combination that the well 16 of upper container 81 and following container 82 is passed through in the said method.

And, shown in Figure 16 (a), stacked have plate 87a through hole, level and smooth identical with the outer circumference portion shape of well and have the plate 87b of aperture than its little through hole, differential part in the hole is inserted chip, and with transparent panel 94 covering kits, with the hole of closing plate 87 with liquid access hole.In this case, for

example plate

87a, 87b are formed by silicon wafer, and transparent panel 94 is to form by grinding quartz glass, by having utilized the surface combination of flatness, do not use coupling agent also can form kit thus.

The method of operating of＜biochip kit 〉

In the method for operating of biochip kit of the present invention, make in the well that is housed in biochip the solution that is dispersed with the probe carrier particulate and; The various solution that for example have analyte, various damping fluid, cleaning fluid, separating agent, labelled reagent, secondary antibodies, emulsion, protein decomposition enzyme, ionization agent etc. that are housed in the container are in the possibility state of contact, by making the circulation of solution in the well and the solution in the container, thereby can make two solution mix expeditiously, spread, react, clean or separate.

As make above-mentioned two solution be in may contact condition method, can be biochip is moved up and down in container and to make its method that contacts with solution in the container (Figure 20 (a-1) → (a-3), Figure 20 (c-1) → (c-3)); With the solution in the container by geat 86 pressurization/decompression or, by inject method such as discharge solution from the outside, liquid level is moved up and down, thus the method (Figure 20 (b-1) → (b-2), Figure 20 (d-1) → (d-2)) that moves it in the well and contact with solution in the well.

Like this by pressurization/decompression or, make solution increase and decrease in the container by inject to discharge methods such as solution from the outside, and the liquid level of solution in the container is moved up and down, make under the state of microparticle residue in biochip that has probe, make solution and the interior solution integrator of container in the well, the solution in the well is moved inside and outside well.By this method of repeatable operation, can make the solution in the container mix expeditiously, spread, react, clean or separate with the interior solution of well.

Perhaps, shown in Figure 20 (e-1)～(e-5), be connected and installed with filter 18 ' upper container 81 and, the following container 82 of the well 16 that has filter 18 in the bottom is installed, and the geat 83,84 (Figure 20 (e-1)) that upper container 81 and following container 82 are added decompression and drop into discharge solution is set.Add decompression by geat 83, according to this plus-pressure or decompression power solution and the well 16 interior solution in the upper container 81 are contacted, and the solution that makes upper container 81 mixes (Figure 20 (e-2)) with solution in the well 16, moves it to descending container 82 (Figure 20 (e-3) again.Add decompression by geat 84 again, solution is discharged to container outer (Figure 20 (e-4)).And, can in well, fill other solution 85 (Figure 20 (e-5)).

In this case, the volume of upper container 81 and following container 82 is equated, the volume that the amount that preferably drops into solution compares each container up and down is big, when therefore container 82 moves solution downwards, upper container 81 also has solution residual, and air can not be entered in the filter, thereby can make plus-minus pressure minimum.

And when moving up and down above-mentioned well and mobile solution, move and do not carry out continuously, preferably in moving process, stop in required time, move off and on.Perhaps, preferably in the positive dirction moving process, the negative direction of interspersed short time moves.According to this method, the particulate in the well does not stick to filter, stops or negative direction moves in well and circulates by moving, and can make solution and particulate contact rate maximum in the biochip.

The input method of＜analyte 〉

Can drop into identical or different analyte to each well of biochip.When each well drops into identical analyte, can identical analyte be dropped into each well from each aboveground by point sample, or with the solution in the container as analyte, by said method it is contacted with solution in the well, can make that solution circulates, mixes, spreads, reacts in itself and the well.

The well quantity of chip is preferably used the method for the analyte in the container for a long time.According to point sample, point sample needs a large amount of point sample times one by one, then needs the point sample arranged side by side of a plurality of sample applicators.

And, when each well drops into different analytes respectively, from each aboveground with sample applicator etc. different analyte point samples is dropped into each well, or corresponding each well is accommodated different analytes respectively at container independently, and it is contacted with solution in the well by said method, can make that solution circulates, mixes, spreads, reacts in itself and the well.

Here can be various nutrient solutions as employed analyte, tissue, bacterium, virus, cell, lymph corpuscle, lipid, sugar chain, nucleic acid, urine, blood, serum, blood cell, people/mouse cell factor, serine/threonine, kinases, the promptly synthetic peptide of the protein of molecular weight 50～1,000,000, memebrane protein, enzyme, conversion of signals albumen, transport protein, range proteins such as phosphorylating protein, cell factor, lymphokine, antibody such as IgA and IgE, various antigens, transcription factor, the low molecule lead compound of molecular weight 50～1,000,000, matrix, mend enzyme, regulatory factor, hemagglutinin, hormone, neurotransmitters, antisense oligonucleotides, ribozyme, parts such as adaptive son, various medical candidate substances, have the various chemical substances that maybe may have physiologically active of physiologically active etc., but be not limited to above-mentioned substance.

And, when using the reaction of competitive hybridization or competitive immunometric assay, use analyte that contains the target material and the labelled analyte that contains standard substance simultaneously.

And, except that analyte, can also be by the different materials of said method input corresponding to each well, for example various damping fluids, cleaning fluid, labelled reagent, secondary antibodies, emulsion etc.

The target material in the＜analyte and the reaction method of probe 〉

In the target material in the analyte that has used biochip kit of the present invention and the reaction method of probe, react by the step of following (1)～(3).

(1) by above-mentioned any method specific particulate is contained in the well of biochip.And this step also can be adjusted by chip manufacturer when making chip in advance.

(2) in the well of biochip, drop into the solution that contains analyte,, make the above-mentioned particulate in this analyte and the whole well be in the possibility state of contact according to the aforesaid operations method.In this step, for example by well being moved up and down in the solution or making that the liquid level of solution moves in the container, make in the container solution integrator in the solution and well in container, carry out the target material in the analyte and the diffusion (and reaction) of probe.

(3) in the above-mentioned solution in being contained in the container of biochip, abovegroundly move down or the liquid level of the above-mentioned solution in the container that is contained in biochip is moved up and down, carry out target material and probe reaction in the analyte by making.In this step,, carry out the reaction of analyte and probe according to the aforesaid operations method.

＜B/F separation method 〉

Use the B/F separation method of biochip kit of the present invention to be undertaken by (1) in the above-mentioned reaction method～(3) step and following (4), (5) step.

(4) liquid level that reduces above-mentioned solution is to the filter lower surface that does not reach bottom, will not remove in each well with the analyte of the probe reaction of above-mentioned particulate carrying.

(5) cleaning fluid is dropped in the well of biochip, by said vesse cleaning fluid is recycled in the well of biochip, cleaning fluid is come in and gone out inside and outside the well of this biochip,, remove probe in conjunction with the material beyond the target material thereby clean by outside well, discharging cleaning fluid.

＜separation stage division 〉

Used the separation stage division of the target material in the analyte of biochip of the present invention to be undertaken by (1) in the above-mentioned reaction method～(3) step and following (4)～(6) step.

(5) cleaning fluid is dropped in the well of biochip, cleaning fluid is recycled in the well of biochip, inside and outside the well of this biochip, come in and go out, remove probe in conjunction with the non-target material beyond the target material thereby clean by making cleaning fluid by said vesse.

(6) corresponding with recess, protuberance or the partes glabra of the well sidepiece lower end that is arranged at above-mentioned biochip, protuberance, recess or partes glabra are set in the upper end, make container chimeric with above-mentioned part, then separating agent solution is dropped in the well of biochip, therefore the target material from above-mentioned separation of particles analyte, and move to the well of said vesse.

Below, an example for the separation stage division of analyte of the present invention specifies according to Figure 21～23.At first, each well 16 of each biochip is accommodated different types of particulate that carries probe respectively.

Then, shown in the (a) and (b) of Figure 21, in the container 12 of biochip 10, drop into the analyte solution that contains detected material 25, make the probe carrier particulates 24 in analyte and the whole well 16 be in the possibility state of contact.At this moment, well 16 interior liquid level of solution height must be not more than the height of sidewall 20.

Containing the method for the solution of analyte as liquid level input so in accordance with regulations, for example can be following method.First method, biochip kit 14 as shown in figure 13, independent chip 10 and the container 12 of using put into analyte solution to container 12 in advance, chip 10 is moved down and floods, and makes analyte solution enter method in each well 16 by filter 18.At this moment, exist under the situation of filter 18 pressure losses, make chip 10 and container 12 interconnective parts form thin slice, apply plus-pressure or decompression power, can make analyte solution pass through filter 18, and import in each well 16 to filter 18.

Second method, biochip kit 14 as shown in figure 12, use and to make the bottom surface of container 12 and the chip that filter 18 has certain distance in advance, by solution reception room 26, drop into the method for analyte solution from the introducing port of the low wall that is formed on container 12 or sidewall.

Analyte solution also can drop into from the peristome 17 of well, in this case, preferably drops into the solution of equivalent with sample applicator etc. to each well 16.And, with analyte solution, when for example dropping into, can use from this introducing port pressurization and send into analyte solution or reduce pressure and send into the method for analyte solution any one in solution reception room 26 1 sides by the introducing port 42 among Figure 21.

According to said method, can make analyte contact (Figure 21 (a)) with whole wells 16 interior probe carrier particulates 24.The method of mixing, spread, reacting as the probe carrier particulate and the detected solution that make in the well 16, under the situation that the sidewall 20 shown in for example Figure 12 (b) and Figure 13 (b) is made of filter 18 and each well of adjacency is cut apart by filter 18, by stirring analyte solution, can replace the solution in each well 16.As this stirring means, the vibration that can use the stirring machine that has stirrer paddle, produce by sound wave or ultrasound wave, by air or moving of normal magnetic and stirring etc. are stirred according to existing method.Wherein preferably use the vibration that produces by sound wave or ultrasound wave, can use container 12 or chip 10 vibrations or oscillator be put into any one of method of the analyte solution of chip 10.In this case, the suitable frequency of vibration can be regulated aptly according to the kind of probe or analyte, can use 100Hz～1GHz, preferred 1kHz～300MHz.In the frequency of this scope, impedance is very low, the damage of object can be dropped to minimum.When being suitable for frequency less than 100Hz, stirring capacity is low; When being suitable for frequency greater than 1GHz, possible breakdown diagnosis thing or probe.And container as shown in figure 13 12 and chip 10 are under the situation independently, with chip 10 after the liquid level of solution in the container 12 are mentioned, use in said method any one to stir these solution in the container 12, then chip 10 is immersed in once more in the solution of container 12 method or by rotation on surface level or move up and down the method that chip 10 relatively moves container 12 and chip 10, can replace solution in each well 16 by said method.When well 16 was cut apart by the sidewall that is not filter 18, the solution importing solution reception room 26 from filter 18 with diapire 22 imported solution in other the well 16 by filter 18 then, carries out the replacement of solution thus.

Perhaps, any chip among Figure 12,13 can import forced air from entrance hole 42, by improving the liquid level of the analyte solution in container 12, imports analyte solution to well 16.

Then, shown in Figure 22 (a), the liquid level that reduces analyte solution is to the lower surface that does not reach the diapire 22 of well 16, will not remove with the non-target material of the probe reaction of above-mentioned probe carrier particulate 24 carryings.Here, can use as the method that the liquid level of solution height is dropped to below the bottom surface of filter 18, fall analyte solution, the filter 18 by diapire 22 is by pump or add the method that modes such as decompression move outside container 12 or to solution reception room 26 from escape hole 44.And container 12 and chip 10 be under the situation independently, can use container 12 and chip 10 to relatively move and make method that filter 18 moves up and down or chip 10 is moved to and the container of biochip 10 adjacency or the method for other container independently.

In above-mentioned, using the open well of a side, and having under the situation of chip of opposed facing filter shown in Figure 14, needn't worry to leak particulate from well, the height that liquid surface can surpass well moves arbitrarily.

And, before or after carry out this operation, as required, also can drop into various clean-out systems and chemical reagent such as various damping fluid or secondary antibodies.These input methods can be identical with the input method of analyte.

Then, as shown in figure 23, prepare to have a plurality of containers 54 of accommodating well 52 corresponding to each well 16.Drop into the separating agent solution from the opening 17 on top to each well 16, from this target material of probe carrier separation of particles of the target substance reaction of analyte, and make the target material move to container 54.

This container 54 can be as shown in figure 24, to be provided with the container of trickle through hole 53 in the bottom surface of well 52.By such through hole is set, can make between the well 52 of the well 16 of chip and container and produce pressure reduction, can make the liquid in the well 16 of chip in the lump dirty.Carry out the method number of times identical, that move to the liquid of the well of container from the well of each chip by geat with the quantity of well, compare according to above-mentioned dirty in the lump with this method, can be with still less mobile number of times, can also prevent that the pollution that caused by geat etc. or analyte substance are owing to the loss that causes attached to geat simultaneously.

The diameter of the through hole that forms at this container bottoms is according to the height in hole, preferred 100～500 μ m, more preferably 150～300 μ m.The aperture is during greater than 500 μ m, and liquid may flow out from the bottom surface; The aperture is during less than 100 μ m, and it is big that the resistance when adding decompression becomes.

Separating agent solution can use sample applicator etc. from well opening 17, to each well 16 respectively or to each well 16 disposable input.

The container 54 that the solution that separated the target material is moved is not particularly limited to being provided with the corresponding respectively a plurality of target materials of each well 16 and accommodates well 52,52 ... container 54.For example, can have and well 16,16 being provided with ... the target material of same size is accommodated well 52,52 ... container 54, be positioned chip 10 under, separating agent solution is dropped into and with the probe carrier separation of particles of target material in this well 16 from well opening 17, and accommodate the target material that into is positioned under it and accommodate well 52.

Exist on bottom and top shown in the (a) and (b), (c) of Figure 14 of filter under the situation, also can be according to the step operation of said method.For example under the situation of the chip shown in Figure 14 (b), shown in Figure 20 (e-2), (e-3), carry out input and output by

geat

83,84 and add the decompression air, make the solution integrator in solution and the well 16 in the upper container 81, little by little move to upper container 81, time container 82.

Analyte according to the separated classification of said method is to use as the initial substance of following reactions steps or the refining body of test.Drop into the analyte of separated classification to each pick-up units such as mass analyzer, Raman analysis meter, surface plasma analysis meter, X-ray analysis meter, electrochemical detection device, quartz-crystal microbalances especially, according to the analytical approach of needs labelled reagent not, the content of Analysis and Identification analyte.

During according to these means analysis, by change reaction conditions such as temperature or, by repeatedly detecting in real time, can carry out dynamics (Kinetic) analysis.And, not only to analyze and whether exist individually and the content of analyte, the part of probe reaction, also to analyze the content of each particulate in each well 16, can obtain the concentration or the amount of the target material that contains analyte thus.

The detection method of＜analyte 〉

In the optical detection authentication method of the target material in analyte of the present invention, carry out with above-mentioned analyte in the target material and the reaction method of probe, or after the step identical, carry out following (1)～(3) step with the B/F separation method.

(1) drops into labelled reagent, labelled reagent is combined with probe and target material, clean afterwards and remove unconjugated labelled reagent.

(2) solution in the well is discharged outside well in well by the filter of bottom, the particulate in the well is positioned the pore of filter.

(3) detect reaction or the interaction that is carried between the probe of particulate and the target material in the analyte.

And, in so-called homology test method, can omit the B/F separating step.

Can use as the labelled reagent in the step (1), for example radioactive isotope, organic fluorescent, terres rares etc. are crosslinked is fluorophor, fluorescin, phosphor, utilize the luminous milimicron particle, chemiluminescence agent, enzyme luminous agent, gold of quantum effect or silver-colored milimicron particle etc.

But also can use secondary probes such as combining these secondary antibodies of missing dose or be used for the fluorescence molecule/delustring molecule of molecule labelling method, the raw material that is used for FRET (FluorescentResonance Energy Transfer) method and acceptor molecule etc.

In step (3), can detect for each particulate, the probe identifying information of particulate and; Probe that is carried by particulate and reaction or the interactional information between the target material in the analyte.And, particulate for each well, identification is carried on the identifying information of the probe of particulate, calculate the correlation behavior of the target matter interaction in measuring probe and the analyte then, according to the interactional status information of gained in the individual particulate, can calculate with the well is the interaction information of unit.

Below, be specifically described about an example of the detection method of analyte of the present invention.At first, each well is accommodated the particulate of different types of carrying probe respectively.When biochip of the present invention is used for the check and analysis thing, the particle number of each probe in each well preferred 1～1000 each, more preferably 1～500, most preferably 1～200.

Then, in the container 12 of biochip 10, drop into the solution contain analyte, make this analyte and each well 16 interior probe carrier particulates contact ((a) and (b) of Figure 21).

Then, the liquid level that reduces analyte solution is to the lower surface that does not reach the filter 18 of well 16, will not remove (Figure 22 (a)) with the analyte of the probe reaction of above-mentioned probe carrier particulate 24 carryings.

These steps are implemented according to the operation in the separation stage division of above-mentioned analyte.And, in removing the step of unreacted analyte, shown in Figure 22 (b), import cleaning fluid and carry out the particulate cleaning, carry out importing, stirring, the discharge of cleaning fluid as required repeatedly, from the probe carrier particulate, remove unreacted analyte.

Then, as shown in figure 25, the solution in the biochip is discharged by the bottom surface filter, the particulates in the well 16 are positioned to detect reaction or interaction between each well 16 middle probe carrier particles and the analyte after the hole portion of bottom surface filter 18.And, represent the probe carrier particulate is positioned actual conditions on the filter hole with electron micrograph, as shown in figure 28.

In the prior art, when detecting the reaction of solution middle probe carrier particles and analyte, part or all of the particulate that will react detects evaluation as an analyte, the liquid level that reduces analyte solution like this is to the lower surface that does not reach the filter 18 of well 16, according to detecting under the state that is positioned at the particulate that will have probe on the filter hole, can be with the particulate that all reacts as detecting the object of identifying one by one, thus reach the attempt that improves detection sensitivity.Figure 28 is positioned the probe carrier particulate for electron micrograph on the filter hole.

Detection method as analyte can be used normally used the whole bag of tricks, for example radioactive isotope detection, fluoroscopic examination, chemiluminescence detection, enzyme luminous detection, Raman detection etc., but be not limited to said method.

When detecting according to above-mentioned detection method, by change reaction conditions such as temperature or, by repeatedly detecting in real time, can carry out dynamics (Kinetic) analysis.And, not only to analyze and whether exist individually and the content of analyte, the part of probe reaction, also to analyze the content of each particulate in each well, can obtain the concentration or the amount of the target material that contains analyte thus.

And, for so-called chemical detection such as fluorescence radiation, chemiluminescence detection, from the container of accommodating the chip that constitutes by well, take out this chip, preferably separately chip is carried out the irradiation of exciting light, the detection of detection light.When chip was taken out from container, exciting light can shine from the upper surface of chip, any one of lower surface.And, detect light and also can shine from the upper surface of chip, any one of lower surface.

By taking out chip from container, get rid of distortion of vessel and from the fluorescence influence of container self, and owing to become near with the distance of object, it is big that the opening number NA in the time of can making optical detection (Numerical Aparture) becomes.

And transparent by container is made as shown in figure 16, needn't take out chip from container just can be with any one taking-up up and down from chip exciting light, the detection light.In this case, the thickness of the transparent panel 94 among Figure 16 and the thickness of plate 87b are approached as much as possible, can make NA become big thus.

And, at (b) of Figure 14 or in the chip (c), detect for being used to react the back, the upper container 81 that will have filter takes out, for down arranging the fine-grained following container 82 of filter down that has on the filter, can carry out rayed and detect from the upside open surface.

In above-mentioned detection step, each well is calculated the correlation behavior of the target matter interaction in measuring probe and the analyte, according to the interactional status information of gained in each particulate, can calculate with the well is the interaction information of unit, and above-mentioned situation is described.At first, calculate to measure each and be contained in the probe carrier particulate in each well the information that the interaction between probe and the analyte is relevant.As the means of carrying out this measurement, can use the whole bag of tricks of above-mentioned record.For example, when using fluoroscopic examination, irradiation is used to detect UV light or the visible light equal excitation light that is arranged in each particulate in the same well, the signals such as detection light that its result obtains with detections such as CCD camera, fluorescent tubes.This method is implemented respectively each particulate, and the particulate in other well is carried out this detecting operation similarly one by one.Then, these data that obtain for each well difference accumulated process, are carried out the statistical standard processing for the data of the particle number N in the well.And as required,, these data are revised or judgment processing with reference to more existing various data of database.

According to this method, the optical detecting method in the biochip of prior art for example to well or unit, with measure the fluorescence intensity of sending in a well or unit, the method for fluorescence spectrum is compared, owing to can obtain the information of the microparticle probes number N in each well, improve susceptibility more so the information quantitative change; Maybe can identify and carry out quantitative detection by the information distribution of a statistical treatment N microparticle probes.

And by changing reaction conditions such as temperature or repeatedly detecting in real time, in what is called action mechanics (Kinetic) was analyzed, by real-time tracing, the high reliability that can obtain a lot of parameter N was analyzed data for the detecting pattern of particulate or particulate in each well.

In biochip of the present invention, except with nucleic acid as the DNA chip of its probe by particulate carrying, also comprise with albumen such as antigen and antibody or with the chemical substance of these albumino reactions by particulate carrier protein molecule chip; Stop interactional chips such as albumen with the carrying low molecular compound; With the sugar chain chip of sugar chain by the particulate carrying; With the cell chip of cell by the particulate carrying.

According to the various probes that are fixed on the carrier, these DNA chips, protein chip, sugar chain chip and cell chip can be applied to: gene function analysis; Gene expression analysis; Functional protein group or structural proteins group; Be used to confirm the clinical examination of patient's situation of diseases such as function plug network nurse (selloum), structure plug network nurse, diseases analysis, various infection diseases; The many types of detection of gene; Be called as symptomatic treatment, selected for patient's gene order treatment medicine; The research of nucleic acid, protein, cell or sugar chain and chemical substance Study of Interaction, isolated receptor part; Be called pharmacy screening or chemical substance security and toxicity screening the pharmacology genomics, that be used for the pharmaceuticals exploitation; Other various screenings etc.And, by being used in combination specific analyte and specific probe, various tests such as enzyme test such as the interaction test, albumen, sugar chain, cell-part test, receptor-ligand test, kinases of immunity test, protein-DNA, protein-protein etc. are carried out in combination.

Below, the present invention will be described according to embodiment, and the present invention is not limited to these embodiment.

[embodiment 1]

Make filter and rib by electroforming

1. the manufacturing of filter

At stainless steel substrate (SUS304, size 120mm * 120mm * thickness 1mm) on, is 5 μ m painting erosion resistant agent THB-110N (trade name, JSR Corp.'s system) with rotary coating machine (Pin Coater) with thickness, carries out 120 ℃, 5 minutes preliminary drying on heating plate.Behind the preliminary drying, use mask aligner M-3LDF (trade name, Mikasa Co., Ltd. system) with 400mJ/cm ²The pattern of exposure sintering regulation make that not electroplate part residual.After imaging liquid uses PD523 (trade name, JSR Corp.'s system) to carry out video picture, on heating plate, carry out 90 ℃, 5 minutes post bake.

Then, to guaranteeing that the pH value is 4.0 to 4.5, keeps and bathe temperature and bathe at 50 ℃ nickel sulfamic acid and (bathe and form: 700g/l, 60% nickel sulfamic acid solution, the nickelous bromide of 5g/l, the boric acid of 35g/l, 1.5g/l the stress correctives, 2.5ml/l depression prevent agent) in, drop into SUS substrate with resist THB-110N system film, electric current and voltage is controlled at voltage 7V, current density 1A/dm ², applied direct current 30 minutes, the thickness 5 μ m that make, minimum-value aperture 3 μ m, minimum aperture spacing 7 μ m nickel films.

After the plating,, all peeled off the filter of resist by electroforming sample dipping in resist stripper THB-S1 (trade name, JSR Corp.'s system) was stirred 30 minutes.

2. the manufacturing of rib

On the nickel filter that obtains according to electrocasting, use step same as described above to make the filter that has rib by stacked rib.Device and electroforming condition are created conditions roughly the samely with above-mentioned, but will obtain the rib of thickness 50 μ m for the filter of thickness 5 μ m, use the rotary coating machine to be coated with the resist of THB-220N (trade name, JSR Corp.'s system), with 650mJ/cm ²Exposure expose.And for obtaining the nickel electroplating film that thickness is 50 μ m, the nickel that carried out 5 hours is electroplated.

[embodiment 2]

There is the silicon wafer of oxide film to make filter and rib by etching tape

1. the manufacturing of filter

Use spinner (CLEAN TRACK MARK8 (trade mark); Tokyo Electron society system) with resist IX1170G (trade name; JSR Corp.) on the silicon wafer that has 6 inches oxide films (thickness 2 μ m); with 3300rpm rotary coating 30 seconds; then at heating plate (CLEAN TRACK MARK8 (trade mark); Tokyo Electron society system) goes up with 90 ℃ of dryings 60 seconds, thereby obtain the diaphragm of thickness 0.86 μ m.Use reduced projection exposure device NSR-2205i12D (trade name to this diaphragm; society of Ricoh system; NA=0.57; δ=0.60) expose with the exposure of 0.4umC/H 1000msec, 0.8C/H 580msec after; use imaging liquid PD523AD (trade name, JSR Corp.'s system) to carry out PADDLE video picture in 1 minute with 23 ℃.Washed then 20 seconds, film figure is protected after the drying.

To through having on the silicon wafer that thickness is 2 μ m oxide films behind the system film, use plasma etching apparatus with CF ₄Plasma is used O after shining with the RIE pattern ₂Plasma is removed resist and is obtained being formed on filter on the oxide film, this filter has the filter bore dia and hole is 0.4/0.8 μ m, 0.8/0.8,2.8/1.0,2.8/2.0,2.8/2.8,2.8/4.0,4.0/1.0,4.0/2.0,4.0/4.0,4.0/6.0,4.0/8.0,8.0/2.0,8.0/4.0,8.0/6.0 μ m, highly is the hole of 2 μ m.

2. the manufacturing of rib

On the face opposite with the face that carries out filter processing; resist (THB-151N) is used spinner (rotary coating machine 1H-DX2 (trade name); Mikasa Co., Ltd. system) with 1700rpm rotary coating 20 seconds, obtains the diaphragm that thickness is 40 μ m with 120 ℃ of dryings 5 minutes with heating plate.After times projection aligner MA-150CC (trade name, SUSS MicroTec society system) such as this diaphragm use expose, use imaging liquid PD523AD (trade name, JSR Corp.'s system) to carry out PADDLE video picture in 90 seconds with 23 ℃.Washed then 30 seconds, film figure is protected after the drying.

To through having on the silicon wafer that thickness is 440 μ m oxide films behind the system film, use plasma etching apparatus with SF ₆Plasma is used O after shining with the RIE pattern ₂Plasma is removed resist and is obtained having the silicon wafer of well, and this aboveground being formed with on silicon highly is that 440 μ m, diameter are the hole of 500 μ m.Cut off the above-mentioned thing that makes with dicing saw, be formed in the square chip of 10mm with 28 wells.

[embodiment 3]

Prepare the solution with 10 times of cow's serum dilutions with PBS (Phosphate Buffered Saline).Using the filter aperture that obtains among the embodiment 2 is that 4.0 μ m, span are the chip of 2.0 μ m, in the container chip is being set shown in (b) of Figure 16, at loam cake pipe is set, connects the groove that is used to import the cow's serum dilution, the height of groove is arranged to have 4gf/cm at the other end of pipe ²And 18gf/cm ²Pressure reduction, the cow's serum dilution is flowed in a part of filter of chip container, discharge the cow's serum dilution from the lower cover of container down then.The discharge rate that day part adds up to and the relation of efflux time are as shown in figure 30.

Shown in same accompanying drawing,, in the assessment period, do not produce and stop up or destroy from the discharge rate constant of filter.

[embodiment 4]

For the 8 kind combinations different of the aperture of embodiment 3 chips, with 18gf/cm with span ²Pressure reduction carry out the test identical with embodiment 3.Its result as shown in figure 31.And in same accompanying drawing, a/r represents the open area ratio of filter.Test findings is: in the regional A of same accompanying drawing (open area ratio is less than 10%), filter stops up easily; In area B (open area ratio is greater than 60%), filter is destroyed.Therefore, open area ratio is 10%～60% o'clock, is not easy to take place to stop up and destroy.

Claims

1, a kind of biochip is characterized in that, has the well that the bottom is provided with filter, and described filter comprises the straight pore of arranging by impartial span with equal equal aperture.

2, biochip as claimed in claim 1 is characterized in that, the thickness of above-mentioned filter is 1～10 μ m.

3, biochip as claimed in claim 1 or 2 is characterized in that, the open area ratio of above-mentioned filter is 15～60%.

4, as any described biochip in the claim 1～3, it is characterized in that the Facing material of above-mentioned filter is silicon dioxide, titania or aluminium oxide.

5, as any described biochip in the claim 1～4, it is characterized in that having a plurality of above-mentioned well that is integral with each other.

6, as any described biochip in the claim 1～4, it is characterized in that having single above-mentioned well.

7, as any described biochip in the claim 1～6, it is characterized in that the upside of the filter in above-mentioned well or downside are provided with the reinforcement flank.

8, biochip as claimed in claim 7 is characterized in that, above-mentioned reinforcement is a shape that is formed with a plurality of through holes with flank.

As claim 7 or 8 described biochips, it is characterized in that 9, above-mentioned reinforcement directly combines with above-mentioned filter with flank.

As claim 7 or 8 described biochips, it is characterized in that 10, above-mentioned reinforcement flank extends to form with identical materials continuously with above-mentioned filter.

11, as any described biochip in the claim 1～10, it is characterized in that,,, the atresia portion that does not form the filter pore is set in its circumferential edges Rack in the bottom of above-mentioned well.

12, as any described biochip in the claim 1～11, it is characterized in that, when the bottom is provided with first filter,, second filter is set in the opposite side of first filter in the mode that well is clipped in the middle.

13, as any described biochip in the claim 1～12, it is characterized in that, in above-mentioned well, accommodate and have the dispersion liquid that has disperseed the probe carrier particulate.

14, biochip as claimed in claim 13 is characterized in that, the ratio in the particle diameter of above-mentioned particulate and the aperture of filter is particle diameter/aperture=1.1～2.5, and particle diameter＜span＜particle diameter * 10.

15, biochip as claimed in claim 13 is characterized in that, the particle diameter of above-mentioned particulate, the aperture of filter and span satisfy the relation of particle diameter＞aperture+span/2.

16, biochip as claimed in claim 13 is characterized in that, accommodates to have the dispersion liquid that has disperseed the probe carrier particulate in above-mentioned well, and this probe carrier particulate has at least a means of identification that is used to provide the probe identifying information.

17, biochip as claimed in claim 16 is characterized in that, above-mentioned means of identification is at least a for what select from color, shape, particle diameter and the gene order of probe carrier particulate.

18, as claim 16 or 17 described biochips, it is characterized in that, the a plurality of probe carrier particulates identical at the probe identifying information of whole above-mentioned means of identification are housed in the same well, and identical for the above-mentioned probe identifying information that is accommodated in a plurality of probe carrier particulates in each well.

19, as claim 16 or 17 described biochips, it is characterized in that, the a plurality of probe carrier particulates identical at the probe identifying information of whole above-mentioned means of identification are housed in the same well, and for the above-mentioned probe identifying information difference that is accommodated in a plurality of probe carrier particulates in each well.

20, as claim 16 or 17 described biochips, it is characterized in that, the a plurality of probe carrier particulates different at the probe identifying information of at least a above-mentioned means of identification are incorporated in the same well, and it is, identical in the formation of the above-mentioned probe identifying information of its whole means of identification for a plurality of probe carrier particulates that are accommodated in each well.

21, as claim 16 or 17 described biochips, it is characterized in that, the a plurality of probe carrier particulates different at the probe identifying information of at least a above-mentioned means of identification are housed in the same well, and for a plurality of probe carrier particulates that are accommodated in each well, in the formation difference of the above-mentioned probe identifying information of its whole means of identification.

22, a kind of biochip kit is characterized in that, has container, and this container or accommodate a plurality of wells or the single well that is integral with each other in any described biochip in the claim 1～21 perhaps is connected with above-mentioned well.

23, biochip kit as claimed in claim 22 is characterized in that, said vesse and above-mentioned well form as one.

24, biochip kit as claimed in claim 22 is characterized in that, said vesse and above-mentioned well form independently.

As any described biochip kit of claim 22～24, it is characterized in that 25, said vesse has and the corresponding well of the well of above-mentioned biochip.

26, biochip kit as claimed in claim 25 is characterized in that, at the bottom formation through hole of said vesse.

27, as claim 25 or 26 described biochip kits, it is characterized in that, above-mentioned biochip is connected with said vesse, so that corresponding well interconnects.

As any described biochip kit of claim 22～27, it is characterized in that 28, said vesse is to use the plate that is formed with through hole and does not form at least a in the plate of through hole, forms by stacked a plurality of above-mentioned plates.

29, a kind of biochip kit is characterized in that, any described a plurality of biochips of claim 1～21 is connected each other, so that corresponding well interconnects.

30, as any described biochip kit of claim 22～29, it is characterized in that, the par of upper end that is arranged on the well sidepiece of the par of well sidepiece lower end of above-mentioned biochip and said vesse that is arranged on separation or above-mentioned biochip directly is connected, well is interconnected.

31, as any described biochip kit of claim 22～29, it is characterized in that, well sidepiece lower end at above-mentioned biochip is provided with, with the recess of the location usefulness of the set protuberance tabling in the well sidepiece of said vesse that separates or above-mentioned biochip upper end; Or with the protuberance of the location usefulness of the set recess tabling in the well sidepiece of said vesse that separates or above-mentioned biochip upper end.

32, a kind of manufacture method of biochip, it is characterized in that, when making any described biochip of claim 1～21, for form with the material of different component at least by the two-layer plate that constitutes, carry out etching by etched pattern, be carved into two layers border and form wellhole, carry out etching by etched pattern simultaneously from a lateral erosion, etch into two layers border and form the filter hole from opposite side, obtain the biochip of well and filter combination thus.

33, a kind of manufacture method of biochip is characterized in that, when making any described biochip of claim 1～21, makes filter, rib and well respectively by the etching silicon wafer, and is then that it is stacked.

34, a kind of method of operating of biochip kit, it is characterized in that, in the solution of this container of any described biochip kit of the claim 22～31 that the well that is housed in container and biochip forms independently, by the aboveground of biochip moved down, interior solution of this container and well interior probe carrier particulate and/or solution are contacted.

35, a kind of method of operating of biochip kit, it is characterized in that, move up and down by the interface that makes the solution in the container that is housed in any described biochip kit of claim 22～31, interior solution of this container and well interior probe carrier particulate and/or solution are contacted.

36, a kind of method of operating of biochip kit, it is characterized in that, the any described biochip kit of claim 22～31 is had be used between the container that connects biochip and the chip or produce pressure reduction each other at the well of this chip, produce contact, liquid the moving between well of interior liquid of well and probe carrier particulate thus, or the two produces simultaneously.

37, a kind of method of operating of biochip kit, it is characterized in that, the any described biochip kit of claim 22～31 is had be used between the container that connects biochip and the chip or the well of this chip produces pressure reduction each other, produce contact, liquid the moving between well of interior liquid of well and probe carrier particulate thus, or the two produces simultaneously.

38, as the method for operating of any described biochip kit of claim 34～37, it is characterized in that, by the solution in the said vesse is contacted with probe carrier particulate and/or solution in the well, the content in the biochip is mixed, spreads, reacts, separates or cleans.

39, as the method for operating of any described biochip kit of claim 34～38, it is characterized in that, in each well of above-mentioned biochip, drop into identical analyte.

40, as the method for operating of any described biochip kit of claim 34～38, it is characterized in that, in each well of above-mentioned biochip, drop into the analyte that differs from one another.

41, the target material in a kind of analyte and the reaction method of probe is characterized in that, may further comprise the steps:

Specific particulate is placed in the well of the biochip in any described kit of claim 22～31, forms any described chip of claim 18～21;

In the above-mentioned solution that the biochip container is accommodated, make and abovegroundly move down or the interface that is housed in the above-mentioned solution in the biochip container is moved up and down, perhaps make solution circulation inside and outside the well, thereby target material and probe in the analyte are reacted by applying pressure reduction.

42, a kind of method that B/F separates the target material from analyte is characterized in that, may further comprise the steps:

Specific particle is placed in the well of biochip of any described kit of claim 22～31, forms any described chip of claim 18～21;

43, a kind of analyte separation stage division of material that hits is characterized in that, may further comprise the steps:

Specific particle is placed in the well of the biochip in claim 30 or the 31 described kits, forms any described chip of claim 18～21;

In the well of biochip, drop into cleaning fluid, cleaning fluid is circulated in the well of this biochip by said vesse, cleaning fluid is discharged in input inside and outside the well of this biochip, and discharges cleaning fluid from well, thereby cleans the material beyond the target material of removing the probe combination; And

44, the interactional detection authentication method of target material in a kind of analyte and probe is characterized in that, may further comprise the steps:

Specific particle is placed in the well of the biochip in any described kit of claim 22～31, forms any described chip of claim 18～21;

In the well of biochip, drop into cleaning fluid, cleaning fluid is circulated in the well of this biochip by said vesse, cleaning fluid is discharged in input inside and outside the well of this biochip, and discharges cleaning fluid from well, thereby cleans the material beyond the target material of removing the probe combination;

Particulate in the well is positioned in the pore of filter; And

45, the interactional detection authentication method of target material in the analyte as claimed in claim 44 and probe, it is characterized in that, for each detection of particulates two aspect information, be the probe identifying information of particulate, and reaction between the target material of probe that above-mentioned particulate carried and analyte or interactional information.

46, the interactional detection authentication method of target material in the analyte as claimed in claim 44 and probe, it is characterized in that, for the particulate in each well, discern the probe identifying information that above-mentioned particulate carries, the interactional state of the target material in relevant probe of instrumentation and the analyte then, with the interactional status information based on each particulate, calculating with the well is the interaction information of unit.