CN1406153A

CN1406153A - Linear probe carrier

Info

Publication number: CN1406153A
Application number: CN01805094A
Authority: CN
Inventors: 陈世平; 罗宇龄; 安东尼·C·陈
Original assignee: GenoSpectra Inc
Current assignee: GenoSpectra Inc
Priority date: 2000-01-10
Filing date: 2001-01-10
Publication date: 2003-03-26
Also published as: IL150434A0; KR20020089323A; JP2003519784A; AU2786801A; EP1248678A1; CA2397069A1; WO2001051207A1

Abstract

The invention relates to a probe carrier in which a flexible substrate carries a one-dimensiona configuration of probes wherein each different type of probe is attached to its own discrete portion of the substrate. The invention also relates to a probe carrier in which a flexible substrate such as a tape or fiber carries a two-dimensional configuration of probes. Furthermore, systems for fabricating and packaging flexible probe carrier threads are presented. Flexible probe carrier threads are packaged in forms of pins, rods, coils and spools to increase efficiency of hybridization and generate compact formats for transportation and use of probe carriers. Novel methods for hybridization of packaged probe carriers are disclosed. Methods for reading results of hybridization to packaged probe carriers are also disclosed.

Description

Linear probe carrier

The priority application that the present patent application requires is the U.S. Patent application 60/175 that proposed on January 10th, 2000,225, the U.S. Patent application 60/190 that proposed on March 20th, 2000, the U.S. Patent application 60/244 that on October 30th, 495 and 2000 proposed, 418, fully set forth as following, these applications are all incorporated text into as a reference at this.

Technical field

Generally speaking, the present invention relates to by being attached to the target analysis field measured on the probe (as what in dna sequence dna is identified, found usually).The invention still further relates to the immobilized nucleic acids probe in the on-chip arrangement of solid phase.More particularly, the present invention relates to the packing of probe carrier fine rule, its middle probe is fixed on the flexible carrier separately with the form of array.

Background of invention

The evaluation of molecular structure had become extremely important already in research and many industries, and the analysis of biomolecule such as nucleic acid and protein has been formed the basis that clinical diagnosis is measured.The method that is adopted generally include the considerable consumption plenty of time repeating step (referring to, for example, Sambrook, people such as J., molecular cloning: laboratory manual (Molecular Cloning:A Laboratory Manual).Publishing house of cold spring harbor laboratory, cold spring port, New York (second edition 1989).The test site array that exploitation forms on planar substrates provided more simple and rapid molecular analysis methods already.Each test site all comprise be applied to device on the probe that combines of sample.Such probe can be oligonucleotides, protein, antibody or cell binding molecule, and being chosen in of probe only is confined to the possibility that combines or react with the sample specificity in theory.Test sample and probe combine and identify this probe, identify sample whereby.At present mainly be around two-dimensional planar array particularly oligonucleotide arrays should be used for development technique, and these arrays are enough small and dense, are enough to be called microarray.

With the ability that effective and economic mode is produced microarray, be that the researcher in the global range is quite interested, and have important commercial value.How to say again and also can not exaggerate microarray technology for biotechnology industry and for the importance of whole sanitation and health-care field.Microarray can significantly improve the efficient of traditional biological chemical experiment.Originally just can finish in the test that may spend several years several hours now even a few minutes.This The Application of Technology not only has influence on sanitation and health-care field, but also has influence on Genotyping, medical diagnosis on disease, drug discovery, the science of law, agronomy, biological warfare, even biocomputer.Various types of microarray production equipments and technology were described already.

Present technological development direction remains the denser two-dimentional probe array of preparation on rigid substrate.But there are many problems in this approach.At first, along with increasing of test site in the array, the complicacy of making an array or a plurality of arrays has also increased greatly.Secondly, will be placed into as the biomolecule of probe conventional method (photolithography, mechanical deposition art and ink-jet art) on the fc-specific test FC site consuming time, expensive, often lack required accuracy and can not satisfy required size restrictions.The original position lithoprinting of probe is synthetic to be a kind of tedious technology that satisfied accuracy can not be provided and have narrow probe length.The mechanical deposition art is a kind of slow technological process, and wherein Zui Xiao test site size is subjected to the restriction of this technological property.Chemistry ink-jet art have with the synthetic similar inaccuracy of original position and with mechanical deposition similarly to the restriction of test site size.The 3rd because make the required complicacy of each array and high accuracy and output lower, so the manufacturing cost of each array is very high, making the array that contains the probe that is enough to the evaluate complicated biological sample often needs several thousand dollars.The 4th, quite high cost and the complicacy of basis that is used for the fetch equipment of detector probe-sample crossbred, all increase along with the increase of array density, and because this fetch equipment must carry out two-dimensional scan with very high spatial precision (10 μ m level), so the processing time of each scanning also prolongs along with the increase of two-dimentional probe array density.

In addition, the basic principle of operation of microarray comprises and makes the specific molecular reaction that is fixed in on-chip probe and the sample liquid.Hybridization need provide enough chances that runs into its complementary molecule for probe.In existing system, pass microarray by diffusion or driving sample liquid and can realize this point.The former is a stochastic process, and the latter needs complicated micro-fluidic system.

Therefore, it is a kind of easy to need, the quick cheap probe carrier that makes up, this carrier can hold thousands of or hundreds thousand of probes, can suppress and store and use, can produce with higher productive rate, be easy to make probe/target to interact expeditiously and need not expensive and high-precision fetch equipment, can be on probe carrying substrates itself each probe or probe groups for details, the probe that can hold different length and complexity in the customization group, and this carrier is pressed, be easy to use and cheap carry out disposable use to the pin-point accuracy that is enough to final formation.

The improved packing that also needs these probe carriers required hybridization liquid measure minimum and a large amount of probes can be fixed on the substrate, and size can not increase thereupon as two-dimentional standard gene chip matrix is required.

Summary of the invention

The invention provides a kind of new direction and approach for preparing probe carrier or probe configurations, it need not to set up dense two-dimensional symmetric array on rigid substrate, also can not limit the size that can be attached to on-chip probe inherently.In addition, by using the technology of similar streamline, can quite easily make product of the present invention.

The invention provides a kind of probe carrier, in this carrier, on a strip flexible substrate, multiple probe stationary is in different zones, there is a kind of probe in each zone, the length of substrate is approximately 5: 1 at least with the ratio of width, be at least 50: 1, be at least 500: 1, be at least 10,000: 1 or be at least 100,000: 1.In one embodiment, each length that contains the zone of probe is no more than 1000 microns, in another embodiment, each length that contains the zone of probe is no more than 500 microns, and in yet another embodiment, each length that contains the zone of probe is no more than 100 microns, in another embodiment, each length that contains the zone of probe is no more than 50 microns, and in yet another embodiment, each length that contains the zone of probe is no more than 20 microns.

The present invention also provides a kind of probe carrier, in this carrier, on a flexible substrate, multiple probe stationary is in different zones, and there is a kind of probe in each zone, and the length of substrate is approximately 5: 1 at least with the ratio of width, wherein substrate has superficial layer, and these probe stationary are on laminar surface.On the other hand, the present invention also has the second layer between ground floor and described substrate.In one embodiment, ground floor comprises silica, and the second layer comprises a kind of metal material.

The present invention also provides the form with ranks to be fixed on the lip-deep linear one dimension probe arrangement of flexible substrate, and the linear density of its middle probe is that every linear centimeter surpasses 10 kinds of probes, and perhaps preferably every linear centimeter is 50 kinds of probes.Another aspect of the present invention is, the linear density of probe is that every linear centimeter surpasses 100 kinds of probes, and another aspect of the present invention is that the linear density of probe is that every linear centimeter surpasses 200 kinds of probes, of the present invention is that the linear density of probe is that every linear centimeter surpasses 500 kinds of probes more on the one hand.

In addition, the invention provides the probe of various fixed on the zones of different of flexibility band shape substrate surface, there is a kind of probe in each zone, and wherein the thickness of this band is no more than 500 microns.Another aspect of the present invention is, the thickness of this band is no more than 100 microns, another aspect, and the thickness of this band is no more than 20 microns.

The present invention also provides the probe of various fixed on the zones of different of flexible fiber substrate surface, and there is a kind of probe in each zone, and wherein the diameter of fiber is no more than 500 microns.Another aspect of the present invention is, the diameter of fiber is no more than 200 microns, another aspect, and the diameter of fiber is no more than 100 microns, and on the one hand, the diameter of fiber is no more than 20 microns again.

All above-mentioned aspects of the present invention also comprise first label and second label that transmits relevant second group of detecting probe information that transmits about first group of detecting probe information.In some embodiments, these labels are for example optical bar code or fluorescent markers of optical markings thing, and in another embodiment, these labels can be magnetic.In comprising an embodiment of label, probe is polynucleotide, in comprising another embodiment of label, probe is a polypeptide, in comprising another embodiment of label, probe is an antibody, and in comprising another embodiment of label, probe is selected from cell surface receptor, oligosaccharides, polysaccharide and lipid.

And in all above-mentioned embodiments, no matter whether they comprise label, and this device all also comprises ground floor, and probe stationary is on this layer; This layer can be made of silica.Or this device comprises the ground floor and the second layer; Ground floor can be a silica, and the second layer can be a metal material.If the second layer is a metal, it still is magnetizable so.

In all above-mentioned embodiments, linear structure that probe can be selected or bar (with the major axis of substrate with an angle) linear structure arrange.

And in all above-mentioned embodiments, these probes can be polynucleotide or polypeptide or antibody or part or be selected from cell surface receptor, oligosaccharides, polysaccharide and lipid.If probe is polynucleotide, they can be DNA so, and if DNA, they can be single stranded DNAs so.

Being used for substrate of the present invention can be quartz glass or plastics or metal material or polymkeric substance, and if polymkeric substance, this polymkeric substance can be selected from polyimide and teflon so.Preferred substrate is an optical fiber.If substrate is a metal material, it can also have a layer between substrate and probe so, thereby makes probe stationary on this layer; This one deck can be a silica.And metal substrate can magnetize.

The present invention can be twined around a rotary drum or a plurality of rotary drum.It can also have or not have on the horizontal support and self is wound up as flat spin one, but also can be fixed on the reel at flat spin center.In addition, the outermost end of the substrate in this spiral can be extended and be fixed on second reel.

A different aspect is, the present invention includes a kind of being used for multiple probe liquid is transported on the substrate so that print the device of probe array.Generally speaking, this device comprises reservoir and the one group of kapillary with a plurality of holes, layout wherein capillaceous makes each end capillaceous all link to each other with a hole in the reservoir, thereby the content in the hole can enter kapillary, and the other end capillaceous is arranged as flat single ranks.In an embodiment of this device, reservoir is a plurality of holes on the titer plate.When using this device of the present invention,, probe liquid is moved in the kapillary in the hole of reservoir by applying a pressure reduction between reservoir and the kapillary and/or a voltage being provided between reservoir and substrate.Kapillary can positioned parallel also can pass the longitudinal axis of strip probe substrate, so that row's probe is deposited on the substrate.Below also want other method of more detailed description probe deposition.

Second kind of probe conveying device has the row's probe container that constitutes in the mode that is similar to travelling belt.This row's container moves with speed and direction, thereby intersects with the substrate that moves with another speed and direction, thus probe is side by side deposited on the substrate.In another kind of configuration, this row's probe container moves, thereby intersects with the sample applicator of being made by the flexible material bundle that a row moves, and each sample applicator intersects with a container thus, thereby probe is transported to sample applicator from container.Conveyor for example also can mobile substrate, thereby that row's sample applicator that makes substrate and carry probe intersects, and each sample applicator is depositing to this probe on the substrate after the pickup probe from container thus.This row's sample applicator can be built into a loop, and these sample applicators can wash at wash plant after depositing to probe on the substrate and before returning container.

The present invention also comprises and some probe is deposited to method on the substrate surface from fluid delivery system.In one approach, probe is coated with slivering on substrate.This probe can be carried on the thin resilient flexible sample applicator, and this sample applicator is with brushing motion contact substrate surface, so that scribble this band.In one embodiment, this sample applicator can be quartz capillary or fiber.Additionally, kapillary or fiber can be made by other material such as the metal that can carry the fluid that contains probe, pottery, polymkeric substance or other material.In other method, these probes as a plurality of or the point deposit in non-contacting mode.These methods comprise magnetic, the electricity, heat, sound with inkjet deposited.In the magnetic sedimentation, probe is attached on the magnetic bead.When carrying the sample applicator that is fixed on the probe on the magnetic bead and intersect with substrate, the electromagnet that is placed on below the substrate is activated.The magnetic field that is produced by electromagnet attracts probe to come out from fluid delivery system, thereby probe is deposited on the surface of substrate.In electro-deposition method, the voltage of suitable polarity is applied between substrate and the conveyer, setting up an electric field, thereby charged probe (for example oligonucleotides) is shifted onto on the substrate surface.In the heat deposition method, quick spot heating is introduced in the fluid passage, thereby produce fast local volume expansion (bubble) probe liquid is shifted onto on the substrate.Can utilize resistance heater wire in the mode of electricity or utilize suitable laser to introduce Fast Heating in the mode of light.In the sound sedimentation, ultrasonic pulse is incorporated in the probe liquid, thereby drop is shifted onto on the substrate surface.In inkjet deposition process, in the probe liquid container, set up a piezoelectric actuator.This piezoelectric actuator is activated by voltage signal, can reduce volume of a container fast, thus probe is shifted onto on the substrate surface.

In another replacement method, utilize the probe precipitation equipment can realize the form of probe with bar is coated on the substrate, in this device, fiber matrix is impregnated in the corresponding matrix of reservoir, each reservoir all contains probe, fiber matrix moves through the first of substrate then, and fiber makes one of each fiber laydown cross the line probe of separating of substrate with the position of substrate, and required substrate is online and line between.In the method, fiber matrix can wash and be impregnated in another matrix of reservoir, moves through another part of substrate then, thereby deposits another group probe bar.

In all said methods, substrate can be a plurality of fibers that are arranged in parallel, thereby a path of several fiber utilization probe precipitation equipments receives probe, perhaps this substrate can be a band, wherein, after the probe deposition, this band can carry out optic cutting along its major axis, thereby produces a plurality of bands that carry probe.

In all said methods, these probes can be received on the substrate by covalent bond.

In all said methods, also can comprise label is applied to on-chip step.

In other factors, the present invention is based on such scientific discovery: promptly, the probe carrier that has the one dimension probe configurations on flexible substrate provides a kind of easy, economic, reliable and special mode of classifying of identifying that target molecule exists in the sample.And the probe on probe carrier of the present invention is not subjected to size restrictions.By the discussion of this paper, these scientific discoveries of the present invention and advantage and further feature all are conspicuous.

The present invention includes the hybridization of a kind of new improvement and target thing or with the mode of the efficient of target thing reaction.This method comprises that the traveling probe carrier makes it to pass sample liquid, thereby has strengthened the chance that the probe that is fixed on the carrier mixes with its target molecule in the sample liquid.Carrier can adopt various forms, and these forms comprise fine rule, band, sheet, coil, rotary drum or pin.This forms of motion comprises translation, rotation or the vibration of probe carrier, and both independent a kind of forms of motion also can be the associating of several forms of motion.

The present invention also comprises several designs of hybridization device, wherein probe carrier is inserted and contains the indoor of sample liquid.Make the slit minimum between probe carrier and the chamber interior walls, to reduce the volume of sample liquid.Drive probe carrier, make it to move, thereby improved probe/interactional efficient of target thing indoor.The another kind of method that strengthens hybridization is included between the wall of probe carrier and hybridization chamber and applies a voltage.At a utmost point, electric field attracts the probe place of target molecule on carrier, thereby has increased the local concentration of target molecule and improved the possibility of hybridizing.At antipole, electric field repels target molecule from probe, thereby helps to increase the specificity of hybridization.By with suitable frequency shift polarity, can improve the hybridization efficiency between target thing and the probe.

The present invention can be used in conspicuous other application of evaluation, sequencing, sequence conclusive evidence, medical diagnosis on disease and those skilled in the art of known point mutation analysis, expression analysis, genomic fingerprinting, polymorphism analysis, linkage analysis, mRNAs and mRNA colony.

The accompanying drawing summary

Fig. 1 shows an embodiment of probe carrier, and its middle probe is fixed on the substrate with the form of selecting, and carrier also carries the label of optical bar code form.Probe can also a plurality of or the form of arranging a little more fix, but and label can be optics, magnetic or any other identification marking;

Fig. 2 a is the sectional view of probe-carrier, and its middle probe is fixed in the groove of carrier; Fig. 2 b is the sectional view of two layers of probe-carrier, and its middle probe is fixed in the groove of carrier, and this figure has set forth fixing probe location is how to make it avoid with following one deck friction taking place in the groove.

Fig. 3 shows the apparatus and method of making probe carrier, and wherein a plurality of pipes are transported to probe substrate and with the form of bar probe " are coated with " on substrate from reservoir.

Fig. 4 schematically shows out a kind of method of making probe carrier, and wherein discrete probe container passes one or more substrates and when each probe container passes through on substrate probe deposited on the substrate.

Fig. 5 shows the polytype probe container that can be used in the aforementioned manufacturing technology, and every kind of probe container is used for probe is deposited to on-chip device from carrier.

Fig. 6 a-6e shows the several different methods of making probe carrier.Fig. 6 a shows its middle probe and is included in a kind of method of making probe carrier in each reservoir with liquid form.Fig. 6 b shows a kind of method of making probe carrier, and the moving belt that wherein has a plurality of sample applicators intersects with reservoir, thereby each sample applicator all picks up a kind of probe of separation.Fig. 6 c shows a kind of method of making probe carrier, wherein has a plurality of moving belts that are combined with the sample applicator of probe and deposits on it mutually and with probe with one or more substrates friendships.Fig. 6 d shows a kind of method of making probe carrier, wherein a plurality of sample applicators can be arranged in a continuous loop, and each sample applicator can be washed and be reused for the new probe that point sample is provided by the reservoir array in this loop.Fig. 6 e has drawn a kind of method of probe being transferred to sample applicator from reservoir.In this structure, sample applicator moves below substrate and with the substrate surface location that faces down, thereby makes sample applicator deposition probe below substrate.

Fig. 7 a and 7b show the another kind of method of structure probe carrier, wherein the matrix of sample applicator is impregnated in the corresponding matrix in hole, a kind of probe is all contained in each hole in the matrix, secondly the sample applicator matrix was brushed one or more substrates with the angle of the line of a separation of each sample applicator deposition, the sample applicator array can be washed and move in the new probe matrix that contains a plurality of holes then, and this cycling of repeated impregnations-brushing-washing on one of substrate new cross section part.

Fig. 8 shows the structure of probe carrier pin and probe carrier rod.

Fig. 9 shows the manufacture method of probe carrier pin and probe carrier rod.

Figure 10 a is the vertical view of the flexible stylet carrier of coil configuration.Figure 10 b is the side view of the flexible stylet carrier of coil configuration.Figure 10 c is the sectional view of two adjacent windings of probe-carrier fine rule, wherein in the groove of probe stationary on carrier.

Figure 11 a shows the flexible stylet carrier that is packaged in the reel structure in the small box.Figure 11 b is the sectional view of two layers of the probe-carrier of reel form, and wherein in the groove of probe stationary on carrier, the probe location that the figure shows the groove internal fixation is how to make it avoid rubbing with following one deck.

Figure 12 shows a kind of method of utilizing the hybridization of electric field controls probe carrier.

Figure 13 shows a kind of method of probe carrier pin hybridization.

Figure 14 shows a kind of method of utilizing probe carrier to carry out parallel hybridization at a plurality of target samples on the standard microtitre plate.

Figure 15 is the viewed view that is used for the hybridization device of probe carrier rod of major axis along rod.

Figure 16 is the side view that is used for the hybridization device of probe carrier coil.

Figure 17 a and 17b show the hybridization device that is used for the probe carrier reel.

Figure 18 shows the instrument that reads that is used for scan-probe carrier pin or probe carrier rod.

Figure 19 shows the instrument that reads that is used for scan-probe carrier coil.

Figure 20 shows the instrument that reads that is used for scan-probe carrier reel.Detailed Description Of The Invention

1, probe carrier device

The A general description

Utilize the one dimension probe array can easily carry out the scanning and the imaging of microarray, this is because such array need not the required high precision of two-dimensional imaging.In following document, can find and utilize the many devices that are attached to the polynucleotide on the optical fiber: people such as " biological nucleic acid sensor diagnostic method " Krull, WO#98/58079 and WO#95/26416; People such as " the fiber optics biology sensor that is used for the oligonucleotides sample of selectivity detection fluid-mixing sample " Walt, WO#98/50782; " be used to detect and measure the analytical approach of specific sequence nucleic acid " people such as Sutherland, EP#0245206; " gene probe method for biosensor " Squirrel, WO#93/06241; " nucleic acid determination method " Hirschfield, US 5,242, and 797; People such as Piunno are used for the fiber optics DNA sensor of fluorescent core acidity test, analytical chemistry (Anal.Chem.) 67:2635-2643,1995; People such as Uddin are used for the fiber optics biology sensor of fluoroscopic examination triple helical DNA, nucleic acids research (NucleicAcids.Res.) 25:4139-4146,1997; People such as Abel are used to detect the optical fiber evanescent wave biosensor of oligonucleotides, analytical chemistry 68:2905-2912,1996; People such as Kleinjung are used for the fiber optics gene sensor that specific assay flies the DNA oligomer of mole, analytical chemistry journal (Anal.Chim.Acta) 150:51-58,1997; People such as Zhang are used to detect the chemiluminescence fiber optics biology sensor that DNA is hybridized, and analyze communication (Anal.Lett.) 32:2725-2736,1999; People such as Ferguson are used for the fiber optics DNA biology sensor microarray that analyzing gene is expressed, Nature Biotechnol (Nature Biotech.), 14:1681-1684,1996.

Yet these devices only generally comprise a kind of probe molecule sequence are attached on the single fibre-optic glass surface.People such as Krull (WO#98/58079) had set up the theory of using more than a kind of not differential potpourri of probe type already, yet, the inorganization distribution significant limitation of probe molecule the number of the different probe sequence in these technology, the inorganization of probe molecule distributes needs each single probe molecule to come mark (people advised as Krull etc.) by for example fluorescent marker, so that identify this probe molecule and the adjacent thing (can be to have not homotactic probe) at it and its one's own department or unit is distinguished.In addition, former method has only been used short fiber (several centimetres or still less), thereby defines number and the kind of the probe that can fix.At last, these former technology are utilized the optical fiber that is fixed with probe on it, and light is conducted to hybridizing label (being generally fluorophore) or the conduction light from the hybridization label.This detection technique depends on and fades luminously from fibre-optic, and therefore this luminous being confined to inherently and fiber surface adjacent areas tightly can not distinguish that many group probes also are restricted in sensitivity.And, utilize optical fiber itself to excite and launch light and limited and utilize optical fiber separately as the substrate that is fixed with probe on it, and hinder and use other substrate for example metal wire or polymkeric substance, and these substrates have the ability of the information of relevant each probe of other advantage such as carrying or probe groups and the advantage of hybridization aspect.

" genetic chip " technology that forms the two-dimensional points matrix with the dna probe of being set up on plain film is different, and " probe carrier fine rule " is along the thin flexible thin wire of the certain-length form stationary probe with one-dimensional array.Probe carrier fine rule system is made of following three fundamentals: the structure of probe carrier fine rule and manufacturing, hybridize and read.The packing of improved probe carrier fine rule by utilizing probe carrier pin, probe carrier rod, probe carrier coil and probe carrier reel technology has increased the density of probe and has strengthened the intrinsic advantage of probe carrier fine rule technology." flexibility " used herein, being meant can be crooked, twine, coiling or bend to the required degree of operation of the present invention in addition and can not fracture.

As shown in Figure 1, in one embodiment of the invention, probe is selected (110) as long and thin flexible substrate (100) center a plurality of or is fixed as the fillet (referring to Fig. 4,404) that runs through substrate (100) width.Or the point that probe can be used as the plurality of continuous ranks fixes, and the major axis of described ranks and substrate is an angle.The length of substrate and the ratio of width are approximately 5: 1 at least, preferably are at least 50: 1, more preferably are at least 500: 1, most preferably are at least 10,000: 1.Substrate contains the length of part of probe and the ratio of width is approximately 5: 1 at least, preferably is at least 50: 1, more preferably is at least 500: 1, most preferably is at least 10,000: 1.The characteristics that the ratio of these length and width, the flexibility of substrate and probe are located with the arrangement of one dimension or approximate one dimension make the method for manufacturing, use and analysis probe carrier novel and easy.

" a kind of probe " used herein is can combine with specific sample or sample segment generation specificity or a kind of molecule of specific reaction or a kind of one group of copy of molecular structure.This group can contain the molecule or the multimolecular structure copy of any number." multiple probe " used herein is meant more than a kind of such molecule or multimolecular structure series.These molecules or multimolecular structure can be the combinations of polynucleotide, polypeptide, oligosaccharides, polysaccharide, antibody, cell receptor, part, lipid, cell, micromolecule (in the screening medicine such as being used for sieving medicine) or these structures, other structure that perhaps arbitrary and interested sample or interested sample segment specificity combine or react.These probes can be fixed on the substrate by the mode of covalently or non-covalently adhering to." flexibility " used herein is meant can be crooked, twine, coiling or bend to the required degree of operation of the present invention in addition and can not fracture." width " of substrate is defined as the whole vertical extreme length of the major axis with substrate that is included in the substrate." width " that contains cylindrical part such as the fine rule substrate of probe is defined as the linear range of the long arc in the major axis part that contains probe vertical, that be included in substrate with the part that contains probe of substrate.The length of the part that contains probe of substrate be along the major axis of substrate from substrate on first kind of probe a kind of linear range of probe to the end of multiple probe, perhaps, if have sizable interval forming between the probe of probe groups, length is the first kind of probe a kind of linear range of probe to the end in this group probe so.

The same with the conventional two-dimentional microarray of arranging in the plane, device of the present invention is used for coming analytic sample by following steps: 1) at first, with with sample or sample fragment in conjunction with or the probe of reaction separate then 2 with the probe region that does not combine sample) evaluation is in conjunction with those probes of sample.

The alternate manner of identifying probe is to utilize to identify single label of planting probe or probe groups.Such label can be used to transmit more information, and not merely is to identify probe.

This length of probe carrier, the thin and flexible character of tool make and itself are suitable for many new mode of loadings and purposes.Can many forms pack probe carrier, these forms include, but is not limited to: pin, rod, coil and reel.Owing to only need few hybridization solution and strengthened mixing, therefore significantly strengthened hybridizing method.Needing large volume, be low to moderate in the application of medium scale microarray and have superiority especially with the flexible stylet of reel packaged, such as related those in the medical diagnosis on disease of large hospital and the treatment.In these are used, number of probes required in the array less (in hundreds of arrives several thousand scope), but can consume the array of considerable same type every day.Utilize this flexible stylet carrier format, tens thousand of copies of some groups of identical probes are along the continuous length repeated arrangement of fine rule and be sealed in a big coil or the reel.The use of full automatic system is integrated with the equipment (such as hybridization station and scanner) in each stage of analytic process.This machine in whole process with full automatic mode suck patient the DNA sample, send the flexible stylet carrier to and produce analysis result, and need not artificial intervention.

B specifically describes

1.1 substrate

Substrate of the present invention can be made by different materials.The requirement of substrate is, its have enough flexibilities bear the manufacturing of this device and use in required structural change, and it can fix particular probe to be used or can modification (for example, by coating), so that can carry out so fixing.Substrate also can comprise the different layer of being made by different materials, and each layer all has function in this device.

Specific embodiment needs flexibility in various degree.Can measure flexibility by bearing the ability that is wound into a certain diameter (for example, the diameter of 10cm, 5cm, 2cm, 1cm, 0.5cm or 0.1cm).The preferred material that is used for substrate of the present invention is the polymkeric substance that quartz glass, metal material, plastics and intensity are enough to bear manufacturing and use.

For fixedly polynucleotide and polypeptide, silica is that pure glass is preferable material, and this is because polynucleotide and polypeptide can be covalently attached on the glass surface of handling, and silica sends the fluorescence noise signal of minimum.Silica can be the one deck on another kind of material, perhaps can be the core or the base material substrate of this device, and perhaps the two has concurrently.One embodiment of the invention comprise utilizes metal wire as core substrate, and is coated with the silica that is used for fixing probe thereon.Another embodiment comprises utilizes plastics or polymer belt as basic unit's substrate, and coating thereon is used for fixing the silica of probe.In this embodiment, can add another metal material layer, this layer both can also can be clipped between silica layer and polymkeric substance or the plastics in the side opposite with silica layer of band.Another embodiment of the present invention is, has the layer of metal material and the silica fibre of another silica layer is arranged on metal material on silica nuclear; Probe can be fixed on the silica layer of outside.

Optical fiber.The probe carrier fine rule can be made by different materials.Preferable material is a silica, and this is because DNA can be covalently attached on the glass surface of handling and silica sends minimum fluorescence noise.With glass is that this common understanding of rigid breakable material is opposite, and the fiber of being made by silica is flexible and has bigger elastic strength.For example, the optical fiber that is used for the radio communication industry of large-scale production is at present made by silica.Optical fiber is the substrate material of mainly being made by silica and satisfies necessary requirement.Fiber although it is so is to make for the purpose of transmitting light, and the present invention does not need this character (though this character is used in some embodiments) of fiber.On the contrary, be that fibre-optic other character makes it have special superiority for the present invention.The fibre-optic physical strength that records is 7Gpa, be approximately 4 times of the strongest steel, and weight only is 1/6 of this steel.Optical fiber also has highly flexible.The fiber of 125 μ m diameters of standard can curl up into the loop of the following diameter of 5mm and can not fracture.

And, make fibre-optic process and make and itself can customize, particularly change a social system by the cross sectional shape of fiber.Optical fiber is made by prefabricated component, and the general length of prefabricated component is that 1 meter, diameter are 3 centimetres and make with silica.The core of prefabricated component is doped with germanium, thereby produces the nuclear core with high index that direct light is passed.Then prefabricated component is installed on the fiber traction tower in the cleaning indoor environment, the latter is heated to fusing point with prefabricated component and fiber is pulled out on the big rotary drum.The cross sectional shape of fiber generally exactly likes the cross sectional shape of prefabricated component, and can utilize drawing velocity to come the diameter of controlling fiber.Most of optical fiber on the market all has the ring section and external diameter is 125 μ m.Yet other diameter and shape particularly " D " cross sectional shape also are useful.If use in order to store and to be easy to, the final probe carrier that forms is around self twining, and so this shape is particularly useful.Shown in Fig. 2 a, can utilize the D shape prefabricated component of tool groove to regulate the cross section of fiber, thereby fiber (200) but have the groove or the groove (202) of stationary probe (110) in it.This design avoids the probe of one deck and the substrate of succeeding layer to rub (shown in Fig. 2 b), wherein the cross section of two successive layerss represented go out be a top at another.

In addition, because the carefulness of the high-purity of material and manufacture process control, optical fiber does not almost have fault of construction.And optical fiber has perfect dimensional accuracy.Diameter is controlled in ± 1 μ m within.At last, fibre-optic cost is very low, is approximately 1-2 U.S. dollar/rice.This is because the manufacture process of fiber is considerably simple and clear and prefabricated component can produce standard radio telecommunications fibers up to 100 kms.

Employing is attached to many devices of the polynucleotide on the optical fiber and all describes to some extent in following document: people such as " biological nucleic acid sensor diagnostic method " Krull, WO#98/58079 and WO#95/26416; People such as " the fiber optics biology sensor that is used for the oligonucleotides sample of selectivity detection fluid-mixing sample " Walt, WO#98/50782; " be used to detect and measure the analytical approach of specific sequence nucleic acid " people such as Sutherland, EP#0245206; " gene probe method for biosensor " Squirrel, WO#93/06241; " nucleic acid determination method " Hirschfield, US 5,242, and 797; People such as Piunno are used for the fiber optics DNA sensor of fluorescent core acidity test, analytical chemistry 67:2635-2643,1995; People such as Uddin are used for the fiber optics biology sensor of fluoroscopic examination triple helical DNA, nucleic acids research 25:4139-4146,1997; People such as Abel are used to detect the fiber optics evanescent wave biosensor of oligonucleotides, analytical chemistry 68:2905-2912,1996; People such as Kleinjung are used for the fiber optics gene sensor that specific assay flies the DNA oligomer of mole, analytical chemistry journal 150:51-58,1997; People such as Zhang are used to detect the chemiluminescence fiber optics biology sensor that DNA is hybridized, and analyze communication 32:2725-2736,1999; People such as Ferguson are used for the fiber optics DNA biology sensor microarray that analyzing gene is expressed, Nature Biotechnol 14:1681-1684,1996.

These devices only generally comprise a kind of probe molecule sequence are attached on the single fibre-optic glass surface, thereby have limited their use value greatly.Method is in the past only used the short part (several centimetres or still less) of fiber, has therefore limited the number and the kind of the probe that can fix.At last, former technology adopts the optical fiber that is fixed with probe on it that light is conducted to hybridizing label (being generally fluorophore) or the conduction light from the hybridization label.This detection technique depends on and fades luminously from fibre-optic, and therefore this luminous being confined to inherently and fiber surface adjacent areas tightly can not distinguish that many group probes also are restricted in sensitivity.And, utilize optical fiber itself to excite and launch light and limited and utilize optical fiber separately as the substrate that is fixed with probe on it, and hinder and use other substrate for example metal wire or polymkeric substance, and these substrates have the ability of the information of relevant each probe of other advantage such as carrying or probe groups, and the advantage (as described below) of hybridization.

Coml radio communication fiber is coated with one deck non-porous polymer, and this is not best for probe stationary.Can utilize technology well known in the art (such as US patent 5,948, those technology described in 202, the document is all incorporated this paper into as a reference at this) to remove this coating.Yet the uncovered fibres of this coating is not easy to be subjected to the attack of water vapor, thereby produces micro-crack and make its intensity degraded at fiber surface.As a result, Luo Lu silica fibre may be unable to bear the environment of crossing phase very severe.Several ways of addressing this issue are arranged.

A kind of method is along strip cylinder or rotary drum fiber to be wound in spiral coil after probe stationary.Fiber is located on the rotary drum side by side and is attached on its solid phase surface.Probe is arranged along a side of fiber, and this side is away from that side that is attached to the fiber on the rotary drum.Rotary drum provides mechanical support for fiber in the testing process of the hybridization of sample and crossing pattern.

Second method is by one or several coatings being applied to the intensity that has increased this fiber on the silica fibre, also keeping bonding probes well simultaneously thereby avoid fiber to be damaged by water vapor.Then reinforcing fiber for example can be wrapped on the custom-designed reel and in the seal box of packing into so that transportation and handling.An example of substrate intensifying method is to use the metal material coated fiber, adds a silica layer then.Suck moisture for fear of exposed silica fibre, can add one or more inner liners.Suitable coating material comprises gold, silver and titanium, and this is because these metals have suitable inertia in chemical solution.In fiber optics wireless telecommunications industry, also use carbon coating widely, to carry out gas-tight seal.The present invention also provides the coating of the another one silica on inner liner in one embodiment, so as with the dna probe covalent bond.Can form such coating by sol-gel process cheaply, and this coating provides one to be used for fixing the particularly surface by the covalent bond stationary probe of probe.

Except silica, other material also can be used as the main body of substrate.It comprises thin metal wire or strength polymer (for example polyimide or polytetrafluoroethylene (PTFE)) band.Moreover sol-gel silica coating can be applied on the substrate, to be convenient to bonding probes.For polymer belt shape substrate, can between band and silica, apply the layer of metal material.

Metal material part in above-described all substrate designs is not only protected and/or is strengthened substrate, and also has other benefit (following will the description to some extent) in the manufacture process of probe carrier and in the process of combined belt electric charge sample.

Substrate is a strip." strip " used herein is meant, the length of substrate and the ratio of width surpass about 5: 1, preferably surpasses 100: 1, more preferably surpasses 1000: 1, most preferably surpasses 10,000: 1.Think length: the ratio of width can be bigger, as at least 100, and 000: 1 or at least 1,000,000: 1.As defined above, " width " of substrate is meant the whole extreme length vertical with the substrate major axis that is included in the substrate.If substrate has different width, the width that is used for the ratio of computational length and width so is the longest width." width " of the part that contains probe of substrate is meant that the longest radian is (for the arc area that contains probe, as usually arriving seen in the substrate of cylindrical spiral being similar to) or the big linear range of planar substrates, it is included in the part that contains probe of substrate and is vertical with the major axis of the part that contains probe of substrate." length " of substrate is meant the length of substrate major axis.If substrate has the length more than one, the ratio of utilizing the shortest length in these length to come computational length and width so.

The cross section of substrate can be an Any shape." cross section " used herein is meant and passes substrate, the planar section vertical with the major axis of substrate.Though the cross section can be an Any shape, there are two kinds of given shapes to represent different embodiments of the present invention.At first, " band " used herein is meant the embodiment of using band shape, strip or sheet-form substrate, and its cross section is strip or is similar to strip or parallelogram.Such band will have the thickness corresponding with the width of cross section.In different embodiments of the present invention, this thickness is no more than 500 microns, perhaps is no more than 100 microns, perhaps is no more than 50 microns, perhaps is no more than 20 microns.Secondly, " fiber " used herein is meant the embodiment of using fibrous, thin-line-shaped or silk thread shape substrate, and its cross section is circular.The cross section can be annular, oval or part annular, and for example fiber has the D tee section.This cross section has footpath always, and this diameter in this article refers to the longest linear dimension in cross section.In a plurality of embodiments of the present invention, the diameter of fiber is no more than 500 microns, perhaps is no more than 200 microns, perhaps is no more than 100 microns, perhaps is no more than 50 microns, perhaps is no more than 20 microns.Term " band " and " fiber " refer to two parts in the table cross sectional shape scope.Yet the present invention can have the cross section of Any shape.One or more grooves that substrate can have approximately and the fiber major axis runs parallel, and probe stationary is at (shown in Fig. 2 a) in these grooves.Such groove is seen as a groove on the cross section.Utilize such groove or groove to reduce or eliminate and be fixed on interior probe of groove and the friction between other surface; For example, when substrate when self being wound in spirality, be fixed on a probe on the winding because interior the avoiding of recessed groove contacts with substrate on next winding.D tee section with this groove is easy to one deck winding is deposited in down on one deck, and the protection probe.Other embodiment can adopt different cross sections, and these cross sections are useful when using this device and are conspicuous for those skilled in the art.

1.2. probe

" probe " used herein is a kind of molecule that can combine with specific sample or sample segment specificity or one group of copy of multimolecular structure." multiple probe " used herein is meant the structure more than such component.Utilize that covalently or non-covalently attachment method can be with probe stationary to substrate.Probe can be the combination of polynucleotide, polypeptide, oligosaccharides, polysaccharide, antibody, cell receptor, part, lipid, cell or these structures, or other structure of combining of arbitrary and interested sample or interested sample segment specificity.The purposes of this device is depended in the selection of this group probe.For example, if this device with polynucleotide as probe, if what carry out so is sequential analysis, then preferred one whole group or near n nucleotide of whole group; U.S. Pat 5,700 has more fully been described the purposes of these probe groups in 637 and 6,054,270, and these two pieces of patents are all incorporated this paper into as a reference at this.On the other hand, if use device comes sudden change or polymorphism in analyzing gene or the genome, for in interested specific gene or a plurality of gene, probe is preferably represented the polynucleotide of a complete sudden change group or selected sudden change group (sudden change is for example replaced, lacked and inserts in sudden change) so.As another example, in the sudden change diagnosis relevant such as cancer, the specific sudden change " focus " in the relevant one group of gene of known and a certain cancer or several cancer is the zone that its complementary polynucleotide is used as probe groups.These examples only show the probe into the selected different customization groups of specific device, and concentrate on polynucleotide, and this is because these are present the most frequently used probe kinds; Should be appreciated that the probe of other kind and other polynucleotide group also are conspicuous for a person skilled in the art.

This paper all " polynucleotide " is meant the polymerized form of the nucleotide of any length, and these nucleotide comprise deoxyribonucleotide, ribonucleotide and/or their analog.Term used herein " polynucleotide " and " nucleotide " can exchange use.Polynucleotide can have any three-dimensional structure and can implement known or unknown any function.That term " polynucleotide " comprises two strands or strand and triple helical molecule.Unless specially refers in addition or need, otherwise the of the present invention any embodiment that comprises polynucleotide as herein described all comprises in two complementary single stranded form of double chain form and known or prediction formation double chain form each.Duan polynucleotide (being less than about 100 nucleotide) also can be called oligonucleotides relatively.

Below be the indefiniteness example of polynucleotide: isolation of RNA, nucleic acid probe and the primer of gene or genetic fragment, extron, introne, mRNA, tRNA, rRNA, ribozyme, cDNA, recombination of polynucleotide, branch's polynucleotide, plasmid, carrier, the DNA isolation of any sequence, any sequence.Polynucleotide can comprise modified nucleotide for example methylated nucleotide and nucleotide analog.The analog of purine and pyrimidine is known in the art, include, but is not limited to: the aziridinyl cytimidine, the 4-acetylcytosine, 5 FU 5 fluorouracil, 5-bromouracil, 5-ethyloic aminomethyl-2-thiouracil, 5-ethyloic-aminomethyl uracil, inosine, the N6-isopentennyladenine, the 1-methyl adenine, 1-methyl pseudouracil, the 1-methyl guanine, the 1-methylinosine, 2, the 2-dimethylguanine, the 2-methyl adenine, the 2-methyl guanine, the 3-methylcystein, 5-methylcytosine, pseudouracil, 5-pentyne uracil and 2, the 6-diaminopurine.Utilize uracil to replace thymine in the DNA (deoxyribonucleic acid), also be considered to the analog form of pyrimidine.

If present, before or after the assembling polymkeric substance, carry out the modification of nucleotide structure.Can utilize non-nucleotide to become to assign to interrupt the sequence of nucleotide.After polymerization such as can also further modifying polynucleotide by combining with marked member.Included other type is modified in this definition, for example " cap ", one or more naturally occurring nucleotide are replaced with analog, modify and for example have the neutral key (methyl phosphonate for example between nucleotide, phosphotriester, the phosphono aminate, carbamate etc.) those modifications and have electric charge key (thiophosphate for example, phosphorodithioate etc.) those modifications, has intercalator (acridine for example, psoralen etc.) those modifications, contain sequestrant (metal for example, radioactive metal, boron, oxidized metal etc.) those modifications, contain those modifications of alkylates, with modifier keys (for example α-end group isomery nucleic acid etc.) those are modified, and the unmodified form of polynucleotide.

In addition, but be present in usually in the sugar any hydroxyl all phosphonate ester group, bound phosphate groups replace, perhaps protected by the blocking group of standard, perhaps be activated from and with other nucleotide or solid support bonding.5 ' and 3 ' end OH base can partly replace by phosphorylation or with organic end-capping group of amine or 1-20 carbon atom.Other hydroxyl also can be derivatized to the blocking group of standard.

Polynucleotide also contain the ribose known usually in this area or the analog form of ribodesose, and it includes, but is not limited to 2 '-O-methyl, 2 '-O-allyl, 2 '-fluorine or 2 '-nitrine-ribose, carbocyclic ring sugar analogue, α-end group isomerized sugar, epimerism sugar such as arabinose, wood sugar or lyxose, pyranose, furanose sedoheptulose, no ring analogues and alkali-free yl nucleosides analog (for example methyl nucleoside).As already pointed out, one or more phosphodiester bonds can replace by replaced linking group.These alternative linking groups include, but is not limited to some embodiments like this, and in these embodiments, phosphate is by P (O) S (" sulfo-"), P (S) S (" two sulfo-s "), (O) NR ₂(" amidation "), P (O) R, P (O) OR ', CO or CH ₂(" formylation ") replaces, wherein respectively do for oneself H or randomly contain ether (O-) replacement of key or not substituted alkyl (1-20 carbon), aryl, alkenyl, naphthenic base, cycloalkenyl group or epoxy radicals of each R or R '.Be not that all keys in the polynucleotide are all essential identical.For example replaceable backbone structure as the polyamide skeleton is benefited in being substituted in when designing final product of the analog form of sugar, purine and pyrimidine.

Term " polypeptide ", " oligopeptides ", " peptide " and " protein " can exchange use in this article, are meant the amino acid polymer of any length.This polymkeric substance linear or branch, it can comprise modified amino acid and can be interrupted by non-amino acid.These terms also comprise natural or interleave the amino acid polymer of modification; For example, the formation of disulfide bond, glycosylation, lipoidization, acetylation, phosphorylation or any other operation or modification (such as combining) with marked member.What also comprise in this definition is for example to contain one or more amino acid analogues polypeptide of (comprising for example non-natural amino acid etc.), and other modification well known in the art.Polypeptide can strand or the existence of association chain.

" part " used herein is the molecule with special receptors bind.This receptor can be the part of cell receptor or another molecule, for example the acceptor of the other structure modifier of enzyme.The example of part includes, but is not limited to activator and antagonist, virus epitopes, haptens, hormone, agglutinin and the medicine (for example opiate and steroids) of other structure modifier, cell-membrane receptor, toxin and the venom of enzyme cofactor, substrate and inhibitor, enzyme.

" cell receptor " used herein is cellular elements, and it both can be positioned at born of the same parents usually and also can link to each other with cell membrane, and had compatibility for given part.The example of these cell receptors includes, but is not limited to hormone receptor, cell traffic albumen, cytokine receptor and neurotransmitter receptor.

1.3. probe is fixing

Utilize any obtainable mode that oligonucleotide probe of the present invention is added, fixes, provides and/or is applied on the surface of solid support, thereby a certain position on the solid support is fixed, locatees, provides and/or be applied to oligonucleotides.Utilize ink jet printing method (US 4,877,745), photolithography (referring to the US patent No. 5,919,523,5,837,832,5,831,070,5,770,722 and 5,593,839), stencil, hectographic printing method, driving, utilize x-y unit and grating technology to use the machinery of micropipet to apply method or when placing binding constituents, can provide required accuracy and any other method of apart degree, different samples are placed on the specific site.

As (the US patent No. 5,770,367,5 people such as Southern, 700,637 and 5,436,327), people's such as people such as Pirrung (the US patent No. 5,143,854), Fodor (the US patent No. 5,744,305 and 5,800,992) and people's such as Winkler (the US patent No. 5,384,261), the combination array method need in the situation of short sequential polymerization thing to be successfully used to already.In these " genetic chips ", the preparation of oligonucleotide probe (20-25 nucleotide) or peptide nucleic acid (PNAs) both can be in the microarray manufacture process original position produce, also can utilize the classic method off line and put on the microarray.People's such as Fodor the US patent No. 5,445,934 and 5,744,305 have described the manufacture process of the substrate (density is every square centimeter and contains 400 or more different probe) that contains a plurality of sequences.Utilize solid state chemistry and photolithography to synthesize these chips.These combined methods have produced tangible biological and chemical diversity, but but can not make up big high molecular microarray and implement also relatively more expensive and difficult.

Utilize inkjet dispenser equipment that droplets of liquid is deposited on the solid phase substrate.Put down in writing in the following document and utilized such technology to make the biological and chemical array: people's (US patent No. 5,658,802) such as Brennan (the US patent No. 5,474,796), Tisone (the US patent No. 5,741,554) and Hayes.These non-contact technologies can not be arranged a large amount of samples easily, can not control the quality of the microarray of final acquisition.

As in Augenlicht (the US patent No. 4,981,783), people's such as Drmanac (the US patent No. 5,525,464), people's such as Roach (the US patent No. 5,770,151), people's such as Brown (the US patent No. 5,807,522) and people's such as Shalon (the US patent No. 6,110,426), the 3rd class array equipment utilization direct surface contact print art is come work.In this form, probe is long complementary DNA s (cDNAs) (the 500-5000 base is long), and it utilized classic method synthetic before fixing.These technology conducts are can not accurately absorb and distribute sample and lack durability based on the existing defective of the sample applicator of quill.

People such as Martinsky (the US patent No. 6,101,946) have described the application of a kind of electron discharge machine (EDM), and this machine can be fixed to the kinetic control system that is used for accurately and carries out three-dimensional motion automatically.As the US patent No. 5,807 of Brown and Shalon, 522 (1998) is described, and Oligonucleolide primers also can be applied on the solid support.In addition, utilize robot system (such as by GeneticMicroSystems (Woburn, MA), GeneMachines (San Carlos, CA) or CartesianTechnologies (Irvine, CA) robot system of Zhi Zaoing) primer can be applied on the solid support.

Can utilize the whole bag of tricks that oligonucleotides is attached on the solid phase substrate.By adopting the solid phase substrate that chemical reaction can take place, the chemical reaction that is present on nucleic acid group can be provided, this group will react with chemical activity solid phase substrate surface.Can form estersil, so that nucleic acid is covalently bound on the surface.Can utilize organic addition polymer (for example styrene, acrylate and methacrylate, vinethene and ester etc.) to replace the functionality of silica, wherein existing functionality can with the functionality reaction that exists on the nucleic acid.Amino, active halogenide, carboxyl, mercapto, epoxide etc. also can be provided in a conventional manner.These keys can be acid amides, amidine, amine, ester, ether, thioether, disulfide etc.The method that is used to form these covalent bonds is described in the US patent No. 5,565,324 and the list of references wherein mentioned to some extent.

Can utilize primer extension or adopt modify the NTPs preparation to have the part that is used for combination and the nucleic acid of sequence flag, wherein primer can have part and/or sequence flag, modifies dNTPs and has part and/or sequence flag.For RNA, can use in-vitro transcription, promptly utilize phage promoter (for example T7, T3 or SP6) and by the sequence flag of dna encoding, and comprise mark NTP (NTPs of biological example element-16-UTP) in the presence of, transcribe with T7, T3 or SP6 polymerase respectively, the RNA that finally obtains thus has oligonucleotide sequence sign and the relative binding partner that is distributed in randomly in the chain on predetermined site.

In the present invention, probe can be fixed on the substrate then in the synthetic or first preparation of original position on the substrate.For polynucleotide, this technology is described in the US patent No. 5,419,966 to some extent, and the document is incorporated herein by reference at this.Or, can synthesize poly probe (such as polynucleotide) from single monomer or less polynucleotide or other subunit with the mode of segmentation.Preferably, probe is fixed on the zones of different of substrate.In addition, be fixed with more than a kind of probe in the specific region, and by carrying out the difference mark such as the fluorized marking that utilizes different colours, a kind of single probe molecule and other probe molecule of particular type made a distinction." fixing " used herein is meant, utilizes mode covalently or non-covalently that probe is attached on the substrate, and the affinity between the two is enough to bear manufacturing, sample combination, these steps of sample analysis, and if necessary, can also reuse.

Be used to derive that solid support is known in the art with the method for fixedly polynucleotide and polypeptide and material and such as the US patent No. 5,744,305 and 5,919, describe to some extent in 523, these two pieces of documents are all incorporated this paper into as a reference at this.Adhere to for non-covalent, preferable methods is to utilize the biotin-streptavidin combined techniques, but can provide any non-covalent combined techniques of essential affinity all to can be used for the present invention.

Except oligonucleotides or any other organic body, can also use molecular aggregate, as be the situation of organelle (for example nucleus, mitochondria, tenuigenin, liposome etc.) or cell (protokaryon with eucaryon).Binding constituents can directly be attached on the solid phase substrate or utilize one or more intermediates to be attached to indirectly on the solid phase substrate, and wherein these intermediates are as the bridge between binding constituents and the solid phase substrate.In general, in molecule is covalently bound to situation on the solid phase substrate surface, can utilize various reactive functional group degree to activate substrate surface, this depends on the character of binding constituents and the character of solid phase substrate surface.

For example, can utilize the whole bag of tricks that oligonucleotides is attached on the solid phase substrate.When using chemical reaction solid phase substrate, a kind of chemical reaction group that is present on the nucleic acid can be provided, this group will react with chemical activity solid phase substrate surface.Can form estersil, so that nucleic acid is covalently bound on the surface.Can utilize organic addition polymer (for example styrene, acrylate and methacrylate, vinethene and ester etc.) to replace the functionality of silica, the existing functionality of wherein organic addition polymer can with the functionality reaction that exists on the nucleic acid.Amino, active halogenide, carboxyl, mercapto, epoxide etc. also can be provided in a conventional manner.Key can be acid amides, amidine, amine, ester, ether, thioether, disulfide etc.The method that is used to form these covalent bonds is described in the US patent No. 5,565,324 and the list of references wherein mentioned to some extent.

Can or adopt modify the NTPs preparation with primer extension and have the part that is used for combination and the nucleic acid of sequence flag, wherein primer can have part and/or sequence flag, modifies dNTPs and has part and/or sequence flag.For RNA, can use in-vitro transcription, promptly utilize phage promoter (for example T7, T3 or SP6) and by the sequence flag of dna encoding, and comprise the NTP of mark (NTPs of biological example element-16-UTP) in the presence of, transcribe with T7, T3 or SP6 polymerase respectively, the RNA that finally obtains thus has oligonucleotide sequence sign and the relative binding partner that is distributed in randomly in the chain on predetermined site.

1.4. label

Can determine that by the label that utilizes probe or probe groups each probe is in on-chip position and the out of Memory of relevant probe and/or probe-sample composites.These labels can be used in the conventional two-dimensional array and a microstructure of the present invention." label " used herein be meant that but on substrate and/or the probe, in substrate and/or probe or any identification marking that links to each other with substrate and/or probe, arrangement or other structure or pattern, their transmit and a certain probe or the relevant information of probe groups.One type label is an optics.Label can also the free token thing (being the interval of probe row on the substrate) and/or bar code, fluorescent marker, chemiluminescent labels or can use up any other label of detection.The label of another kind of type is a magnetic marker.Because substrate contains magnetizable hardware, so the present invention is originally as such label.These labels can be positioned on the same side of substrate with probe, also can be positioned on the another side of substrate at probe place, perhaps can be clipped in the middle or mix substrate inside.A kind of method that is used for free token and/or out of Memory is the opposite sides that covers the substrate at probe place with one deck magnetic thin film.In manufacture process, utilize the mode record space or the probe of magnetic to identify then.A significant advantage of this method is, crossing phase can write on the out of Memory relevant with the target thing substrate from one's body.And at sweep phase, also sweep parameter and other digital output value can be write on same being with, so that with further reference to.The label of other type is conspicuous for the person of ordinary skill of the art.

1.5 probe groups

Probe can be fixed on the substrate in groups, and each group has some common features.For example,, need one group of nucleotide of common hybridization conditions to fix so, and need another group of different with it hybridization conditions to fix along another length of substrate along a certain length of substrate if probe is a nucleotide.Utilize this mode, each the group probe all can under a different set of condition, be exposed to sample, thereby make sample in conjunction with optimization.

Additionally, single probe carrier can carry not probe on the same group, so that diagnose different diseases.For example, one group of probe of locating along one section carrier is used to diagnose HIV, and another group probe is used to diagnose diseases such as bleb.Another example is, the part of carrier or carrier is exclusively used in the HER-2/neu gene.The HER-2 gene is also referred to as HER-2/neu and c-erbB2, and it plays a crucial role in the adjusting of normal cell growth, but in the growth course of cancer, this gene obtains amplification.The HER-2 gene of amplification causes the protein acceptor on tumor cell surface excessively to produce.These specific proteins combine with other cycling deposition factor, thereby cause growth of tumor out of control.This probe carrier contains the probe of HER-2/neu gene and variant thereof.

In another embodiment, probe groups is repetition, thereby makes a carrier can be recycled and reused for identical mensuration, and one group of new probe can be used for each subsequent measurements.

1.6 the structure of device

Probe can be fixed on the substrate by any structure, and the probe that these structures make it possible to be combined with sample separates with those probe region that are not combined with sample or identifies.The plain mode of realizing this point is, probe is placed on different zones, and there is a kind of probe in each zone.These zones can be a plurality of points (as shown in Figure 1) or many lines (as Fig. 4, shown in 404).These zones can be used as along the major axis of substrate or a parallel with it row and constitute.Probe can directly be attached on the substrate, and perhaps, in another embodiment, probe can be attached to earlier on the globule, then globule is attached on the substrate.Make probe be attached to that method on the globule of different materials is known in the art and describe to some extent in such as WO 99/60170, the document is all incorporated this paper into as a reference at this.Another possible alternative structure is to have to arrange a little more, thereby contains multiple probe on a given row.

As shown in Figure 1, fix dna probe 110 with the form of fillet of selecting or running through the width of substrate 100 that is positioned at long and thin flexible thin wire substrate 100 centers.By being imprinted on the evaluation that free token thing in the space 130 between the probe groups and/or bar code 120 are realized probe.Or, these labels are imprinted on the opposite side of fine rule substrate.If necessary, fine rule 100 can have specific cross sectional shape.Shown in Fig. 2 a, the cross section of scalable fiber, so that make fiber 200 have a groove or groove 202, and probe 110 is fixed therein.This design avoids the probe of one deck and the substrate of succeeding layer to rub (shown in Figure 11 B), the cross section of two successive layerss is represented go out be a top at another.

The length of probe carrier and thin and soft characteristic makes it be suitable for many new mode of loadings, setting and use-pattern.Can many forms pack this probe carrier, these forms include, but is not limited to pin, rod, coil and reel.Owing to only need hybridization solution seldom and strengthened mixing, therefore quite strengthened hybridizing method.Needing large volume, be low to moderate in the application (such as those application relevant) of medium scale microarray and have advantage especially with the flexible stylet carrier of reel packaged with medical diagnosis on disease.In these are used, number of probes required in the array few (at hundreds of to several thousand scope), but but need to consume the array of quite a large amount of same types every day.Utilize this flexible stylet carrier format, tens thousand of copies of same probe group are arranged repeatedly and are sealed in the big coil or reel along a continuous length of fine rule.

Twine the ready-made flexible stylet carrier of certain-length by a part of fine rule that centers on cylinder or pipe, and prepare pin or clavate packing.Fine rule tightly is positioned on the outside surface of support cylinder abreast in preferred embodiments, and can utilize glue, cement or alternate manner for good and all to be fixed to the upper.Difference between probe carrier pin and the probe carrier rod is size.The probe carrier pin has the diameter less than 10mm usually, and the probe carrier rod is wanted bigger and can be had bigger diameter, can hold very many probes thus.For example, 1.5 meters long, the fine rule of 50 μ m diameters only occupy the shorter part of a 5mm after on being wound into the probe carrier pin of 5mm diameter, but supposition has the probe space of 100 μ m along fine rule, and it can carry about 15,000 probes.On the other hand, the probe carrier rod of wide, the 40mm diameter of 30mm can hold 700,000 probes along 70 meters long, the fine rule of 50 μ m diameters.

In a probe carrier coil, ready-made flexible stylet carrier fine rule is wound in the square position shape coil.In a preferred embodiment of the invention, the probe on the fine rule is exposed to a side of dish, and opposite side utilizes epoxy, cement or other suitable mode for good and all to be fixed on the support of solid phase dish type plane.Randomly, probe is deposited in the lip-deep groove of probe carrier fine rule.Can cover this plane support in advance with a conductive layer, to be convenient to hybridization control.Suppose the fine rule of 50 μ m diameters, the probe carrier coil of 40mm diameter can hold the probe carrier fine rule up to 24 meters, carries 240,000 probes.

The structure of probe carrier reel is very similar to the probe carrier coil.Yet different with the probe carrier coil is that the probe carrier fine rule can for good and all not be attached on the stayed surface, in order to hybridize, to read or other purpose, can make that fine rule launches from reel thus.In addition, because each circle fine rule all is deposited in the top of each other in reel, so the design of the cross sectional shape of probe carrier fine rule will avoid the probe carrier fine rule in dna probe and the adjacent turn to rub.Still as shown in figure 17, can prepare a box, so that protect the probe carrier reel and make it be easy to twine and launch.In addition, a plurality of reels can be deposited in the box.

2. the manufacturing of probe carrier

In some embodiment of probe deposition technique as herein described, the substrate that is similar to fiber or band is fit to continuously really, large-lot production at a high speed.Fig. 3-7 shows the example of Manufacturing System Design.Though conventional point sample technology can be used to produce different zone (a kind of probe is contained in each zone), an aspect of of the present present invention is to adopt the technology of brushing or spraying probe on substrate.Such technology combines with the character of the one dimension in fact of substrate, makes its suitable manufacturing system, in these manufacturing systems, can also once produce a plurality of copies of identical band or fiber at a high speed accurately.

2.1 multiply brush

What Fig. 3 represented is an exemplary embodiment of this device and manufacture method thereof.A kind of manufacture method (as shown in Figure 3) comprise with in suitable liquid or itself be exactly liquid (for example, at room temperature be the liquid or the lipid that under suitable temperature, can liquefy) probe be sent in the pipe, and by moving pipe with respect to substrate, simultaneously probe liquid is driven on the substrate from pipe, thereby probe is deposited on the substrate from pipe.Should be appreciated that, this motion be relative to and all move and finish by moving pipe assembly, mobile substrate or the two.In deposition process, the capillary tip can contact with substrate surface.In addition, this tip can on the substrate surface or under move one section short distance.Utilize one of noncontact deposition process that probe liquid is deposited on the substrate.These methods comprise and probe are attached on the magnetic bead that is suspended in the probe liquid and an electromagnet is placed on below the substrate.This magnet is activated when kapillary and substrate intersect (kapillary is through the contiguous place of substrate), thereby magnetic bead and continuous probe thereof are attracted on the substrate surface.Another noncontact deposition process is, a metal level is covered on the support below two end faces capillaceous and substrate surface or the substrate, applies a high pressure then between the support of kapillary and substrate or substrate.The electric field that is produced is moved charged probe (for example oligonucleotides) on the substrate surface to.Utilize any of above-mentioned two kinds of methods, if electric excitation signal is a very short pulse, probe will be as a spot deposition to substrate so.If the most of times or the All Time that to be kapillary intersect with substrate of signal representative, the result will be an on-chip probe bar so.Can alternative condition to guarantee that probe stationary is to substrate.A plurality of pipes can be merged together, thereby make " brush " that can deposit a plurality of probe bars simultaneously.And, can arrange a plurality of such brushes, move with respect to substrate and during the deposition probe bar with the different brush of box lunch, the number of probes that can make deposition is simultaneously or sequentially be multiplied.In addition, if substrate is a fiber, then several fiber substrates can be placed on the appropriate location so that the stroke action of a brush is deposited on the probe bar on all substrates.Or, utilize the band shape substrate of a broad to receive the probe bar, then will be with being cut into a plurality of independent thinner bands in the vertical.Be appreciated that a kind of like this manufacture method increased widely can produced simultaneously probe carrier number, thereby increased output and reduced cost.Be also to be understood that the production in enormous quantities method (such as the use of travelling belt) that is easy to adopt standard is with automatic enforcement and control this manufacture method mentioned in this article and other manufacture method.

In Fig. 3, the flexible kapillary 300 of a row is glued in the hole below each hole of standard microtitre plate 302.Kapillary 300 also can be in the patchhole of top.Kapillary 300 is lined up a linear array then, thereby forms " brush " 304.Every kind of independent dna probe stored in the hole of separating on the plate and by pressure reduction or by between the tip of hole and brush 304, applying a voltage and it is driven in the kapillary 300 that links to each other with the hole.Because dna molecular is electronegative, so should apply negative pole to nose end.Can make up a plurality of such kapillary brushes.After the filled capillary pipe, capillary array is moved, with " brush " fixing excessively probe carrier band shape substrate 306 and deposited dna probe 110 arrays, shift to new position with shape substrate 306 then, so that second kapillary " brush " 304 can be deposited to more probe 110 on the position of postorder.Or, utilize same brush along the more multicopy of band shape substrate 306 sedimentary facies with probe array.In addition, with a large amount of fine rule substrate parallel be placed into brush below so that make each " brushing " action can both on different thin line or belts, produce a plurality of copies of one dimension probe array.

Another elite of this technology and all technology of mentioning is, also deposition or obtain the label (referring to, Fig. 1 for example) of probe 110.These labels can be between the probe or the interval around it, perhaps they can be optical bar code (Fig. 1,120) or fluorescent marker or be coded in magnetic marker on the metal parts of substrate, or be used to identify any alternate manner of particular probe or probe groups.These labels can in that side that deposits probe of substrate or in contrast that side, perhaps both sides have.Should be appreciated that substrate can have only a surface (for example, having the fiber of ring section), and the word in the context " side " is meant the specific region on the surface that deposits probe.In having the band shape substrate of more definite top surface and backplate surface, " side " is meant one of these tops or backplate surface.

Can utilize variety of way to provide is driven into probe in the pipe and is driven into on-chip power from reservoir.For example, can set up pressure reduction.Or, if probe electrically charged (for example when probe is DNA) can set up potential difference (PD) so between reservoir and substrate, thereby probe moves on the substrate from reservoir and pipe.This substrate can contain the metal parts (for example metal level) that forms electrode.

2.2 probe print head

Fig. 4 shows second kind of design of probe carrier fine rule manufacturing system, and wherein every kind of probe is stored in " print head " 410 of print system 408.With constant speed V _hOn the travelling belt 400 that moves a large amount of such print heads 410 are arranged in one-dimensional array.Travelling belt can be wound in the reel 406 by belt wheel or capstan winch 412, so that save the space.Space between the print head has several millimeters big and be enough to hold the reservoir of every kind of probe.Probe carrier band shape substrate 402 is placed on below the printhead array, and substrate 402 also with constant than jogging speed V _tMove.When print head intersects with probe carrier band shape substrate 402, its one is selected or a bar " printing " on probe carrier band shape substrate 402.Suppose that the space between two adjacent print heads on the travelling belt is L _h, and required space is L between two adjacent probe on the probe carrier band shape substrate 402 _p, then can accurately control the speed V of print head travelling belt _pSpeed V with probe carrier band shape substrate 402 _t, so that satisfy V _p/ V _t=L _p/ L _tIn this mode, the linear array of DNA bar 404 in a continuous manner high speed deposition to probe carrier band shape substrate 402.Since substrate is moving or substrate and travelling belt causing a line probe angular cross vertical with the substrate major axis, so line probe substrate athwart.Otherwise, substrate is stopped, and print head print, under applying, advance to next printing position then before a kind of probe.

Intrasystem each print head comprises the reservoir that holds a certain amount of probe sample and probe is sent to probe carrier or the on-chip device of band shape fine rule.Probe is distributed to probe transmits and is fixed to (perhaps probe itself is exactly a liquid) in the liquid that necessary condition is provided on the substrate, and the definite character of this liquid depends on specific probe and specific substrate.

Fig. 5 show print head some may design.In Fig. 5 a, an extremely thin flexible fiber 500 is attached to a little opening 502 below the probe reservoir 504.Fiber 500 is hydrophilic, thereby by surface tension or capillary effect probe liquid is moved on its surface.In addition, fiber 500 is necessary for the flexible and indeformable material of plasticating of thin (＜80 μ m).The solid phase or the hollow silica fibre that are coated with metal or nylon are a kind of candidate materials preferably.When print head intersected with probe carrier band shape substrate 402, it was used as probe " ink " and " is coated with " surface of probe bar to probe carrier band shape substrate 402.When being the metal coated fibres, negative pressure can be applied on the fiber, so that shift the DNA sample onto the probe carrier fine rule or be with.

Fig. 5 (b)-(h) shows the different designs based on the ink-jet principle, wherein produces a pin hole in the bottom of probe reservoir.Pulse energy is introduced reservoir, and this energy will be ejected into from the drop of pin hole on the probe carrier fine rule below it.In Fig. 5 b, pressure ring 506 is adhesive on the wall of memotron, this ring extruded tube and liquid droplets under a voltage.In Fig. 5 c, press mold 506 covers on the barrier film 508 at reservoir 504 tops, and this press mold and pressure ring have identical functions, but press mold is more cheap when adopting a large amount of reservoirs 504.In Fig. 5 d, the current impulse of process resistive conductor 510 utilizes spot heating to produce bubble, and bubble is released drop again.In Fig. 5 e, introduce the injection energy by external ultrasound converter 512.In Fig. 5 f, memotron is transparent and realizes heating by laser instrument 514 being focused on the optical absorption film in the pipe.In Fig. 5 g, reservoir 516 is made of metal.Between reservoir and probe carrier band shape substrate 402 (the perhaps object under the probe carrier band shape substrate 402), apply a high pressure and negative pole is positioned at reservoir.Because dna vector is electronegative, so electric field is ejected into sample on the surface of probe carrier band shape substrate 402.In Fig. 5 h, probe molecule is attached on the magnetic bead in the fluid 520 that is suspended in the reservoir 518.Current impulse is applied on the electromagnet 519 below the substrate, so that the little opening of probe below reservoir is attracted on the substrate surface 402.Notice that in design 5e-5h, actuator externally and not moves with print head.Because each reservoir (504 or 516 or 518) only intersects once at fixed position and probe carrier band shape substrate 402, therefore in system, only need such actuator.Suppose the 2mm that is spaced apart of reservoir, then 150,000 reservoir arrays are exactly 300 meters long and can be contained in the reel of diameter less than 80cm.

2.3 sample applicator and reservoir

Fig. 6 shows another probe carrier Manufacturing System Design, and wherein the print head structure of previous designs is divided into " sample applicator structure " and " reservoir configuration ".Every kind of probe all has the reservoir of oneself, and in order to reduce cost, it is simple that the structure of reservoir keeps.Fig. 6 a shows one of possible reservoir design, and wherein liquid internal pressure and the capillary summation liquid 600 that causes containing probe 110 expands slightly at opening 612 places.The a large amount of reservoir 602 of assembling on travelling belt, thus a linear array formed.Shown in Fig. 6 b, the linear structure by for example metal strip being made miniature tooth or short silica fibre 606 is glued on the flexible metal band 608 can be made sample applicator structure 604.Can use any material of the fiber that produces the suitable transmission of row probe or the combination of some materials.The sample applicator tip is suspended on the reservoir opening, and a bit of distance is arranged between the two, so that most advanced and sophisticated contact probe liquid.When sample applicator by highly elastic material (for example silica fibre) when making, in fact the sample applicator tip can reduce opening slightly, thereby sample applicator can littlely be contacted in the opening, so that collect probe liquid.According to arrow 614 and 616 indicated different directions to move such as constant speed drive sample applicator and reservoir configuration.When the opening 612 of sample applicator and reservoir 602 intersects, a probe liquid 600 will be shifted out from the reservoir transfer, thereby form drop 610 on sample applicator.The shape and size of utilization intersection duration and sample applicator are controlled the liquid measure in the drop.(will describe) Move Mode that designs sample applicator and reservoir configuration by this way later on to some extent: promptly, the sample applicator that makes each consecutive intersects with the reservoir of corresponding consecutive, so that make the different probe drop of each sample applicator carrying.Then, shown in Fig. 6 c, sample applicator structure 604 moves and intersects with the substrate 618 that moves according to the direction that sample applicator structure and substrate are intersected.The same with being provided with of reservoir, the tip of each sample applicator 606 can physical form contact substrate or can be suspended on a bit of distance on the substrate (tens millimeters), and this distance makes drop 610 contact substrates 618.The probe drop is sent on the substrate from the sample applicator structure, so that on substrate, form linear probe structure 630.If probe is charged, so when a specific sample applicator and the probe reservoir that has electric charge (will attract probe) when intersecting, can make it charged by the electronics mode, then when this sample applicator intersects with substrate subsequently, make the electric charge changeabout electric charge on it, so that probe is repelled on the substrate from sample applicator.This superb design makes it possible to the drop size on the accurate control points sample device consistently.Thisly probe material is sent to on-chip method is called " point sample ", and manual use the in the laboratory widely.

In addition, shown in Fig. 6 d, sample applicator structure 604 can move in a ring, and the number of sample applicator is far smaller than the probe sum in the structure.Sample applicator leaves after the substrate 618, and it can wash at scrubbing section 620, carry out drying and by be used for making 626 to intersect with probe reservoir configuration 624 again around moving at dry section 622.Because finish parallel with point sample of washing, so it does not influence manufacturing output.Be easy to clean according to sample applicator of the present invention.

In another structure shown in Fig. 6 e, the probe reservoir can be straight tube 632 or the hole 634 that has little opening 636 in its bottom 638.Because the combined action of gravity and capillary force, probe liquid 600 expands downwards at opening part.Sample applicator 606 intersects with it below the probe reservoir and some probe liquid 600 is collected in its tip.Then, sample applicator move and intersect with probe carrier substrate 610 and with the similar structure of Fig. 6 c (just sample applicator carrier 406 be now substrate below by rather than thereon by) on substrate, scribble a fillet.Substrate face is located, so that scribble downwards with sample applicator.The structure that comprises probe collection, point sample, washing and dry whole manufacturing system is similar with shown in Fig. 6 d all.

In above-mentioned two kinds of manufacturing systems, every kind of composition moves with the constant speed of predesignating.The complicacy that has reduced motion control like this and accurately required.In addition, can make a large amount of probe carriers 100 continuously and need not artificial intervention.As a result, manufacturing output is very high.And if adopt silica fibre or thin line as the probe carrier substrate, so many fibers can parallelly be attached to the wide carrier band that is used for the fabrication phase.So, can make a plurality of copies of same probe carrier fine rule simultaneously.

Can adopt broad-band chip in processing factory, and the probe drop is deposited on the line that runs through band (shown in the zone 628 of Fig. 6 d).After the probe deposition, cut this broadband, thereby produce many copies (as shown in Figure 1) of same probe carrier fine rule 100.In addition, also significantly increased output.Therefore these two system designs are suitable for producing in enormous quantities in special center microarray manufacturing plant.In a system of the present invention, the interval between interval between the probe on the fine rule and the reservoir (and sample applicator) is respectively 100 μ m and 5mm.Suppose that the fine rule substrate moves with the speed of 1cm/s, then reservoir and sample applicator array move (this is easy to accomplish) with the speed of 50cm/s.And, being divided in the situation of 20 fine rule substrates at carrier band 618, above-mentioned two system designs should be able to produce 150,000 probe arrays in per 7.5 seconds.

2.4 sample applicator matrix

Fig. 7 shows the 4th kind of Manufacturing System Design, and this system has greater flexibility and is particularly suitable for the customization of probe carrier on a small scale.Fig. 7 a is the vertical view of system, and Fig. 7 b is the front view of system.Herein, probe is stored in the titer plate or similar matrix container 700 of standard.Coupling sample applicator matrix 702 has identical distance with hole matrix on the plate.Different with the spotting needle of routine, as used in last system, each sample applicator all is the flexible hydrophilic fibers 704 that approaches.The sample applicator matrix at first is impregnated in the hole matrix, moves then to intersect with probe carrier band shape substrate 706, wherein the probe carrier band shape substrate 706 temporary transient stationary states that keep.The direction that sample applicator moves is vertical with probe carrier band shape substrate 706, but matrix row's direction tilts with low-angle α.Each point sample fiber will produce the line 708 (referring to enlarged drawing) of the separation of penetration probe carrier band shape substrate 706, herein: α=arc.sin[L _C/ (C+1) L _R] (1)

L wherein _CAnd L _RBe respectively the fiber spacing in sample applicator matrix column and the row, and C is the number of the row in the sample applicator matrix.

The sample applicator matrix array washs and cleans and 712 carry out drying at the drying station at cleaning area 710.Simultaneously, probe carrier band shape substrate 706 advances to a new part and carries new hole matrix 712, so that prepare next " dipping and point sample " circulation.In this design, the spacing between the probe on the probe carrier band shape substrate 706 is by L _C/ (R+1) provide, and be approximately 250 μ m.When the probe carrier band shape substrate 706 that carries 10,000 kinds of probes with the probe spacing of 259um has 2.5 meters long (it can be wound in the reel of diameter less than 3cm), such density is useful for custom arrays on a small scale.

3. the packing of probe carrier

It is various multi-form that the flexibility of probe carrier fine rule platform can be packaged into the probe carrier fine rule of manufacturing, and these forms include, but is not limited to probe carrier pin, probe carrier rod, probe carrier coil and probe carrier reel.The superior strength of probe carrier fine rule substrate, precision and flexible for the manufacturing of probe carrier fine rule with to read in the process desired accurate probe location and transmit be desirable.Spacing and the fine rule thickness of supposing probe all are 100 μ m, then can hold whole human genome (about 150 along 15 meters fine rule, and this fine rule can be wound in the three-dimensional coil of 1.5cm height, 3cm diameter or 0.1mm is thick, diameter is less than the reel of 4cm 000 gene).So probe carrier fine rule packing is preferred for the moulding of big probe.Several modes of packing probe carrier fine rule and band have below been described.

3.1 probe carrier pin and probe carrier rod

As shown in Figure 8, by around the three-dimensional probe carrier fine rule 100 that twines the manufacturing of certain-length of strip support member 804 (for example solid phase cylinder or pipe), can be made into probe carrier pin 810 and probe carrier rod 820.Tightly the fine rule 100 of Chan Raoing 806 is positioned on the part 802 of support cylinder 804 outside surfaces side by side, and for good and all is attached on it by jointing compound, bonding agent or alternate manner.In order to control hybridization, before winding process, be coated with cylinder 804 with conductive material.Probe 110 is positioned on the side of probe carrier fine rule 100, and this side is away from that side that contacts with support member 804 of probe carrier fine rule 100.

Discussed as former, the difference between probe carrier pin 810 and the probe carrier rod 820 is relative size and shape.Probe carrier pin 810 has the diameter less than 10mm usually, and probe carrier rod 820 is bigger and therefore hold more probe.For example, 1.5 meters long, the fine rule of 50 μ m diameters only occupy the so short part of 5mm after on being wound into the pin 810 that diameter is 5mm, but but approximately can carry 15,000 kinds of probes (supposing that along the probe spacing of fine rule 100 be 100 μ m).On the other hand, the probe carrier rod 820 of wide, the 40mm diameter of 30mm can hold the probe of 700k along 70 meters long, the fine rule 100 of 50 μ m diameters.In one embodiment, the flexible stylet carrier can be the band shape substrate of carrying with the fixing probe of two-dimensional array.For example in Fig. 3 and 4, express the manufacturing of this array.This flexible stylet carrier band can coil around pin 810 or rod 820, rather than twines probe carrier fine rule 100.

Can effectively produce above-mentioned probe carrier pin 810 and probe carrier rod 820 in high yield ground.As shown in Figure 9, in the fine rule manufacture process and before being put into thin line or belt on the support cylinder, between any two groups of probes of thin line or belt 100, insert " sky " spacing 904 of certain-length along probe carrier.Twine probe carrier fine rule 100 continuously along long support cylinder 804 then.Be coated with cylinder 804 in some position in advance with epoxy or other bonding agent, so that adhere to the part probe carrier fine rule 100 of carrying probe 110.After epoxy solidifies, cut the long cylinder 804 that has fine rule 100 on it with proper spacing, thereby produce a plurality of probe carrier pin 810 or probe carrier rods 820 with probe carrier fine rule 100, wherein fine rule 100 is attached with probe 100 and twines around cylinder, and on certain part 902 of cylinder.Because be not coated with 904 parts of the accompanying support cylinder of blank fine rule 804 in advance with epoxy, so after cutting, should will unclamp and disconnect by the blank fine rule from this cylinder, 904 parts of original support cylinder are come out, and this part can be used for being fitted in the adapter in crossover process.

3.2 probe carrier coil

In the probe carrier coil shown in Figure 10, the probe carrier fine rule 100 of manufacturing is wound in flat dish type coil 1012.Figure 10 a shows the vertical view of probe carrier coil 1012 assemblies, and Figure 10 b shows the side view of this assembly.Probe 110 on the fine rule 100 is exposed to a side of dish 1012, and opposite side for good and all is attached on the solid phase planar support dish 1010 by epoxy, bonding agent or other suitable mode.Notice that in Figure 10 c (this figure is the enlarged drawing in the cross section 1000 of probe carrier coil 1012), probe 110 is deposited in the probe carrier fine rule 100 lip-deep grooves 202.This characteristic is chosen wantonly in this packaged form.With conductive layer overlay planes supporter 1010 in advance, to be convenient to control hybridization, this will go through following.The diameter of supposing fine rule is 50 μ m, and then diameter is that the probe carrier coil 1012 of 40mm can hold the fine rule up to 24 meters that carries 240,000 kinds of probes.

3.3 probe carrier reel

The structure of probe carrier reel 1110 is very similar to the structure of probe carrier coil.Yet different with probe carrier coil 1012 is, probe carrier fine rule 100 can permanent attachment to stayed surface 1010, thus in order to hybridize, to read and other purpose, make fine rule launch (though the end of fine rule can be attached on the substrate) from cylinder.In addition, shown in Figure 11 b, because each circle of fine rule 100 all is deposited in the top of each other in reel 1110, cross sectional shape that therefore can designing probe carrier fine rule 100 rubs with the fine rule of avoiding dna probe and adjacent turn.Can select to be used for to produce the cross section of the substrate of probe carrier fine rule 100, so that fiber 200 has groove or groove 202 (probe 110 fixing within it).This design avoids the substrate of one deck probe and postorder layer to rub.And, shown in Figure 11 a, can prepare box 1100, to protect reel 1110 and to make it be easy to twine and launch.In addition, a plurality of reels 1110 can be deposited in the box 1100.

4. hybridization

The purposes of device of the present invention comprises: 1) preparation sample; 2) form probe-sample composites; And 3) analyze binding pattern, so that identify the various probes of sample institute combination.

The preparation of sample is different along with the difference of sample type.The specimen preparation scheme that is used for analysis of polynucleotide comprises that with fluorized marking mark sample to be convenient to carry out step 3), promptly analyze binding pattern, this preparation scheme is known in the art.Referring to for example, the US patent No. 5,800,992, the document is all incorporated this paper into as a reference at this.Under the situation that is polynucleotide, utilize known technology (for example digestion with restriction enzyme method) to make the sample segmentization, be translated into single stranded form then and with suitable this single-chain fragment of fluorized marking mark.

When sample contacted with this device, the sample or the sample fragment that particular probe are had affinity combined with those probes.Microarray of the present invention utilizes the hybridization of complementary strand of DNA as associated methods usually.Hybridization conditions is depended in the hybridization of DNA to a great extent, and these conditions have obtained extensive studies and description (referring to for example, the US patent No. 6,054,270 and 5,700,637, these two pieces of documents are all incorporated this paper into as a reference at this).

Yet the present invention also comprises the sample-probe combination of any kind, this combination make can from measure with probe that sample or sample fragment combine obtain information.These examples comprise by utilizing the characteristic of different antibody or antigen antigen or antibody as probe and in the working sample respectively, perhaps utilize the acceptor that combines with hormone to identify hormone in the sample etc.The tabulation right to sample/probe can expand any matched group to, and these matched groups can be with affinity and the specificity combination each other that is enough to identify, and the right example of sample/probe also is conspicuous for a person skilled in the art.

The hybridization of nucleic acid generally includes with high degree of specificity and detects minority target nucleic acid (DNA and RNA) in a large amount of non-target nucleic acids.Strict hybridization conditions is to keep the specificity of required degree necessary, and can be used for this purpose such as the multiple combination of these reagent of salt, temperature, solvent, denaturant and detergent and condition.On various forms of solid supports, carried out the hybridization (referring to for example, Beltz, people's such as G.A. " Enzymology method " (Methods in Enzymology), 100 volumes, B part, 19:266-308, Academic Press, NY (1985)) of nucleic acid already.

Exploitation to the dna microarray technology recently makes the extensive mensuration of carrying out multiple target molecule on a solid support become possibility.Usually, comprise that the DNA chip of oligonucleotide arrays is made up of many single oligonucleotides that link to each other with solid support with the pattern of rule, thereby every kind of oligonucleotides is positioned at all on the known position.After array produced, the sample that will contain target sequence was exposed in the array, and the complementary oligonucleotide that combines with institute on array hybridization, utilize then the whole bag of tricks, the most frequently used be that radioactivity or fluorescent marker detect.In the US patent No. 5,837,832 people such as () Chee and the relevant patented claim oligonucleotide probe array that is fixed for hybridizing has been described all, and the specific nucleic acid sequence in the test sample.

The present invention also provides some custom-designed equipment that are used for based on the probe carrier fine rule hybridization of microarray.In existing system, by spreading naturally or forcing liquid to circulate and realize hybridization.Device form more slowly and system subsequently manufacture complicated.In one embodiment of the invention, the design of hybridization chamber can guarantee to have only an extremely thin target fluid layer between probe carrier fine rule or its packaged form and hybridization chamber inwall.By this way, hybridization only needs very small amount of target liquid, thereby has improved contacting between probe molecule and the target molecule.Drift and moving hybridization is quickened by for example making probe carrier fine rule 100 or its packaged form pass target thus.

In addition, by between the support of probe carrier fine rule and hybridization chamber inwall, applying a voltage, may command crossover process also.In this process,, hybridization is quickened by adding kation, volume exclusion and chaotropic agent.When an array comprised several thousand addresses, importantly, the correct product sequence that connects had the chance with proper address hybridization.By making oligonucleotides under used high temperature, carry out thermal motion, perhaps make mechanical movement by making with the array surface fluid in contact, perhaps move by utilizing electric field to make oligonucleotides pass array, can realize this point.After the hybridization, wash array successively with low severity lavation buffer solution and high severity lavation buffer solution.

As shown in figure 12, when probe carrier fine rule 100 had positive pole, the dna molecular in the target liquid was drawn towards probe carrier fine rule 100, produced temporary transient local concentration at the fine rule near surface, thereby hybridization is strengthened.If polarity is opposite, then electric field will repel unmatched nucleic acid molecules and make it away from probe 110, and hybridization probe keeps its target molecule, and the specificity of hybridization is increased.Therefore, can between probe carrier fine rule 100 or its support 1200 and hybridization locular wall 1210, apply AC oscillating voltage 1220, to improve the efficient of this process.Support 1200 and hybridization locular wall 1210 have conductive coating 1212, so that make this process be easy to carry out.As described below, all probe carrier fine rule forms can also be rotated in crossover process, stir and mix and make the improvement that contacts between probe and the target molecule thus thereby increased.Can utilize slip ring conduct electricity on traveling electrode of brush to press.The design of this electric slip ring is known in the art.

As shown in figure 13, by probe carrier pin 810 is directly filled in the hole 1300 of containing target liquid 1310, can make 810 hybridization of probe carrier pin.The diameter in hole 1300 is only slightly greater than the external diameter of probe carrier pin 810.Owing to have only the extremely thin target liquid 1310 of one deck between the inwall in probe carrier pin 810 and liquid hole 1300, therefore required target liquid is minimum.For example, the diameter of supposing the hole is 8mm, and the diameter of probe carrier pin has only 50 μ m, and there is the 5mm height in the cross section of winding, and then the target liquid of 3 μ l just is enough to cover the whole free area of probe carrier pin.The probe carrier pin can move up and down 1330 or front and back 1332 rotate, the perhaps combination of these two kinds of motions is so that increase hybridization speed.Because the spiral winding pattern on probe carrier pin 810, probe carrier rod 820 and the probe carrier coil 1012, so rotational motion not only drives target liquid along the annular direction of packing but also the axial or radial direction of edge packing.Its molecule in the running target liquid effectively on the whole surf zone that covers by probe carrier fine rule 100.

A plurality of probe carrier pins 810 can be filled in the adapter 1400, so that the matrix of the pattern match of formation and space pitch and standard microtitre plate 1420.By this way, can finish a plurality of crossover process by following steps are directly parallel in standard microtitre plate 1420: at first each probe carrier pin 810 is impregnated in the respective aperture 1410 of standard microtitre plate 1420 (as shown in Figure 4), moves up and down adaptation board then arbitrarily or rotate each probe carrier pin.

Probe carrier rod 820 can with the probe carrier pin similarly but hybridize in the big hybridization chamber.Or, adopt hybridization chamber design 1500 shown in Figure 15.Rotation probe carrier rod 820 to 1520 is sentenced target liquid 1510 is moved on probe.Because the spiral of the probe carrier fine rule 100 on the probe carrier rod 820 twines pattern, so target liquid 1510 covers all the probe positions on the probe carrier rod 820 thus not only along the annular direction of rod 820 but also move along its axial direction.Between probe carrier fine rule 100 and hybridization locular wall 1500, apply AC oscillating voltage 1530, so that improve the efficient of this process.

The hybridization chamber design 1600 of probe carrier coil 1012 is rotated 1620 by Mechanical Driven or magnetic driving with front and back and is incorporated in this coil, and apply AC oscillating voltage conversion 1630 between coil support and chamber, to strengthen hybridization efficiency as shown in figure 16.

Figure 17 a shows the chamber that is used for from the probe carrier fine rule 100 that probe carrier rotating cylinder 1110 launches is hybridized and designs 1700, wherein covers on the narrow groove on the substrate 1780 by covering 1770, forms fluid-tight basically kapillary 1760.The sectional dimension of groove is slightly greater than probe carrier fine rule (shown in Figure 17 b).Before closing the lid,, target liquid is introduced by the little opening 1790 that covers with the center section of target liquid 1750 lead-ingrooves.Probe carrier fine rule 100 is moved forward and backward, to strengthen hybridization efficiency by hybridization chamber.Because fine rule is hydrophobic, therefore target liquid is retained in the groove by capillary force.Moreover, by between kapillary 1760 inwalls of metal level on the probe carrier fine rule 100 and hybridization chamber 1700, applying alternating voltage 1730, can further improve hybridization efficiency.

5. read instrument

The flying-spot microscope that utilization has laser or broadband excitation can read all above-mentioned probe carrier fine rule packings.Utilize scanning electron microscope, Laser Scanning Confocal Microscope, charge-coupled device (CCD), scanning tunneling microscope, infrared microscope, atomic force microscope, electricity to lead and fluorescence or phosphorus imaging, can finish scanning.Yet,, preferably provide special scanning motion being used for the instrument that reads of different probe carrier fine rule form.

Shown in the meaning property, probe carrier pin 810 and probe carrier rod 820 all can be inserted in the adapter that reads instrument just as shown in Figure 18, but this instruments design is the end of locking pin or rod and rotates 1810 and/or move 1812 with predetermined speed along the longitudinal axis.This motion makes all probes along optical excitation with read probe carrier fine rule 100 distributions below the lens 1800.Or probe carrier pin 810 or probe carrier rod 820 turn 1810, and optical head 1800 is mobile along the axle of pin or rod, so that scanning is fixed on the length of the probe carrier fine rule 100 on probe carrier pin 810 or the probe carrier rod 820.

Equally, as shown in figure 19, the rotation 1910 by introducing coil 1012 and along radial direction the relatively moving between coil 1012 and optical read head 1900 (1912 move) of coil, but scan-probe carrier coil 1012.

In probe carrier rotating cylinder scanner shown in Figure 20, be included in probe carrier reels 1110 in the box 1100 and make and read at optical read head 2002 and label that one section probe carrier fine rule that does not twine 100 of process launches under the instrument 2004.The probe carrier fine rule 100 that this does not twine is carried on all probe groups that move under the optical read head 2002 and can keeps static.The probe carrier fine rule 100 that this can not twined is collected in second reel 2012.

6. the using method of probe carrier

This device makes it be applicable to many fields.Utilize polynucleotide to can be used for evaluation, sequencing, medical diagnosis on disease and the polymorphism analysis of mutation analysis, genomic fingerprinting, linkage analysis, mRNAs and the mRNA colony of known point as the device of probe.Utilizing antibody is useful especially in diagnosis as the device of probe.Other purposes relevant with other probe is conspicuous for those skilled in the art.

The purposes of these devices comprises: 1) preparation sample (if necessary); 2) form probe-sample composites; 3) analyze binding pattern so that identify the various probes that are combined with sample.

6.1. the preparation of sample

The preparation of sample is different along with the difference of sample type.The specimen preparation scheme that is used for the polynucleotide analysis comprises with fluorized marking mark sample promptly analyzes binding pattern to be convenient to carrying out step 3), and this scheme be known in the art (referring to for example, the US patent No. 5,800,992, the document is all incorporated this paper into as a reference at this).In the situation of polynucleotide, utilize known technology for example the digestion with restriction enzyme method make sample fragmentization, make it be transferred to single stranded form again, and with suitable this single-chain fragment of fluorized marking mark.

6.2. the formation of probe-sample composites

When sample contacted with device, the sample or the sample fragment that particular probe are had affinity combined with those probes.The hybridization of the complementary strand of present microarray general using DNA is as associated methods.Hybridization conditions is depended in the hybridization of DNA to a great extent, and these conditions have obtained extensive studies and description (referring to for example, the US patent No. 6,054,270 and 5,700,637, these two pieces of documents are all incorporated this paper into as a reference at this).

Yet the present invention also comprises the sample-probe combination of any kind, and this combination makes from determining which kind of probe with sample or sample fragment combine obtains information.As just an example, sample can comprise many kinds of molecules, and some of them are enzymes.The probe that is used for analyzing the device of this sample be plurality of enzymes substrate (herein, the meaning of " substrate " speech is, reactant when enzyme works as catalyzer), and by determining which kind of substrate-probe combines the characteristic that obtains enzyme in the sample with enzyme after contact.Other example comprises by utilizing different antigen to determine the characteristic of antibody in the sample as probe, perhaps utilizes the acceptor that combines with hormone to identify hormone in the sample etc.Sample/probe can expand any matched group to catalogue listing, and these pairings can be with affinity and the specificity combination each other that is enough to identify, and the right example of sample/probe also is conspicuous for a person skilled in the art.

One aspect of the present invention can strengthen the combination of charged sample greatly.This performance is the result who applies voltage on substrate, and wherein substrate contains hardware or conduction.For example, if DNA is a sample to be analyzed, the oscillating voltage that passes substrate so will alternately attract electronegative DNA to probe carrier, and then repel it.Attraction makes complementary strand be easy to combination, and will help the release of non-specific binding or imperfect hybridization sample during repelling.Identical principle is applicable to the charged sample of any kind of, and has increased the efficient and the fidelity of sample combination.

6.3 the analysis of binding pattern

Two steps are generally arranged in the analysis of binding pattern: the location is in conjunction with the probe of sample, and those probes are identified.Possible is, if specific sample and combining of its correspondent probe produce for this sample/probe being only one variation, right for specific sample/probe so, these two steps can be kept to one.

Can utilize any way that allows the location that probe-sample pair is made a distinction with the probe that does not combine sample.Many such technology all are known in this area.For example, utilize the standard method that detects used label type, can carry out the detection of the sample polynucleotide of mark.So, for example can directly detect fluorescent marker or radioactively labelled substance.It is to mix in the sample in the preparation process of sample that other labelling technique requires label (biological example element or digitophyllin), and utilize antibody or other binding molecule (for example streptavidin) to detect this label, wherein said binding molecule be labeled or self can combine anti--streptavidin antibody that for example labeled molecule can be and fluorescence molecule (for example fluorescein isothiocynate, texas Red and rhodamine) combines or combines with the enzymatic activity molecule or anti--digitophyllin antibody with labeled molecule.No matter newly what the label on the synthetic molecules is, and no matter label be directly in sample or be connected with molecule that sample combines on (perhaps in conjunction with the molecule that combines with sample), other suitable device that these labels (for example fluorescence, enzyme, chemiluminescent or colorimetric) all available laser scanner or ccd video camera or X-ray film (depending on label) or be used to detects particular marker detects.For example, in the major applications of the polynucleotide microarray that is used for genetic analysis,, use each sample fragment of fluorescence labeling substance markers then with the sample polynucleotide passageization.After probe polynucleotide array contacts, utilize on the sample fluorized marking location with the sample fragment of complementary probe multi-nucleotide hybrid.The probe in conjunction with sample fragment does not just have such fluorescent marker.Can for example antibody, enzyme etc. carry out similar fluorescence labeling to the molecule of other type.The sign of other type for example radioactively labelled substance, chemiluminescent labels, phosphorescent labels, magnetic marker etc. is conspicuous for a person skilled in the art.

Be combined with the probe of sample for detection, the light detection mode is preferred, though also can adopt other detection method for example radiometric method, atomic spectroscopy etc.For the light detection mode, can adopt fluorescence method, phosphorimetry, absorption process, chemoluminescence method etc.Fluorescence method the most advantageously, it can have many forms.Can utilize independent fluorescer or paired fluorescer, particularly when hope has a plurality of emission wavelengths (having big stokes drift (20nm at least)).The fluorescer that is exemplified comprises fluorescein, rhodamine, texas Red, Hua Jing, rhodophyll, thiazole orange and indigo plant or the like.When using paired dyestuff, can on a kind of molecule, use a kind of dyestuff, and with another kind of molecule that first kind of molecule combines on the another kind of dyestuff of use.Important factor is, when two kinds of compositions in conjunction with the time these two kinds of dyestuffs lean on enough closely, be enough to carry out effective power transfer.

The invention provides second step (promptly identifying the step of the specific probe that is combined with sample or sample fragment) pipelining that makes in the analysis and the chance that enlarges.In the probe microarray of routine (for example polynucleotide array), can identify probe by determining the x-y position of probe in array; The x-y position of every kind of probe is known.Utilize known technology to determine that the position of probe needs complicated and expensive imaging device.Because probe of the present invention is with the arrangement of one dimension ranks, so position analysis is wanted much easier and do not needed complex apparatus, this is because only there is one dimension (rather than two dimension) to need to follow the tracks of (as in the quilt fine rule of reeling).

In any embodiment of the present invention, the label that use and probe or probe groups link provides the mode of grasping the probe clue.This point was discussed in front.Label can be simple or complicated, can with probe homonymy in the same side of substrate or not, can be more than one type, can contain more, but not only contain the information of this probe or multiple probe characteristics.

Probe carrier of the present invention can be used to make up the very large probe array with the minimum volume packing, and these arrays are hybridized with target nucleic acid subsequently.The analysis of the crossing pattern of chip provides the fingerprint identification result immediately of target nucleotide sequences.Can carry out artificial or Computer Analysis to pattern, but obviously, the position order-checking of being undertaken by hybridization is suitable for Computer Analysis and robotization.Algorithm that the sequence that can be applicable in the methods described herein rebuilds and software (people such as R.Drmanac, J.Biomol.Struc.﹠amp have been developed; Dyn.5:1085-1102,1991; P.A.Pevzner, J.Biom0l.Struc.﹠amp; Dyn.7:63-73,1989, they are quoted as a reference specially at this).

Can be used for the extensive hybridization analysis of many genes in using according to the flexible stylet carrier that contains the immobilized nucleic acids sequence of the present invention preparation, these analyses comprise evaluation, sequencing, medical diagnosis on disease and the polymorphism analysis of mutation analysis, genomic fingerprinting, linkage analysis, mRNAs and the mRNA colony of known point.Utilizing antibody is useful especially in diagnosis as the device of probe.Other purposes relevant with other probe is conspicuous for a person skilled in the art.

For gene mapping, with oldered array hybridization of gene or cloned DNA fragment and dna fragmentation, and the pixel by detected array or pixel mode and clearly determine to be applied to DNA characteristic on the array.People's such as Nelson " natural genetics (Nature Genetics) " described a kind of application that this type of is used to produce the array of gene map among the 4:11-18 (1993).When making up genomic physical map, make the hybridization of immobilized cloned DNA fragment array and other cloned DNA fragment so that determine cloned sequence in the probe mixture whether overlapping and therefore the immobilization on the array clone.For example, genome analysis, I volume: genome and physical map (Genome Analysis, vol.I:Genetic and Physical Mapping.) (K.E.Davies and S.M.Tilghman edit) publishing house of cold spring harbor laboratory, people such as 39-81 page or leaf Lehrach " the hybridization fingerprint analysis in genomic mapping and the order-checking " (1990) have described this process.

The flexible stylet carrier of immobilized DNA fragment also can be used for genetic diagnosis.For the purpose of illustrating, can survey the mutator containing various ways or the probe carrier of a plurality of genes with the mark potpourri of patient's DNA, and patient's dna marker potpourri only interacts with one of fixed form of gene.This interactional detection can cause the medical diagnosis result.And, diagnosable some medical conditions of the detection of some expression of gene level.For example, in about 25% early-stage breast cancer, can amplify HER-2/neu (c-erbB-2) gene that causes the overexpression of p185HER-2 growth factor receptors.Determined that HER-2/neu is an important independent prognostic factor of early-stage breast cancer in large numbers of patients and the colony with follow-up period of growing very much (30 years).New data have begun hint, and HER-2/neu not only can be used as the prognosis factor, but also can be used as foresight HER-2/neu monoclonal antibody.HER-2/neu coding 185kD strides the film oncogene protein, and this gene is amplified in some breast cancer disease people and/or crosses and express, and comparing it with the women of no HER-2/neu amplification is usually a characteristic relevant with poorer prognosis.Though to this locus after deliberation many year, the technical matters relevant with the most frequently used method (Southern blotting and immunohistochemistry staining method) caused data to have some inconsistent already.People's such as Pegram M.D. " HER-2/neu is the foresight mark of breast cancer treatment in response ", breast cancer research and treatment BreastCancer Research and Treatment 52 (1-3): 65-77,1998.The fast processing of a plurality of samples that obtained by the present invention makes can test a plurality of control groups fast, thereby has avoided the inconsistent of data.

7. the application of probe carrier

The flexible stylet carrier of immobilized DNA fragment also can be used for the diagnosis of dna probe.For example, hybridize, can clearly identify the characteristic of pathogen microorganism by DNA sample that makes unknown pathogen and the probe carrier that contains the known pathogen DNA of numerous species.Similar techniques also can be used for any organic clear and definite Genotyping.Other molecule that hereditary interest arranged is cDNAs and RNAs can be fixed on the probe carrier or additionally as the potpourri that is labeled probe that is applied on the probe carrier for example.

In one application, represent a plurality of genes cDNA clone probe carrier with hybridize from organic total cDNA so that in order to study and gene expression with diagnostic purpose.With a kind of coloured fluorophore mark from Normocellular total cDNA, and with the total cDNA of another kind of coloured fluorophore mark from pathological cells, then these two kinds of cDNA samples are hybridized to simultaneously on the identical cDNA clone array, make can these two kinds of fluorophore intensity recently measure differential gene expression.This double-colored experiment can be used to monitor histological types, morbid state, to the reaction of medicine or to the gene expression in the reaction of environmental factor.

This method is as just being not limitation of the scope of the invention for example, and it can be used to screen many patients simultaneously at all known mutations in the disease gene.The present invention can be for example the form of 96 same probe carrier pin in matrix use, wherein each probe carrier pin all contains 1500 dna fragmentations of all known mutations of for example representing given gene.Can be to from interesting areas amplification, the mark of each DNA sample of 96 patients and hybridize in 96 independent arrays, and each mensuration all is to carry out in the hybridization solution of 10 microlitres.With this linking matrix as one piece material hatch, rinsing and utilize the radioactivity, fluorescence of standard or colour comparison detection apparatus to detect and analyze (people such as Maniatis, 1989), wherein said linking matrix contains whole 96 identical probe carrier pins of useful 96 patients' sample determination.In the past, such method be included in the closed chamber of 96 separation to the film of 96 separation handle, processing and spike.By in a step, handle whole 96 patients' sample with minimum hybridization solution, can save time significantly and cost.

This mensuration mode can be reversed, and wherein patient or organic DNA are fixed as the probe composition, and each probe carrier is hybridized with different mutation allele or genetic marker.Also available probe carrier matrix carries out parallel non-DNA ELISA and measures.And the present invention can adopt all standard detecting methods.

One aspect of the present invention comprises that the ligase detection reaction (LDR) and the polymerase chain reaction (PCR) that utilize coupling detect nucleotide sequence difference (as people's such as Baranyi the US patent No. 6 that is entitled as " utilizing the ligase detection reaction of coupling and polymerase chain reaction to detect nucleotide sequence difference ", 027, disclosed in 889, the document is all incorporated this paper into as a reference at this).

Except above-mentioned listed gene is used, utilize device of the present invention can make all cells, peptide, enzyme, antibody, antigen, acceptor, part, phosphatide, polymkeric substance, pharmaceutical preparation or chemical substance array, so that the extensive Screening test in medical diagnosis, medicine exploration, molecular biology, immunology and the toxicology.

All publications mentioned in this instructions and patented claim are can same degree incorporated herein by reference, although independently publication or patented claim are all pointed out to quote as a reference specially and separately for each.

Though for the purpose of being aware and understand, by setting forth and for example the preferred embodiments of the invention are described, this does not also mean that the present invention is exhaustive or be defined as disclosed these exact form.Many changes and the modification done under the situation that does not break away from marrow of the present invention all are that those of ordinary skill in the art realizes under instruction of the present invention easily.Therefore scope of the present invention is only limited by back appending claims and equivalent thereof.

Claims

1, a kind of device with probe specificity evaluation sample, it comprises: the flexible strip shape substrate with first substrate surface, certain-length and width; And being fixed on multiple different probe on the isolated area that contains probe portion of substrate surface, each described isolated area contains a kind of probe.

2, according to the device of claim 1, wherein said device also comprises second label of the information of first label of the information that transmits relevant first group of described probe and the relevant second group of described probe of transmission.

3, a kind of device with probe specificity evaluation sample comprises:

Substrate;

Be fixed on the multiple different probe on the isolated area that contains probe portion of substrate surface, each described isolated area contains a kind of probe; And

Transmit second label of the information of first label of information of relevant first group of described probe and the relevant second group of described probe of transmission.

4, according to the device of claim 3, wherein substrate and probe are encapsulated in the container.

5, according to device any among the claim 2-4, wherein first and second labels are magnetic.

6, according to device any among the claim 2-4, wherein first label is a magnetic, and second label is a kind of optical markings thing.

7, according to any described device among the claim 2-4, wherein first and second labels are optical markings things.

8, according to the device of claim 7, wherein said optical markings thing is an optical bar code.

9, according to the device of claim 7, wherein said optical markings thing is a fluorescence.

10, a kind of it is constructed to carry the band shape device of probe according to any described device among the claim 1-9, and wherein said substrate comprises that thickness is no more than 500 microns, has the flexibility band shape substrate on a surface; And wherein said multiple different probe stationary is on the isolated area that contains probe portion of substrate surface, and each described isolated area contains a kind of probe.

11, according to the device of claim 10, wherein the thickness with the shape substrate is no more than 100 microns.

12, according to the device of claim 10, wherein the thickness with the shape substrate is no more than 20 microns.

13, a kind of according to device any among the claim 1-9, it is constructed to carry the fiber plant of probe, wherein said substrate comprise have length, the flexible fiber substrate on diameter and a surface, this diameter is no more than 500 microns; And wherein said multiple different probe stationary is on the isolated area that contains probe portion of substrate surface, and each described isolated area contains a kind of probe.

14, according to the fiber of the carrying probe of claim 13, wherein the diameter of fiber substrate is no more than 200 microns.

15, according to the fiber of the carrying probe of claim 13, wherein the diameter of fiber substrate is no more than 100 microns.

16, according to the fiber of the carrying probe of claim 13, wherein the diameter of fiber substrate is no more than 20 microns.

17, a kind of according to any described device among the claim 1-16, wherein said device comprises the linear one dimension of probe to be arranged, and wherein said multiple probe stationary is arranged in the single ranks on the surface of substrate and with the linear density of every linear centimeter above 50 kinds of probes.

18, according to the device of claim 17, the linear density that wherein is arranged in the probe in the on-chip described single ranks surpasses 100 kinds of probe/linear centimeter.

19, according to the device of claim 17, the linear density that wherein is arranged in the probe in the on-chip described single ranks surpasses 200 kinds of probe/linear centimeter.

20, according to the device of claim 17, the linear density that wherein is arranged in the probe in the on-chip described single ranks surpasses 500 kinds of probe/linear centimeter.

21, a kind of according to any described device among the claim 1-20, wherein this device also comprises the ground floor on the substrate surface; And wherein said multiple different probe stationary is on the part that contains probe on described ground floor surface, the described part that contains probe has certain length and width, to such an extent as to the ratio that makes the length that contains probe portion and the width that contains probe portion was above 5: 1.

22, according to the device of claim 21, wherein said ground floor comprises silica.

23, according to device any among the claim 21-22, it also is included in the second layer between described ground floor and the described substrate.

24, according to the device of claim 23, the wherein said second layer comprises a kind of metal material.

25, according to the device of claim 24, the wherein said second layer is magnetizable.

26, a kind of according to device any among the claim 1-25, wherein substrate comprises a kind of material that is selected from quartz glass, plastic material, polymeric material and metal material.

27, according to the device of claim 26, wherein substrate comprises optical fiber.

28, according to the device of claim 26, wherein substrate comprises described quartz glass.

29, according to the device of claim 26, wherein substrate comprises described metal material.

30, according to the device of claim 29, wherein metal substrate is magnetizable.

31, according to the device of claim 26, wherein substrate comprises described polymeric material.

32, according to the device of claim 31, wherein polymeric material is selected from polyimide and polytetrafluoroethylene (PTFE).

33, a kind of according to device any among the claim 1-32, each isolated area that wherein contains a kind of probe has and is no more than 1000 microns length.

34, according to the device of claim 33, the described length that wherein contains a kind of each isolated area of probe is no more than 500 microns.

35, according to the device of claim 33, the described length that wherein contains a kind of each isolated area of probe is no more than 100 microns.

36, according to the device of claim 33, the described length that wherein contains a kind of each isolated area of probe is no more than 50 microns.

37, according to the device of claim 33, the described length that wherein contains a kind of each isolated area of probe is no more than 20 microns.

38, a kind of according to device any among the claim 1-37, wherein the ratio of the length of substrate and width was above 5: 1.

39, according to the device of claim 38, wherein the ratio of the length of substrate and width was above 100: 1.

40, according to the device of claim 38, wherein the ratio of the length of substrate and width surpasses 10,000: 1.

41, according to the device of claim 38, wherein the ratio of the length of substrate and width surpasses 100,000: 1.

42, a kind of according to device any among the claim 1-41, its middle probe comprises polynucleotide.

43, according to the device of claim 42, wherein polynucleotide comprise DNA.

44, according to the device of claim 43, wherein DNA comprises single stranded DNA.

45, a kind of according to device any among the claim 1-41, its middle probe comprises polypeptide.

46, a kind of according to device any among the claim 1-41, its middle probe comprises antibody.

47, a kind of according to device any among the claim 1-41, its middle probe comprises part.

48, a kind of according to device any among the claim 1-41, its middle probe is selected from cell surface receptor, oligosaccharides, polysaccharide and lipid.

49, a kind of according to device any among the claim 1-48, its middle probe is to arrange as the linear structure of point.

50, a kind of according to device any among the claim 1-48, its middle probe is to arrange as the linear structure of bar, and described the major axis with substrate is an angle.

51, a kind of according to device any among the claim 1-50, wherein substrate self is wound in a screwed pipe.

52, a kind of device according to claim 51, wherein substrate self is wound in a flat screwed pipe.

53, a kind of according to device any among the claim 51-52, wherein also comprise first reel that substrate screwed pipe center is accompanying.

54, according to the device of claim 53, it also comprises second reel, and wherein the outermost end of substrate extends out from screwed pipe, and wherein the outermost end of substrate is attached on second reel.

55, a kind of according to device any among the claim 51-52, it also comprises the flat substrate that substrate is accompanying.

56, a kind of according to device any among the claim 1-51, and it also comprises first rotary drum, wherein substrate twines and is attached on it around described rotary drum.

57, a kind of according to device any among the claim 1-52, and it also comprises a plurality of rotary drums, wherein substrate sections twines and is attached on it around described rotary drum, and be fixed on the probe that contains on the probe section of described substrate sections can be identical or different in the different piece of substrate.

58, a kind of target molecule that makes is attached to device on the immobilization probe, and it comprises:

The flexible stylet carrier that spirals; And

Be fixed on the lip-deep multiple probe of probe carrier.

59, a kind of device according to claim 58, the wherein said flexible stylet carrier that spirals is a flat screwed pipe.

60, a kind of according to device any among the claim 58-59, wherein said probe carrier comprises flexible band shape substrate.

61, a kind of device according to claim 60, wherein said flexible band shape substrate bearing two dimension probe array.

62, a kind of according to device any among the claim 58-59, wherein said probe carrier comprises the flexible thin wire substrate.

63, a kind of device according to claim 62, wherein said flexible thin wire substrate bearing one dimension probe array.

64, a kind of according to device any among the claim 62-63, wherein the cross sectional shape of flexible thin wire substrate is a D shape, and multiple probe is locked in the groove on the fine rule substrate surface, thereby makes the friction force between the adjacent windings of the flexible stylet carrier that multiple probe avoids spiraling.

65, a kind of according to device any among the claim 58-64, wherein the flexible stylet carrier comprises the part of the one or more carrying probes that alternately exist with one or more blank parts of not carrying probe.

66, a kind of according to device any among the claim 58-64, it also comprises the strip support member, and the probe carrier surface that wherein is fixed with a kind of probe at least is away from described support member.

67, a kind of according to device any among the claim 58-64, it also comprises a plurality of flexible stylet carriers that spiral and a plurality of strip support member, the corresponding strip support member that each probe carrier in wherein said a plurality of probe carrier that spirals all centers in described a plurality of support member curls up separately, the probe carrier surface that is fixed with at least a probe is away from support member, and wherein a plurality of strip support member is fixed on the plane support.

68, a kind of according to device any among the claim 66-67, wherein the diameter of strip support member is less than about 10mm.

69, a kind of according to device any among the claim 66-67, wherein the diameter of strip support member is approximately between the 10mm-150mm.

70, a kind of device according to claim 69, wherein said diameter is approximately between the 10mm-40mm.

71, a kind of according to device any among the claim 58-64, it also comprises the plane disc support member with an axle, the flexible stylet carrier curls up on this support member, the probe carrier surface that wherein is fixed with at least a probe is away from support member, and multiple probe annulate shaft distributes with the receiving target molecule.

72, a kind of device according to claim 71, wherein the plane disc support member has the axial screw shape groove on the plane disc support surface, and the flexible stylet carrier is attached on the dish type support member along this spiral slot.

73, a kind of device according to claim 71 or 72, wherein the diameter of plane disc support member approximately between the 10-100mm and carrying up to about 1,000,000 probe.

74, a kind of according to device any among the claim 66-73, wherein said flexible stylet carrier adheres at least a portion of support member by bonding agent.

75, a kind of device according to claim 74, wherein bonding agent is a permanent adhesives.

76, a kind of device according to claim 74, wherein bonding agent comprises the epoxy cement.

77, a kind of according to device any among the claim 66-73, wherein support member comprises magnetic material, the flexible stylet carrier comprises magnetic material, and the flexible stylet carrier by probe carrier magnetic material and the magnetic force between the magnetic material of support member fix.

78, a kind of device according to claim 77, wherein the magnetic material of support member comprises at least a magnetic bead.

79, a kind of according to device any among the claim 66-73, wherein said flexible stylet carrier is to curl up on support member removably.

80, a kind of according to device any among the claim 66-79, wherein the flexible stylet carrier comprises the part of the one or more carrying probes that alternately exist with one or more blank parts of not carrying probe, and wherein at least one described blank parts is not adhered on the support member.

81, a kind of according to device any among the claim 66-80, wherein support member has a conductive coating.

82, a kind of according to device any among the claim 66-80, wherein support member is formed by a kind of metal material.

83, a kind of according to device any among the claim 66-82, wherein probe carrier has groove on the probe carrier surface, the position that this groove contacts with support member away from probe carrier, and multiple probe is encapsulated in this groove.

84, a kind of according to device any among the claim 66-83, wherein support member is included in the box.

85, a kind of according to device any among the claim 58-84, its middle probe is selected from: the combination of polynucleotide, oligonucleotides, protein, polypeptide, oligosaccharides, polysaccharide, antibody, cell receptor, part, lipid, cell and these materials.

86, a kind of according to device any among the claim 58-84, its middle probe can be incorporated on a kind of target thing, and this target thing is to be selected from: the combination of polynucleotide, oligonucleotides, protein, polypeptide, oligosaccharides, polysaccharide, antibody, cell receptor, part, lipid, cell and these materials.

87, any one device among a kind of 5-86 according to Claim 8, wherein polynucleotide are selected from: isolation of RNA, nucleic acid probe and the primer of gene or genetic fragment, extron, introne, mRNA, tRNA, rRNA, ribozyme, cDNA, recombination of polynucleotide, branch's polynucleotide, plasmid, carrier, the DNA isolation of any sequence, any sequence.

88, a kind of according to device any among the claim 58-87, wherein probe carrier comprises and is selected from following substrate: silica, glass, optical fiber, metal, magnetizable metal, plastics, polymkeric substance, polyimide and teflon.

89, a kind of according to device any among the claim 58-88, its middle probe is fixed on the probe carrier surface by being selected from following method: ink jet printing method, lithographic plate autotype, stencil, hectographic printing method, driving, utilize x-y unit and grating technology to use the machinery of micropipet to apply method.

90, a kind of according to device any among the claim 58-89, wherein at least a label is positioned on the probe carrier and is adjacent with one or more immobilization probes, and each label all carries at least one information corresponding at least a adjacent probe.

91, a kind of device according to claim 90, wherein at least a label is to be selected from: optical markings thing, free token thing, bar code, fluorescent marker, chemiluminescent labels and magnetic marker.

92, a kind of multiple probe is deposited to on-chip device, comprising:

The reservoir that comprises the hole array; And

One row's kapillary, wherein each kapillary has first end and second end, each described first end capillaceous links to each other with a hole of reservoir like this, thereby the content in the hole can enter in the kapillary, and the layout of each described second end capillaceous makes all second ends capillaceous form smooth single ranks.

93, make the method for the device of carrying probe, comprise probe is deposited on the substrate that wherein said substrate is flexible, and be strip, and have a surface, a length and a width.

94, according to the method for claim 93, wherein by carrying out point sample with the form of selecting and probe is deposited on the substrate, wherein every bit comprises a kind of probe.

95, according to the method for claim 93, wherein by scribbling and probe is deposited on the substrate with the form of bar, wherein each bar all comprises a kind of probe.

96, make the method for the device of carrying probe, comprising:

To contain by scribbling article one on first liquid deposition to a substrate of first probe, wherein said substrate has a surface, a length and a width; And

Second liquid deposition that will contain second probe by scribbling second is to this substrate.

97, a kind of according to method any among the claim 93-96, wherein the ratio of the length of substrate and width was above 5: 1.

98, according to the method for claim 97, wherein the ratio of the length of substrate and width was above 100: 1.

99, a kind of according to method any among the claim 93-98, wherein probe is deposited on the substrate by following steps:

Various probes are transferred in the pipe from reservoir; And

Described pipe is placed the place enough near apart from substrate surface, so that various probes are deposited on the described surface.

100, the method for claim 99 also is included in and applies a pressure reduction between reservoir and the pipe, so that probe is driven in the pipe from reservoir.

101, according to method any among the claim 99-100, wherein also be included in and apply a voltage between reservoir and the substrate, this voltage plays probe is attracted to on-chip effect like this.

102, a kind of according to method any among the claim 99-101, wherein said reservoir is included in a plurality of holes on the titer plate.

103, a kind of according to method any among the claim 93-102, its middle probe Covalent Immobilization is on substrate.

104, a kind of according to method any among the claim 93-102, its middle probe is non-covalent to be fixed on the substrate.

105, a kind of according to method any among the claim 93-104, wherein probe is deposited on the substrate by following steps:

Move row's probe deposition head with first speed, and with the second speed mobile substrate, the position of substrate makes substrate and this row's probe deposition head intersect, wherein each described probe deposition head all comprises the reservoir accommodating the liquid that contains a kind of probe and the hydrophilic fibre of a thin softness, wherein said fiber is used for probe is sent out the probe deposition head from reservoir, and wherein said first speed and second speed can be identical or different; And

When the first probe deposition head of described a plurality of probe deposition heads intersects with substrate, probe is deposited on the substrate from the described first probe deposition head.

106, the method for claim 105, wherein said probe deposition head is brushed to probe on the substrate.

107, a kind of according to method any among the claim 105-106, the row of its middle probe deposition head is single row.

108, a kind of according to method any among the claim 105-107, the hydrophilic fibre of wherein said thin softness comprises the silica fibre with the material coating that is selected from metal material and nylon.

109, the method for claim 108, wherein this material is a kind of metal material.

110, the method for claim 109 wherein applies a voltage between metal material on the hydrophilic fibre of thin softness and substrate, wherein said voltage plays probe is moved on to on-chip effect from fiber.

111, a kind of according to method any among the claim 93-104, wherein probe is deposited on the substrate by following steps:

Move the probe deposition head that a plurality of print nozzles are drawn together in a package with first speed, and move substrate with second speed, wherein the position of substrate makes it to intersect with this typesetting and printing brush nozzle, in addition, metallic nozzle comprises a reservoir and an interior pin hole of this reservoir that holds a kind of liquid, contain a kind of probe in the described liquid, and first and second speed are identical or different; And

When first print nozzles of described a plurality of print nozzles is intersected with substrate, probe is deposited on the substrate from first print nozzles.

112, the method for claim 111, wherein said first print nozzles deposits to described probe on the substrate with a bar.

113, a kind of according to method any among the claim 111-112, wherein said print nozzles also comprises a pressure rings and a power supply, wherein described pressure rings is fixed on the wall of print nozzles reservoir and pushes this reservoir when applying voltage.

114, a kind of according to method any among the claim 111-112, wherein said print nozzles comprises that also a vibrating diaphragm, one add press mold and a power supply, wherein said vibrating diaphragm is positioned at the top of reservoir, and the described press mold that adds is coated on the vibrating diaphragm and makes the distortion of this vibrating diaphragm when applying voltage.

115, a kind of according to method any among the claim 105-114, wherein said probe deposition head comprises a power supply and a resistive conductor in reservoir.

116, a kind of according to method any among the claim 99-108, wherein when transmitting probe, comprise the ultrasonic tr-ansducer that activates the reservoir outside.

117, a kind of according to method any among the claim 99-108, wherein when transmitting probe, comprise an optical absorption film of using from the rayed reservoir of the lasing light emitter of reservoir outside.

118, a kind of according to method any among the claim 99-108, wherein when transmitting probe, comprise the power supply that activation and first conductive material and second conductive material all link to each other, wherein said first conductive material links to each other with reservoir wall, second conductive material links to each other with substrate, thereby apply a voltage between two conductive materials probe is moved on the substrate from reservoir.

119, a kind of according to method any among the claim 93-98, wherein probe is deposited on the substrate by following steps:

Move row's reservoir with first speed, and move row brush with second speed, the layout of brush makes that can arrange reservoir with this intersects, wherein each described reservoir all comprises a kind of probe, and each described brush all comprises one flexible material, described first speed can be identical or different with described second speed, and the selection of described first and second speed makes each brush contact the probe that comprises in reservoir, thus a part of probe is transferred on the brush from reservoir; And

With the third speed mobile substrate, described third speed can with described first and/or second speed identical or different, described substrate makes each brush this probe can be deposited on the substrate pick up described probe from reservoir after with respect to the position of this row brush.

120, the method for claim 119, wherein the ranks with brush are configured to annular.

121, a kind of according to method any among the claim 119-120, its also be included in brush probe is deposited on the substrate after and turn back to reservoir before brush is washed.

122, a kind of according to method any among the claim 119-121, wherein brush comprises metal material, and wherein this method also comprises off and on to each brush charging, so that probe is attracted to probe on the brush when the probe reservoir is transferred on the brush at brush, and under when brush deposits to probe on the substrate, probe being repelled from brush.

123, a kind of according to method any among the claim 119-122, wherein described probe is deposited on the substrate with one.

124,, wherein probe is coated with the form of bar and signs on the substrate by following steps according to method any among the claim 93-98:

With one group of fiber impregnation in first group of hole, a kind of probe is all contained in each hole in described first group of hole, be first matrix wherein, and described group hole is arranged as second matrix, so that the fiber of first matrix is aimed at the hole of second matrix described group fibre placement; And

Make fiber move through the first of substrate, and fiber makes one of each fiber laydown run through the probe bar that separates of substrate with the position of substrate, and have required interval between bar and the bar.

125, the method for claim 124 also comprises

Washing the fibre;

With fiber impregnation in second group of hole, a kind of probe is all contained in each hole in described second group of hole, be first matrix wherein, and described second group hole is arranged as the 3rd matrix, so that the fiber of first matrix is aimed at the hole of the 3rd matrix described group fibre placement; And

Make fiber move through the second portion of substrate, and fiber makes one of each fiber laydown run through the probe bar that separates of substrate with the position of substrate, and have required interval between bar and the bar.

126, according to method any among the claim 93-125, wherein substrate is a plurality of fibers that are arranged in parallel.

127, according to method any among the claim 93-125, wherein substrate is a band.

128, the described method of claim 127, it also is included in after the probe deposition, band is divided into a plurality of copies of the band that carries probe along the major axis of band.

129, according to method any among the claim 93-128, it also comprises the probe that is deposited covalently bound to substrate.

130, according to method any among the claim 93-129, it comprises that also first label with first group of probe is placed on the substrate, and second label of second group of probe also is placed on the substrate.

131, a kind of being used for detected the method that the hybridization solution target molecule exists, and comprising: make device any among the claim 1-91 contact one section time enough with described hybridization solution, thereby make described target molecule and the hybridization of described device; And the existence that detects target molecule on the described device.

132, a kind of method that makes target molecule and stationary probe hybridization, this method comprises: the multiple probe that is fixed on the flexible strip shape probe carrier that twines is provided; And at least a probe in the multiple probe is contacted with the hybridization solution that contains described target molecule.

133, a kind of method according to claim 132, wherein probe carrier is fixed on the strip support member, thereby form a probe carrier pin or probe carrier rod, and wherein when described probe contacts with hybridization solution, comprise described probe placed it is contacted with hybridization solution in the hybridization chamber.

134, a kind of according to method any among the claim 132-133, wherein when contacting with hybridization solution, described probe comprises multiple probe is impregnated in the hybridization solution.

135, a kind of method according to claim 132, wherein with multiple probe stationary to the flexible strip shape probe carrier of a plurality of discrete windings, described each probe carrier is fixed on the strip support member separately, thereby form a plurality of probe carrier pins or probe carrier rod, and a plurality of probe carrier pins or rod are fixed on the adaptation board, thereby form matrix with spatial separation, and be the pattern corresponding, and wherein when multiple probe contacts with hybridization solution, comprise a plurality of probe carrier pins or excellent being impregnated in the hole of titer plate with the hole on the titer plate.

136, a kind of method that is used to read the results of hybridization of target molecule and immobilization probe, this method comprises utilizes method any among the claim 132-135 to make target molecule and probe hybridization, and probe carrier spare is rotated around the longitudinal axis of this probe carrier, move and between described probe carrier spare and read head, carry out opposing parallel along the described longitudinal axis simultaneously.

137, a kind of method according to claim 132, wherein probe carrier is fixed on the square position shape support member, and comprises when described probe contacts with hybridization solution probe carrier is impregnated in the hybridization solution in the hybridization chamber.

138, a kind of method that is used to read the results of hybridization of target molecule and stationary probe, this method comprises that the method for utilizing claim 137 makes target molecule and probe hybridization, and make this disc spins motion, move and between described dish and read head, carry out opposing parallel along the radial direction of dish simultaneously.

139, a kind of according to method any among the claim 132-138, wherein utilize permanent adhesives that probe carrier is adhered on the support member.

140, the method for claim 139, wherein this permanent adhesives comprises the epoxy cement.

141, a kind of method according to claim 132, wherein the flexible strip shape probe carrier of Chan Raoing forms a flat spin on a strip support member, thereby form a smooth-drum shape probe carrier, and when described probe contacts with hybridization solution, comprise the flexible strip shape probe carrier that launches winding and make this flexible strip shape probe carrier translate across hybridization solution.

142, the method for claim 141 comprises when this flexible strip shape probe carrier translates across hybridization solution that wherein extracting this flexible strip shape probe carrier passes the groove that contains hybridization solution, and described groove comprises one slightly greater than the kapillary of probe carrier.

143, the method for claim 132, wherein with multiple probe stationary on the flexible strip shape probe carrier of a plurality of discrete windings, the flexible strip shape probe carrier of each winding forms a flat spin on a discrete strip support member, thereby form a plurality of smooth-drum shape probe carriers, and when described probe contacts with hybridization solution, comprise the flexible strip shape probe carrier that launches a plurality of windings and make these flexible strip shape probe carriers translate across hybridization solution.

144, a kind of method that is used to read the results of hybridization of target molecule and immobilization probe, this method comprises utilizes method any among the claim 141-143 to make target molecule and probe hybridization, and probe carrier is launched, so that drive probe carrier through read head.

145, a kind of according to method any among the claim 132-144, wherein probe carrier is included in the box.

146, a kind of according to method any among the claim 132-145, and it also comprises by making probe carrier carry out at least a rotation and translation motion to improve the efficient of hybridization.

147, a kind of method according to claim 146 wherein drives by at least a mechanical adapter and magnetic, gives described motion.

148, a kind of according to method any among the claim 132-147, and it also comprises by exchanging oscillating voltage and being applied in the hybridization solution, improves the efficient of hybridization.

149, a kind of method according to claim 148 wherein is applied to described interchange oscillating voltage the support member that probe carrier or probe carrier contact and between the hybridization locular wall that contains hybridization solution.

150, a kind of method according to claim 149, it also is included in a conductive coating is provided on the support member.

151, a kind of according to method any among the claim 132-150, wherein said immobilization probe is selected from: the combination of polynucleotide, oligonucleotides, protein, polypeptide, oligosaccharides, polysaccharide, antibody, cell receptor, part, lipid, cell and these materials.

152, a kind of according to method any among the claim 132-150, wherein said target molecule is selected from: the combination of polynucleotide, oligonucleotides, protein, polypeptide, oligosaccharides, polysaccharide, antibody, cell receptor, part, lipid, cell and these materials.

153, a kind of according to method any among the claim 151-152, wherein polynucleotide are selected from: isolation of RNA, nucleic acid probe and the primer of gene or genetic fragment, extron, introne, mRNA, tRNA, rRNA, ribozyme, cDNA, recombination of polynucleotide, branch's polynucleotide, plasmid, carrier, the DNA isolation of any sequence, any sequence.

154, a kind of according to method any among the claim 132-153, wherein probe carrier comprises and being selected from: the substrate of silica, glass, optical fiber, metal material, magnetizable metal material, plastics, polymkeric substance, polyimide and teflon.

155, a kind of according to method any among the claim 132-154, wherein probe carrier is a wire.

156, a kind of according to method any among the claim 132-154, wherein probe carrier is a band.

157, a kind of according to method any among the claim 132-156, wherein probe carrier has a groove on the probe carrier surface, the zone that this groove contacts with its support member away from probe carrier, and multiple probe is locked in this groove.

158, a kind of according to method any among the claim 132-157, wherein utilize following technology with probe stationary to the probe carrier surface: ink jet printing method, photolithography, stencil, hectographic printing method, driving, utilize x-y unit and grating technology to use the machinery of micropipet to apply method.

159, a kind of according to method any among the claim 132-158, wherein probe carrier comprises one or more the adjacent labels at least a and described multiple probe, and wherein said label carries the probe corresponding information adjacent with label.

160, a kind of method according to claim 159, wherein at least a label is selected from: optical markings thing, free token thing, bar code, fluorescent marker, chemiluminescent labels and magnetic marker.

161, a kind of method that is used to read the results of hybridization of target molecule and immobilization probe, this method comprises utilizes method any among the claim 132-160 to make target molecule and probe hybridization, and detects the existence of target molecule.