CN1242047C

CN1242047C - Method and device for creating micro-arrays

Info

Publication number: CN1242047C
Application number: CNB008199582A
Authority: CN
Inventors: S·B·莱顿
Original assignee: Beecher Instruments
Current assignee: Beecher Instruments
Priority date: 2000-10-10
Filing date: 2000-10-10
Publication date: 2006-02-15
Anticipated expiration: 2020-10-10
Also published as: JP2004511761A; KR20040007402A; CA2425634C; AU2000280042B2; EP1325108A4; CA2425634A1; EP1325108A1; KR100891217B1; AU8004200A; CN1454253A

Abstract

The present invention provides a method and a device for simultaneously manufacturing a plurality of identical micro arrays for biological samples. The present invention adopts a plurality of blocks of substrates, each substrate comprises a top surface, a bottom surface and a through hole model, and each through hole has a wide upper cross section, a narrow lower cross section and a step or a platform formed in a transition region. When the substrates are stacked, the through holes are aligned, and form passages penetrated through the substrate stack. A chemical agent flows through the passages and are deposited on the step or the platform region. A barrier layer is provided in order to avoid the leakage between two adjacent through holes. After the deposition of the chemical agent, the substrates are separated. When the method is used, a series of micro arrays (each micro array has hundreds or thousands of biological samples such as cDNA fragments) can be simultaneously formed.

Description

Make the method and apparatus of microarray

Technical field

The present invention relates to make simultaneously the method and apparatus of series of identical microarray, each microarray comprises the hundreds of or thousands of analyte determinations district on the solid phase carrier, and each analyte specific reagent is used for for example detecting the cDNA of cross experiment mark.

Background technology

There is the microarray in hundreds of or thousands of biological analyte determinations district to be widely used for bioanalysis.Make droplet (every contains different known agent, and the normally different polynucleotide or the biopolymer of polypeptide be dna segment as is known) with the array deposition of rule and be fixed on the solid-phase matrix sheet such as the microscope glass microscope slide on.It is unknown that exsiccant array of droplets contact is contained, for example in advance with the pulsating solution of complementary DNA (cDNA) of fluorescence or radio chemistry sign mark.When being complementary between any place complementary sequence polynucleotide and the cDNA in this array, association reaction or hybridization will take place.Optics then or radiosensitivity scanning can determine that place contains sign, identify thus to have complementary compound in the solution.

Be used for the useful means that quick bio is analyzed though microarray provides, the method for making microarray is still time-consuming and expensive.

For example, known United States Patent (USP) 5,807,522 people such as () Shalon adopts the kapillary nib of different shape to print or the point sample droplet substrate on substrate tablet.Though can under robot control, use many nibs (generally being 8 or 16) simultaneously usually, each nib or respectively organize every at nib a kind of reagent only is housed.Then, nib is contacted one by one with substrate, sedimentary on every substrate is much at one droplet.After every group of substrate that will prepare (generally being several hundreds of sheets of getting to) was with the first group reagent droplet point sample, washing and the dry group nib that is somebody's turn to do reloaded next group reagent, and next group droplet are printed on the contiguous zone of same substrate.This process is time-consuming and need expensive and accurate equipment to reach precision and speed.

Another known method comprises with long flexible kapillary the liquid of each storage hole is carried to tip capillaceous, these tips are put on one connects on one the substrate to be similar to the described method of people such as Shalon.This method has the described same shortcoming of people with Shalon etc., and requires a large amount of expensive reagent storage in kapillary.

United States Patent (USP) 5,800,992 people such as () Fodor disclose another known method, and it comprises with the associativity chemical substance by series of chemical synthetic oligonucleotide (Affymetrix) on substrate.This method is limited to oligonucleotide and is unsuitable for long-chain cDNA.In addition, the sequence of oligonucleotide must be known in advance.This method also has shortcoming to need heavy expensive device and time-consuming reactions steps.

For the fluorescence that amplifies association reaction or radiated signal indication, United States Patent (USP) 5,843,767 (Beattie) propose, and provide and pass substrate, arrange in groups, and have a plurality of pipelines that separate of the binding reagents that is fixed on the conduit wall.These pipelines have increased the combined surface area of substrate, have improved detection sensitivity and efficient theoretically.It is so big unlike what expect to find that these improve, and can only directly receive from projection in zone because the detection optical device still is subject to but in actual applications.

Summary of the invention

In view of known manufacture method and the above-mentioned shortcoming of microarray type inherent, the purpose of this invention is to provide a kind of improved method and apparatus that is used for making simultaneously a plurality of identical microarray of biological sample.

The invention provides a kind of substrate that is used for making microarray, described substrate have end face, bottom surface and a plurality of at described substrate end face and the bottom surface between the through hole that extends, each through hole has the cross section of a broad, a narrower cross section and a platform that is parallel to substrate top surface that is formed in the zone of transition; Described substrate is glass or silicon; Described substrate comprises and is positioned at that its at least one surface is gone up and connects the water impervious hydrophobic viscous substance or the weak tackiness agent on boundary with described through hole.

One preferred embodiment in, described substrate has at least 100 through hole/cm ²

Another preferred embodiment in, described at least the first platform has the first analyte specific reagent, and at least the second platform has the second analyte specific reagent different with the described first analyte specific reagent.

Another preferred embodiment in, described analyte specific reagent can detect the cDNA of mark in the cross experiment.

The present invention also provides a kind of stack of substrates of being made up of the above-mentioned substrate that is used for making microarray, and described stack of substrates comprises at least two above-mentioned substrates, and wherein, the alignment of the through hole of correspondence position forms the continuous passage that connects described stack of substrates in described stack of substrates; Be bonded together with tackiness agent between each substrate; Binder layer have with substrate in the corresponding through hole in hole so that reagent flows through passage, and as level shielding to prevent the seepage of reagent between the passage; Tackiness agent on the position in hole to stop that mode is coated on one of substrate top surface and bottom surface or both flat surfaces.

One preferred embodiment in, described stack of substrates comprises at least 10 substrates.

The present invention also provides a kind of device that is used for forming simultaneously one group of identical microarray, and described device comprises:

Dismountable clamping device,

By the above-mentioned stack of substrates of described dismountable clamping device support,

Be used for reagent is introduced device in the through hole that separates of described stack of substrates upper surface or lower surface.

One preferred embodiment in, described device also comprises the vacuum unit that is communicated with the substrate face relative with the reagent lead-in surface.

Another preferred embodiment in, described being used for comprises two or more tubular channels with the device that reagent is introduced in the through hole separately, each passage is communicated with the supply groove that contains reagent.

The present invention also provides a kind of method of making series of identical microarray simultaneously, and it comprises:

Form an above-mentioned stack of substrates,

Introduce in the second passage to major general's first reagent introducing first channel and with second reagent, and

Separate described substrate.

One preferred embodiment in, described method also comprises to the stack of substrates face relative with the reagent lead-in surface and applies vacuum.

Another preferred embodiment in, described method also comprises the step that described substrate and binder layer are separated.

Another preferred embodiment in, described at least two kinds of different reagent have passed through at least two different passages.

Another preferred embodiment in, described binder layer is formed by the material that is selected from wax.

Another preferred embodiment in, the diameter of described binder layer through hole is at least three times of described substrate through-hole diameter.

Another preferred embodiment in, described heap comprises at least 10 dismountable binder layers of alternative and substrate.

Another preferred embodiment in, at least 100 passages are by described heap.

Description of drawings

In order to understand character of the present invention and purpose more fully, should come reference in conjunction with the accompanying drawings by following detailed description:

Fig. 1 is the stereographic map of stack of substrates, shows the hole and the depression of coupling.

Fig. 2 is the sectional view of exhausting hole part in the stack of substrates.

Fig. 3 is the sectional view of stack of substrates and with the method for transfer pipet filling substrate.This figure also shows anchor clamps.

Fig. 4 is the sectional view of stack of substrates and uses the method for vacuum take-off filling substrate and the pipeline that leads to the microtitre groove.

Fig. 5 is among Fig. 2, but the sectional view of the stack of substrates of arranging in another way.

Fig. 6 a is the exploded view of the alternating layer stacked arrangement of wide hole pad and narrow hole substrate, and these layers not to scale (NTS) is as used in the interchangeable embodiment of the present invention.

Fig. 6 b is by two pads of Fig. 6 a and the sectional view of two substrate layers.

Fig. 6 c is to use the part of microarray of the parts preparation of Fig. 6 a and 6b, shows six that are formed in hundreds of on the solid phase carrier or the thousands of kinds of analyte determination districts.

Embodiment

The present invention relates to form on solid phase carrier the method for analyte determination district microarray, wherein, each zone in the array has the selected analyte specific reagent of known quantity.More put it briefly, the invention provides one or more bonded substrates in a kind of polynucleotide that are used for certification mark and the numerous different sequences of fixed polynucleotide.

Microarray, and the reagent that is used to form microarray knows in this area, so just do not need to describe in detail in this article.These reagent preferably are fixed in on-chip different polynucleotide or polypeptide bioabsorbable copolymer.

Use the method for microarray, contact as under the cDNA by the fluorescent reporter molecule mark and the condition that contains the complementary sequence multi-nucleotide hybrid of the pulsating polynucleotide microarray of a plurality of known dnas in the cDNA that causes mark and array, under the fluorescence excitation condition, detect fluorescence then, also be known in the art, so do not need to describe in detail in this article.To the discussion of technology, can be with reference to United States Patent (USP) 5,800,992 people such as () Fodor and United States Patent (USP) 5,807,522 people such as () Brown, its disclosure is with reference to being incorporated into this.

Specifically, the present invention relates to make quickly and easily the method and apparatus of a plurality of identical microarraies of biological sample.

An important and outstanding employing polylith substrate that is characterised in that of the present invention, each substrate has end face, bottom surface and through hole model.Each through hole has upper section, the narrower lower section of broad and preferably is formed on step or the platform that is parallel to substrate top surface in the zone of transition.When some substrate stack, corresponding through hole alignment also forms the passage that connects stack of substrates.Make used reagent flow through these passages and be deposited on step or platform area on.Thus, the reagent accurately measured of all substrates that pile up " point sample " simultaneously on accurate position.Barrier layer between the substrate can prevent the seepage between the adjacent holes.

After reagent deposition, separate substrate.Form a series of microarraies (can contain hundreds of or thousands of biological samples such as cDNA segment separately) by this way simultaneously.

Like this, (this device is designed to only once deposit a kind of reagent on substrate surface with the planar array substrate of prior art, substrate of primary treatment) compares, the present invention includes a pile and have the preferably identical substrate of the through hole of same model separately, each hole in every substrate is corresponding to each point of the analyte specific reagent of estimating in the final array.For example, make 100 identical arrays if desired, every substrate has an array, each array has 10,000 different points, then will use 100 identical substrates, every produces 10,000 through holes, and these through hole alignment are embarked on journey when substrate stack.

The communicating pores of substrate represented to adjoin in the heap simply in term used herein " through hole ".In a better embodiment of the present invention, formation makes the horizontal bench of each communicating pores post or platform area look overlapping when piling up.These horizontal bench or platform area can abbreviate discontinuous mensuration district or " point sample district " as.In order replacedly to use the microarray carrying substrates, preferably each on-chip point sample district is identical, and Shi Yan point sample can be carried out by robot like this, by program point sample on concrete x, y coordinate of setting.Narrower cross-sectional area should be positioned at a side in point sample district, for example at the edge in this point sample district.Better, the narrower cross-sectional area of even number microscope slide through hole is positioned at a side (for example right side) in point sample district in the heap, and be positioned at the opposite side (for example left side) in this point sample district with the narrower cross-sectional area of odd number microscope slide through hole in a pile, flow through like this before and after the reagent of passage " circling round " flow, so available reagent washs each point sample district, and guarantees not carry secretly in the passage bubble.

In a better embodiment of the present invention, " point sample district " is formed on the substrate with 180 ° of symmetric patterns, promptly rotates about 180 ° and when being stacked on non-rotary second substrate top when first substrate, and through hole keeps alignment also to form passage.So just may form all substrates with a manufacturing process, and when piling up by 180 ° of the loading agreements that contracts a film or TV play to an actor or actress of numbering alternately of rotation simply, on a side of odd number microscope slide, form zone, and on the opposite side of even number microscope slide, form the zone of narrow diameter than narrow diameter.

Size to substrate does not have theoretic restriction, but their transverse diameter is generally 0.5-5cm, thickness is 0.05-3mm, and can be the size of conventional slide.Different sizes is suitable for different application.The quantity of point, size and interval, and the quantity of substrate depends on the number and the amount of the reagent that uses in the array.

Arrange the eclipsed through hole alignment and when forming the passage that connects stack of substrates of making in heaps when identical substrate, be preferably in and form barrier seal between the microscope slide, make and take office what from a passage that its passage does not produce horizontal seepage.In addition, each hole in each substrate can be counterbore, boring or other (may the be eccentric) depression in this substrate.When cross-section, these depressions produce the small volume towards the hole pipeline side that connects substrate.In addition, these depressions provide and each substrate general surface substrate surface zone of parallel small area substantially.Preferably, each through hole has upper section, the narrower lower section of broad and preferably is formed on step or platform in the zone of transition that is parallel to this substrate top surface.

Sealing method can be the tackiness agent or the sealing agent of the used concrete chemical substances compatible (having inertia) of hydrophobic viscous substance such as grease, wax, weak tackiness agent or any other and array.For other purposes, extremely thin elastomer layer (pad) will satisfy the demands.The surface property that has been found that the dead smooth that the micromachining method that is used for making substrate produces can make that sealing is comparatively simple.In fact, for some purposes, the ultra-smooth surface of the substrate that may place one's entire reliance upon as glass slide, wherein, adjoins microscope slide contact critically each other except through hole.

Be injected in an end of each passage with being used for all ingredients of manufacturing array, the upper end of this passage normally, then each passage receives (usually) different reagent.But injection a kind of reagent of a shot or grouping injection perhaps preferably are injected in all passages simultaneously.Inject available syringe, pipe or other device, manual or automatic, assisting down, carrying out at various pumps with kapillary or vacuum.

Flow through the passage that connects stack of substrates along with reagent, comprise the side-facing depressions that forms than the large through-hole district by diameter, they will also combine with the surface in contact reaction of the described passage with depression, this depends on the chemical property and the surface of the concrete reagent of use, with the method for droplet physical deposition on flat surfaces that is similar to available technology adopting.Drying can be carried out after deposition, and the similar approach that adopts in also can routine techniques is carried out.Like this, all same chemical substances used in the art and combination now can be adopted method and apparatus of the present invention, and this helps its concrete application.

After reaction required in the whole stack of substrates is finished, the dry substrate heap.Can remove sealing material (if any) simply stack of substrates is separately become the monolithic substrate.Obtained the identical single substrate of polylith can be used for routine techniques and hybridizes etc. this moment.But, only carry out the processing of point sample, rather than point sample is some beats or become hundred microscope slides.

The hole of model identical (and relevant side depression or larger-diameter zone) preferably uses silicon or the micro-lithographic printing art of glass and micromachining technology to make.This technology is ideally suited in produce a plurality of identical models at an easy rate with required reduced size.But, can adopt other technology to include but not limited to the mechanical workout or the abrasion of laser engine processing, plasma etching and spendable routine, and the technology that comprises the arrangement of different glass material, a kind of is acid etching (channel glass) and the fibers form corresponding with through hole to be formed, another kind is an inert, carries out chemical milling subsequently to remove etched glass.

In the one side of each substrate, outstanding (lug boss) on the substrate top surface for example, and at opposite face, the depression of the correspondence on the bottom surface for example, can be used to the alignment substrate with form porose stack of substrates of all aliging.Perhaps, latch can be set by being positioned at all on-chip collimating apertures to reach same effect.In addition, if mechanical workout is enough accurate, then substrate can be by providing by the fairlead of all layers justified margin along them.Other is used for gauged method and will be apparent to those skilled in the art.

Importantly, the position of point sample should be determined in this preliminary production step, rather than determine by robot sample deposition.So just may make the position at height of center volumetrical substrate and obtain high tolerance range with lower cost.Injecting step does not subsequently need expensive device, and it can carry out with different reagent in a plurality of different laboratories.The center manufacturing step of making substrate does not comprise or the choice of location or the arrangement of definite all ingredients or point sample that they can be selected by each customer laboratory.

Can use various schemes that the reagent injection device is connected in stack of substrates.The passive little funnel or the simpler array of passage can change more coarse injection device into meticulousr array crack.Perhaps, can use simple but accurate x-y locating device to remove the array heap about injection device.Because one-tenth hundred or more substrate are injected simultaneously, can reach rational throughput rate, and needn't spend the high cost of using quick robot in this area.

Below, the illustrative embodiment shown in is described the present invention in more detail with reference to the accompanying drawings.In Fig. 1, substrate 1 usefulness intermediary binder layer 4 is stacked.Binder layer have with substrate in the corresponding through hole in hole so that reagent flows through passage, and as level shielding to prevent the seepage of reagent between the passage.Each substrate layer has the through hole 3 that a row aligns with the through hole that adjoins substrate.Tackiness agent on the position in hole to stop that mode is coated on one of substrate top surface and bottom surface or both flat surfaces.Perhaps, tackiness agent can be coated on the substrate before the step that forms the hole, and the same area that should form in the hole is removed tackiness agent in the identical time in both cases.

As shown in Figure 1, each through hole has upper section, the narrower lower section of broad and preferably is formed on step or the platform 2 that is parallel to substrate top surface in the zone of transition.This step or the platform 2 final submission districts that form the analyte determination zone.Fastening screw 14 and/or lug boss 15 can be used for initial alignment or keep heap to be in line.

Referring to Fig. 2, have the bore region alignment in narrower cross section 3 and be connected to form passage, this passage connects to the bottom fully from the top of heap, comprises through-bonding agent layer 4.Recess side forms in step or platform 2 zones.Tackiness agent can maybe can not be coated in the substrate platform district.

Fig. 3 shows a kind of device, and it is used for injecting stack of substrates 1 of the present invention.This stack of substrates is clipped between liner plate 6 and the vacuum manifold 9.Clamping pressure is provided by clip 10 and frame 8.The injection shield that tapered hole 7 is arranged in liner plate forms, tapered hole have bigger upper diameter so that injection device such as pipette tip enter easily, and the narrower base diameter that aligns with through hole.Injecting the process of reagent, will wait branch reagent to inject liner plate and flow downward, or applying slight vacuum to each passage bottom and produce pressure difference and come auxiliary flow by gravity or capillary action.Consider the cost of reagent, be careful and guarantee in enough reagent suction passages all filling up passage, but do not have or have only unnecessary reagent seldom to discharge from channel bottom.This can transfer to vacuum usually enough greatly helping and reach in the reagent suction passage, but vacuum can not be greatly to the capillary force that surpasses the reagent in the reserve channel.

Fig. 4 represents an interchangeable embodiment of the present invention, shows the stack of substrates 1 that is clipped between top vacuum manifold 9 and the lower tubular liner plate 13.Pipe 11 that stretches out and the span in the microtitre groove 12 mate, and communicate with the reagent in the hole that is arranged in microtitre groove 12.

Fig. 5 is the sectional view of the stack of substrates of Fig. 2.But in an interchangeable device, wherein, the pattern of through hole 3 be stagger or alternate mode, determined the flow passage that circles round of reagent.As long as the path by each through hole post and depression post is a successive, adjoin or alternating layer in just do not need accurately to repeat the exact position of each post.The advantage of this alternate path is can cover concave bottom better under some mixed filling fluidic situation.Can between two different models, exchange the most simply, but also can just have repeat pattern or random pattern by per three microscope slides.All depressions of embarking on journey are carried out the simplest later observation and automation data obtain also very simply, but need the proper handling method anything but.Its a plurality of possible changes will be apparent to those skilled in the art.

The present invention also can adopt the embodiment shown in Fig. 6 a (separating) and the 6b (assembling) to carry out, and only shows two in staggered substrate that possible dozens of piles up and the spacer among these figure.In this embodiment, substrate 17a, 17b only have the narrower through hole 18a, the 18b that pass substrate through mechanical workout, compare with ladder shown in 5 or boring substrate with Fig. 1,2, and manufacture method is simplified.Each smooth deposition region is determined by hole 19a, 19b among spacer 16a, the 16b on the substrate.

Spacer can separate with substrate, perhaps, spacer layer can be coated on the substrate, carry out the through hole etching subsequently, perhaps spacer layer can carry out silk screen printing, offset printing or other method of printing on substrate, perhaps can will have the solid elastic film that is pre-formed through hole or other film lamination on substrate layer.For the waste with reagent reduces to minimum, preferably spacer is formed as plastics (preferably flexible polymer plastics), rubber, wax, glass and metal by not adsorbing reagent, being more preferably the material of anti-reagent deposition.The above-mentioned any material relevant with first embodiment of the present invention can be used for second embodiment of the present invention.

Spacer is inserted between the substrate layer, and the hole in the spacer that separates is greater than through hole 18a, 18b among substrate 17a, the 17b.The thickness of spacer layer limits, and is thinner than substrate usually, so just produced depression between the substrate adjoining, and this is recessed in and reagent is contacted and is deposited on the substrate.Above-mentioned arbitrarily the stagger substrate through-hole relevant with Fig. 5 can randomly be used for the embodiment of Fig. 6 a and 6b.

Fig. 6 b is the sectional view of two spacers and two substrate layers, is illustrated in the two-layer of assembled condition figure below 6a.Significantly, reagent flow through serially among spacer 16a, the 16b spacer hole 19a, 19b and by hole 18a, 18b among substrate 17a, the 17b.

Fig. 6 c is the synoptic diagram of a microarray part that adopts the device preparation of Fig. 6 a and 6b, shows six in the hundreds of or thousands of analyte determinations district that is retained in after spacer 16a, 16b remove on the solid phase carrier.Significantly, under the coating or the situation of lamination interval thing on the substrate, spacer layer will remain adhered on each substrate or the end face or bottom surface of substrate on.Say that exactly because reagent will be coated on the two sides of substrate, any one side in the substrate two sides all can be regarded end face or available as.

Preferably spacer keeps thin as far as possible, reduces to reagent minimum and that will be deposited on the spacer with the amount with required reagent and reduces to minimum.The selection of spacer and substrate material and their thickness, those skilled in the art are not difficult to determine.

About above description, recognize the optimal size relation of parts of the present invention, comprise the various changes of size, material, shape, form, function and working method, assembling and purposes, and this be really to those skilled in the art be not difficult to understand, conspicuous, all with shown in the accompanying drawing and the described content of specification sheets relation of equal value mutually all comprise in the present invention.

Therefore, foregoing is regarded as merely explanation of the principles of the present invention.In addition, because a large amount of modifications and change are carried out to those skilled in the art easily, so do not wish to limit the invention in illustrated and described precise structure and the operation, therefore, adoptable all suitable modifications and Equivalent are all within the scope of the present invention.

Now, the present invention describes.

Claims

1. substrate that is used for making microarray, described substrate have end face, bottom surface and a plurality of at described substrate end face and the bottom surface between the through hole that extends, each through hole has the cross section of a broad, a narrower cross section and a platform that is parallel to substrate top surface that is formed in the zone of transition; Described substrate is glass or silicon; Described substrate comprises and is positioned at that its at least one surface is gone up and connects the water impervious hydrophobic viscous substance or the weak tackiness agent on boundary with described through hole.

2. substrate according to claim 1 is characterized in that: described substrate has at least 100 through hole/cm ²

3. substrate according to claim 1 is characterized in that: described at least the first platform has the first analyte specific reagent, and at least the second platform has the second analyte specific reagent different with the described first analyte specific reagent.

4. substrate according to claim 3 is characterized in that: described analyte specific reagent can detect the cDNA of mark in the cross experiment.

5. stack of substrates of forming by the described substrate that is used for making microarray of claim 1, it is characterized in that: described stack of substrates comprises at least two described substrates of claim 1,

Wherein, the alignment of the through hole of correspondence position forms the continuous passage that connects described stack of substrates in described stack of substrates; Be bonded together with tackiness agent between each substrate; Binder layer have with substrate in the corresponding through hole in hole so that reagent flows through passage, and as level shielding to prevent the seepage of reagent between the passage; Tackiness agent on the position in hole to stop that mode is coated on one of substrate top surface and bottom surface or both flat surfaces.

6. stack of substrates according to claim 5 is characterized in that: described stack of substrates comprises at least 10 substrates.

7. one kind is used for forming simultaneously the device of one group of identical microarray, and described device comprises:

Dismountable clamping device,

By the described stack of substrates of claim 5 of described dismountable clamping device support,

8. device according to claim 7 is characterized in that: it also comprises the vacuum unit that is communicated with the substrate face relative with the reagent lead-in surface.

9. device according to claim 8 is characterized in that: described being used for comprises two or more tubular channels with the device that reagent is introduced in the through hole separately, and each passage is communicated with the supply groove that contains reagent.

10. method of making series of identical microarray simultaneously, it comprises:

Form a described stack of substrates of claim 5,

Separate described substrate.

11. method according to claim 10 is characterized in that: it also comprises to the stack of substrates face relative with the reagent lead-in surface and applies vacuum.

12. method according to claim 10 is characterized in that: it also comprises the step that described substrate and binder layer are separated.

13. method according to claim 10 is characterized in that: described at least two kinds of different reagent have passed through at least two different passages.

14. method according to claim 10 is characterized in that: described binder layer is formed by the material that is selected from wax.

15. method according to claim 10 is characterized in that: the diameter of described binder layer through hole is at least three times of described substrate through-hole diameter.

16. method according to claim 10 is characterized in that: described heap comprises at least 10 dismountable binder layers of alternative and substrate.

17. method according to claim 10 is characterized in that: at least 100 passages are by described heap.