CN103954786B - Semi-contact under-oil continuous droplet sample applying and liquid adding method - Google Patents
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Abstract
The invention provides a semi-contact under-oil continuous droplet sample applying and liquid adding method, which is suitable for sequentially operating a droplet array system. According to the method, the distance between the pointed end of a capillary tube sample applying needle and the lower surface of a micro-hole (or a generated droplet) is controlled accurately, and the affinity or interface tension interaction between the droplet and the surface (or the generated droplet) is utilized, so that rapid and reliable continuous droplet sample applying or continuous liquid adding is realized, and the problem of cross infection during sample applying is solved effectively. The method is suitable for biochemical analysis screening researches such as high-flux medicament screening, protein crystallization condition screening, enzyme kinetics research and drug toxicity determination.
Description
Technical field
The field that the present invention relates to is liquid drop analysis field, the continuous point sample of drop and liquid feeding method under the oil of particularly a kind of semi-contact.
Background technology
Microflow control technique (Droplet-based microfluidics) based on drop achieves development fast and applies widely.The major advantage of drop microflow control technique comprises: 1) drop reactor volume can receive upgrading flexible flying to upgrade to, and thus sample and reagent consumption can be down to extremely low level; 2) a large amount of drop reactors can be generated at short notice, thus improve Analysis and Screening flux significantly; 3) rapid mass transfer of component, heat transfer can be realized and mix in drop, effectively improve biochemical reaction efficiency; 4) parcel of oil phase, effectively reduces the evaporation of micro biochemical reaction, diffusion and cross-contamination issue; 5) the self assembly effect of bio-compatibility surfactant molecule in oil phase, the microenvironment providing a gentleness homogeneous can to the biochemical reaction in drop, is conducive to the accuracy improving Analysis and Screening.
At present, most drop analysis system adopts T-shaped interface or cross to focus on interface method and generates drop.But the drop that these class methods generate generally has identical chemical composition and concentration, because which limit its application in extensive different sample analysis screens.In recent years, in order to realize the generation of a large amount of different chemical composition drop in liquid droplet system, the people such as Zhejiang University Fang Qun have developed a kind of high density drop array screening method (Fang Qun, Du Wenbin, Sun Meng, based on Microfluidic droplet generation system and the using method of drop der group packing technique, Chinese invention patent application, application number: 201010250945.0; Fang Qun, Zhu Ying, Zhang Yunxia, a kind of using method with the robotization microlayer model array screening system of skin upgrading precision, Chinese invention patent application, application number: 201210589055.1).The cardinal principle of the method is, first utilizes three-dimensional machinery system to complete the sample hose of robotization and the quick switching of Reagent Tube, is extracted by the sample of trace and form drop to kapillary sample probe.Then, above micropore capillary probe being moved to micro-fluidic chip, long-time static reaction will be carried out in drop one by one point sample to chip micropore and next step detects.The method has used for reference the advantage of micro-array biochip liquid deposition techniques, and can realize the Analysis and Screening of different sample in enormous quantities on the chip of several square centimeters, sample reagent consumption also significantly reduces.Further, compared with micro-array biochip screening technique, its advantage is; in the process of drop point sample and incubation reaction; due to immiscible oil phase parcel and protective effect, the biochemical reaction in drop carries out in the homogeneous phase environment of gentleness, closer to it in natural state.Therefore, its selection result more accurately and reliably.
Find in our further investigation, because whole drop deposition process carries out in oil phase, conventional micro-sampling technology, as contact point sample and contactless deposition techniques (Rose, D.DrugDiscov.Today 1999,4,411-419), be all difficult to realize drop point sample in reliable oil phase.This is because, adopt contact deposition techniques to generate drop in oil phase, easily generate the uncontrollable small volume drops of a large amount of volume.And adopt contactless deposition techniques in oil phase, generate drop, there is the problem that drop is difficult to depart from spotting needle.In addition, in order to the drop reactor improved containing sample and reagent in biochemical screening process generates flux, the people such as Zhejiang University Fang Qun have been developed a kind of method inserting drop based on capillary tip and sample or reagent have been injected continuously drop (Fang Qun on chip, Zhu Ying, Zhang Yun's rosy clouds, there is a using method for the robotization microlayer model array screening system of skin upgrading precision, Chinese invention patent application, application number: 201210589055.1).But, due to capillary tip continually from the drop contact of different sample, easily cause cross pollution, cause false-positive the selection result.
Summary of the invention
The object of this invention is to provide and be a kind ofly applicable to the continuous point sample of drop and liquid feeding method under the semi-contact oil of sequential operation droplet array system.The method is by accurately controlling the tip of kapillary spotting needle and the distance of micropore lower surface (or generating drop), utilize the affine or interfacial tension effect on drop and surface (or generating drop), the continuous point sample of drop or the continuous liquid that realize fast and reliable add, and efficiently avoid the cross-contamination issue in deposition process.The method is applicable in the biochemical analysis screening studies such as high-flux medicaments sifting, protein crystallization condition screening, enzyme dynamics, drug toxicity mensuration.
Concrete technical scheme of the present invention is as follows:
The continuous point sample of drop and a liquid feeding method under the oil of semi-contact, comprise kapillary, with micropore microwell array chip, for driving the liquid driven system of kapillary dropping liquid, comprise the steps:
Step 1: be full of the sample solution or reagent solution for the treatment of point sample in kapillary, and cover one deck oil phase on microwell array chip;
Step 2: utilize kapillary to form the array of sample drop or reagent droplet on microwell array chip;
Step 3: reagent solution to be added or sample solution will be full of in kapillary;
Step 4: control capillary tip and substantially aim at the center of the micropore at drop place to be added, and to keep at a distance d2 with the upper surface of the sample drop in micropore or reagent droplet;
Step 5: start liquid drive system, releases the reagent solution of preset vol or sample solution from the tip of kapillary, contacts merge with the existing sample drop in micropore or reagent droplet;
Step 6: control kapillary and depart from micropore, the reagent droplet be pushed out or sample drop merge completely with existing sample drop in micropore or reagent droplet, the drop reactor that formation is deposited in micropore;
Repeat step 3 to step 6, microwell array chip is formed drop reactor array.
Described step 2 can adopt existing additive method to form the array of sample drop or reagent droplet, and as preferably, the concrete steps of described step 2 are:
Step 2-1: the center of a certain micropore (3) on microwell array chip (2) is aimed at the tip controlling kapillary (1) substantially, and to keep at a distance d1 with the lower surface of this micropore (3);
Step 2-2: start liquid drive system, releases the sample solution (5) of preset vol or reagent solution (7) from the tip of kapillary (1), contacts with the lower surface of micropore (3);
Step 2-3: control kapillary (1) tip and depart from micropore (3), form the sample drop (6) or reagent droplet (8) deposited in micropore (3);
Repeat step 2-1 to step 2-3, microwell array chip (2) is formed the array of sample drop (6) or reagent droplet (8).
In step 2-1, during d1 setting, contact with micropore inside surface, to ensure that the drop of formation can enter in micropore smoothly before needing at least to meet sample solution that kapillary in step 2-2 releases or the drop that reagent solution has been formed.For improving the contact area of drop and micropore inside surface, ensure that drop enters into rapidly in micropore, improve point sample efficiency, as preferred further: during d1 setting, demand fulfillment, the sample solution that in step 2-2, kapillary is released or reagent solution volume are a half of preset vol, start to contact with micropore inside surface.
In step 2-1, step 2-3, step 4 and step 6, control by mobile microwell array chip or by the relative position of mobile kapillary to kapillary and micropore; The relative position of transfer table to both generally preferably by mobile carrying microwell array chip controls automatically.
In step 2-2, step 5, liquid driven system generally adopts liquid charge pump or fluid filling pump, realizes automatically controlling by computing machine etc.The volume of sample solution or reagent solution is generally determined according to actual needs.
In step 2-3, step 6, after capillary tip departs from micropore, at micropore inside surface under the suction-operated of micropore, drop is separated with capillary tip, forms independently sample drop in micropore, completes once complete sample drop generating run process.
What be full of in kapillary in exchonge step 1, step 4 in the present invention treats the sample solution of point sample or the kind of reagent solution, can generate different types of sample drop or reagent droplet, to meet the demand that various occasion detects.Experimentally object is different, can select first inject reagent solution or first inject sample solution; And then inject other sample solution or reagent solution; The solution be filled with in kapillary in step 1 and step 4 can only be the one of reagent solution and sample solution, and different.
In step 4, during d2 setting, need at least to meet before reagent solution that kapillary in step 5 releases or the drop that sample solution has been formed with the sample drop formed or reagent droplet exterior surface, to ensure to release from the tip of kapillary the reagent droplet that formed or smooth to contact with the existing sample drop micropore or reagent droplet of sample drop is merged.
Point sample of the present invention and liquid feeding method can be applied to Multi-example system, namely utilize step 1 to the method for step 2 to carry out point sample to different sample respectively; Then point sample is carried out to difference is actual respectively by step 4 to the method for step 6.Its concrete operation step is:
1) in the different micropores on microwell array chip, form the sample drop with the different samples of same volume, form sample drop array;
2) utilize kapillary above each sample drop, release the reagent solution of same volume, form reagent droplet, and merges with the sample drop on chip and form drop reactor, formation reactor droplet array.
When needing to carry out having the experiment of the sample of multiple concentration or reagent, also can adopt method of the present invention, what now in described step 2-2, kapillary released be the sample drop of volume difference, same sample; What in described step 5, kapillary was released is the reagent solution of different volumes; The cumulative volume of described drop reactor is identical.Its concrete operation step is:
1), in the different micropores on microwell array chip, same sample drop or the reagent droplet with different volumes is formed; Such as volume increases gradually or reduces gradually;
2) utilize kapillary above each drop, release reagent solution or the sample solution of different volumes, and merge with the sample drop on microwell array chip or reagent droplet, form drop reactor.By controlling the volume of the drop formed in micropore and the follow-up volume adding solution, make the cumulative volume of the drop reactor after fusion identical, and in drop reactor, the concentration of sample or reagent or the concentration proportioning of sample and reagent are that certain rule distributes.
In step 2-1 according to point sample generate the volume of drop, adjust the distance d1 between the tip of kapillary and the lower surface of micropore, setting range is 1 micron to 5 millimeters, ensures that the drop that point sample generates contacts with the lower surface of micropore.
In step 4, according to the volume of the drop generated in the volume of liquid feeding and micropore, adjust the distance d2 between the drop upper surface generated in capillary tip and micropore, setting range is 1 micron to 5 millimeters, ensures that the drop that liquid feeding generates contacts with drop upper surface in micropore.
The material of described kapillary is quartz or glass or metal or high molecular polymer; Capillary inner diameter at 1 micron within the scope of 5 millimeters; Microcapillary tube wall thickness at 1 micron within the scope of 5 millimeters; Capillary pipe length at 1 millimeter within the scope of 10 meters.
The depth range of the micropore on described microwell array chip is 1 micron to 5 millimeters, and receiving fluids volume range 1 to be ascended to heaven to 100 microlitres.
For ensureing that drop enters in micropore smoothly, the acting force between described micropore inside surface and drop should be greater than or equal to the acting force between capillary tip and drop.Hightail kapillary, as preferably: the tip of described kapillary carries out drawing point process, and carries out hydrophobizing surface process to the inside and outside wall of kapillary for ensureing that drop is formed rapidly further simultaneously; Further reduction drop and intercapillary acting force.In addition, also can select to carry out hydrophobization process to the surface of microwell array chip.For avoiding drop along the drift of microwell array chip surface, preferred as another kind, can carry out the hydrophobic and hydrophilicity-imparting treatment of selectivity to microwell array chip, make micropore inside surface keep water wettability, the chip surface beyond micropore keeps hydrophobicity.That is, hydrophilicity-imparting treatment is carried out to micropore inside surface, hydrophobization process is carried out to the remaining surface of the microwell array chip beyond micropore.Described hydrophobization process, comprises silanization or the method such as fluothane or polymer coating.
Compared with prior art, advantage of the present invention is mainly:
(1) drop point sample and liquid feeding method under the oil of semi-contact, can improve reliability and the homogeneity of drop point sample on microwell array chip effectively;
(2) adopt continous way drop point sample and liquid feeding method, considerably improve the speed of drop formation, be beneficial to the raising analyzed and screen flux;
(3) in point sample and liquid feeding process, capillary tip not with chip or existing drop contact, avoid cross pollution;
(4) utilize variable volume drop point sample and liquid feeding method, on chip, the sample or reagent droplet array with concentration gradient can be generated fast.
Accompanying drawing explanation
Fig. 1 adopts the continuous point sample of drop and liquid feeding method under semi-contact oil to carry out sample drop to generate and reagent adds the principle schematic of operation.
Fig. 2 adopts the continuous point sample of drop and liquid feeding method under semi-contact oil to carry out reagent droplet to generate and sample adds the principle schematic of operation.
Fig. 3 is the fluorescein droplet array picture utilizing drop continuity point quadrat method to generate, and utilizes continuous liquid feeding method to carry out equal-volume to fluorescein droplet array to dilute the droplet array picture obtained.
Fig. 4 utilizes the continuous point sample of variable volume drop and liquid feeding method to generate the principle schematic with the droplet array of concentration gradient.
Fig. 5 is the fluorescein droplet array picture with concentration gradient utilizing variable volume drop point sample and liquid feeding method to generate.
Fig. 6 is the linear diagram of the corresponding fluorescence intensity of fluorescein concentration in drop.
In figure: 1-kapillary, 2-microwell array chip, 3-micropore, 4-oil phase, 5-sample solution, 6-sample drop, 7-reagent solution, 8-reagent droplet, 9-drop reactor.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described further:
With reference to accompanying drawing, below will describe in detail according to a preferred embodiment of the invention.
Embodiment 1
Fig. 1 adopts the continuous point sample of drop and liquid feeding method under semi-contact oil to carry out sample drop to generate and reagent adds the principle schematic of operation.The specific operation process (Fig. 1 (1)-(4)) that the continuous point sample of drop generates sample drop 6 is as follows: draw point to process the cross pollution reduced in sampling process to the sampling end of kapillary 1, and carry out hydrophobizing surface process to prevent sample and the absorption of reagent on its surface to the inwall of kapillary 1.One deck and the immiscible oil phase 4 of water is covered above microwell array chip 2.In kapillary 1, suck the sample solution 5 that certain volume treats point sample, mobile translation stage, kapillary 1 is placed in directly over micropore 3 center of microwell array chip 2.According to the volume size of point sample drop, the adjustment tip of kapillary 1 and the spacing d1 of micropore 3 lower surface, make the sample drop 6 pointed out can contact with micropore 3 lower surface.Start liquid drive system, releases the sample solution 5 of certain volume from kapillary 1, and first form sample drop 6 at capillary tip, then this sample drop 6 contacts with the lower surface of micropore 3, forms sample drop 6 in micropore 3.Mobile translation stage, makes kapillary 1 leave micropore 3.Because micropore 3 lower surface is to the suction-operated of sample drop 6, sample drop 6 is separated with kapillary 1 tip, forms independently sample drop 6 in micropore 3.So far, complete sample drop generating run process is completed once.Repeat aforesaid operations, the sample drop array be made up of multiple sample drop 6 can be formed on microwell array chip 2.
Specific operation process (as Fig. 1 (5)-(7)) to the continuous liquid feeding method of the drop generated is as follows: in kapillary 1, suck certain volume reagent solution to be added 7, and mobile translation stage, make kapillary 1 be placed in micropore 3 and generated directly over sample drop 6.Adjustment kapillary 1 tip and the distance d2 of the upper surface of sample drop 6, make the reagent droplet 8 pointed out can contact with sample drop 6 upper surface.Start liquid drive system, releases the reagent solution 7 of certain volume from kapillary 1, first forms reagent droplet 8 at kapillary 1 tip, then contacts with the sample drop 6 in micropore 3, merge.Mobile translation stage, makes kapillary 1 leave micropore 3, and reagent droplet 8 merges completely with the sample drop 6 in micropore 3 and forms drop reactor 9.So far, complete sample drop generating run process is completed once.Repeat aforesaid operations, in each sample drop 6 in the sample drop array generated, a certain amount of reagent solution 7 can be added, form drop reactor 9 array.
Embodiment 2
Fig. 2 adopts the continuous point sample of drop and liquid feeding method under semi-contact oil to carry out reagent droplet to generate and sample adds the principle schematic of operation.The specific operation process (Fig. 2 (1)-(4)) that the continuous point sample of drop generates reagent droplet 8 is as follows: draw point to process the cross pollution reduced in sampling process to the sampling end of kapillary 1, and carry out hydrophobizing surface process to prevent sample and the absorption of reagent on its surface to the inwall of kapillary 1.One deck and the immiscible oil phase 4 of water is covered above microwell array chip 2.In kapillary 1, suck the reagent solution 7 that certain volume treats point sample, mobile translation stage, kapillary 1 is placed in directly over micropore 3 center of microwell array chip 2.According to the volume size of point sample drop, the adjustment tip of kapillary 1 and the spacing d1 of micropore 3 lower surface, make the reagent droplet 8 pointed out can contact with micropore 3 lower surface.Start liquid drive system, releases the reagent solution 7 of certain volume from kapillary 1, and first form reagent droplet 8 at capillary tip, then this reagent droplet 8 contacts with the lower surface of micropore 3, forms reagent droplet 8 in micropore 3.Mobile translation stage, makes kapillary 1 leave micropore 3.Because micropore 3 lower surface is to the suction-operated of reagent droplet 8, reagent droplet 8 is separated with kapillary 1 tip, forms independently reagent droplet 8 in micropore 3.So far, complete reagent droplet generating run process is completed once.Repeat aforesaid operations, the reagent droplet array be made up of multiple reagent droplet 8 can be formed on microwell array chip 2.
Specific operation process (as Fig. 2 (5)-(7)) to the continuous liquid feeding method of the reagent droplet generated is as follows: in kapillary 1, suck certain volume sample solution to be added 5, and mobile translation stage, make kapillary 1 be placed in micropore 3 and generated directly over reagent droplet 8.Adjustment kapillary 1 tip and the distance d2 of the upper surface of reagent droplet 8, make the sample drop 6 pointed out can contact with reagent droplet 8 upper surface.Start liquid drive system, releases the sample solution 5 of certain volume from kapillary 1, first forms sample drop 6 at kapillary 1 tip, then contacts with the reagent droplet 6 in micropore 3, merge.Mobile translation stage, makes kapillary 1 leave micropore 3, and reagent droplet 8 merges completely with the sample drop 6 in micropore 3 and forms drop reactor 9.So far, complete sample drop generating run process is completed once.Repeat aforesaid operations, in each reagent droplet 8 in the reagent droplet array generated, a certain amount of sample solution 5 can be added, form drop reactor 9 array.
Embodiment 3
The fluorescein droplet array picture that Fig. 3 utilizes drop continuity point quadrat method to generate, and utilize continuous liquid feeding method to carry out equal-volume to fluorescein droplet array to dilute the droplet array picture obtained.The internal diameter that kapillary is chosen in experiment is 150 microns, and external diameter is 200 microns.Draw point process to its sampling end, most advanced and sophisticated size is about 30 microns.Octadecyl trichlorosilane alkane is adopted to carry out hydrophobization process to the inwall of kapillary and tip.To be equipped with the syringe pump of 1 microliter syringe for liquid driven system, and syringe is connected with kapillary.Adopt the method for photoetching and wet etching to process microwell array on a glass substrate, each micropore is of a size of diameter 260 microns, dark 60 microns, and the distance of center circle between micropore is 400 microns.Same employing octadecyl trichlorosilane alkane carries out hydrophobizing surface process to the surface of microwell array chip.Microwell array chip is loaded on D translation platform, and is the mineral oil of 2 millimeters in its surface coverage a layer thickness.First in kapillary draw 100 receive rise Fluress (10mM), according to the drop continuity point quadrat method described in Fig. 1, move translation stage respectively, the distance keeping capillary tip and micropore lower surface is 100 microns, in each micropore, click and enter 1.98 receive the Fluress risen, formed and receive containing 50 1.98 the droplet array of uranin drop risen.Then, release Fluress unnecessary in kapillary in sewer pipe, and kapillary is cleaned.Finally, be full of in kapillary 100 receive rise damping fluid (50mM borax), according to the continuous liquid feeding method of the drop described in Fig. 1, move translation stage respectively, the distance keeping capillary tip and micropore lower surface is 150 microns, above each uranin drop, release 1.98 in kapillary receive the damping fluid drop risen, and to merge with the sample drop in micropore, obtaining 50 by volume is the droplet array that the 3.96 dilution drops to receive liter are formed.Carry out fluorescence to two kinds of droplet arrays respectively to take pictures, and calculate the relative standard deviation RSD of liquid-drop diameter.The liquid-drop diameter RSD of uranin droplet array and dilution droplet array is respectively 1.11% and 1.00%, shows that the continuous deposition techniques of drop and continuous liquid feeding technology have higher liquid handling precision.
Embodiment 4
Fig. 4 utilizes the continuous point sample of variable volume drop and liquid feeding method to generate the principle schematic with the droplet array of concentration gradient.The specific operation process of the continuous point sample of variable volume drop is as follows: in kapillary 1, first suck the sample solution 5 that certain volume treats point sample, then according to the drop continuity point quadrat method described in Fig. 1, move translation stage respectively, in each micropore 3, point out the different sample solution of volume 5, form sample drop 6.The volume V of sample drop 6
sampleincrease gradually according to certain rule.In the process of point sample, drop 6 volume V per sample
sampledistance that is most advanced and sophisticated to kapillary 1 and micropore 3 lower surface carries out dynamic conditioning, and the sample drop 6 pointed out can be contacted with the lower surface of micropore 3.The specific operation process of the continuous liquid feeding method of variable volume drop is as follows: in kapillary 1, suck certain volume reagent solution to be added 7, and mobile translation stage, make kapillary 1 be placed in the top of sample drop 6.Above each sample drop 6, in kapillary, release the reagent droplet 8 of different volumes respectively, and adjust kapillary 1 tip and the distance of micropore 3 lower surface, reagent droplet 8 is contacted with sample drop 6 and merges, form drop reactor 9.The volume V of reagent droplet 8
reagentthe volume V of drop per sample
samplecarry out dynamic conditioning, make the volume V of drop reactor 9
always=V
reagent+ V
sampleremain unchanged, thus obtain the drop reactor array with different sample/reagent concentration and proportioning.
Embodiment 5
Fig. 5 is the fluorescein droplet array picture with concentration gradient utilizing variable volume drop point sample and liquid feeding method to generate.Adopt 10 μMs of uranins as sample solution.According to the continuous point sample operation steps of the variable volume drop described in embodiment 4, first in microwell array, generate the uranin droplet array of different volumes, droplet size is from left to right respectively 0.42 to be received liter, 0.84 to receive liter, 1.26 to receive liter, 1.68 to receive liter, 2.10 to receive liter, 2.52 to receive liter, 2.94 to receive liter, 3.36 receive and rise and 3.78 receive liter, and each volume repeats three drops.In the process of drop point sample, the distance of capillary tip and micropore lower surface carries out dynamic adjustments according to droplet size, from a left side to by being respectively 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 110 microns, 120 microns and 130 microns.Then according to the operation steps of the continuous liquid feeding of variable volume drop described in embodiment 4, in each uranin drop, add the borate buffer solution drop of different volumes respectively, and merge with uranin sample drop.The borax solution drop volume added in each uranin drop is from left to right respectively 3.78 to be received liter, 3.36 to receive liter, 2.94 to receive liter, 2.52 to receive liter, 2.10 to receive liter, 1.68 to receive liter, 1.26 to receive liter, 0.84 receive and rise and 0.42 receive liter.Mixed drop cumulative volume is 4.20 receive liter, thus after making mixing in drop uranin concentration increase progressively according to the gradient of 1 μM, 2 μMs, 3 μMs, 4 μMs, 5 μMs, 6 μMs, 7 μMs, 8 μMs and 9 μMs, form concentration gradient droplet array.As shown in Figure 6, fluorescence imaging is carried out to droplet array, and get the fluorescence intensity level of drop, obtain the linearity curve of the corresponding fluorescence intensity of concentration.Linearly dependent coefficient is 99.96%, demonstrates and utilizes variable volume drop point sample and liquid feeding method to have higher liquid manipulation precision.
Claims (10)
1. drop continuity point quadrat method under the oil of a semi-contact, comprising kapillary (1), with the microwell array chip (2) of micropore (3) and for driving the liquid driven system of kapillary (1) dropping liquid, it is characterized in that: comprise the steps:
Step 1: be full of the sample solution (5) or reagent solution (7) for the treatment of point sample in kapillary (1), and at microwell array chip (2) upper covering one deck oil phase (4);
Step 2: utilize kapillary (1) to form the array of sample drop (6) or reagent droplet (8) on microwell array chip (2);
Step 3: be full of the reagent solution (7) or sample solution (5) for the treatment of point sample in kapillary (1);
Step 4: control the most advanced and sophisticated basic center aiming at the micropore (3) at drop place to be added of kapillary (1), and to keep at a distance d2 with the upper surface of the sample drop (6) in micropore (3) or reagent droplet (8);
Step 5: start liquid drive system, the reagent solution (7) of preset vol or sample solution (5) are released from the tip of kapillary (1), contacts with the existing sample drop (6) in micropore (3) or reagent droplet (8) and merge;
Step 6: control kapillary and depart from micropore (3), the reagent droplet (8) be pushed out or sample drop (6) merge completely with existing sample drop (6) in micropore or reagent droplet (8), form the drop reactor (9) deposited in micropore (3);
Repeat step 3 to step 6, at microwell array chip (2) upper formation drop reactor (9) array.
2. drop continuity point quadrat method under the oil of semi-contact according to claim 1, is characterized in that: the concrete steps of described step 2 are:
Step 2-1: the center of a certain micropore (3) on microwell array chip (2) is aimed at the tip controlling kapillary (1) substantially, and to keep at a distance d1 with the lower surface of this micropore (3);
Step 2-2: start liquid drive system, releases the sample solution (5) of preset vol or reagent solution (7) from the tip of kapillary (1), contacts with the lower surface of micropore (3);
Step 2-3: control kapillary (1) tip and depart from micropore (3), form the sample drop (6) or reagent droplet (8) deposited in micropore (3);
Repeat step 2-1 to step 2-3, microwell array chip (2) is formed the array of sample drop (6) or reagent droplet (8).
3. drop continuity point quadrat method under the oil of semi-contact according to claim 2, is characterized in that: the sample drop (6) different for volume that in described step 2-2, kapillary (1) is released; The reagent solution (7) for different volumes that in described step 5, kapillary (1) is released; The cumulative volume of described drop reactor (9) is identical.
4. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, it is characterized in that: in step 2-1 according to point sample generate the volume of drop, distance d1 between the tip of kapillary (1) and the lower surface of micropore (3) is adjusted, setting range is 1 micron to 5 millimeters, ensures that the drop that point sample generates contacts with the lower surface of micropore (3).
5. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, it is characterized in that: in step 4, according to the volume of the drop generated in the volume of liquid feeding and micropore (3), distance d2 between the drop upper surface generated in kapillary (1) tip and micropore (3) is adjusted, setting range is 1 micron to 5 millimeters, ensures that the drop that liquid feeding generates contacts with drop upper surface in micropore (3).
6. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, is characterized in that: the internal diameter of described kapillary (1) at 1 micron within the scope of 5 millimeters.
7. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, it is characterized in that: the depth range of the micropore (3) on described microwell array chip (2) is 1 micron to 5 millimeters, receiving fluids volume range 1 to be ascended to heaven to 100 microlitres.
8. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, is characterized in that: the tip of described kapillary (1) carries out drawing point process, and carries out hydrophobizing surface process to the inside and outside wall of kapillary (1).
9. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, is characterized in that: carry out hydrophobization process to the surface of microwell array chip (2).
10. drop continuity point quadrat method under the oil of the semi-contact according to Claims 2 or 3, it is characterized in that: hydrophilicity-imparting treatment is carried out to micropore (3) inside surface, hydrophobization process is carried out to the remaining surface of the microwell array chip (2) beyond micropore (3).
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| CN104492508B (en) * | 2014-11-13 | 2016-08-24 | 浙江大学 | A kind of ultramicron liquid drop control device and method based on liquid residue |
| WO2016078340A1 (en) | 2014-11-17 | 2016-05-26 | 中国科学院微生物研究所 | Apparatus, system, and method for dispensing/mixing a small quantity of liquid |
| CN104450891B (en) * | 2014-11-17 | 2017-06-06 | 中国科学院微生物研究所 | Digital nucleic acid amplification quantitative analysis method and system based on microlayer model |
| CN104324769B (en) * | 2014-11-17 | 2016-06-22 | 中国科学院微生物研究所 | Generation method based on the drop of microchannel |
| CN104826673B (en) * | 2015-03-16 | 2017-06-23 | 中国科学院微生物研究所 | Write two dimension Microfluidic droplet array device, purposes and its application method |
| CN104849111B (en) * | 2015-04-14 | 2018-04-20 | 浙江大学 | The forming method of gradient microlayer model array based on sequential injection and microflow control technique |
| CN104774747B (en) * | 2015-04-14 | 2017-03-01 | 浙江大学 | Microfluidic droplet chip apparatus for cell migration assay experiment and method |
| CN106568982B (en) * | 2016-10-31 | 2018-05-29 | 浙江大学 | A kind of device and its application method for being formed and being screened for two-dimentional droplet array |
| CN108267570B (en) * | 2016-12-27 | 2023-09-12 | 北京达微生物科技有限公司 | Porous plate for pre-storing reagent microbeads and preparation and use methods thereof |
| CN109337963B (en) * | 2018-10-24 | 2022-04-19 | 四川大学华西医院 | Continuous volume gradient capillary digital PCR method and kit |
| CN111665111A (en) * | 2020-07-03 | 2020-09-15 | 上海百傲科技股份有限公司 | Sample applicator |
| CN113189358B (en) * | 2021-05-06 | 2023-05-30 | 上海迈振电子科技有限公司 | Semi-contact sample application instrument and preparation method of micro-cantilever sensing chip |
| CN116169005B (en) * | 2023-02-28 | 2023-07-28 | 厦门金诺花科学仪器有限公司 | Disposable hydrophobic nano-liter sample application needle head and sample application test method |
| CN116465955B (en) * | 2023-03-10 | 2023-10-24 | 厦门金诺花科学仪器有限公司 | Nanoliter sample application instrument, reusable needle head and testing and regenerating methods thereof |
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