CN208621619U - A multi-well plate with pre-stored reagent microbeads - Google Patents
A multi-well plate with pre-stored reagent microbeads Download PDFInfo
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- CN208621619U CN208621619U CN201721806867.1U CN201721806867U CN208621619U CN 208621619 U CN208621619 U CN 208621619U CN 201721806867 U CN201721806867 U CN 201721806867U CN 208621619 U CN208621619 U CN 208621619U
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- porous plate
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- 239000003153 chemical reaction reagent Substances 0.000 title abstract 9
- 239000011325 microbead Substances 0.000 title abstract 7
- 238000012216 screening Methods 0.000 abstract 6
- 238000006243 chemical reaction Methods 0.000 abstract 2
- 239000007788 liquid Substances 0.000 abstract 2
- 206010013710 Drug interaction Diseases 0.000 abstract 1
- 238000002425 crystallisation Methods 0.000 abstract 1
- 230000008025 crystallization Effects 0.000 abstract 1
- 230000000813 microbial effect Effects 0.000 abstract 1
- 239000004005 microsphere Substances 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
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Abstract
本实用新型提供一种预储存试剂微珠的多孔板：多孔板的容器中充满第一种液体，并在其底部预先引入一个负载试剂的微珠。负载试剂的微珠可以是固态微球、微液滴或两者的任意组合。所述的第一种液体与负载试剂的微珠不互溶。所述多孔板封膜或加盖保存，用户使用时无需配制和添加筛选试剂。该预存储试剂微珠可以大大减少试剂和样品的消耗量，试剂微珠方便与后续引入微量样品的混合反应，可以将常规筛选反应的体积降低2至3个数量级，可广泛应用于蛋白质结晶筛选、药物互作筛选、微生物耐药筛查、培养条件筛选、阵列PCR、单细胞分析等领域。
The utility model provides a perforated plate for pre-storing reagent microbeads: the container of the perforated plate is filled with a first liquid, and a microbead loaded with reagents is pre-introduced at the bottom of the perforated plate. The reagent-loaded microbeads can be solid microspheres, microdroplets, or any combination of the two. The first liquid is immiscible with the reagent-loaded microbeads. The multi-well plate is sealed or stored with a cover, and users do not need to prepare and add screening reagents when using. The pre-stored reagent microbeads can greatly reduce the consumption of reagents and samples. The reagent microbeads are convenient for mixing reactions with the subsequent introduction of trace samples, which can reduce the volume of conventional screening reactions by 2 to 3 orders of magnitude, and can be widely used in protein crystallization screening. , drug interaction screening, microbial resistance screening, culture condition screening, array PCR, single cell analysis and other fields.
Description
Technical field
The present invention relates to high throughput analysis and screening technique field, and in particular to a kind of micro-volume based on pre-stored reagent
Reaction screening porous plate.
Background technique
Combinatorial chemistry, biology, clinical examination, the reaction screening of materia medica and Mass-analysis of samples sorting, need mostly
Manual reagent preparation is wanted, a large amount of reagents and consumptive material are expended, the error generated in operating process is big.For the bottleneck that, and develop
That gets up is previously added the porous plate of reagent, avoids the solution-operated that user prepares a large amount of reagents by hand, easy to use, guarantees
The precision of amount of reagent, is of great significance.However, the existing porous plate that reagent is stored in advance, most of is dry-storage,
Reagent consumption is larger, and cost is high, and the sample volume for carrying out screening reaction needs is big, and the reagent of storage is easy to happen change
Matter is easy to happen cross contamination between porous plate.The it is proposed that the present invention the innovates microballon reagent of oil mutually package, reagent can be with
The form long-term preservation of liquid or solid-state microballon, cooperation automation micro sample-adding technology, reaction system can be contracted to by we
Nanoliter rank, it is low in cost, in such as antibiotics susceptibility test, Multiplex PCR screening, the screening of cell drug interaction, protein crystal screening
Etc. application fields have wide popularization and use prospect.
Utility model content
The object of the present invention is to provide a kind of porous plates of preparatory storage reagent for micro-volume reaction screening, porous
In plate, in advance in each hole in the form of supported reagent microballon introduce reaction screening reagent, while add protection volatilization prevention and
The oil-based liquid of cross contamination, is stored with long-time stable.Reagent wells are stored in advance when in use in this, cooperation automation
The micro-volume reaction screening experiment on common porous plate can be completed in micro liquid feeding technology.The porous of reagent is stored in advance in this
Plate, easy to use, reagent consumption is small, the volume of conventional screening reaction can be reduced by 2 to 3 orders of magnitude, can be steady for a long time
It is fixed to save, effectively prevent oxidation deterioration and the cross contamination of reagent.In crystallization of protein screening, the screening of drug interaction, micro- life
The fields such as the screening of object drug resistance, condition of culture screening, array PCR, single cell analysis have broad application prospects.
The present invention provides a kind of porous plate of pre-stored reagent in the form of microballon, comprising: a) provides and stores reagent in advance
Porous plate is full of the first liquid in each container of the porous plate；B) a previously-introduced examination in the hole of the porous plate
Agent microballon, reagent microballon and the first described liquid are immiscible；C) porous plate can be open containers, be also possible to seal
Container.
Further, the first described liquid includes following characteristics: 1) specific gravity is less than reagent microballon；It is oil-based liquid
Or with liquid immiscible with second of liquid in the step c).Further, the oil-based liquid includes mineral
And the specific gravity immiscible with water phase such as oil, liquid alkane, esters, silicone oil, alkane alkene or saturated hydrocarbons semi liquid state mixture are less than
The liquid of water.
Further, the alkane alkene or saturated hydrocarbons semi liquid state mixture refer to its state at room temperature between solid and
Between liquid, fusing point is at 4 degrees Celsius or more, including solid paraffin, vaseline.
Further, the reagent microballon, microballon include the combination of solid-state-microspherical, drop or both, are not involved in porous
The reaction carried out when plate use.
Further, the solid-state-microspherical includes glass microballoon, polymer microballoon, porous microsphere, gel micro-ball, magnetism
Microballoon.
Further, the reagent microballon, reagent are the substances that reaction is participated in when porous plate uses.
Preferably, the reagent microballon volume range is 1 nanoliter to 5 microlitres.
Further, the porous plate include 96 holes, 384 holes and 1536 orifice plates, orifice plate bottom include it is flat, round,
Taper and any of the above combination.
Invention also provides a kind of method of the pre-stored reagent microballon porous plate of the preparation of simple possible, feature exists
In the screening reagent aqueous solution can be manipulated technology batch by conventional microlitre of volume of liquid and is added on porous plate, be passed through
Low-temp low-pressure volatilization processing removes moisture, is retained in reagent in solid-state-microspherical, difficult solvent flashing or macromolecular water-soluble substance,
Realize the preparation that the micro-volume supported reagent microballon manipulated is difficult to using conventional method.
Further, the porous plate preparation method includes: second a) be added in porous plate containing surfactant
Kind liquid；B) reagent and microballon are added in second of liquid of the container of porous plate by liquid-transfering device；C) centrifugation makes to try
Agent and microballon fall to container bottom, contact the two；D) reagent makes moisture in reagent and the in low-temp low-pressure processing
Two kinds evaporate, and the reagent after drying is incorporated on microballon；E) the first liquid is added in porous plate；It f) is porous plate
Sealer obtains the pre-stored reagent microballon porous plate of nanoliter volumes.
Further, second of the liquid, which is characterized in that the liquid has volatility, is oil-based liquid
Liquid, including petroleum ether, hexamethylene, pentamethylene, isopentane, normal heptane etc. either immiscible with reagent microballon.
Further, the preparation method of the pre-stored reagent microballon porous plate, the low temperature range be -80 to
50 degrees Celsius, the low pressure refers to that vacuum degree reaches 0.5 to 0.05.
Invention also provides the application methods of pre- storage reagent microballon porous plate, which comprises the following steps:
A) porous plate sealer is thrown off；B) it crosses micropipette device and sample or second is added in the first liquid of the container of porous plate
Reagent microlayer model；C) porous plate is placed in a centrifuge centrifugation, pre-stored reagent microballon and sample drop is made to fall to container bottom
Portion, with reagent microballon hybrid reaction；D) b)-c is repeated) step, second of reagent is added or sample drop carries out multistep reaction sieve
Choosing.
Detailed description of the invention
Composition, the side of making and using in order to illustrate more clearly of the porous plate of the pre-stored reagent proposed in the present invention
Method, the drawings to be used in the description of the embodiments or prior art will be briefly described below.
Fig. 1 is pre-stored the preparation method schematic diagram of reagent microballon porous plate.
Fig. 2 reacts screening technique schematic diagram based on the micro-volume of pre-stored reagent microballon.
Porous plate reaction experiment result micro-image of Fig. 3 based on pre-stored solid-state-microspherical.
Porous plate reaction experiment result micro-image of Fig. 4 based on pre-stored microlayer model.
Fig. 5 is suitable for the configuration schematic diagram of pre-stored microlayer model porous plate.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments, cannot
They are interpreted as the restriction to the application protection scope.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
Embodiment 1
Such as Fig. 1, for the preparation method schematic diagram for being pre-stored reagent microballon porous plate.The porous plate used is 96 hole PCR
Plate illustrates a row of 96 orifice plates in diagram, totally 8 containers；I~VIII refers to that screening reagent dilute aqueous solution, dilution ratio are
40 times；Eight channel pipettors (1)；Liquid relief reagent dilutions (2)；Petroleum ether (3) containing 0.01% Span80；Water phase volatilizees it
Supported reagent microballon (4) afterwards；Mineral oil (5).The preparation method of pre-stored reagent microballon porous plate, the specific steps are as follows:
A) 100 microlitres of petroleum ethers (containing 0.01%Span80) is previously added in each hole of 96 orifice plates, porous plate is placed in 4
On DEG C constant-temperature metal bath；
B) by eight logical pipettors (1-10 microlitres of liquid relief amount), the 40 times of dilutions 5 μ L of reagent to be screened is drawn in batches, it is dilute
It releases in liquid containing 0.3%PEG400, is added on 384 orifice plates of prepackage petroleum ether；
C) centrifugation makes reagent to be screened fall to 96 orifice plate bottoms；
D) orifice plate being put into vacuum oven, set temperature is 37 DEG C, and vacuum degree 0.08, low-temp low-pressure is handled 3 hours,
The moisture in petroleum ether and reagent is set sufficiently to volatilize, and Span80 retains, while screening reagent is micro- in the PEG400 of hardly possible volatilization
In drop, the volume-diminished of droplet is to about 15-20 nanoliters；
E) 100 microlitres of mineral oil are added in porous plate；
F) plastic packaging film is sealed on porous plate, is pre-stored the preparation of reagent microballon porous plate and is completed.
Make using porous plate, using (the digital nucleic acid amplification quantitative analysis side based on microlayer model of method described in patent
Method and system, Du Wenbin；Xu Peng Chinese patent, grant number: ZL 20140655309.4,2014) 230 nanoliters of sample liquids are added
Drop, and be centrifuged and merged with pre-stored reagent microballon, end reaction volume is about 250 nanoliters.Above method be utilized water and
PEG400 has different boiling points, and petroleum ether and Span80 have different boiling points.Supported reagent has been obtained by low-temp low-pressure
Microballon facilitates preparation.The automatical and efficient completion of conventional micro updating liquor removing workstation can be used in the preparation of porous plate.By with top
The pre-stored reagent wells that method obtains, the reagent consumption that reaction can be made to screen substantially reduce.
Embodiment 2
Other conditions are with embodiment 1, e) step is changed to 100 microlitres of mineral oil containing 6% vaseline are added that (room temperature is liquid
Body).This pre-stored reagent wells covers the protective layer of microballon in 4 DEG C of preservations as solidification state.Microballon in porous plate
Position is fixed, and during storage and transport, is resistant to unexpectedly shake overturning etc. without changing.Using porous plate
When, porous plate needs to be restored to room temperature, and oily mutually melt is made to carry out micro-example filling operation again later.
Embodiment 3
As shown in Fig. 2, the micro-volume screening of the porous plate based on pre-stored reagent microballon provided for the embodiment of the present application 1
Method flow schematic diagram.The porous plate of the pre-stored reagent microballon include: the sealer (1) of seal container, porous plate (2),
The first liquid (3) and supported reagent microballon (4).The container of used porous plate have V-arrangement bottom, make microballon or
Sample droplet converges at V-arrangement center and realizes fusion, is conducive to the progress of reaction screening.The first liquid used is mineral
Oil.The step of use includes:
A) sealer (1) is thrown off；
B) by (Chinese patent, application number: 201410655191.5) the microlayer model generation method passes through publication
Reagent or sample drop (6) is added in micropipette device (5) in the first liquid (3) of the container of porous plate；
C) centrifugation makes sample drop (6) fall to container V-Bottom portion, is mixed with pre-stored reagent microballon, certain
Under the conditions of complete screening reaction.
As shown in figure 3, being the experimental result micro-imaging figure of the embodiment of the present invention 1.Using Stereo microscope, observation is wherein
One micropore.Before sample is added, it can be seen that V-arrangement container bottom contains a transparent reagent microballon, and diameter is 200 micro-
The glass microsphere of rice, the adsorbing chlorinated iron of microsphere surface；Intermediate picture shows the pure water drop of 50 nanoliters of addition, makes pre-stored chlorine
Change ferron dissolution；50 nanoliters of potassium rhodanate aqueous solution is added in the right picture presentation, and iron chloride is complexed with potassium rhodanate
Reaction, generates red thiocyanation ferrous solution, and the color of drop reddens color.Chlorination is stored using in glass microsphere dry tack free
Iron avoids the rotten of solution chlorination iron, store reagent can steadily in the long term in porous plate.Scale bar is as shown in the figure
200 microns.
Embodiment 4
As shown in figure 4, the screening effect of the porous plate based on pre-stored reagent microballon provided for the embodiment of the present invention 2
Figure.Using the transparent porous plate of flat polystyrene material, drop is observed with facilitating；The first liquid used
For mineral oil.
Steps are as follows for the use of this porous plate:
A) 50 nanoliters of ferric chloride solution droplet (reagent) is introduced in porous plate container in advance；
B) by micro sample-adding device, 50 nanoliters of potassium rhodanate aqueous solutions (sample) are added in mineral oil into container；
C) centrifugation mixes iron chloride with potassium rhodanate droplet, obtains the mixing drop of 100nL, and reaction generates red sulphur
Ferricyanide.
Embodiment 5
It is the porous plate of bottom various configuration such as Fig. 5.Wherein, cylindrical micro- hole, reagent microballon a) are had for conical lower portion
It is stored in central dimple.Using elongated micro- hole, using microplate reader, realized based on absorption spectrophotometry or fluorescence method
Detection is conducive to the sensitivity for improving detection；It b) is the conical bottom with slight flat, the drop for being conducive to fall into micro- hole collects in
The absorption anchoring of bottom microballon drop is cheated, convenient react microlayer model carries out imaging；It c), is d), e), f) existing on the market
Orifice plate Solid suspension, be followed successively by round bottom, flat, U-shaped bottom, V-type bottom.
Claims (1)
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| Application Number | Priority Date | Filing Date | Title |
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| CN201621446811 | 2016-12-27 | ||
| CN2016214468115 | 2016-12-27 |
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| CN208621619U true CN208621619U (en) | 2019-03-19 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110559710A (en) * | 2019-08-29 | 2019-12-13 | 中国科学院苏州生物医学工程技术研究所 | Heating sealing filtering device and high-flux drug eutectic screening system thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110559710A (en) * | 2019-08-29 | 2019-12-13 | 中国科学院苏州生物医学工程技术研究所 | Heating sealing filtering device and high-flux drug eutectic screening system thereof |
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Effective date of registration: 20200529 Address after: 100176 No. 2 middle Sheng Road, Daxing District economic and Technological Development Zone, Beijing Patentee after: HOLLYSYS AUTOMATION TECHNOLOGIES Ltd. Address before: 100101 No. 1, West Beichen Road, Beijing, Chaoyang District, 3 Patentee before: INSTITUTE OF MICROBIOLOGY, CHINESE ACADEMY OF SCIENCES |
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Effective date of registration: 20200812 Address after: B106, 1 / F, No.5 Kaifa Road, Haidian District, Beijing 100085 Patentee after: Beijing Da Microbiology Technology Co.,Ltd. Address before: 100176 No. 2 middle Sheng Road, Daxing District economic and Technological Development Zone, Beijing Patentee before: HOLLYSYS AUTOMATION TECHNOLOGIES Ltd. |
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Effective date of registration: 20231115 Address after: 611731 building 4, No.8, Anhe 2nd Road, hi tech Zone, Chengdu, Sichuan Province Patentee after: MACCURA MEDICAL ELECTRONICS Co.,Ltd. Address before: 100085 B106, 1st floor, No.5 Kaifa Road, Haidian District, Beijing Patentee before: Beijing Da Microbiology Technology Co.,Ltd. |
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Effective date of registration: 20231230 Address after: 611731, 16 Chengdu, hi tech Zone, Sichuan, China Patentee after: MACCURA BIOTECHNOLOGY Co.,Ltd. Address before: 611731 building 4, No.8, Anhe 2nd Road, hi tech Zone, Chengdu, Sichuan Province Patentee before: MACCURA MEDICAL ELECTRONICS Co.,Ltd. |
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