CN107502648A - Unicellular drop high-throughput screening method based on micro-fluidic chip - Google Patents
Unicellular drop high-throughput screening method based on micro-fluidic chip Download PDFInfo
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- CN107502648A CN107502648A CN201610415873.8A CN201610415873A CN107502648A CN 107502648 A CN107502648 A CN 107502648A CN 201610415873 A CN201610415873 A CN 201610415873A CN 107502648 A CN107502648 A CN 107502648A
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Abstract
The invention discloses a kind of unicellular drop high-throughput screening method based on micro-fluidic chip, comprise the following steps：(1) microbial single-cell drop is generated on micro-fluidic chip；(2) throw aside and do not wrap up microorganism drop；(3) the microbial single-cell drop after culture is merged with micromolecular compound biology sensor drop on micro-fluidic chip；(4) metabolite in biosensor response fusion drop, carries out fluoroscopic examination and sorting.High-throughput screening method provided by the invention, by carrying out unicellular drop culture on micro-fluidic chip, equivalent to building thousands of individual miniature organism reactors on chip, it is possible to achieve treat the tracking and screening of sieve growth of microbial cells characteristic；By droplet coalescence technology, small molecule metabolite biology sensor is realized to the fluorescence labeling containing microbial cell drop to be sieved, and then realize the screening to target extracellular products.
Description
Technical field
The present invention relates to a kind of cell screening method, more particularly to a kind of unicellular drop high pass based on micro-fluidic chip
Measure screening technique.
Background technology
In recent years, technology flourishes micro-fluidic chip (Microfluidic Chip, MC), and steps into industrialization
Epoch.Microflow control technique has the advantages that quick, efficient, low consumption, in crowds such as biology, high-flux medicaments sifting, medicals diagnosis on disease
More application fields have broad prospects.Wherein, the microlayer model fluorescent screening technology based on microflow control technique
(Fluorescence-Activated DropletSorting, FADS) is by the height of high flux screening area research personnel
Concern.Its core is that measuring samples and fluorescent marker are wrapped in drop into (Water-In-Oil) jointly, so as to realize measuring samples
Flow detection and sorting.In China, the technology is applied to by the researcher of Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences
The parcel of microculture extracellular products, so as to realize the high flux screening of metabolin, its screening efficiency can reach 10~100
Individual drop is per second.Solve to still suffer from following technological deficiency in terms of industrial microorganism high flux screening however, these are operated in：
(1) unicellular culture technique is lacked, the incubation of microorganism is performed in deep-well plates system, and its flux is integrally formed screening
The bottleneck of efficiency；(2) fluorescence labeling system uses traditional dual-enzyme coupling method, and cost is high, system poor compatibility, and application has
Limit.
The content of the invention
It is an object of the invention to provide a kind of unicellular drop high-throughput screening method based on micro-fluidic chip.
Technical scheme is as follows：
A kind of unicellular drop high-throughput screening method based on micro-fluidic chip, it is characterised in that comprise the following steps：
(1) microbial single-cell drop is generated on micro-fluidic chip；
(2) throw aside and do not wrap up microorganism drop；
(3) the microbial single-cell drop after culture and micromolecular compound biology sensor drop are melted on micro-fluidic chip
Close；
(4) metabolite in biosensor response fusion drop, carries out fluoroscopic examination and sorting.
Wherein, microbial single-cell drop described in step (1) extremely diluting, it is real by way of oil phase parcel aqueous phase
It is existing.This method by need not lower yardstick micro-fluidic chip, greatly reduce the processing cost of chip.
Wherein, microbial single-cell drop produces extracellular products after culture described in step (2).
Preferably, the extracellular products is micromolecular compound.
It is further preferred that the micromolecular compound includes organic acid.
Above-mentioned steps also include the separation of the fusion drop.Microlayer model and small molecule biology sensor liquid after culture
Drop, is merged, forms new detection drop in the chips；After co-cultivation, new detection drop will be gone forward side by side by fluoroscopic examination
Row evaluation, and separated；Mixed Microbes after separation can obtain pure mesh by means such as dilution spread flat board or resistance screenings
Mark production bacterial strain.
Beneficial effect：Method provided by the present invention, by carrying out unicellular drop culture on micro-fluidic chip, quite
In building thousands of individual miniature organism reactors on chip, it is possible to achieve treat tracking and the sieve of sieve growth of microbial cells characteristic
Choosing；By droplet coalescence technology, realize small molecule metabolite biology sensor to the fluorescence containing microbial cell drop to be sieved
Mark, and then realize the screening to target extracellular products.This unique design principle, it ensure that in screening purpose product phenotype
While do not lose growth vigor phenotype, greatly improve screening efficiency；Droplet coalescence technology need not import biology sensor
(this is the necessary step of FACS technologies) is treated in sieve cell, genetic manipulation is avoided, particularly suitable for largely not having genetic manipulation work
The efficient seed selection of the industrial strain of tool.
Brief description of the drawings
Fig. 1 is the schematic diagram of the unicellular drop high-throughput screening method based on micro-fluidic chip of the embodiment of the present invention.
Embodiment
With reference to embodiment, the present invention is expanded on further.
Fig. 1 gives the schematic diagram of the unicellular drop high-throughput screening method based on micro-fluidic chip.
1st step, microbial single-cell drop is generated on micro-fluidic chip.By extremely diluting, oil phase parcel aqueous phase
Mode is realized embeds individual cells in micro-fluidic chip each microlayer model, and then in micro-fluidic chip dissolved oxygen control device
Cultivated, realize the generation of the amplification of microorganism, purpose product to be measured.
2nd step, throw aside and do not wrap up microorganism drop.
3rd step, microorganism produces extracellular products, such as the micromolecular compound such as organic acid after culture.
4th step, merges micromolecular compound biology sensor drop, microbial single-cell drop after will cultivating with it is small
Molecular compound biology sensor drop merges on micro-fluidic chip.Using the technology road for being different from FACS intracellular fluorescence labelings
Line, small molecule biology sensor is incorporated into detecting system by droplet coalescence technology, genetic manipulation is avoided, is particularly suitable for
In the screening of a large amount of industrial strains without genetic manipulation instrument.
5th step, biosensor response small molecule metabolite forms fluorescence, for determining and sorting.Such as by grinding
Specific small molecule biology sensor processed, the concentration of microlayer model small molecular and fluorescence intensity coupling are got up, realized small to this
The real-time high flux screening of molecule yield.The modulin system is placed in Escherichia coli, makes specific organic acid molecule
Biology sensor, it is put into each microlayer model, then can produces the production of organic acid to flora in each microlayer model by fluorescence intensity
Amount is characterized and realizes high flux screening.This method will not produce interference, high specificity, spirit to the growth conditions of strain to be tested
Sensitivity is high, while is suitable to the whole detection of flora again.
In addition, gained mixed bacterial after blended drop fluoroscopic examination, sorting, can by simple dilution spread or
The methods of resistance screening, it is relatively easy to obtain the single bacterium colony of target production bacterial strain.
Claims (6)
1. the unicellular drop high-throughput screening method based on micro-fluidic chip, it is characterised in that comprise the following steps：
(1) microbial single-cell drop is generated on micro-fluidic chip；
(2) throw aside and do not wrap up microorganism drop；
(3) the microbial single-cell drop after culture and micromolecular compound biology sensor drop are melted on micro-fluidic chip
Close；
(4) metabolite in biosensor response fusion drop, carries out fluoroscopic examination and sorting.
2. high-throughput screening method as claimed in claim 1, it is characterised in that microbial single-cell liquid described in step (1)
Drop is extremely diluting, is realized by way of oil phase parcel aqueous phase.
3. high-throughput screening method as claimed in claim 1, it is characterised in that microbial single-cell liquid described in step (2)
Drop produces extracellular products after culture.
4. high-throughput screening method as claimed in claim 3, it is characterised in that the extracellular products is micromolecular compound.
5. high-throughput screening method as claimed in claim 4, it is characterised in that the micromolecular compound includes organic acid.
6. the high-throughput screening method as described in claim any one of 1-5, it is characterised in that the step also includes described melt
The separation of drop is closed, the Mixed Microbes after separation can obtain pure target production by means such as dilution spread flat board or resistance screenings
Bacterial strain.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108485984A (en) * | 2018-02-08 | 2018-09-04 | 中国科学院天津工业生物技术研究所 | The high-throughput screening method of cellulase high-yield |
| CN110713922A (en) * | 2018-09-20 | 2020-01-21 | 浙江达普生物科技有限公司 | Real-time monitoring of single cells or activities of single cells |
| CN112430528A (en) * | 2020-11-24 | 2021-03-02 | 华中科技大学 | High flux microorganism inoculation device based on spraying is supplementary |
| CN112574937A (en) * | 2020-12-31 | 2021-03-30 | 江南大学 | Ultramicro injection method based on micro-fluidic chip and application thereof in high-throughput screening |
| CN112852915A (en) * | 2019-11-28 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Application of high-throughput screening method using droplet microfluidic chip in actinomycetes |
| CN113337573A (en) * | 2021-05-31 | 2021-09-03 | 江南大学 | Construction method and application of micro-droplet biosensor |
| CN114062679A (en) * | 2021-11-16 | 2022-02-18 | 中国科学院上海微系统与信息技术研究所 | Single-cell secretion high-throughput detection method and system based on droplet microfluidics |
| CN114669337A (en) * | 2022-03-25 | 2022-06-28 | 天津大学 | Micro-fluidic device and application thereof in bacteria separation |
| CN115521882A (en) * | 2021-06-24 | 2022-12-27 | 清华大学 | Micro-upgrading single-cell droplet generation and culture method and device |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108485984A (en) * | 2018-02-08 | 2018-09-04 | 中国科学院天津工业生物技术研究所 | The high-throughput screening method of cellulase high-yield |
| CN110713922A (en) * | 2018-09-20 | 2020-01-21 | 浙江达普生物科技有限公司 | Real-time monitoring of single cells or activities of single cells |
| CN112852915A (en) * | 2019-11-28 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Application of high-throughput screening method using droplet microfluidic chip in actinomycetes |
| CN112852915B (en) * | 2019-11-28 | 2024-04-09 | 中国科学院天津工业生物技术研究所 | Application of high-throughput screening method using droplet microfluidic chip in actinomycetes |
| CN112430528A (en) * | 2020-11-24 | 2021-03-02 | 华中科技大学 | High flux microorganism inoculation device based on spraying is supplementary |
| CN112574937A (en) * | 2020-12-31 | 2021-03-30 | 江南大学 | Ultramicro injection method based on micro-fluidic chip and application thereof in high-throughput screening |
| CN113337573A (en) * | 2021-05-31 | 2021-09-03 | 江南大学 | Construction method and application of micro-droplet biosensor |
| CN115521882A (en) * | 2021-06-24 | 2022-12-27 | 清华大学 | Micro-upgrading single-cell droplet generation and culture method and device |
| CN114062679A (en) * | 2021-11-16 | 2022-02-18 | 中国科学院上海微系统与信息技术研究所 | Single-cell secretion high-throughput detection method and system based on droplet microfluidics |
| CN114062679B (en) * | 2021-11-16 | 2023-09-08 | 中国科学院上海微系统与信息技术研究所 | Single-cell secretion high-flux detection method and system based on droplet microfluidic |
| CN114669337A (en) * | 2022-03-25 | 2022-06-28 | 天津大学 | Micro-fluidic device and application thereof in bacteria separation |
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