Single bacterium separation method based on digital microfluidic system and application

Abstract

The invention relates to a single bacterium separation method based on a digital micro-fluidic system, which comprises the following steps: injecting diluted bacterium liquid into a digital micro-fluidic chip to form bacterium liquid drops with 2 < N > power as electrode bases, and distributing 18-22 bacteria per 64 electrode bases in each bacterium liquid drop; the liquid drops with the same volume are controlled to move in the opposite direction, and half-and-half tearing is carried out; according to the mode, the liquid drops after half-and-half tearing are subjected to half-and-half tearing again, N rounds are carried out in total, and 2 < N > single-electrode liquid drops are formed; the single bacterium liquid drops are obtained. The separation method has the advantages that the separation efficiency is high, the separation speed is high, the separation flux is high, and the final separation success rate can reach 100%, and the single-bacterium separation method can be well applied to bacterium culture, polypeptide drug or antibody screening and genomics analysis in a digital microfluidic system.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a single bacteria separation method based on a digital microfluidic system and an application thereof. Background Art

[0002] In the past 10 years, the application of microfluidics in the field of biotechnology has become relatively mature. For example, Bio-Rad relies on a microfluidics platform to develop a digital PCR chip that can achieve absolute quantitative detection of nucleic acids; the American single-cell sequencing company 10XGenomics uses a similar oil-in-water method to generate single cell samples for library construction and sequencing.

[0003] Microfluidic systems can integrate multiple sorting methods based on the physical parameters of cells. The micro size of microfluidic systems provides a perfect interface for the operation of single cells and can be adjusted and controlled in a variety of ways. They can also reduce the volume and cost of reagents and have potential portability. Based on these advantages, microfluidic technology has become the best candidate for high-throughput cell sorting technology in the future. Compared with traditional microfluidic systems, digital microfluidic systems have the advantages of simple structure, high throughput, and low sample loss rate. However, at this stage of digital microfluidic technology, there are still various technologies that need to be improved. For example, due to the limited chip area but the large electrode size, there are few droplets that can be operated and the flux is low. Due to the small number and large size of electrodes and the large volume of single droplets, it is relatively unfavorable to separate single bacteria, and the success rate of separating single bacteria is low.

[0004] Chinese patent application CN114669337A discloses a microfluidic device and its application in separating bacteria, which involves using the common microfluidic micro-channel technology to embed droplets in an oil-in-water manner, and then using mathematical probability methods to separate and embed single bacteria. However, this patented technology still cannot separate all cells that want to be separated, the loss rate is large, and the probability of obtaining single cells is low, which affects the subsequent operations on the chip.

[0005] Chinese patent application CN115197840A discloses a single bacterial genome sequencing method based on digital microfluidics technology, in which the on-off control of the electrode is performed in a preset order, so that a bacterial suspension droplet is generated from the electrode unit in the bacterial storage area, and the droplet is moved to the hydrophilic site area by control, so that the cell suspension droplet passes through the hydrophilic site, and small droplets are left at the hydrophilic site under the action of surface tension. The small droplets may contain different numbers of bacteria, and they pass through the hydrophilic site repeatedly until only a single bacterium is contained in the small droplet, and the single bacterium is captured. By controlling the electrode drive circuit, the original cell suspension droplet is moved to the waste liquid area for removal. However, the throughput and efficiency of this traditional single bacteria separation method are not high. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a single bacteria separation method based on a digital microfluidics system and its application, which can achieve high-throughput separation of single bacteria with high separation efficiency.

[0007] The above-mentioned objectives are achieved by means of the following technical solutions.

[0008] The first aspect of the present invention is to provide a single bacteria isolation method based on a digital microfluidics system, comprising the following steps:

[0009] S1. Cultivate bacteria in a culture medium;

[0010] S2. dilute the bacterial solution with culture medium;

[0011] S3. Inject the diluted bacterial solution onto the digital microfluidic chip to form bacterial solution droplets with 2 to the power of N as the electrode base, with the volume of each single electrode droplet being 3 nL, the amount of bacteria in each bacterial solution droplet being 18-22 bacteria per 64 electrode bases, and the number of bacterial solution droplets being at least 1; N is a positive integer equal to or greater than 1;

[0012] S4. The droplets are controlled by controlling the power on and off of the electrodes on the chip, and the droplets of equal volume are controlled to move in the opposite direction, and the droplets are torn in half to obtain droplets torn in half; according to the above method, the droplets torn in half are torn in half again for several times to form 2N single-electrode droplets;

[0013] S5. Through the fluorescence imaging system, the single-electrode droplet is subjected to fluorescence detection and judgment, and the droplet containing only one bacterium is marked. The electrode containing the droplet containing only one bacterium is continuously powered to keep the droplet in place, thus obtaining a single-bacteria droplet.

[0014] In some embodiments, the separation method further comprises:

[0015] S6. For droplets that do not contain bacteria or contain more than one bacterium, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and re-merged into an independent empty droplet that does not contain bacteria and a droplet with multiple bacteria, respectively. The empty droplet is removed and the droplet with multiple bacteria is recovered.

[0016] In some embodiments, the separation method further comprises:

[0017] S7. By controlling the power on and off of the electrodes on the chip, the droplets containing the single bacteria are moved again and rearranged into a droplet matrix with one electrode interval;

[0018] S8. After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet with 2 to the power of N as the electrode base. The above steps S3-S7 are repeated until the recovered droplets do not contain bacteria and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria.

[0019] The second aspect of the present invention is to provide the application of the above-mentioned single bacteria isolation method in the cultivation of bacteria, the screening of polypeptide drugs or antibodies, and genomics analysis in a digital microfluidic system.

[0020] The single bacteria separation method of the present invention can quickly form a single bacteria droplet by tearing the droplet in half on the digital microfluidic chip. Moreover, the researchers of the present invention found in the experiment that the reason why the single bacteria separation efficiency is not high is the amount (concentration and volume) of the bacterial liquid injected into the digital microfluidic chip. After a lot of exploration, it was found that the separation efficiency is highest when the number of bacteria allocated to each 64 electrode base is 18-22, based on the volume of each single electrode droplet of 3nL. The single bacteria obtained through such rapid and efficient separation can continue to be cultured on the chip, or library construction, as well as a series of other experimental operations, such as antibody screening and genomic analysis, which will greatly reduce R&D costs and improve R&D efficiency.

[0021] In summary, the efficient single bacteria isolation method based on the digital microfluidic system described in the present invention has the following advantages:

[0022] 1. Throughput: up to 19-21 single bacterial droplets can be obtained per 64 droplets.

[0023] 2. The sample retention rate can reach 100%. For samples that wish to obtain single bacteria, all bacteria can be separated into single bacteria without losing any bacteria.

[0024] 3. Through the circulation separation method, it can be achieved that all separated droplets contain single bacteria, that is, the theoretical separation success rate can reach 100%.

[0025] 4. Fast separation speed, circulating separation method and high success rate of separation each time make the time to reach 100% separation rate short. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the structure of a digital microfluidic chip.

[0027] Figure 2 Schematic diagram of the half-tearing process in the single bacteria isolation method of the present invention.

[0028] Figure 3In Example 6, the protein expression in the isolated single bacterial droplet was observed by fluorescence microscopy, wherein A is wild-type intern-GFP, which does not emit green light when no inducer is added, and B is wild-type intern-GFP, which emits green light when an inducer is added. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0031] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0032] In some embodiments of the present invention, a single bacteria separation method is provided, which is performed in a digital microfluidic system, wherein the digital microfluidic chip comprises an upper electrode plate and a lower electrode plate, wherein a fluid channel layer is formed between the upper electrode plate and the lower electrode plate; and the lower electrode plate comprises an electrode layer, wherein the electrode layer comprises a plurality of electrodes arranged in an array. The following steps are included:

[0033] S1. Cultivate bacteria in a culture medium and dilute the bacterial solution to an OD600 of about 0.3 using the culture medium;

[0034] S2. Dilute the bacterial solution to a bacterial concentration of 70-120 cfu / uL;

[0035] S3. Inject the diluted bacterial solution onto the digital microfluidic chip to form a plurality of bacterial solution droplets with an electrode base number of 2 to the power of N; the amount of bacteria in each bacterial solution droplet is: 18-22 bacteria are allocated per 64 electrode bases; N is a positive integer equal to or greater than 1;

[0036] S4. The droplets will move toward the adjacent powered electrode and away from the powered-off electrode. By controlling the power on and off of the electrodes on the chip, the droplets can be manipulated to control droplets of equal volume to move in the opposite direction. The droplets are torn in half to obtain droplets torn in half. According to the above method, the droplets torn in half are torn in half again, and this is repeated N times to form an array of 2 to the power of N single-electrode droplets. See Figure 1 ;

[0037] S5. The single electrode droplet array is detected and judged by the recognition system, and the droplet containing only one bacterium is marked, and the electrode containing the droplet containing only one bacterium is continuously powered to keep the droplet in place, that is, the single bacterial droplet after separation is obtained for the first time; according to the method of the present invention, the single bacteria is continued to be separated:

[0038] S6. For droplets without bacteria or with more than one bacteria, the droplets are manipulated by controlling the power on and off of the electrodes on the chip, and empty droplets and droplets containing multiple bacteria are collected from one side, respectively, and re-merged into an independent empty droplet without bacteria and a droplet with multiple bacteria, respectively. The empty droplet is removed and the droplet with multiple bacteria is recovered. The sample recovery rate here reaches 100%, which is used to continue to separate single bacteria;

[0039] S7. By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a droplet matrix with an interval of one electrode to optimize the available space of the chip for the next step of separation; (Through software recognition, the droplets with a single bacterium can move by themselves and rearrange into a droplet matrix with an interval of one electrode. The remaining droplets without bacteria will be removed, and the droplets containing two or more bacteria will be recycled; if 10-15 single bacterial droplets can be successfully obtained from 64 droplets at a time, then there are about 50 droplets that are invalid. After these droplets are recycled or removed, the space on the chip can be reused);

[0040] S8. After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment (according to the efficient separation method of the present invention, generally 0-6 bacteria remain after separation of a 64-droplet matrix), and multiple recovered bacterial droplets are merged into a new droplet with 2N as the electrode base, and the above steps S3-S7 are repeated until the recovered droplets contain no bacteria, and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria, and the empty droplets are discharged from the chip. At this time, the success rate of single bacteria separation of a sample reaches 100%.

[0041] It is clear to those skilled in the art that the steps included in the method of the present invention do not need to be limited to a specific order, and those skilled in the art can make appropriate adjustments to the order based on their common sense.

[0042] In some of the embodiments, the Nth power of 2 is 2 to the power of 1-8, for example, 2 grids, 4 grids, 8 grids, 16 grids, 32 grids, 64 grids, 128 grids, 256 grids, more preferably 16 grids, 32 grids, 64 grids, or 128 grids, and most preferably 32 grids or 64 grids.

[0043] In some embodiments, in step S1, when the bacteria are cultured to an OD600 of about 1.5-2.5, the culture is stopped, and the bacterial solution is diluted with a culture medium to an OD600 of about 0.2-0.4 to maximize the efficiency of single bacteria isolation.

[0044] In some embodiments, the volume of a single electrode droplet is 0.3nL-3nL. The separation method described in the present application has good separation effects and strong adaptability for chips of different specifications. Preferably, the volume of a single electrode droplet is 1nL-3nL, preferably 2nL-3nL, which has stronger adaptability to the concentration of bacteria and stronger operability.

[0045] In some preferred embodiments, in step S2, the bacterial concentration after dilution is 70-120 cfu / uL, more preferably 70-110 cfu / uL, 90-120 cfu / uL, and further preferably 90-110 cfu / uL, so that 18-22 bacteria are distributed per 64 electrodes.

[0046] In some preferred embodiments, the amount of bacteria is 19-21 bacteria per 64 electrodes to achieve better separation efficiency.

[0047] In some preferred embodiments, the number of droplets of each group of bacterial liquid is 2 to the power of the electrode base, for example, the range is 4-992, preferably 16-248, more preferably 32-124, and most preferably 64. The separation method of the present invention can be operated in accordance with the specifications of the chip, and has strong adaptability.

[0048] The separation method of the present invention has the following advantages:

[0049] 1. Different from the traditional microfluidic system that uses the oil-in-water method to embed bacteria in aqueous droplets, the digital microfluidic system can directly manipulate droplets containing bacteria and directly separate large droplets into small droplets, thereby achieving the separation of single bacteria. At the same time, the digital microfluidic system can operate multiple droplets simultaneously to achieve high-throughput single bacteria separation operations.

[0050] 2. The traditional microfluidic system uses a water-in-oil droplet generation method, and the droplets can only be collected in another sample pool after generation, and the generated droplets cannot be recovered. However, the digital microfluidic system can control all separated droplets, and can recover and separate multi-bacteria droplets, thereby achieving a 100% sample retention rate.

[0051] 3. By recycling or removing sample droplets that fail to separate, the space on the chip can be well utilized to ensure that all separated droplets contain a single bacterium, thereby achieving a single bacteria separation rate close to 100%.

[0052] 4. After completing a series of preliminary experiments on the digital microfluidic chip, there is no need to export the bacteria out of the chip for single clone separation and identification. Single bacteria can be separated directly at the cellular level and the isolated clones can be identified.

[0053] In order to better understand the present invention, the digital microfluidic platform used in the following embodiments is introduced: it is equipped with a panoramic camera for observing the overall operation of the chip and the droplets; a microscopic camera for observing the contents in the droplets (such as magnetic beads); a micro-optical measurement module for measuring the OD of bacteria on the chip 600 absorbance; magnetic adsorption module, which can be programmed according to the preset and automatically move to the area that needs magnetic adsorption with 3 axes; chip structure, the sandwich structure of the chip currently used is displayed; temperature control module, used to control the temperature of the chip area; electric three-axis control platform, used to digitally control the movement of the chip platform under different lenses.

[0054] In the following examples, the power-on conditions (light-proof) of the digital microfluidic chip used are:

[0055] Line voltage: 45V,

[0056] Column voltage: 40V,

[0057] Hold time: 10us,

[0058] Frame delay time: 1ms,

[0059] The row and column voltages are set to a lower voltage overnight to ensure the performance of the chip the next day.

[0060] Interval power on: Power on and off alternately at the final resting position of the single bacterial droplet, 1000ms on, 1000ms off.

[0061] Temperature control: Maintain at 37°C overnight.

[0062] The present invention is further described in detail below with reference to specific embodiments.

[0063] Example 1

[0064] 1. Cultivate DH5α E. coli containing eGFP fluorescent protein plasmid in LB medium. Stop culturing when the OD600 of E. coli reaches about 2.0;

[0065] 2. Use LB medium to dilute the bacterial solution to an OD600 of about 0.2;

[0066] 3. The bacterial solution with an OD600 value of about 0.2 was further diluted 1000 times with LB medium to make the bacterial concentration about 70cfu / uL-90cfu / uL; the diluted bacterial solution was injected into a chip with a total number of 16384 electrodes, and each chip formed 64 groups of 64 grids (2 to the sixth power of the electrode base) of droplets. The volume of each single electrode droplet was 3nL. On average, each group was allocated 19 bacteria per 64 electrode bases;

[0067] 4. The droplets are manipulated by controlling the power on and off of the electrodes on the chip; the droplets will move toward the adjacent powered electrode and away from the powered-off electrode; in this way, the droplets of equal volume are controlled to move in the opposite direction and are torn in half with equal volume, and each group obtains two droplets after being torn in half; in accordance with the above method, the droplets torn in half are torn in half again for a second round to obtain four droplets, and then the four droplets are torn in half for a third round to obtain eight droplets, and a total of six rounds of half-tear are performed to quickly form 64 groups of 64 single-electrode droplets;

[0068] 5. Through the fluorescence imaging recognition system, the droplet array is fluorescently detected and judged, and the droplets containing only one bacterium are marked. The electrode where the droplet is located is continuously powered to keep the droplet in place. Among the 64 groups, 15-18 single bacterial droplets are basically separated, and an average of about 17 single bacterial droplets are separated in each group, with a separation rate of 89.4%. The success rate of single bacterial separation in 64 electrodes is 26.5%;

[0069] 6. The remaining droplets without bacteria or with more than one bacteria are controlled by turning on and off the electrodes on the chip, respectively, to realize the manipulation of the droplets, collect the empty droplets and the droplets containing multiple bacteria from one side, and re-merge them into two independent large droplets, i.e., one independent empty droplet without bacteria and one droplet containing multiple bacteria; the empty droplets are removed and the droplets containing multiple bacteria are effectively recovered;

[0070] 7. By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a droplet matrix with one electrode interval, thus optimizing the available space on the chip;

[0071] 8. After recovery, the droplets containing bacteria are subjected to fluorescence detection and judgment (generally, 0-3 bacteria are separated from a 64-drop matrix), and multiple recovered bacterial droplets are merged into a new droplet, maintaining the bacterial concentration at 70-100 cfu / uL, and repeating the above steps 5-9 until the recovered droplets contain no bacteria. The empty droplets are discharged from the chip (at this time, the success rate of single bacterial separation of a sample is 100%);

[0072] 9. In the end, up to 4096 single bacterial droplets can be obtained, which can be continued to be cultured on the chip or exported to the outside of the chip for further experiments. If the number of isolated single bacterial droplets decreases, more experimental operations can be performed on the chip by controlling other reagent droplets.

[0073] Example 2

[0074] 1. Cultivate wild-type DH5α E. coli in LB medium. Stop culturing when the OD600 of E. coli reaches about 2.0.

[0075] 2. Use LB medium to dilute the bacterial solution to an OD600 of about 0.2.

[0076] 3. Add FM 1-43 fluorescent dye at a final concentration of 5ug / mL to stain the bacteria and incubate at room temperature for 30min.

[0077] 4. Dilute the fluorescently labeled bacterial solution 100 times with LB medium to make the bacterial concentration about 700-1200 cfu / uL;

[0078] 5. The diluted bacterial solution was injected into a chip with a total number of 179,200 electrodes. The volume of a single-grid electrode droplet on the chip was about 0.3 nL, forming 700 (groups) of 64-grid droplets. On average, each group was allocated 18 bacteria per 64-grid electrode base.

[0079] 6. The droplets are manipulated by controlling the power on and off of the electrodes on the chip. The droplets will move toward the powered electrode and away from the powered-off electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction and are torn in half. A total of 6 rounds of half-tear are performed to quickly form 700 groups of 64-grid single-electrode droplets.

[0080] 7. Through the fluorescence imaging system, the droplet array is detected and judged by fluorescence, and the droplets containing only one bacterium are marked. The electrode where the droplet is located is continuously powered to keep the droplet in place; among the 700 groups, 14-16 single bacterial droplets were basically separated, and an average of about 15 single bacterial droplets were separated in each group, with a separation rate of 83.3%. The success rate of single bacterial separation in 64 electrodes was 23.4%.

[0081] 8. The remaining droplets that do not contain bacteria or contain more than one bacteria are controlled by turning on and off the power of the electrodes on the chip. The empty droplets and droplets containing multiple bacteria are collected from one side and re-merged into two independent large droplets for the removal of empty droplets and the effective recovery of samples.

[0082] 9. By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a droplet matrix with one electrode interval, thus optimizing the available space on the chip;

[0083] 10. After recovery, the droplets containing bacteria are subjected to fluorescence detection and judgment (generally, 0-6 bacteria are separated from a 64-drop matrix), and multiple recovered bacterial droplets are merged into a new droplet, maintaining the bacterial concentration at 700-1200 cfu / uL, and repeating the above steps 6-10 until the recovered droplets contain no bacteria. The empty droplets are discharged from the chip. (At this time, the success rate of single bacterial separation of a sample is 100%).

[0084] 11. In the end, up to 44,800 single bacterial droplets can be obtained, which can be continued to be cultured on the chip or exported to the outside of the chip for further experiments.

[0085] 12. If the number of droplets is small, more experimental operations can be performed on the chip by controlling other reagent droplets.

[0086] Example 3

[0087] 1. Cultivate Lactobacillus casei in MSR medium. Stop culturing when the OD600 of Lactobacillus casei reaches about 0.15-0.2.

[0088] 2. Add FM 1-43 fluorescent dye at a final concentration of 5ug / mL to stain the bacteria and incubate at room temperature for 30min.

[0089] 3. Dilute the fluorescently labeled bacterial solution 1000 times with MSR medium to make the bacterial concentration about 90-110 cfu / uL.

[0090] The diluted bacterial solution was injected into a chip with a total of 16,384 electrodes to form 64 droplets of 64 grids. On average, each group was allocated 20 bacteria per 64 electrodes.

[0091] 4. Control the droplets by turning on and off the electrodes on the chip. The droplets will move toward the powered electrode and away from the powered electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction and split in half. A total of 6 rounds of splitting in half are performed to quickly form 64 groups of 64 single-electrode droplets, with a single electrode droplet volume of about 3nL.

[0092] 5. Through the fluorescence imaging system, the droplet array was fluorescently detected and judged, and the droplets containing only one bacterium were marked. The electrode where the droplet was located was continuously powered to keep the droplet in place; among the 64 groups, 16-19 single bacterial droplets were separated in each group, and an average of about 18 single bacterial droplets were separated in each group, with a separation rate of 90%. The success rate of single bacterial separation in 64 electrodes was 28.1%;

[0093] 6. According to the separation method of Examples 1 and 2, up to 4096 single bacterial droplets can be obtained, and the culture can continue on the chip: the temperature control system is used to set the chip to 37°C. The anaerobic environment manufacturing device is started, the area where the chip is located is adjusted to an anaerobic state, and the Lactobacillus casei on the chip is cultured for 24 hours. After the culture is completed, it can be clearly observed that the droplets are obviously turbid, and it can be observed under a microscope that the droplets are full of Lactobacillus casei.

[0094] Example 4

[0095] 1. Cultivate infant Bifidobacterium in BBL medium. Stop culturing when the OD600 of infant Bifidobacterium reaches about 0.2-0.25.

[0096] 2. Add FM 1-43 fluorescent dye at a final concentration of 5ug / mL to stain the bacteria and incubate at room temperature in an anaerobic environment for 30 minutes.

[0097] 3. Dilute the fluorescently labeled bacterial solution 1000 times with BBL medium to make the bacterial concentration about 90-120 cfu / uL.

[0098] 4. The diluted bacterial solution was injected into a chip with a total of 16,384 electrodes to form 64 droplets of 32 grids. On average, each group was allocated 11 bacteria per 32 electrodes.

[0099] 5. Control the droplets by turning on and off the electrodes on the chip. The droplets will move toward the powered electrode and away from the powered electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction, and the half-tear is performed for a total of 5 rounds to quickly form single-electrode droplets. 64 groups of 32 single-electrode droplets are quickly formed, and the volume of a single droplet is about 3nL.

[0100] 6. Through the fluorescence imaging system, the droplet array was fluorescently detected and judged, and the droplets containing only one bacterium were marked. The electrode where the droplet was located was continuously powered to keep the droplet in place; among the 64 groups, 7-10 single bacterial droplets were separated in each group, and about 9 single bacterial droplets were separated in each group on average, with a separation rate of 81.8%. The success rate of single bacterial separation in 32 electrodes was 28.1%;

[0101] 7. The remaining droplets that do not contain bacteria or contain more than one bacteria are controlled by turning on and off the power of the electrodes on the chip. The empty droplets and droplets containing multiple bacteria are collected from one side and re-merged into two independent large droplets for the removal of empty droplets and the effective recovery of samples.

[0102] 8. By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a single bacterial droplet matrix with one electrode interval, thereby optimizing the available space on the chip.

[0103] 9. After recovery, the droplets containing bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet, and the bacterial concentration is maintained at 90-120 cfu / uL. The above steps 5-9 are repeated until the recovered droplets contain no bacteria. The empty droplets are discharged from the chip.

[0104] 10. Cultivation: Set the temperature of the chip to 37°C through the temperature control system, start the anaerobic environment manufacturing device, adjust the area where the chip is located to an anaerobic state, and culture the single bacterial droplet matrix of infant Bifidobacterium separated by one electrode on the chip for 24 hours. After the cultivation, a significant increase in Bifidobacterium infantis in the droplets can be observed under a microscope.

[0105] Example 5

[0106] 1. Cultivate DH5α E. coli containing eGFP fluorescent protein plasmid in LB medium. Stop culturing when the OD600 of E. coli reaches about 2.0;

[0107] 2. Use LB medium to dilute the bacterial solution to an OD600 of about 0.2;

[0108] 3. The bacterial solution with an OD600 value of about 0.2 was diluted with LB medium at different times to make the bacterial concentration in different ranges;

[0109] 4. The diluted bacterial solution was injected into a chip with a total of 16,384 electrodes in the same volume to form five droplets with 64 grids. The average number of bacteria allocated to different groups was different, and other operations were the same;

[0110] 5. Referring to the method of Example 1, the droplets are controlled by controlling the power on and off of the electrodes on the chip. The droplets will move toward the powered electrode and away from the powered-off electrode. In this way, the droplets of equal volume are controlled to move in the opposite direction, and multiple rounds of half-tear are performed to quickly form single-electrode droplets, and the volume of a single droplet is about 3nL;

[0111] 6. Through the fluorescence imaging system, the droplet array is fluorescently detected and judged, and the droplets containing only one bacterium are marked. The electrode where the droplet is located is continuously powered to keep the droplet in place.

[0112] The proportion of droplets containing a single bacterium was counted.

[0113]

[0114] 7. The remaining droplets that do not contain bacteria or contain more than one bacteria are controlled by turning on and off the power of the electrodes on the chip. The empty droplets and droplets containing multiple bacteria are collected from one side and re-merged into two independent large droplets for the removal of empty droplets and the effective recovery of samples.

[0115] 8. By controlling the power on and off of the electrodes on the chip, the droplets containing a single bacterium are moved again and rearranged into a droplet matrix with one electrode between them, thereby optimizing the available space on the chip.

[0116] 9. After recovery, the droplets containing bacteria are subjected to fluorescence detection and judgment (generally, 0-6 bacteria are separated from a 64-drop matrix). Multiple recovered bacterial droplets are merged into a new droplet, and the above steps 5-9 are repeated. At this time, the success rate of single bacterial separation of a sample is 100%. The fastest way is to repeat the above separation steps about three or four times to recover droplets without bacteria. For other situations with different bacterial contents, it may take up to more than ten times to achieve 100% separation success.

[0117] 10. In the end, up to 320 single bacterial droplets can be obtained, which can be continued to be cultured on the chip or exported to the outside of the chip for further experiments. If the number of isolated single bacterial droplets decreases, more experimental operations can be performed on the chip by controlling other reagent droplets.

[0118] Because it involves the re-separation of the recovered droplets, after multiple rounds of separation, based on the separation rate of the single bacterial droplets obtained in the initial separation, it can be seen that the separation effect of the entire separation method is very high due to the reasonable control of the bacterial liquid concentration.

[0119] Example 6

[0120] 1. Use LB medium containing 100ug / mL ampicillin to culture the mixed bacterial liquid of wild-type intein-GFP and mutant intein-GFP Escherichia coli until OD600 is about 2.0.

[0121] 2. Dilute the mixed bacterial solution with LB culture medium to an OD600 of about 0.2.

[0122] 3. Dilute the mixed bacterial solution with an OD600 value of about 0.2 by 1000 times with LB medium to make the bacterial concentration about 90-110 cfu / uL.

[0123] 4. Inject the bacterial solution into a chip with a single-electrode droplet volume of 3 nL to form a droplet with 64 grids (2 to the sixth power of the electrode base), and the droplet is allocated to 20 wild-type intein-GFP and mutant intein-GFP Escherichia coli in total;

[0124] 5. According to Example 1, the droplets are controlled by controlling the power on and power off of the electrodes on the chip; the droplets will move toward the adjacent powered electrode and away from the powered off electrode; in this way, the droplets of equal volume are controlled to move in the opposite direction, split in half, and quickly form single electrode droplets, obtaining 20 single bacterial droplets;

[0125] 6. Inject 10ug / mL of ampicillin on the chip, control the antibiotic droplets to mix with the separated single electrode droplets (single bacteria droplets), and the concentration of the mixed antibiotics of ampicillin and kanamycin in the mixed droplets is 5ug / mL;

[0126] 7. Set the chip to 37°C through the temperature control system and culture E. coli in air environment for 18 hours;

[0127] 8. After the culture is completed, add 1*2 grid LB medium containing 10uM IPTG, mix with the resistant E. coli, and induce protein expression at 37℃ for 3h;

[0128] 9. The protein expression was then observed under a fluorescence microscope. Under FITC excitation light, it was observed that the E. coli in some droplets emitted green light. The genotypes of the E. coli in the droplets that emitted green light and those in the droplets that did not emit green light were analyzed by sequencing, corresponding to the wild-type intein-GFP E. coli and the mutant intein-GFP E. coli, respectively.

[0129] from Figure 3 It can be seen that under the 10x eyepiece and 40x objective lens, the wild-type intern-GFP does not emit green light without the addition of inducers, and no luminescence is observed under a high-power microscope, and the single bacteria separation is 100% successful. When the wild-type intern-GFP is added with inducers, it emits green light under a high-power microscope, and the single bacteria grow well after culture.

[0130] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A single bacteria isolation method based on a digital microfluidics system, characterized in that: The following steps are involved: S1. Cultivate bacteria in a culture medium; S2. dilute the bacterial solution with culture medium; S3. Inject the diluted bacterial solution onto the digital microfluidic chip to form bacterial solution droplets with 2 to the power of N as the electrode base, the amount of bacteria in each bacterial solution droplet is 18-22 bacteria per 64 electrode bases, and the number of bacterial solution droplets is at least 1; N is a positive integer equal to or greater than 1; S4. The droplets are manipulated by controlling the power on and off of the electrodes on the chip, and the droplets of equal volume are controlled to move in the opposite direction, and the droplets are torn in half to obtain droplets torn in half; according to the above method, the droplets torn in half are torn in half several times to form 2N single-electrode droplets; S5. Through the recognition system, the number of bacteria in the single-electrode droplet is detected and judged, and the droplet containing only one bacterium is marked. The electrode containing the droplet containing only one bacterium is continuously powered to keep the droplet in place, so as to obtain a single-bacteria droplet.

2. The single bacteria isolation method according to claim 1, characterized in that: The separation method also includes: S6. For droplets containing no bacteria or containing more than one bacteria, the movement of the droplets is controlled by controlling the power on and off of the electrodes on the chip, and the empty droplets and droplets containing multiple bacteria are collected from different sides, respectively, and they are re-fused into an independent empty droplet containing no bacteria and a droplet containing multiple bacteria, the empty droplet is removed, and the droplet containing multiple bacteria is recovered.

3. The single bacteria isolation method according to claim 2, characterized in that: The separation method further comprises: S7. By controlling the power on and off of the electrodes on the chip, the droplets containing the single bacteria are moved again and rearranged into a droplet matrix with one electrode interval; S8. After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are merged into a new droplet with 2 to the power of N as the electrode base. The above steps S3-S7 are repeated until the recovered droplets do not contain bacteria and the bacteria contained in each droplet in the entire array of chip droplets are single bacteria.

4. The single bacteria isolation method according to any one of claims 1 to 3, characterized in that: The Nth power of 2, N is 1-8, preferably 5-6.

5. The single bacteria isolation method according to any one of claims 1 to 3, characterized in that: In S3, the amount of bacteria in each bacterial liquid droplet is 19-21 bacteria per 64 electrode bases.

6. The single bacteria isolation method according to any one of claims 1 to 3, characterized in that: The single electrode droplet volume is 0.3nL-3nL.

7. The single bacteria isolation method according to claim 6, characterized in that: The single electrode droplet volume is 1nL-3nL, preferably 2nL-3nL.

8. The single bacteria isolation method according to any one of claims 1 to 3, characterized in that: In step S2, the bacterial concentration after dilution is 70 cfu / uL-120 cfu / uL, preferably 90 cfu / uL-110 cfu / uL.

9. The single bacteria isolation method according to any one of claims 1 to 3, characterized in that: In step S1, when the bacteria are cultured to an OD600 of about 1.5-2.5, the culture is stopped, and the bacterial solution is diluted with a culture medium to an OD600 of 0.2-0.

4.

10. Application of the single bacteria isolation method according to any one of claims 1 to 9 in bacterial culture, polypeptide drug or antibody screening, and genomics analysis in a digital microfluidic system.

Images