Phage panning method based on digital microfluidics, single-bacterium culture method and use thereof

Abstract

The present invention relates to a phage panning method based on digital microfluidics. In the phage panning method, shut-off washing is used, so that the yield of positive phages is greatly increased, the process is optimized, and the pollution is reduced; and the used agent is only about 0.6% of the agent in the original process, which greatly saves the cost of the agent. Further provided in the present invention is a method for screening a polypeptide drug or a monoclonal antibody according to the phage panning method. The digital microfluidic platform based on the phage panning method, single-bacterium separation method and bacterial screening and culture method of the present invention can be well used for the screening of peptide drugs or monoclonal antibodies in the phage display technology.

Description

Phage screening method and single bacteria culture method based on digital microfluidics and their applications Technical Field

[0001] The present invention belongs to the field of biotechnology, and further to phage display technology, and specifically relates to a phage screening method and a single bacteria culture method based on digital microfluidics and applications thereof. Background Art

[0002] In the past decade, the application of microfluidics in the field of biotechnology has become relatively mature. For example, Bio-Rad has developed a digital PCR chip based on a microfluidics platform, which can achieve absolute quantitative detection of nucleic acids. The American single-cell sequencing company 10X Genomics 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 the microfluidic system provides a perfect interface for the operation of single cells and can be adjusted and controlled in a variety of ways. Microfluidic systems can also reduce the amount of experimental reagents and reduce experimental costs, and have potential portability. Moreover, unlike traditional microfluidic systems that use an oil-in-water method to embed bacteria in aqueous droplets, digital microfluidic systems can directly manipulate droplets containing bacteria and separate a large number of small droplets, thereby achieving high-throughput separation of single bacteria. Based on these advantages, microfluidic technology has become the best candidate for future high-throughput cell sorting technology. However, at this stage, digital microfluidic technology still has various technologies that need to be improved.

[0004] Since its creation in the early 1990s, phage display techniques (PDT) have become an important research tool in biological research. It has fundamentally changed the traditional monoclonal antibody preparation process and is widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, and cell signal transduction research.

[0005] Currently, traditional phage display technology suffers from the drawbacks of requiring large volumes of reagents and high costs. Furthermore, the experimental procedures are cumbersome and time-consuming, with three rounds of panning requiring at least six days, requiring high operator skills. Furthermore, the lengthy experimental process presents the risk of experimental failure due to contamination introduced by improper operation.

[0006] Traditional microfluidic technology relies on the use of mechanical pumps and valves. The integration of multifunctional modules is difficult, and it is difficult to achieve continuous multi-step processing of samples. At the same time, it is not easy to achieve precise control of the positions and reaction times of multiple reagents. It is also difficult to meet the application requirements of high-throughput detection and analysis of multiple reaction systems or multiple reaction conditions in parallel.

[0007] There are a few digital microfluidic phage display technologies available, but overall, the current digital microfluidic phage screening methods still have defects such as poor screening effect, low phage positive yield and high reagent cost.

[0008] In addition, the existing digital microfluidics technology has a low success rate for single bacterial separation and a low throughput, making it impossible to complete high-throughput effective single bacterial separation in one go, and it is impossible to recover and re-isolate bacteria that have failed single separation. With the improvement of technology, even if the problem of liquid separation flux has been solved to a certain extent, due to the characteristics of the system, bacteria that have failed single separation cannot be recovered and re-isolated, so the sample loss rate is relatively high. Existing microfluidics systems are basically unable to screen and continuously culture the single bacteria isolated in situ on the chip, that is, they cannot be screened and continuously cultured in situ, and can only be transferred outside the chip for culture and identification, resulting in the inability to perform subsequent complex reaction operations, the single screening conditions that can be used are single, and the contamination rate is relatively high.

[0009] Summary of the Invention

[0010] Based on this, the purpose of the present invention is to provide a phage screening method based on digital microfluidics and its application,

[0011] The panning method has the advantages of good panning effect and high phage positive yield.

[0012] The first aspect of the present invention provides a phage screening method based on digital microfluidics, which is performed on a digital microfluidic chip and includes the following steps:

[0013] S1. Cultivate bacteria in the continuous bacterial culture area;

[0014] S2. In the panning experimental area, negative screening magnetic beads are mixed with the constructed phage library. After incubation, the negative screening magnetic beads are removed by magnetic separation to obtain screened phage droplets.

[0015] S3. Add positive screening magnetic beads and mix with the remaining phage after screening in step S2. Incubate. After the positive screening magnetic beads are adsorbed by the automated magnetic adsorption module, the droplet containing the positive screening magnetic beads is torn to remove the excess phage library.

[0016] S4. Using a wash solution, perform interception-type washing on the positive screening magnetic bead droplets: For a droplet containing the positive screening magnetic beads to be washed, connect it with an adjacent wash solution droplet in the same direction of movement to form a complete droplet. Place a magnet in the direction of movement to move the positive screening magnetic beads to the desired position. Then, move the droplet in the middle of the complete droplet to disconnect it from the entire droplet. Continue with the next wash step in the same manner. Repeat this cycle multiple times to complete one round of washing.

[0017] S5. After washing, add E. coli hosts in the logarithmic growth phase, allow the phages on the magnetic beads to infect the hosts, and culture them to restore the vitality of the E. coli.

[0018] S6. In the reagent preparation area, separate the helper phage droplet and move it to the revived E. coli droplet for mixing, allowing the helper phage to superinfect the E. coli. After infection is complete, tear the solution and remove the magnetic beads.

[0019] S7. In the liquid reserve area, remove the antibiotic and add it to the superfected E. coli for resistance screening. Cultivate the resistance-screened E. coli to produce phage, completing the first round of panning and obtaining a phage library.

[0020] S8. As needed, the phages produced by panning are placed in the panning experimental area and the phage library obtained in step S7 is used for the next round of panning according to the above steps until the panning is completed.

[0021] The second aspect of the present invention is to provide the application of the above-mentioned phage panning method in the screening of polypeptide drugs or antibodies based on digital microfluidic phage display technology, or in the development of anti-pathogenic microorganism vaccines.

[0022] The digital microfluidics-based phage panning method of the present invention, by adopting a self-developed interception-type washing method, greatly improves the yield of phage positives, optimizes the procedure, reduces pollution, and uses only about 0.6% of the reagents of the original process, greatly saving reagent costs. And by obtaining a surfactant suitable for bacterial culture, as well as the particle size of negative screening magnetic beads and positive screening magnetic beads, the panning effect is further optimized (fewer non-specific binding clones, and fewer rounds of panning are required. The number of required washes is reduced, and the total time used is reduced). The digital microfluidics-based phage panning method of the present invention can be well applied to the screening of peptide drugs or monoclonal antibodies in phage display technology.

[0023] The third aspect of the present invention is to provide a single bacteria isolation method based on a digital microfluidic system, comprising the following steps:

[0024] S1. Cultivate bacteria in a culture medium;

[0025] S2. Dilute the bacterial solution with culture medium;

[0026] S3. Inject the diluted bacterial solution onto the digital microfluidic chip to form bacterial solution droplets with a base number of electrodes equal to 2 (N), with a single electrode droplet volume of 3 nL. The number of bacteria in each bacterial solution droplet is 18 to 22 per 64 electrodes, and there is at least one bacterial solution droplet. N is a positive integer equal to or greater than 1.

[0027] S4. By controlling the power on and off of the electrodes on the chip, the droplets are manipulated, controlling the movement of droplets of equal volume in opposite directions, splitting the droplets in half and obtaining split droplets. The split droplets are split in half again several times in the same manner to form 2N single-electrode droplets.

[0028] S5. Use a fluorescence imaging system to perform fluorescence detection and judgment on single-electrode droplets, mark droplets containing only one bacterium, and continuously power the electrode where the droplet containing only one bacterium is located to keep the droplet in place, thus obtaining a single-bacteria droplet.

[0029] In some embodiments, the separation method further comprises:

[0030] S6. For droplets containing no bacteria or more than one bacterium, the droplets are manipulated by controlling the power on and off of the electrodes on the chip. Empty droplets and droplets containing multiple bacteria are collected from different sides and re-merged 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.

[0031] In some embodiments, the separation method further comprises:

[0032] S7. By controlling the power on and off of the electrodes on the chip, the droplets containing the single bacteria are moved and rearranged into a matrix of droplets separated by one electrode.

[0033] S8. After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. The multiple recovered bacterial droplets are merged into a new droplet with an electrode base of 2N. The above steps S3 to 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.

[0034] The fourth aspect of the present invention provides the application of the above-mentioned single bacteria isolation method in digital microfluidic systems for bacterial culture, polypeptide drug or antibody screening, and genomic analysis.

[0035] The single-bacteria isolation method described in the present invention can quickly form single bacterial droplets by splitting the droplets in half on a digital microfluidic chip. Moreover, the researchers of the present invention discovered in experiments that the reason for the low efficiency of single-bacteria separation is the amount (concentration and volume) of bacterial liquid injected into the digital microfluidic chip. After extensive exploration, it was found that the separation efficiency was highest when the number of bacteria distributed per 64 electrodes was 18 to 22, with a volume of 3nL per single-electrode droplet. The single bacteria obtained through such rapid and efficient separation can be further 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.

[0036] A fifth aspect of the present invention provides a single bacteria screening and culturing method based on a digital microfluidic system. The single bacteria screening and culturing method is performed on a digital microfluidic chip. The digital microfluidic chip includes an upper plate and a lower plate, wherein a fluid channel layer is formed between the upper plate and the lower plate; the lower plate includes an electrode layer, wherein the electrode layer includes a plurality of electrodes arranged in an array;

[0037] The single bacteria screening and culture method comprises the following steps:

[0038] S1. Injecting the diluted bacterial solution into a digital microfluidic chip and controlling the power supply of the electrodes on the chip to form single-electrode droplets. Detection and determination of the single-electrode droplets are performed, marking droplets containing only one bacterium to obtain single-bacteria droplets.

[0039] S2. Injecting the screening material onto the chip of the digital microfluidic system to separate and obtain single-electrode screening material droplets, controlling the single-electrode screening material droplets, and mixing them with single bacterial droplets to obtain mixed droplets;

[0040] S3. Setting the culture temperature, culturing the bacteria in the mixed droplets, and screening the bacteria;

[0041] S4. The culture medium is then injected into the digital microfluidic chip. By controlling the power on and off of the electrodes on the digital microfluidic chip, a single-electrode culture medium droplet is formed, which is mixed with the bacterial droplet and culture is continued.

[0042] According to actual needs, the following steps may be optionally further included:

[0043] S5. Continue culturing for a period of time, until the bacteria have grown for at least one generation. Then, add a new droplet of culture medium. By controlling the power on and off of the electrodes on the digital microfluidic chip, the bacterial droplet is split in half with equal volume, obtaining a bacterial droplet infused with the new culture medium.

[0044] S6. Repeat step S5 0 or more times to achieve continuous perfusion culture of bacteria on the microfluidic chip.

[0045] According to experimental requirements, subsequent experiments can be performed on the torn bacterial droplets (including single bacterial droplets) on the chip or output from the chip at different time points.

[0046] The single-bacteria screening and continuous culture method based on a digital microfluidic system described in the present invention has the following advantages: after obtaining single bacterial droplets in a simple, efficient, and high-throughput manner, screening is performed, and the separated single bacterial droplets can be continuously cultured in situ on the chip without transferring them off the chip to achieve bacterial proliferation, ensuring that the obtained single bacteria will not be unable to grow due to other reasons such as transfer, which ultimately leads to sample loss. Moreover, the continuous in situ culture reduces the contamination rate and greatly saves costs.

[0047] The sixth aspect of the present invention is to provide a method for screening polypeptide drugs or monoclonal antibodies based on phage display technology of a digital microfluidic system.

[0048] A method for screening peptide drugs or monoclonal antibodies based on digital microfluidics, comprising the following steps:

[0049] Obtaining a panned library by any of the above-mentioned phage panning methods;

[0050] Any of the above single bacteria isolation methods, obtaining a single bacteria droplet;

[0051] Any of the above single bacterial droplet screening and culture methods, performing bacterial culture;

[0052] Screening of peptide drugs or monoclonal antibodies.

[0053] The digital microfluidics-based method for screening peptide drugs or monoclonal antibodies of the present invention, the digital microfluidics platform based on phage panning method, single bacteria isolation method, and bacterial screening and culture method can be well applied to the screening of peptide drugs or monoclonal antibodies in phage display technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a side schematic diagram of the digital microfluidic phage display platform.

[0055] Figure 2 is a schematic diagram of the digital microfluidic chip partitioning.

[0056] FIG3 is a schematic diagram of the structure of a digital microfluidic chip.

[0057] FIG4 is a flow chart of phage display on a digital microfluidic chip according to the present invention.

[0058] Figures 5.1 to 5.4 are schematic diagrams of the washing process using the intercepting washing method described in Example 1.

[0059] FIG6 is a schematic diagram of the results of three rounds of panning of positive phages in the alpaca library of Example 1.

[0060] Figures 7.1 and 7.2 are schematic diagrams showing the effects of different particle sizes of negative screening magnetic beads on panning in Example 2.

[0061] FIG8 is a schematic diagram of the half-tearing process in the single bacteria isolation method of the present invention.

[0062] Figure 9 shows the protein expression in a single bacterial droplet isolated by fluorescence microscopy in Example 9. A is a wild-type intern-GFP, which does not emit green light when no inducer is added, and B is a wild-type intern-GFP, which emits green light when an inducer is added.

[0063] FIG10 is a schematic diagram showing the effects of magnetic beads with different luminescence intensities in Example 11, wherein A is a negative control group, and B to F are different single bacteria isolated.

[0064] FIG11 is a schematic diagram of the results of bacterial screening and continuous culture in Example 14.

[0065] Explanation of the accompanying reference numerals: 10, upper electrode plate, 20, lower electrode plate, 101, fluid channel layer, 201, microdroplets, 21, hydrophobic layer, 22, dielectric layer, 23, electrode layer, 24, electrode, 11, upper cover, 12, dielectric layer, 13, hydrophobic layer. DETAILED DESCRIPTION

[0066] To facilitate 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 present disclosure more thorough and comprehensive.

[0067] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those belonging to the technical

[0069] The terms "and / or" and "and / or" used in this specification are intended to be used to describe specific embodiments and are not intended to limit the present invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the related listed items.

[0070] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0071] Infection: The process by which a bacteriophage injects its genetic material into the host bacterium, E. coli.

[0072] Superinfection: The process by which a helper phage injects genetic material into an E. coli host that has already been infected by the phage.

[0073] Some embodiments of the present invention relate to a method for screening polypeptide drugs or monoclonal antibodies based on digital microfluidics, which is performed on a digital microfluidic chip and includes the following steps:

[0074] Obtaining a panned library by a phage panning method;

[0075] The panned library is subjected to single bacterial isolation to obtain single bacterial droplets;

[0076] Screening and cultivation of single bacterial droplets;

[0077] Screening of peptide drugs or monoclonal antibodies.

[0078] In some embodiments of the present invention, the phage panning method based on digital microfluidics comprises the following steps:

[0079] 1) Culturing bacteria in a continuous bacterial culture area; during the bacterial culture, adding a surfactant to increase bacterial migration time, preferably F68, more preferably, adding 0.05-0.15 μl of F68 surfactant per 100 μl of bacterial solution; in one embodiment, adding 0.09-0.11 μl of F68 surfactant per 100 μl of bacterial solution to increase the migration time to 5 hours;

[0080] 2) In the panning area, negative screening magnetic beads are mixed with the constructed phage library. After incubation, non-specifically bound phages are removed. After the negative screening magnetic beads are adsorbed by an automated magnetic adsorption module, the droplets containing the negative screening magnetic beads are torn apart to remove the negative screening magnetic beads.

[0081] 3) Add positive screening magnetic beads (positive screening magnetic beads are coupled with antigens that can bind to bacterial polypeptides or monoclonal antibodies), mix with the remaining phages after screening in step S2, incubate, and adsorb the positive screening magnetic beads through an automated magnetic adsorption module, tear the droplets containing the positive screening magnetic beads, and remove excess phage library; in some embodiments, to increase the phage positive ratio, the number of positive screening magnetic beads added is 10 to 500; more preferably, the number of positive screening magnetic beads is 50 to 200;

[0082] 4) Using a washing solution, perform interception-type washing on the positive screening magnetic bead droplets: For the droplets containing positive screening magnetic beads that need to be washed, connect the droplet containing positive screening magnetic beads with adjacent droplets of washing solution that are in the same direction of movement to form a whole droplet. Place a magnet in the direction of movement, move the positive screening magnetic beads to the desired position, and then move the middle droplet of the whole droplet to separate it from the whole droplet. Then proceed to the next step of washing as described above, and repeat the cycle to complete one round of washing.

[0083] 5) After washing, add E. coli hosts in the logarithmic growth phase, allow the phages on the magnetic beads to infect the hosts, and culture them; after the E. coli recovers, add antibiotics for resistance screening and culture; tear off the solution to remove the magnetic beads;

[0084] 6) In the reagent preparation area, separate the helper phage droplets and move them to the revived E. coli droplets for mixing, allowing the helper phage to superinfect the E. coli;

[0085] 7) In the liquid reserve area, antibiotics are removed and added to the superstained E. coli for resistance screening. The E. coli after resistance screening is cultured to produce phages, completing the first round of panning;

[0086] 8) If needed, the phages produced by panning are subjected to the next round of panning in the panning experimental area according to the above steps until the panning is complete. Specifically, if needed, the enriched phages produced after the first round of panning can be used as the phage library for the second round of panning and subjected to a new round of panning according to steps 1 to 7.

[0087] In some embodiments, in order to improve the screening effect, the particle size of the positive screening magnetic beads is 10um to 20um, and more preferably, the particle size of the positive screening magnetic beads is 20um.

[0088] In some embodiments, the incubation time is 0.5 h to 2.5 h, further 1 h to 2 h.

[0089] In some embodiments, the final concentration of the antibiotic added in step 5 is 90-110 ug / mL.

[0090] In some embodiments, the final concentration of the antibiotic added in step 5 is 30-40 ug / mL.

[0091] In some embodiments of the present invention, the single bacteria isolation method includes: injecting a diluted bacterial solution into a digital microfluidic chip to form bacterial solution droplets with an electrode base number of 2 to the power of N, wherein the volume of a single electrode droplet is 3 nL, the number of bacteria in each bacterial solution droplet is 18 to 22 bacteria per 64 electrode bases, and there is at least one bacterial solution droplet; N is a positive integer equal to or greater than 1.

[0092] In some preferred embodiments, the single bacteria isolation method comprises the following steps:

[0093] S1. Cultivate bacteria in culture medium and dilute the bacterial solution to an OD600 of approximately 0.3.

[0094] S2. Dilute the bacterial solution to a concentration of 70–120 cfu / µL.

[0095] S3. Inject the diluted bacterial solution onto the digital microfluidic chip to form multiple bacterial solution droplets with an electrode base number of 2 to the power of N. The number of bacteria in each bacterial solution droplet is: 18 to 22 bacteria per 64 electrode bases, where N is a positive integer equal to or greater than 1.

[0096] S4. The droplet moves toward the adjacent powered electrode and away from the de-energized electrode. By controlling the power on and off of the electrodes on the chip, the droplet is manipulated, controlling droplets of equal volume to move in opposite directions, splitting the droplet in half and obtaining a split droplet. This split droplet is split in half again, repeating N times, to form an array of 2 to the power of N single-electrode droplets; see Figure 1.

[0097] S5. Using the recognition system, the single-electrode droplet array is detected and evaluated for bacterial count. Droplets containing only a single bacterium are marked, and power is continuously supplied to the electrode containing the single-bacterium droplet to maintain the droplet in place. This is the initial acquisition of a single bacterial droplet after separation. The single bacterial droplet is then further separated according to the present method.

[0098] S6. For droplets containing no bacteria or more than one bacterium, the chip is powered on and off to manipulate the droplets. Empty droplets and droplets containing multiple bacteria are collected from one side, respectively, and re-merged into a separate empty droplet containing no bacteria and a droplet containing multiple bacteria. The empty droplet is then removed, and the droplet containing multiple bacteria is recovered. The sample recovery rate here reaches 100% and is then used to isolate single bacteria.

[0099] S7. By controlling the power on and off of the electrodes on the chip, droplets containing single bacteria are relocated and rearranged into a droplet matrix with one electrode between them, optimizing the available space on the chip for further separation. (Software identifies droplets containing single bacteria and allows them to move and rearrange themselves into a droplet matrix with one electrode between them. Droplets without bacteria are removed, and droplets containing two or more bacteria are recovered. If only 10 to 15 of the 64 droplets can successfully capture a single bacterial droplet, then approximately 50 droplets remain ineffective. After these droplets are recovered or removed, the space on the chip can be reused.)

[0100] S8. After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection (according to the efficient separation method described in the present invention, typically 0 to 6 bacteria remain after separation in a 64-droplet matrix). The multiple recovered bacterial droplets are then merged into a new droplet with a base number of electrodes equal to 2(N). Steps S3 to S7 are repeated until the recovered droplets are free of bacteria, and each droplet in the entire array of chip droplets contains a single bacterium. Empty droplets are then ejected from the chip. At this point, the success rate for single-bacteria isolation in a single sample reaches 100%.

[0101] In some embodiments, the Nth power of 2 is 2 to the power of 1 to 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.

[0102] In some embodiments, in step S1, the bacterial culture is stopped when the OD600 reaches about 1.5-2.5, and the bacterial solution is diluted with culture medium to an OD600 of about 0.2-0.4 to maximize the efficiency of single bacterial isolation.

[0103] In some embodiments, the single-electrode droplet volume is 0.3nL to 3nL. The separation method described in this application has good separation effects and strong adaptability for chips of different specifications. Preferably, the single-electrode droplet volume is 1nL to 3nL, and more preferably 2nL to 3nL, which has greater adaptability to different bacterial concentrations and greater operability.

[0104] In some preferred embodiments, in the above 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 electrode bases.

[0105] In some preferred embodiments, the amount of bacteria is 19 to 21 bacteria per 64 electrode bases to achieve better separation efficiency.

[0106] In some preferred embodiments, the number of droplets in each group of bacterial liquid is 2 to the power of an electrode basis, for example, ranging from 4 to 992, preferably 16 to 248, more preferably 32 to 124, and most preferably 64. The separation method of the present invention can be operated in accordance with chip specifications, and is highly adaptable.

[0107] In some embodiments of the present invention, the single bacteria screening and culture method based on the digital microfluidics system comprises the following steps:

[0108] S1. Injecting the diluted bacterial solution into a digital microfluidic chip and controlling the power supply of the electrodes on the chip to form single-electrode droplets. Detection and determination of the single-electrode droplets are performed, marking droplets containing only one bacterium to obtain single-bacteria droplets.

[0109] S2. Injecting the screening material onto the chip of the digital microfluidic system to separate and obtain single-electrode screening material droplets, controlling the single-electrode screening material droplets, and mixing them with single bacterial droplets to obtain mixed droplets;

[0110] S3. Setting the culture temperature, culturing the bacteria in the mixed droplets, and screening the bacteria;

[0111] S4. The culture medium is then injected into the digital microfluidic chip. By controlling the power on and off of the electrodes on the digital microfluidic chip, a single-electrode culture medium droplet is formed, which is mixed with the bacterial droplet and culture is continued.

[0112] According to actual needs, the following steps may be optionally further included:

[0113] S5. Continue culturing for a period of time, until the bacteria have grown for at least one generation. Then, add a new droplet of culture medium. By controlling the power on and off of the electrodes on the digital microfluidic chip, the bacterial droplet is split in half with equal volume, obtaining a bacterial droplet infused with the new culture medium.

[0114] S6. Repeat step S5 0 or more times to achieve continuous perfusion culture of bacteria on the microfluidic chip.

[0115] According to experimental requirements, subsequent experiments can be performed on the torn bacterial droplets (including single bacterial droplets) on the chip or output from the chip at different time points.

[0116] In some embodiments, the bacteria are oxygen-tolerant or anaerobic bacteria, and nitrogen is continuously ventilated into the chip culture chamber before screening and continuous culture to adjust the culture environment to an anaerobic state.

[0117] In some embodiments, the bacteria are antibiotic-resistant bacteria or auxotrophic bacteria.

[0118] In some embodiments, the antibiotic-resistant bacteria is ampicillin-resistant Escherichia coli.

[0119] In some embodiments, the culture temperature is 36° C. to 37.8° C., and the antibiotic-resistant bacteria are cultured in an air environment for 16 h to 20 h.

[0120] In some embodiments, the screening agent is an antibiotic, or an auxotrophic culture medium, or a bacterial metabolite.

[0121] In some embodiments, the screened substance is an antibiotic, and the screened bacteria is Escherichia coli. Preferably, the concentration of the antibiotic in the mixed droplets is 4ug / mL to 6ug / mL, and further, the concentration of the antibiotic in the mixed droplets is 4.5ug / mL to 5.5ug / mL.

[0122] 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. Those skilled in the art can make appropriate adjustments to the order based on their common sense. For example, bacterial culture is carried out continuously and there is no strict order with other steps, as long as the corresponding purpose can be achieved.

[0123] To better understand the present invention, the automated phage display platform used in the following examples is described: a panoramic camera is configured to observe the entire chip and droplet operation; a microscopic camera is used to observe the contents of the droplets (such as magnetic beads); a micro-optical measurement module is used to measure the OD of bacteria on the chip 600 absorbance; a magnetic adsorption module that can automatically move to the area requiring magnetic adsorption in conjunction with three axes according to preset programming; a chip structure that shows the sandwich structure of the chip currently used; a temperature control module for temperature control of the chip area; and an electric three-axis control platform for digitally controlling the movement of the chip platform under different lenses. Specifically, referring to Figure 3: the digital microfluidic chip includes an upper plate 10 and a lower plate 20, with a fluid channel layer 101 formed between the upper plate 10 and the lower plate 20; the lower plate 20 includes an electrode layer 23, which includes a plurality of electrodes arranged in an array.

[0124] The digital microfluidic chip for phage panning of the present invention consists of 16,384 electrodes, each of which is a 500um*500um square, with a chip gap of 100um. Adjustable injection holes are distributed on the chip for injecting reagents into the chip. Referring to Figure 2, Area A is the panning experimental area, which is used for the magnetic bead incubation, washing, phage breeding and other processes in multiple rounds of panning experiments; Area B is the reagent storage area, which is used to store spare reagents such as antibiotics and helper phages during the experimental process; Area C is the continuous bacterial culture area, which is used to effectively culture bacteria on the chip.

[0125] The present invention is further described in detail below with reference to specific embodiments.

[0126] Example 1

[0127] The phage screening method based on digital microfluidics described in this embodiment includes the following steps.

[0128] The digital microfluidics chip is operated according to conventional methods. Various reagents required for the experimental process (e.g., a natural alpaca phage antibody library, negative-screening BSA-modified carboxyl magnetic beads, positive-screening GFP-modified carboxyl magnetic beads, bacterial culture medium, ampicillin, kanamycin, helper phage, E. coli, etc.) are injected into the chip. By controlling the power on and off of each electrode, each component is moved to the appropriate partition to continue the reaction or standby. See Figure 2 for a schematic diagram of the partitions.

[0129] 1) In the bacterial culture area, the culture medium and bacteria (Escherichia coli TG1) are mixed by electrode control, and the injected bacterial droplets are cultured at 37°C for about 3 hours to the logarithmic growth phase for subsequent experimental infection processes. At the same time, the bacterial droplets are torn apart by electrode control and mixed with the spare culture medium for transfer culture. The transfer step can be repeated multiple times to keep the bacteria in the logarithmic growth phase at the specified time. The logarithmic phase bacteria used subsequently are all from the bacteria cultured in the chip and do not need to be added separately. At the same time, the bacterial culture area can be integrated with an absorbance detection module to monitor the growth status of bacteria.

[0130] During incubation, 0.1 μl of F68 surfactant was added to every 100 μl of bacterial culture. Observation revealed that the addition of only 0.1% F68 increased bacterial migration time on the chip to 5 hours. Since experiments typically last longer than 2 hours, increasing the bacterial migration time to 5 hours eliminates the need for additional bacteria.

[0131] 2) In the panning area, a certain amount of negative screening magnetic beads (1 grid original concentration, typically 2 mg / mL, calculated based on the concentration of commercially available magnetic beads after coupling) and 10 μL of phage library were moved to a designated area for mixing and incubated at room temperature for 30 minutes to remove non-specifically bound phage. After the magnetic beads were adsorbed by the automated magnetic adsorption module, a droplet containing the magnetic beads (0.025 μL) was ruptured to remove the beads.

[0132] 3) Add a certain number of 20 μm positive screening magnetic beads (100) and mix them with the remaining phage after negative screening. Incubate at room temperature for 2 hours. Then, after the positive screening magnetic beads are adsorbed by the automated magnetic adsorption module, the droplets containing the positive screening magnetic beads are torn to remove the excess phage library.

[0133] 4) Wash the positive screening magnetic beads using the interception washing method (10 times) with washing solution to remove non-specifically adsorbed phages on the positive screening magnetic beads:

[0134] Refer to the arrangement in Figure 5.1. The droplets in the two grids on the left are the initial droplets containing magnetic beads that need to be washed and need to be moved to the right.

[0135] See Figure 5.2: Liquid connection, by connecting two adjacent droplets of washing solution that are consistent with the moving direction to form a whole droplet, a magnet is placed on the right side of the whole droplet to attract the magnetic beads to the right side;

[0136] See Figure 5.3: Continue moving the middle droplet of the entire droplet so that it is separated from the entire droplet. The magnetic bead is now on the right.

[0137] Then continue with the next step of washing according to the above method, repeat the cycle several times to complete a round of washing;

[0138] The entire process uses the fact that the movement speed of magnetic beads is faster than the free diffusion speed of solution molecules, and the molecules in the droplet are retained in the front droplet before they are completely diffused.

[0139] The intercepting washing method greatly saves chip space and washing time while still maintaining good washing effects.

[0140] 5) After washing the magnetic beads, add 0.2 μL of the E. coli host in the logarithmic growth phase cultured in step 1) and allow the phage on the magnetic beads to infect the host at 37°C.

[0141] According to the method described in this example, it was found that there was little difference in the effect of magnetic beads eluted with HCl and directly infected with TG1. Therefore, there was no need to perform the HCl elution step, and the process was optimized.

[0142] The host is cultured at a suitable temperature (37° C. in this embodiment) for a certain period of time (30 minutes in this embodiment) to restore the vitality of the host;

[0143] Add an appropriate amount of antibiotic (Amp in this example, final concentration 100 μg / mL) and perform resistance screening culture under certain conditions (37°C, 1 hour in this example);

[0144] The magnetic beads were adsorbed using an automated magnetic adsorption module and the beads (0.025 uL) were removed by tearing the solution.

[0145] 6) In the reagent preparation area, the helper phage droplets are separated and moved to the revived E. coli droplets for mixing. The helper phage is then allowed to superinfect the E. coli at a suitable temperature (37° C. in this embodiment).

[0146] 7) Transfer antibiotics (final concentration: Kana 35 μg / mL) to the liquid reserve area and add them to the super-infected E. coli. Incubate the double-infected E. coli at an appropriate temperature (30°C in this example) for a certain period of time (3-5 hours in this example) to produce phages. (Total reaction volume at this time: 0.3 μL)

[0147] This completes the first round of screening.

[0148] 8) In the reserve area, a certain amount of negative screening magnetic beads (one grid of 2 mg / mL magnetic beads, approximately 25 nL) and phage (0.3 μL) from the first round of panning were moved to a designated area for mixing. Incubate at room temperature for 30 minutes to remove non-specifically bound phage. After the magnetic beads are adsorbed by the automated magnetic adsorption module, the droplet containing the magnetic beads is ripped apart to remove the beads.

[0149] Repeat the above steps to complete the second round of screening.

[0150] Subsequently, as needed, the first round of screening is repeated to complete 3 or more rounds of screening.

[0151] This example uses the alpaca phage library (GFP-Phage) as an example, and phage panning is performed according to the above method.

[0152] Two anti-GFP phages (GFP-enhancer and GFP-minimizer) known to bind to GFP protein were mixed into the alpaca phage library that cannot bind to GFP as positive input. The input ratio was 10:1.5×10 8 pfu (5 of each of the two Anti-GFP phages).

[0153] Three rounds of panning were performed using BSA-Beads as negative screening beads and GFP-Beads as positive screening beads.

[0154] The amount of Anti-GFP phage obtained in each round of panning was measured by colony PCR to determine the enrichment effect of each round.

[0155] Reaction system:

[0156] Reaction conditions:

[0157] The final results showed that after three rounds of panning, the proportion of anti-GFP phages increased from less than 0.0001% at the beginning to 100% at the end (see Figure 6).

[0158] The present invention's interception-type washing method not only saves chip space but also makes three rounds of panning feasible and stable. For digital microfluidic chips, this reduces reliance on chip electrodes and reduces the probability of losing control of droplets due to prolonged operation.

[0159] Furthermore, it was found that if 20 μm magnetic beads were not used, bead counting would be more difficult, and aggregation would affect capture efficiency, increasing the probability of losing valid samples. Furthermore, according to the panning method described in this invention, the total amount of reagents used in the experiment was only approximately 0.6% of that used in traditional phage display panning experiments, significantly saving reagent costs. Except for the first round, which required a large volume of 10 μL, the total reaction volume for each subsequent round was only 0.3 μL.

[0160] Example 2 Selection of particle size for screening magnetic beads

[0161] During the study, it was accidentally discovered that the particle size of the positive screening magnetic beads has a great influence on the effect of the magnetic beads in capturing phages in the phage screening method described in the present invention: it not only causes agglomeration and low magnetic attraction efficiency, but also affects the capture effect of positive phages and the infection effect after capture.

[0162] The phage panning method described in Example 1 was followed, and experiments were performed with different particle sizes.

[0163] Single-bead capture efficiency test: After incubating a single positive screening magnetic bead with positive phage, unbound phage is removed by washing. The positive screening magnetic bead bound to the positive phage is then mixed with the TG1 host bacteria, allowing the adsorbed phage to infect the TG1 host. The number of colonies is then counted on a resistance plate to determine the number of phages adsorbed by a single positive screening magnetic bead.

[0164] In addition to microscopic observation, the capture effect and post-capture infection effect are supported by the number of plate clones.

[0165] From Figure 7.1, from top to bottom, the positive screening magnetic beads of 4.5um, 10um, and 20um are shown. The 4.5um positive screening magnetic beads can be seen to be obviously aggregated and not in a monodisperse state.

[0166] As can be seen from Figure 7.2, under the same time, the effect of the positive screening magnetic beads being adsorbed to one side shows that all the 20um positive screening magnetic beads are adsorbed, a small amount of 4.5um positive screening magnetic beads remain, and a large amount of 10um positive screening magnetic beads remain.

[0167] Example 3

[0168] We found in the experiment that the number of positive screening magnetic beads has a significant impact on the phage positive ratio. The experiment was conducted according to the method described in Example 1. The results are shown in Table 3.1.

[0169] Captured phage count: After incubating positive screening magnetic beads with phage (both positive and negative), unbound phage are removed by washing. Positive screening magnetic beads bound to positive phage are mixed with the TG1 host bacteria, allowing the adsorbed phage to infect the TG1 host. Colonies are then counted on resistance plates to determine the number of phages adsorbed by a single positive screening magnetic bead.

[0170] Phage positive ratio: Perform colony PCR verification on the plate clones to detect whether the clones are positive clones to determine the phage positive ratio.

[0171] Residual amount determination: After washing, the droplets containing positive screening magnetic beads are first adsorbed to one side with a magnet, and the opposite side is torn apart. The phage content in the half of the droplet that does not contain positive screening magnetic beads is detected to determine the washing residual rate.

[0172] Table 3.1 Effect of the number of positive screening magnetic beads

[0173] It can be seen from Table 3.1 that, taking all factors into consideration, the best panning effect is achieved when more than 100 positive screening magnetic beads are used, while when the number of positive screening magnetic beads reaches 500 or more, the residual rate increases, but the phage positive ratio decreases.

[0174] Example 4

[0175] 1. Cultivate DH5α E. coli containing the eGFP fluorescent protein plasmid in LB medium. Stop culturing when the OD600 of the E. coli reaches approximately 2.0.

[0176] 2. Dilute the bacterial solution with LB medium to an OD600 of approximately 0.2;

[0177] 3. The bacterial solution, with an OD600 value of approximately 0.2, was further diluted 1000-fold with LB medium to a bacterial concentration of approximately 70 to 90 cfu / uL. The diluted bacterial solution was then injected onto a chip with a total of 16,384 electrodes. Each chip formed 64 groups of 64 droplets (2 to the power of 6 electrode bases). The volume of each single electrode droplet was 3 nL. On average, each group received 19 bacteria per 64 electrode bases.

[0178] 4. Controlling the droplets is achieved by switching the electrodes on the chip on and off. Droplets will move toward the adjacent powered electrode and away from the de-energized electrode. In this way, equal-volume droplets are controlled to move in the opposite direction, split in half, and each group obtains two droplets after splitting in half. Following this method, the split droplets are split in half again for a second round to obtain four droplets. These four droplets are then split in half for a third round to obtain eight droplets. A total of six rounds of splitting are performed, quickly forming 64 groups of 64 single-electrode droplets. See Figure 8.

[0179] 5. Using a fluorescence imaging recognition system, the droplet array was fluorescently detected and identified, marking droplets containing only a single bacterium. The electrodes containing the droplets were continuously powered to keep the droplets in place. Across 64 groups, 15 to 18 single bacterial droplets were isolated, with an average of approximately 17 per group, for a separation rate of 89.4%. The success rate for single bacterial isolation across all 64 electrodes was 26.5%.

[0180] 6. The remaining droplets containing no bacteria or more than one bacteria are manipulated by controlling the power on and off of the electrodes on the chip. Empty droplets and droplets containing multiple bacteria are collected from one side and re-merged into two independent large droplets, namely one independent empty droplet containing no bacteria and one droplet containing multiple bacteria. This removes the empty droplets and effectively recovers the droplets containing multiple bacteria.

[0181] 7. By controlling the power on and off of the electrodes on the chip, the droplets containing individual bacteria are moved and rearranged into a droplet matrix with one electrode between them, optimizing the available space on the chip.

[0182] 8. After recovery, the bacteria-containing droplets are subjected to fluorescence detection (generally, a 64-droplet matrix will separate 0-3 bacteria). Multiple recovered bacterial droplets are merged into a new droplet, maintaining the bacterial concentration at 70-100 cfu / uL. Steps 5-9 are repeated until the recovered droplets contain no bacteria. Empty droplets are discharged from the chip (at this point, the success rate of single bacterial isolation in a single sample is 100%).

[0183] 9. Ultimately, up to 4096 single bacterial droplets can be obtained, which can be cultured on the chip or exported off-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.

[0184] Example 5

[0185] 1. Cultivate wild-type DH5α E. coli in LB medium. Stop culturing when the OD600 reaches approximately 2.0.

[0186] 2. Use LB medium to dilute the bacterial solution to an OD600 of approximately 0.2.

[0187] 3. Add FM 1-43 fluorescent dye at a final concentration of 5 μg / mL to stain the bacteria and incubate at room temperature for 30 minutes.

[0188] 4. Dilute the fluorescently labeled bacterial solution 100-fold with LB medium to a bacterial concentration of approximately 700-1200 cfu / uL.

[0189] 5. The diluted bacterial solution was injected onto a chip with a total of 179,200 electrodes. The volume of the droplet per electrode on the chip was approximately 0.3 nL, forming 700 groups of 64 droplets. On average, each group was assigned 18 bacteria per 64 electrodes.

[0190] 6. By controlling the power on and off of electrodes on the chip, the droplets are manipulated; the droplets move toward the adjacent powered electrode and away from the de-energized electrode. In this way, droplets of equal volume are controlled to move in opposite directions, splitting in half. A total of six rounds of splitting are performed, quickly forming 700 groups of 64 single-electrode droplets.

[0191] 7. By using a fluorescence imaging system to identify and identify droplet arrays, the team performed fluorescence detection and identification, marking droplets containing only a single bacterium. The electrodes containing the droplets were then continuously powered to keep the droplets in place. Among the 700 groups, 14 to 16 single bacterial droplets were isolated, with an average of approximately 15 single bacterial droplets per group, for a separation rate of 83.3%. The success rate for single bacterial isolation among the 64 electrodes was 23.4%.

[0192] 8. The remaining droplets containing no bacteria or more than one bacteria are manipulated by controlling the power on and off of the electrodes on the chip. 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.

[0193] 9. By controlling the power on and off of the electrodes on the chip, the droplets containing individual bacteria are moved and rearranged into a droplet matrix with one electrode between them, optimizing the available space on the chip.

[0194] 10. After recovery, the bacterial droplets are subjected to fluorescence detection (typically, a 64-droplet matrix will isolate 0 to 6 bacteria). Multiple recovered bacterial droplets are then merged into a single droplet, maintaining a bacterial concentration of 700 to 1200 cfu / µL. Steps 6 to 10 are repeated until the recovered droplets are free of bacteria. Empty droplets are then ejected from the chip. (At this point, the success rate for single-bacteria isolation in a single sample is 100%).

[0195] 11. Ultimately, up to 44,800 single bacterial droplets can be obtained, which can be cultured on the chip or exported off-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.

[0196] Example 6

[0197] 1. Cultivate Lactobacillus casei in MSR medium. Stop culturing when the OD600 reaches approximately 0.15-0.2.

[0198] 2. Add FM 1-43 fluorescent dye at a final concentration of 5 μg / mL to stain the bacteria and incubate at room temperature for 30 minutes.

[0199] 3. Dilute the fluorescently labeled bacterial solution 1000-fold with MSR medium to a bacterial concentration of approximately 90-110 cfu / uL.

[0200] The diluted bacterial solution was injected onto a chip with a total of 16,384 electrodes, forming 64 droplets of 64 grids. On average, each group was allocated 20 bacteria per 64 electrodes.

[0201] 4. By controlling the power on and off of the electrodes on the chip, the droplets are manipulated. Droplets will move toward the adjacent powered electrode and away from the de-energized electrode. In this way, droplets of equal volume are controlled to move in the opposite direction, splitting in half. A total of six rounds of splitting are performed, quickly forming 64 groups of 64 single-electrode droplets, with a single electrode droplet volume of approximately 3nL.

[0202] 5. Using a fluorescence imaging system, the droplet array was fluorescently detected and identified, marking droplets containing only a single bacterium. The electrodes containing the droplets were continuously powered to keep the droplets in place. Among the 64 groups, 16 to 19 single bacterial droplets were isolated in each group, with an average of approximately 18 single bacterial droplets per group, for a separation rate of 90%. The success rate of single bacterial isolation among the 64 electrodes was 28.1%.

[0203] 6. Using the re-isolation method described in Examples 1 and 2, a maximum of 4096 single bacterial droplets were ultimately obtained, allowing for continued on-chip culture. The chip was set to 37°C via the temperature control system. The anaerobic environment creation device was activated, adjusting the chip area to an oxygen-free state, and the Lactobacillus casei on the chip was cultured for 24 hours. After the culture was completed, the droplets were noticeably turbid, and microscopic observation revealed that the droplets were filled with Lactobacillus casei.

[0204] Example 7

[0205] 1. Cultivate Bifidobacterium infantis in BBL medium. Stop culturing when the OD600 of Bifidobacterium infantis reaches approximately 0.2-0.25.

[0206] 2. Add FM 1-43 fluorescent dye at a final concentration of 5 μg / mL to stain the bacteria and incubate at room temperature in an anaerobic environment for 30 minutes.

[0207] 3. Dilute the fluorescently labeled bacterial solution 1000-fold with BBL medium to a bacterial concentration of approximately 90-120 cfu / uL.

[0208] 4. The diluted bacterial solution was injected onto a chip with a total of 16,384 electrodes, forming 64 droplets of 32 cells. On average, each group received 11 bacteria per 32 electrodes.

[0209] 5. By controlling the power on and off of the electrodes on the chip, the droplets are manipulated. Droplets move toward the adjacent powered electrode and away from the de-energized electrode. In this way, droplets of equal volume are controlled to move in opposite directions, splitting in half for a total of five cycles, rapidly forming single-electrode droplets. Sixty-four groups of 32 single-electrode droplets are quickly formed, with a single droplet volume of approximately 3 nL.

[0210] 6. Using a fluorescence imaging system, the droplet array was fluorescently detected and identified, marking droplets containing only a single bacterium. The electrodes containing the droplets were continuously powered to keep the droplets in place. Across 64 groups, 7 to 10 single bacterial droplets were isolated per group, with an average of approximately 9 single bacterial droplets per group, for an isolation rate of 81.8%. The success rate for single bacterial isolation among the 32 electrodes was 28.1%.

[0211] 7. The remaining droplets without bacteria or containing more than one bacteria are manipulated by controlling the power on and off of the electrodes on the chip. 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.

[0212] 8. By controlling the power on and off of the electrodes on the chip, the droplets containing single bacteria are moved and rearranged into a matrix of single bacterial droplets separated by one electrode, optimizing the available space on the chip.

[0213] 9. After recovery, perform fluorescence detection on the bacterial droplets. Multiple recovered bacterial droplets are merged into a new droplet, maintaining a bacterial concentration of 90-120 cfu / uL. Repeat steps 5-9 above until the recovered droplets are free of bacteria. Empty droplets are discharged from the chip.

[0214] 10. Cultivation: The chip was set to 37°C using the temperature control system. The anaerobic environment creation device was activated to adjust the chip area to an anaerobic state. The single bacterial droplet matrix of infant Bifidobacterium on the chip, separated by one electrode, was cultured for 24 hours. After the culture was completed, a significant increase in the number of infant Bifidobacterium in the droplets was observed under a microscope.

[0215] Example 8

[0216] 1. Cultivate DH5α E. coli containing the eGFP fluorescent protein plasmid in LB medium. Stop culturing when the OD600 of the E. coli reaches approximately 2.0.

[0217] 2. Dilute the bacterial solution with LB medium to an OD600 of approximately 0.2;

[0218] 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 fall within different ranges;

[0219] 4. The diluted bacterial solution was injected into a chip with a total of 16,384 electrodes at the same volume, forming five droplets with 64 cells. The average number of bacteria distributed to different groups was different, and other operations were the same.

[0220] 5. Using the same method as in Example 1, droplet manipulation is achieved by controlling the power on and off of the electrodes on the chip. Droplets will move toward the powered electrode and away from the de-powered electrode. In this way, droplets of equal volume are controlled to move in opposite directions, undergoing multiple rounds of splitting in half, quickly forming single-electrode droplets with a volume of approximately 3 nL.

[0221] 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.

[0222] The proportion of droplets containing single bacteria was counted.

[0223] Table 8.1

[0224] 7. The remaining droplets without bacteria or containing more than one bacteria are manipulated by controlling the power on and off of the electrodes on the chip. 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.

[0225] 8. By controlling the power on and off of the electrodes on the chip, the droplets containing single bacteria are moved and rearranged into a droplet matrix with one electrode interval, optimizing the available space on the chip.

[0226] 9. After recovery, the bacteria-containing droplets are subjected to fluorescence detection and judgment (generally, a 64-droplet matrix will separate 0 to 6 remaining bacteria). Multiple recovered bacterial droplets are merged into a new droplet, and steps 5 to 9 above are repeated. At this time, the success rate of single bacterial isolation in a sample is 100%. The fastest way to recover bacteria-free droplets is to repeat the above separation steps about three or four times. For other cases with different bacterial content, it may take up to more than ten times to achieve 100% successful isolation.

[0227] 10. Ultimately, up to 320 single bacterial droplets can be obtained, which can be cultured on the chip or exported off-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.

[0228] Because it involves the re-separation of the recovered droplets, after multiple rounds of separation, based on the separation rate of the single bacterial droplet 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.

[0229] Example 9

[0230] 1. Culture a mixed culture of wild-type intein-GFP and mutant intein-GFP E. coli in LB medium containing 100 μg / mL ampicillin to an OD600 of approximately 2.0.

[0231] 2. Dilute the mixed bacterial solution with LB medium to an OD600 of about 0.2.

[0232] 3. Dilute the mixed bacterial solution with an OD600 value of about 0.2 with LB medium 1000 times to make the bacterial concentration about 90-110 cfu / uL.

[0233] 4. The bacterial solution was injected into a chip with a single-electrode droplet volume of 3 nL, forming a 64-grid (2 to the power of 6 electrode base) droplet. A total of 20 wild-type intein-GFP and mutant intein-GFP E. coli were distributed into the droplet.

[0234] 5. Following the method described in Example 4, the droplets were manipulated by controlling the power on and off of the electrodes on the chip. The droplets moved toward the powered electrode and away from the de-powered electrode. In this way, droplets of equal volume were controlled to move in opposite directions, splitting in half and quickly forming single-electrode droplets. Twenty single bacterial droplets were obtained.

[0235] 6. Inject 10 μg / mL of ampicillin onto the chip and control the mixing of the antibiotic droplet with the separated single electrode droplet (single bacteria droplet). The concentration of the mixed antibiotics, ampicillin and kanamycin, in the mixed droplet is 5 μg / mL.

[0236] 7. Set the chip to 37°C through the temperature control system and culture E. coli in air for 18 hours;

[0237] 8. After the culture is completed, add 1*2 grid LB medium containing 10uM IPTG, mix with the resistant E. coli cultured, and induce protein expression at 37℃ for 3h;

[0238] 9. Protein expression was then observed using a fluorescence microscope. Under FITC excitation light, E. coli in some droplets were observed to emit green light. Sequencing analysis revealed the genotypes of E. coli in droplets that emitted green light and those that did not, corresponding to wild-type intein-GFP E. coli and mutant intein-GFP E. coli, respectively.

[0239] As shown in Figure 9, the wild-type intern-GFP did not emit green light under the 10x eyepiece and 40x objective lens without the addition of an inducer, and no luminescence was observed under high magnification, indicating 100% success in single-bacterial isolation. The wild-type intern-GFP, when the inducer was added, emitted green light under high magnification, and the single bacterium grew well after culture.

[0240] Example 10

[0241] The single bacteria screening and culture method based on the digital microfluidic system described in this embodiment includes the following steps:

[0242] 1. Use LB medium containing 100 μg / mL ampicillin to culture ampicillin-resistant E. coli to an OD600 of approximately 2.0. Use LB medium to culture wild-type E. coli to an OD600 of approximately 2.0.

[0243] 2. Dilute the ampicillin-resistant E. coli and wild-type E. coli using LB medium to an OD600 of approximately 0.2.

[0244] 3. Dilute the two bacterial solutions with an OD600 value of about 0.2 1000 times with LB medium to make the bacterial concentration about 70-120 cfu / uL.

[0245] Two bacterial solutions were injected into a chip with a single-electrode droplet volume of 3 nL, forming a 64-grid (2 to the sixth power electrode base) droplet. This droplet contained 20 wild-type intein-GFP and mutant intein-GFP Escherichia coli. By controlling the power on and off of the electrodes on the chip, the droplets were manipulated, moving toward the adjacent powered electrode and away from the de-powered electrode. In this way, droplets of equal volume were controlled to move in opposite directions, splitting in half and quickly forming single-electrode droplets. A fluorescence imaging recognition system was used to detect and identify fluorescence on the droplet array, marking droplets containing only a single bacterium. Twenty single-bacteria droplets were obtained.

[0246] 4. Separating and obtaining a droplet containing a single ampicillin-resistant E. coli and a single wild-type E. coli;

[0247] 5. Different concentrations of ampicillin were injected onto the chip to isolate single-electrode ampicillin droplets. These antibiotic droplets were then mixed with droplets of ampicillin-resistant E. coli and wild-type E. coli. The final concentration of ampicillin in the mixed droplets of ampicillin, ampicillin-resistant E. coli, or wild-type E. coli was 2.5 μg / mL to 10 μg / mL, as shown in the table below.

[0248] 6. Set the chip temperature to 37°C through the temperature control system, culture E. coli in an air environment for 18 hours, and observe the growth of E. coli in the droplets.

[0249] Table 10.1. Statistics of E. coli growth status

[0250] When the concentrations of 4ug / mL, 5ug / mL, and 6ug / mL were used as the critical concentrations for antibiotic screening, the results showed that the bacterial growth was greatest at 4ug / mL and least at 6ug / mL. Overall, the best growth was observed at 5ug / mL.

[0251] When the concentration of the antibiotic in the mixed droplets is 4ug / mL to 6ug / mL, Amp-resistant Escherichia coli can be well screened.

[0252] Example 11

[0253] The antibody screening method based on the digital microfluidics system described in this embodiment includes the following steps:

[0254] 1. According to the phage panning method described in Examples 1 to 3, the phage antibody library was panned three times using phage display technology on a digital microfluidic chip. The E. coli produced after panning was diluted 800-1000 times with LB medium (containing 0.1% F68) on the chip to a bacterial concentration of approximately 70 cfu / uL to 90 cfu / uL. According to the single bacteria isolation method described in Example 4, the diluted bacterial solution was injected into a chip with a total number of 16,384 electrodes. Each chip formed 64 groups of 64 droplets (2 to the power of 6 electrode bases). The volume of each single electrode droplet was 3 nL. On average, each group was assigned 20 bacteria per 64 electrode bases.

[0255] 2. Droplets were manipulated by controlling the power on and off of electrodes on the chip. Droplets moved toward the adjacent powered electrode and away from the de-powered electrode. This method controlled the movement of equal-volume droplets in the opposite direction, resulting in a splitting process. Each group of droplets obtained two split droplets. Following this process, the split droplets were split again in half, yielding four droplets. These four droplets were then split in half again, yielding eight droplets. Six rounds of splitting were repeated, rapidly forming 64 groups of 64 single-electrode droplets. Droplets containing a single E. coli were isolated. A fluorescence imaging recognition system detected and identified the droplet array through fluorescence detection, marking droplets containing only a single bacterium. The electrode containing the droplet was continuously powered to maintain the droplet in place. Across the 64 groups, 16 to 19 single-bacterial droplets were isolated, with an average of approximately 18 single-bacterial droplets per group, for a separation rate of 90.0%. The success rate for single-bacterial isolation across the 64 electrodes was 28.1%. The experiment found that when the number of bacteria assigned to each group was less than 18 or greater than 22, the separation rate and separation success rate dropped significantly. The separation results can be seen in Table 2.

[0256] Table 11.1

[0257] For droplets containing no bacteria or more than one bacterium, the chip controls the power on and off of the electrodes to manipulate the droplets. Empty droplets and droplets containing multiple bacteria are collected from different sides, and then re-merged into an independent empty droplet containing no bacteria and a droplet containing multiple bacteria. The empty droplet is then removed and the droplet containing multiple bacteria is recovered.

[0258] By controlling the power on and off of the electrodes on the chip, the droplets containing individual bacteria are moved and rearranged into a droplet matrix with one electrode between them.

[0259] After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are fused into a new droplet with a base number of electrodes of 2 to the power of N. The above steps 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.

[0260] 3. Inject helper phage M13K07 (titer: 10 11 pfu / mL), and the equal volume was torn in half to separate the single-electrode auxiliary phage droplets, and the auxiliary phage droplets and Escherichia coli droplets were controlled to mix;

[0261] 4. Set the chip to 37°C through the temperature control system and incubate for 2 hours to allow the helper phage to infect E. coli.

[0262] 5. Inject 10 μg / mL of ampicillin and kanamycin mixed antibiotics onto the chip, separating into 1x2 electrode droplets. Control the mixing of the antibiotic droplets with the E. coli droplets that have been infected with the helper phage. The concentration of the mixed antibiotics in the droplets is 5 μg / mL.

[0263] 6. E. coli was then cultured in air for 18 hours, and the software marked the successfully grown droplets. The ungrown droplets were removed from the chip, the temperature was adjusted to 30°C, and the cells were cultured for 3 hours to produce more phage progeny.

[0264] 7. Inject antigen-coupled carboxyl magnetic beads (20 μm) onto the chip, separate and obtain droplets containing single magnetic beads, control the mixing of single magnetic bead droplets and grown E. coli droplets, and incubate at room temperature for 2 h;

[0265] 8. Inject M13-PE antibody (1:1000, Sino Biological) containing 0.1% F68 onto the chip to isolate single droplets. Mix the control antibody droplets with the E. coli droplets producing phage progeny to obtain antigen (magnetic beads)-(phage)-(M13-PE) and incubate at room temperature for 30 minutes.

[0266] 9. Use fluorescence microscopy to detect whether the magnetic beads in the droplets emit fluorescence and the intensity of the fluorescence to determine the affinity differences between different clones;

[0267] 10. Control the droplets through electrodes and output the desired droplets (e.g., with higher fluorescence intensity) for cultivation.

[0268] See Figure 10. A is the negative control group, which theoretically emits no fluorescence. B through F show different isolated bacteria, whose phages display different antibodies. The varying affinities between the antibodies and antigens ultimately result in varying phage capture by the magnetic beads within the same timeframe, manifesting as varying fluorescence intensities.

[0269] The more phages bind to the magnetic beads, the higher the fluorescence intensity of the phage dye. Among them, the fluorescence intensity of B, C, and D in Figure 10 is higher.

[0270] Example 12

[0271] This embodiment involves isolating and culturing crowded Corynebacterium, comprising the following steps:

[0272] 1. Cultivate the crowded bacterium in a medium containing BHI + Tween 80 (concentration 0.0625%) until the OD600 is about 1.0.

[0273] 2. Use BHI + Tween 80 (concentration 0.0625%) culture medium to dilute the crowded bacterium to an OD600 of 0.2-0.3.

[0274] 3. Dilute the bacterial solution with an OD600 value of about 0.2-0.3 1000-fold with BHI + Tween 80 (concentration 0.0625%) culture medium to a bacterial concentration of about 70-100 cfu / uL;

[0275] The bacterial solution was injected into a chip with a single-electrode droplet volume of 3 nL, forming a droplet with 64 grids (2 to the power of 6 electrode bases), and 20 bacteria were distributed to the droplet;

[0276] 4. By controlling the power on and off of the electrodes on the chip, the droplets are manipulated, moving toward the adjacent powered electrode and away from the de-energized electrode. In this way, droplets of equal volume are controlled to move in opposite directions, splitting in half and quickly forming single-electrode droplets. This separation yields 19 droplets containing a single, crowded Corynebacterium.

[0277] For droplets containing no bacteria or more than one bacterium, the chip controls the power on and off of the electrodes to manipulate the droplets. Empty droplets and droplets containing multiple bacteria are collected from different sides, and then re-merged into an independent empty droplet containing no bacteria and a droplet containing multiple bacteria. The empty droplet is then removed and the droplet containing multiple bacteria is recovered.

[0278] By controlling the power on and off of the electrodes on the chip, the droplets containing individual bacteria are moved and rearranged into a droplet matrix with one electrode between them.

[0279] After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are fused into a new droplet with a base number of electrodes of 2 to the power of N. The above steps 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.

[0280] 5. The chip was set to 37°C through the temperature control system, and the crowded Corynebacterium was cultured in an air environment for 20 hours. After the culture was completed, the droplets were obviously turbid, and under a microscope, it was observed that the droplets were full of crowded Corynebacterium.

[0281] Example 13

[0282] In this embodiment, Bacillus subtilis is isolated and cultured, comprising the following steps:

[0283] 1. Grow Bacillus subtilis in NB broth to an OD600 of approximately 2.0.

[0284] 2. Using NB broth, dilute Bacillus subtilis to an OD600 of 0.15-0.25;

[0285] 3. Dilute the bacterial solution with an OD600 value of about 0.15-0.25 1000-fold with NB broth to a bacterial concentration of about 80 cfu / uL-120 cfu / uL;

[0286] 4. The bacterial solution is injected into a chip with a single-electrode droplet volume of 3nL, forming a droplet with 64 grids (2 to the power of 6 electrode base). The droplet is distributed to 20 bacteria. According to the method of Example 1 or Example 2, the droplet is controlled by controlling the power on and off of the electrodes on the chip. The droplet 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 a single-electrode droplet. Droplets containing 18 single crowded rod bacteria are separated;

[0287] For droplets containing no bacteria or more than one bacterium, the chip controls the power on and off of the electrodes to manipulate the droplets. Empty droplets and droplets containing multiple bacteria are collected from different sides, and then re-merged into an independent empty droplet containing no bacteria and a droplet containing multiple bacteria. The empty droplet is then removed and the droplet containing multiple bacteria is recovered.

[0288] By controlling the power on and off of the electrodes on the chip, the droplets containing individual bacteria are moved and rearranged into a droplet matrix with one electrode between them.

[0289] After recovery, the droplets containing multiple bacteria are subjected to fluorescence detection and judgment. Multiple recovered bacterial droplets are fused into a new droplet with a base number of electrodes of 2 to the power of N. The above steps 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.

[0290] 5. Then, NB broth culture medium is injected into the digital microfluidic chip. By controlling the power on and off of the electrodes on the digital microfluidic chip, single-electrode culture medium droplets are formed, which are mixed with the screened bacterial droplets and continue to be cultured;

[0291] 6. The chip was set to 37°C through the temperature control system, and Bacillus subtilis was cultured in air for 16 hours. After the culture, the droplets were obviously turbid, and under a microscope, it was observed that the droplets were full of Bacillus subtilis.

[0292] Example 14

[0293] The method for screening polypeptide drugs or monoclonal antibodies based on digital microfluidics described in this embodiment includes the following steps:

[0294] 1. Use LB medium containing 50ug / mL ampicillin to transfer E. coli containing the anti-GFP protein nanobody gene at a ratio of 1:50; the concentration of the bacterial solution after dilution with LB medium is 11cfu / uL~14.5cfu / uL;

[0295] The Escherichia coli expressing the anti-GFP protein nanobody gene was obtained by the digital microfluidics-based phage panning method described in Example 1, and the panning method included the following steps:

[0296] S1) In the bacterial culture area, electrodes control the mixing of culture medium and bacteria (E. coli TG1). The injected bacterial droplets are incubated at 37°C for approximately 3 hours until the logarithmic growth phase, which is used for subsequent experimental infection procedures. Simultaneously, electrodes control the breaking of bacterial droplets and mixing with spare culture medium for transfer culture.

[0297] During the culture, 0.1 μl of F68 surfactant was added to every 100 μl of bacterial solution.

[0298] S2) In the panning area, a certain amount of negative screening magnetic beads (1 grid original concentration, typically 2 mg / mL, calculated based on the concentration of commercially available magnetic beads after coupling) and 10 μL of phage library are moved to a designated area and mixed. Incubate at room temperature for 30 minutes to remove non-specifically bound phage. Magnetic separation is then performed to remove the negative screening magnetic beads, yielding the screened phage droplets.

[0299] S3) Add 100 20 μm positive screening magnetic beads (GFP protein is coupled to the positive screening magnetic beads) and mix them with the phage droplets remaining after negative screening. Incubate at room temperature for 2 hours. Then, after the positive screening magnetic beads are adsorbed by the automated magnetic adsorption module, the droplets containing the positive screening magnetic beads are torn to remove the excess phage library.

[0300] S4) Wash the positive screening magnetic beads with a washing solution in a cut-off flow washing method to remove non-specifically adsorbed phages on the positive screening magnetic beads:

[0301] Then continue with the next step of washing according to the above method, repeat the cycle several times to complete a round of washing;

[0302] S5) After washing the magnetic beads, transfer the beads to 0.2 μL of the E. coli host in the logarithmic growth phase cultured in step S1) and allow the phage on the magnetic beads to infect the host at 37°C.

[0303] The host was cultured at 37°C to restore its vitality;

[0304] Antibiotics (Amp) were added at a final concentration of 100 μg / mL and cultured for resistance screening at 37°C for 1 h;

[0305] Use the automated magnetic adsorption module to adsorb the magnetic beads and tear off the liquid to remove the magnetic beads.

[0306] S6) In the reagent preparation area, the helper phage droplet is separated and moved to the revived E. coli droplet for mixing, and the helper phage is allowed to superinfect the E. coli at 37°C.

[0307] S7) The antibiotic (final concentration Kana 35ug / mL) is moved in the liquid reserve area and added to the super-infected E. coli. The double-infected E. coli is cultured at a suitable temperature of 30°C for 3-5 hours to produce phages, completing the first round of panning and obtaining a phage library containing anti-GFP protein nanoantibody genes.

[0308] S8) In the standby area, move the negative screening magnetic beads and 0.3 μL of phage from the first round of panning to a designated area for mixing. Incubate at room temperature for 30 minutes to remove non-specifically bound phage. After the magnetic beads are adsorbed by the automated magnetic adsorption module, the droplet containing the magnetic beads is ripped apart to remove the beads.

[0309] Repeat the above steps to complete the second round of screening.

[0310] Repeat the first round of panning, complete 3 or more rounds of panning, and obtain an E. coli library containing the nanobody gene against the GFP protein.

[0311] 2. Using the separation method described in Example 4, single bacteria were isolated from the E. coli library obtained above. The diluted E. coli solution was injected into a chip with a single electrode droplet volume of 25 nL to form a 16-grid droplet. The droplet was distributed to 5 bacteria and cultured on the chip at 37°C for 1 hour.

[0312] 3. Cultivate the positive bacteria obtained above according to the cultivation method described in Example 10: control the power on and off of the electrodes on the chip to achieve control of the droplets; control the tearing of 16 grids of bacterial solution, tear out 2 grids of droplets without bacteria and discard them, and control the electrodes to control the mixing of 2 grids of LB medium containing 5 μg / mL ampicillin with the remaining 14 grids of bacterial solution, and incubate at 37°C; repeat the medium exchange operation once every 15 minutes to achieve continuous perfusion culture, replacing the antibiotic concentration in the droplets, and continue for 4 times for a total of 1 hour;

[0313] 4. Manipulate the droplets by controlling the power on and off of the electrodes on the chip as in Example 4 or Example 10; inject 80 μM IPTG (isopropyl-β-D-thiogalactopyranoside) solution into two electrodes on the chip and mix with the bacterial solution to a final concentration of 10 μM;

[0314] 5. Manipulate the droplets by controlling the power on and off of the electrodes on the chip as in Example 4 or Example 10; tear the 18-grid bacterial solution, discard two droplets, and mix the remaining 16-grid bacterial solution with two LB medium containing 5 μg / mL ampicillin and 100 μM IPTG controlled by the electrodes. Repeat the medium change every 15 minutes to achieve continuous perfusion culture, maintaining the concentration of antibiotics and inducers through perfusion culture;

[0315] 6. Control the droplets by turning on and off the electrodes on the chip as in Example 4 or Example 10. After 1 hour of continuous perfusion culture, tear 18 cells of bacterial solution every 15 minutes, tear out 2 cells of droplets for testing, and continue perfusion culture with the remaining droplets.

[0316] 7. Use electrodes to control the droplets and add two droplets containing GFP protein-coupled magnetic beads to the two droplets torn out in step 6. Incubate at 37°C for 1 hour.

[0317] 8. After the incubation, use a magnet to adsorb the magnetic beads and control the electrodes to wash the magnetic beads in the droplets by injecting new droplets containing PBS buffer and tearing off the old droplets;

[0318] 9. After washing, add Anti-Flag-Cy3 and incubate at 37°C for 1 hour; then observe the protein expression under a fluorescence microscope. Under 546nm excitation light, the fluorescence intensity of the magnetic beads is used to determine when E. coli begins to produce the target nanoantibody and when the target nanoantibody concentration reaches the best.

[0319] Please refer to Figure 11. Figure 11A shows the first sample added 1 hour and 30 minutes after perfusion culture. The beads emit weak fluorescence, indicating that the secreted antibody has reached the detection limit. Figures 11B through G show samples added every 15 minutes, showing a continuous increase in fluorescence. The fluorescence intensity in Figure H is consistent with that in Figure G, indicating that the secreted antibody concentration has reached its peak.

[0320] The present invention's single-bacterial screening and continuous culture method based on a digital microfluidic system can significantly reduce the probability of contamination. For example, the probability of contamination per culture is approximately 1%. For 10 consecutive cultures using conventional methods, the probability of contamination is 1% + [(100% to 1%) * 1%]. The contamination rate is 0.099, as shown in Table 3. However, even with 10 consecutive cultures, the probability of contamination using the separation and continuous culture method described herein is approximately 1%. Automated screening depends on the chip area. For example, for A1A, the throughput is approximately 1000. For A3B4, the prior art throughput is approximately 3000, and the theoretical throughput is close to 10,000. This represents a 1000-fold difference compared to conventional methods, and a greater than 1000-fold cost savings compared to some more expensive reagents.

[0321] Table 14.1. Contamination rates of 10 consecutive cultures using the traditional method

[0322] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A phage screening method based on digital microfluidics, wherein: The process is carried out on a digital microfluidic chip and includes the following steps: S1. Cultivate E. coli in the continuous bacterial culture area; S2. In the panning experimental area, negative screening magnetic beads and the constructed phage library are mixed, and after incubation, the negative screening magnetic beads are removed by magnetic separation to obtain screened phage droplets; S3. Add positive screening magnetic beads, mix with the phage droplets screened in step S2, incubate, adsorb the positive screening magnetic beads through the automated magnetic adsorption module, tear the droplets containing the positive screening magnetic beads, and remove the excess phage library; S4. Use a washing liquid to perform interception-flow washing on the positive screening magnetic bead droplets: connect the droplets containing the positive screening magnetic beads to be washed with adjacent washing liquid droplets in the same direction as the moving direction to form a whole droplet, place a magnet at the position in the direction to be moved, move the positive screening magnetic beads to the position to be moved, and then move the middle part of the whole droplet to disconnect it from the whole droplet; then continue the next step of washing in the above manner, repeat the cycle multiple times to complete a round of washing; S5. After washing, add the E. coli host in the logarithmic growth phase, allow the phages on the magnetic beads to infect the host, and culture it to restore the vitality of the E. coli; S6. In the reagent preparation area, separate the helper phage droplets, move them to the revived E. coli droplets for mixing, and allow the helper phage to superinfect the E. coli. After the infection is complete, tear the liquid and remove the magnetic beads; S7. In the liquid reserve area, antibiotics are moved and added to the superstained Escherichia coli for resistance screening, and the Escherichia coli after resistance screening are cultured to produce phages, completing the first round of panning and obtaining a phage library; S8. As needed, the phages produced by panning are subjected to the next round of panning in the panning experimental area using the phage library obtained in step S7 according to steps S1 to S7 until the panning is completed.

2. The phage screening method based on digital microfluidics according to claim 1, wherein: During bacterial culture, a surfactant is added, and preferably, the surfactant is F68.

3. The phage screening method based on digital microfluidics according to claim 2, wherein: 0.05ul to 0.15ul F68 surfactant was added to every 100ul bacterial solution.

4. The phage screening method based on digital microfluidics according to claim 3, wherein: 0.09ul~0.11ul F68 surfactant was added to every 100ul bacterial solution.

5. The phage screening method based on digital microfluidics according to claim 1, wherein: The particle size of the positive screening magnetic beads is 10um to 20um.

6. The phage screening method based on digital microfluidics according to claim 5, wherein: The particle size of the positive screening magnetic beads is 20 um.

7. The phage screening method based on digital microfluidics according to claim 1, wherein: The number of positive screening magnetic beads added to S3 is 10 to 500.

8. The phage screening method based on digital microfluidics according to claim 7, wherein: The number of positive screening magnetic beads added to S3 is 50 to 200.

9. The phage screening method based on digital microfluidics according to claim 1, wherein: The final concentration of the antibiotic added to S5 is 90 ug / mL to 110 ug / mL, and / or the final concentration of the antibiotic added to S5 is 30 ug / mL to 40 ug / mL.

10. A method for screening peptide drugs or monoclonal antibodies based on digital microfluidics, wherein: The process is carried out on a digital microfluidic chip and includes the following steps: Obtaining a bacterial library produced after panning by the phage panning method according to any one of claims 1 to 9; Performing single bacterial separation on the bacterial library obtained above to obtain single bacterial droplets; Screening and culturing the obtained single bacterial droplets; Screening of peptide drugs or monoclonal antibodies.

11. The screening method according to claim 10, wherein: The single bacteria separation comprises the following steps: S21. diluting the bacterial solution of the bacterial library obtained with a culture medium; S22. 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 to 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; S23. 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; S24. Through the recognition system, the number of bacteria in the single-electrode droplet is detected and determined, 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, thereby obtaining a single-bacteria droplet.

12. The screening method according to claim 11, wherein The single bacteria isolation method also includes: S25. For droplets that do not contain bacteria or contain more than one bacterium, 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-merged into an independent empty droplet that does not contain bacteria and a droplet with multiple bacteria, respectively. The empty droplets are removed and the droplets with multiple bacteria are recovered.

13. The screening method according to claim 12, wherein: The single bacteria isolation method also includes: S26. 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; S27. After recovery, the droplets containing multiple bacteria are detected and judged, and multiple recovered bacterial droplets are fused into a new droplet with 2 to the power of N as the electrode base; S28. Repeat the above steps S22 to S26 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.

14. The screening method according to any one of claims 11 to 13, wherein: The Nth power of 2, N is 1-8, preferably 5-6.

15. The screening method according to any one of claims 11 to 13, wherein: In S22, the amount of bacteria in each bacterial liquid droplet is 19 to 21 bacteria per 64 electrode bases.

16. The screening method according to any one of claims 11 to 13, wherein: The volume of a single electrode droplet is 0.3nL to 3nL.

17. The screening method according to claim 16, wherein: The volume of a single electrode droplet is 1 nL to 3 nL, preferably 2 nL to 3 nL.

18. The screening method according to any one of claims 11 to 13, wherein: In step S21, the bacterial concentration after dilution is 70 cfu / uL to 120 cfu / uL, preferably 90 cfu / uL to 110 cfu / uL.

19. The screening method according to any one of claims 11 to 13, wherein: In step S21, the bacterial solution is diluted with culture medium to an OD600 of 0.2 to 0.

4.

20. The screening method according to claim 10, wherein The single bacterial droplet screening and cultivation comprises the following steps: S31. Injecting the screening material onto the chip of the digital microfluidic system, separating and obtaining single-electrode screening material droplets, controlling the single-electrode screening material droplets, and mixing the single bacterial droplets obtained after separation to obtain mixed droplets; S32. Setting the culture temperature, culturing the bacteria in the mixed droplets, and screening the bacteria; S33. Inject the culture medium into the digital microfluidic chip, and form single-electrode culture medium droplets by controlling the power on and off of the electrodes on the digital microfluidic chip, and mix them with the screened bacterial droplets to continue culturing.

21. The screening method according to claim 20, wherein: The following steps are also included: S34. After the bacteria have been cultured for at least one generation, a new culture medium droplet is added, and the bacterial droplet is torn into two halves of equal volume by controlling the power on and off of the electrodes on the digital microfluidic chip to obtain a bacterial droplet injected with the new culture medium; S35. Repeat step S34 for more than 0 times to achieve continuous perfusion culture.

22. The screening method according to claim 20, wherein: The bacteria are bacteria containing antibiotic resistance or auxotrophic bacteria, or bacteria capable of producing certain metabolites.

23. The screening method according to claim 20, wherein The screening agent is an antibiotic, or a nutrient-deficient culture medium, or a bacterial metabolite.

24. The screening method according to claim 23, wherein The screening substance is an antibiotic, and the screened bacteria is Escherichia coli. Preferably, the concentration of the antibiotic in the mixed droplet is 4 to 6 ug / mL, and more preferably 4.5 to 5.5 ug / mL.

25. The screening method according to claim 1, 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; the lower electrode plate comprises an electrode layer, wherein the electrode layer comprises a plurality of electrodes arranged in an array.

26. A single bacteria isolation method based on a digital microfluidics system, wherein: 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 to 22 bacteria per 64 electrode bases, and the bacterial solution droplet 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.

27. The single bacteria isolation method according to claim 26, wherein: The separation method further comprises: S6. For droplets that do not contain bacteria or contain more than one bacterium, 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-merged into an independent empty droplet that does not contain bacteria and a droplet with multiple bacteria, respectively. The empty droplets are removed and the droplets with multiple bacteria are recovered.

28. The single bacteria isolation method according to claim 26, wherein: 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 to 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.

29. The method for isolating a single bacterium according to any one of claims 26 to 28, wherein: The Nth power of 2, N is 1-8, preferably 5-6.

30. The method for isolating a single bacterium according to any one of claims 26 to 28, wherein: In S3, the amount of bacteria in each bacterial liquid droplet is 19 to 21 bacteria per 64 electrode bases.

31. The method for isolating a single bacterium according to any one of claims 26 to 28, wherein: The volume of a single electrode droplet is 0.3nL to 3nL.

32. The single bacteria isolation method according to claim 31, wherein: The volume of a single electrode droplet is 1 nL to 3 nL, preferably 2 nL to 3 nL.

33. The method for isolating a single bacterium according to any one of claims 26 to 28, wherein: In step S2, the bacterial concentration after dilution is 70 cfu / uL to 120 cfu / uL, preferably 90 cfu / uL to 110 cfu / uL.

34. The method for isolating a single bacterium according to any one of claims 26 to 28, wherein: In step S1, when the bacteria are cultured to an OD600 of about 1.5 to 2.5, the culture is stopped, and the bacterial solution is diluted with a culture medium to an OD600 of 0.2 to 0.

4.

35. Application of the single bacteria isolation method according to any one of claims 26 to 34 in bacterial culture, polypeptide drug or antibody screening, and genomics analysis in a digital microfluidic system.

36. A single bacteria screening and cultivation method based on a digital microfluidics system, wherein: This single bacteria screening and culture method is carried out on a digital microfluidic chip. The single bacteria screening and culturing method comprises the following steps: S1. Inject the diluted bacterial solution into the digital microfluidic chip, and tear the electrode on the digital microfluidic chip into half with equal volume by controlling the power on and off to form a single electrode droplet. Detect and judge the single electrode droplet, mark the droplet containing only one bacterium, and obtain a single bacteria droplet; S2. Injecting the screening material onto the chip of the digital microfluidic system, separating and obtaining single-electrode screening material droplets, controlling the single-electrode screening material droplets, and mixing them with the single bacterial droplets to obtain mixed droplets; S3. Setting the culture temperature, culturing the bacteria in the mixed droplets, and screening the bacteria; S4. Inject the culture medium into the digital microfluidic chip, and form single-electrode culture medium droplets by controlling the power on and off of the electrodes on the digital microfluidic chip. Mix the culture medium droplets with the screened bacterial droplets and continue culturing.

37. The single bacteria screening and culturing method according to claim 36, wherein: The following steps are also included: S5. Continue culturing for a period of time. After the bacteria have multiplied for at least one generation, add a droplet of new culture medium, and tear the bacterial droplet into half of equal volume by controlling the power on and off of the electrodes on the digital microfluidic chip to obtain a bacterial droplet injected with the new culture medium; S6. Repeat step S5 for more than 0 times to achieve continuous perfusion culture.

38. The single bacteria screening and culturing method according to claim 36, wherein: The bacteria are bacteria containing antibiotic resistance or auxotrophic bacteria, or bacteria capable of producing certain metabolites.

39. The single bacteria screening and culturing method according to claim 36, wherein: The screening agent is an antibiotic, or a nutrient-deficient culture medium, or a bacterial metabolite.

40. The single bacteria screening and culturing method according to claim 39, wherein: The screening object is an antibiotic, and the screened bacteria is Escherichia coli. Preferably, the concentration of the antibiotic in the mixed droplet is 4ug / mL to 6ug / mL, and more preferably 4.5ug / mL to 5.5ug / mL.

41. The method for screening and culturing a single bacterium according to any one of claims 36 to 40, wherein: Step S1 includes: injecting 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, and based on the volume of a single electrode droplet being 3 nL, the amount of bacteria in each bacterial solution droplet is 18 to 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.

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