Single bacterium liquid drop wrapping method capable of breaking through Poisson distribution and application of single bacterium liquid drop wrapping method

Abstract

The invention belongs to the technical field of biology and microfluidics, and particularly relates to a single bacterium liquid drop wrapping method capable of breaking through Poisson distribution and application. The preparation method comprises the following steps: (1) preparing the micro-fluidic chip by a soft printing method; (2) bacteria death and viability dyeing; (3) counting bacteria; (4) adjusting different densities of bacteria; (5) preparing a viscoelastic fluid, namely a polyoxyethylene (Polyethylene Oxide PEO (6000 KDa) mother solution (0.05%); (6) preparing a sample solution; (7) constructing an experimental operation platform; and (8) observing. The micro-fluidic chip is provided with a spiral focusing area, one end of the spiral focusing area is provided with a sample inlet tube, the sample inlet tube is provided with a sample inlet connected with a disposable syringe, the other end of the spiral focusing area is provided with a liquid drop collecting tube, the tail end of the liquid drop collecting tube is provided with a liquid drop collecting port, and the liquid drop collecting port is provided with a liquid drop outlet. A continuous phase inlet pipe is arranged on the liquid drop collecting pipe, and a continuous phase inlet is formed in the continuous phase inlet pipe.

Description

Technical Field

[0001] The present invention belongs to the field of biology and microfluidics, and particularly relates to a single bacterial droplet encapsulation method that breaks through Poisson distribution and its application. Background Art

[0002] Bacteria are quintessential single-celled organisms, but traditional culture methods often overlook their individual variability. For example, details such as gene expression noise, metabolic heterogeneity, and environmental response specificity are often hidden from traditional methods. Current single-bacteria sorting techniques, such as flow cytometry and microplate-based methods, offer excellent characterization of individual bacteria. However, they still suffer from low throughput, cross-contamination, high risk, and inability to handle hydrophobic strains. In recent years, droplet microfluidics has emerged as a promising alternative. This technology encapsulates individual microorganisms within nanoliter-scale droplets, creating independent reaction units. This offers unprecedented opportunities for high-throughput screening, dynamic monitoring of live organisms, and single-cell phenotyping. However, this technology also faces a challenge: particles / cells randomly approach the encapsulation region in the dispersed phase, resulting in a Poisson distribution of encapsulation populations within the droplets. This results in a single encapsulation rate below 30%, compromising analytical reliability. During droplet generation using microfluidic devices, numerous environmental factors can influence droplet formation. Therefore, precise control and monitoring of the droplet encapsulation process are crucial. Digital microfluidics, a widely used technology, dynamically manipulates discrete droplets on planar substrates based on the electrowetting effect. This technique, used for the detection of single biological particles, offers high sensitivity and multiplexed analysis. The principle is to disperse biological particles into independent chambers, magnetic beads, or droplets after limiting dilution, and then achieve random encapsulation of single particles through probabilistic statistics. While limiting dilution provides a probabilistic solution for single-cell sorting, its potential for application in single-cell omics is severely limited by its low throughput, false-positive rate, and impairment of cell viability. To address the limitations of traditional random encapsulation, Kemna et al. achieved high-yield single-cell droplet encapsulation (cell diameter approximately 13 μm) in a microchannel in 2012 using Dean flow. This study demonstrated that, in a passive mode without the need for external field control, the inertial effects and geometric constraints of the fluid within the microfluidic chip enable natural sorting and dynamic capture of particles in the dispersed phase. However, inertial focusing technology relies heavily on precise flow field control. Recently, Yue et al. proposed a BOAD system that achieves efficient alignment of 6μm microspheres through the three-dimensional synergy of Dean vortices, inertial focusing, and compressive flow confinement. However, this system currently operates only on microspheres and has not yet been further explored with biological particles. Furthermore, bacteria exhibit heterogeneous interference and are easily aggregated or lost in conventional inertial focusing due to their small size (micrometer level). This makes bacterial separation and sorting difficult. Summary of the Invention

[0003] In order to solve the problems of the prior art, the present application provides a single bacterial droplet encapsulation method and application that breaks through the Poisson distribution. The present application is achieved through the following scheme:

[0004] A single bacterial droplet encapsulation method that breaks through the Poisson distribution is prepared in the following steps: (1) preparing a microfluidic chip by soft printing;

[0005] n1. Treat the silicon wafer mold with trimethylchlorosilane vapor for 20 minutes, then mix the PDMS prepolymer and curing agent in a mass ratio of 5:1-15:1 to obtain a PDMS mixture for later use; n2. Pour the PDMS mixture onto the mold and place it in a vacuum pump for degassing for 20 minutes; n3. Use an ear bulb to blow away the bubbles on the surface until the bubbles above the channel disappear; n4. Place the mold in an oven and dry and cure at 80°C for 20 minutes; n5. Cut, punch, and thermally bond the chip according to the distance around the structure;

[0006] (2) Bacterial live or dead staining:

[0007] a. Bacterial culture: Grow bacteria in a suitable liquid medium until the logarithmic growth phase;

[0008] b. Bacterial preparation: Centrifuge the bacterial solution at room temperature for 5 minutes, discard the supernatant, wash the lower layer with physiological saline once, and then adjust the bacterial solution concentration to 10 8 bacteria / mL (OD670≈0.3), and then diluted 100 times with normal saline to make the final bacterial solution concentration 10 6 bacteria / mL;

[0009] c. Dilute DMAO (1000X) and PI (1000X) to 100X using the detection buffer in the bacterial live / dead staining kit. Add 1 μL of DMAO (1000X) and PI (1000X) to 8 μL of detection buffer and mix thoroughly to obtain 10 μL of working solution (100X). For a bacterial suspension containing 5-10 million bacteria, the required amount of bacterial live / dead staining working solution (100X) is 1 μL.

[0010] d. Staining: Add 1 μL of staining working solution (100×) to every 100 μL of bacterial solution, mix thoroughly, and incubate at 37°C in the dark for 20 min.

[0011] (3) Bacterial count

[0012] The stained bacterial solution (OD = 0.3) was aspirated and dropped onto a hemocytometer. The grayscale image and fluorescence image were counted using Image-Pro-Plus software to determine the number of original bacterial solution.

[0013] (4) Adjusting different bacterial densities

[0014] Based on the optimized droplet diameter of 133 μm, the values ​​of λ were calculated to be 0.5, 0.75, 1, 1.25, 1.5, and 2, and the corresponding numbers of bacteria (Escherichia coli E. coli) were 3.3×10 5 / mL, 5×10 5 / mL, 6.67×10 5 / mL, 8.37×10 5 / mL, 1×10 6 / mL, 1.33×10 6 / mL;

[0015] (5) Viscoelastic fluid - Polyethylene oxide (PEO) (6000 kDa) mother liquor preparation (0.05%): Weigh 0.001 g of PEO sample, add ultrapure water to 20 mL and dissolve in a 50 mL round-bottom centrifuge tube. Vortex shaker 3-4 times. Place in a 70°C oven for 45 min. Repeat the above operation three times. After removal, cool to room temperature, filter using a 0.45 μm water filter, and store at -4°C.

[0016] (6) Prepare sample solution

[0017] Bacterial solutions of different densities resuspended in PBS were added to 200 ppm PEO solution (10 μl, 15 μl, 20 μl, 35 μl, 40 μl, 55 μl), and 0.1% glucose aqueous solution was added to maintain the carbon source necessary for bacterial growth, and finally a 1 ml sample solution was prepared;

[0018] (7) Construction of experimental operation platform

[0019] The particle sample suspensions prepared above were loaded into 1mL disposable syringes and connected to silicone tubing (0.5mm ID, 1.5mm OD) via corresponding adapter needles (0.56mm ID, 0.82mm OD). Because the needle's OD is slightly larger than the microtubule's ID, and the microtubule is elastic, a seal between the needle and microtubule is achieved. The silicone tubing and PDMS chip are connected via the needle's steel tube, enabling pressure-resistant introduction of the sample liquid. After passing through the microfluidic chip, the sample liquid is introduced into a waste liquid collection bottle via the outlet silicone tubing. The syringe filled with the sample liquid is then loaded into a precision syringe pump to provide a stable flow rate set for the sample.

[0020] Furthermore, the microfluidic chip is provided with a spiral focusing area, one end of the spiral focusing area is provided with a shallow spiral channel 1, the shallow spiral channel 1 is provided with a sample inlet connected to a disposable syringe, the other end of the spiral focusing area is provided with a shallow spiral channel 2, the shallow spiral channel 2 is connected to a droplet collection tube, the end of the droplet collection tube is provided with a droplet collection port, the droplet collection tube is provided with a continuous phase inlet tube, and the continuous phase inlet tube is provided with a continuous phase inlet.

[0021] Furthermore, a double helix pipe is provided in the spiral focusing area, and the double helix pipe is an Archimedean spiral, the angular velocity of the spiral line is constant, the width of the double helix pipe is 0.1 mm, the interval between the double helix pipes is 0.1 mm, the inner radius of the double helix pipe is 1.05 mm, the outer radius of the double helix pipe is 2.25 mm, there are 7 turns in total, and the center of the double helix pipe is S-shaped.

[0022] Furthermore, a concave structure is provided on the outer wall of the double helix pipe, and the width of the concave structure is 1 / 2 of the width of the double helix pipe; the same number of concave structures are evenly provided on each circle of the double helix pipe, and the number of concave structures on a circle of the double helix pipe is 6-10; the S-shaped surface in the center of the double helix pipe is provided with the same or different number of concave structures as on a circle of the spiral pipe.

[0023] Preferably, the concave structures on the double helix pipe are on a straight line.

[0024] Furthermore, one end of the continuous phase inlet tube is connected to the droplet collection tube, and the other end of the continuous phase inlet tube is connected to the droplet collection tube; the two ends of the continuous phase inlet tube are respectively connected to the same horizontal position of the droplet collection tube, and the connection positions correspond.

[0025] Furthermore, the continuous phase inlet is arranged at the middle position of the continuous phase inlet pipe; the continuous phase inlet coincides with the vertical center line of the droplet collection port.

[0026] Furthermore, the flow rate of the bacteria phase is 1 to 4 μL / min; the flow rate of the fluorinated oil phase is 1 to 16 μL / min.

[0027] Furthermore, the height of the shallow spiral channel 2 and the continuous phase inlet tube is 10 μm and the width is 100 μm;

[0028] Furthermore, the height 100 of the droplet collecting tube is μm, the droplet collecting tube is provided with a droplet collecting tube contraction opening, and the width of the droplet collecting tube contraction opening is 75 μm.

[0029] A single bacterial droplet encapsulation method that breaks through the Poisson distribution is applied. The microfluidic chip is placed on an inverted fluorescence microscope, and the corresponding objective lens and fluorescence excitation module are used to observe bright field and fluorescence images of particle motion on the horizontal center plane of the flow channel. The excitation light is focused on the sample, and the fluorescence signal emitted by the sample is collected. Nikon's high-performance objective lens can provide high resolution and high numerical aperture, which helps to obtain clear fluorescence images. A high-speed CCD camera and supporting software are used to record the dynamic motion of particles and store them as sequence frames or AVI format image videos. Bright field real-time particle motion photos use 4000fps and a 20ms shutter speed, so the movement of single particles can be distinguished. The motion trajectories are then superimposed using Image Pro Plus 6.0 software to obtain a particle trajectory distribution map. The dark background fluorescence spectrum uses a long exposure time of 600ms.

[0030] Beneficial effects:

[0031] 1. This invention combines viscoelastic-inertial bacterial focusing with integrated droplet generation using step emulsification to construct a microfluidic chip integrated system. This system enables precise manipulation of single bacteria and efficient droplet encapsulation. This provides a high-activity, high-throughput single-cell analysis platform for synthetic biology, pathogen detection, and precision medicine, and promotes the practical application of in vivo single-cell research.

[0032] 2. By breaking through the limitations of traditional Poisson distribution, highly monodisperse and efficient single bacterial droplet encapsulation was achieved. The single bacterial encapsulation rate was approximately 42.7%, breaking through the limitations of Poisson distribution, and the droplet coefficient of variation was less than 4%, confirming the effectiveness of viscoelastic-inertial focusing control for encapsulating living microorganisms. Furthermore, the system can stably generate droplets; high sensitivity: viscoelastic fluids enhance the inertial migration efficiency of small bacteria (such as nanobacteria);

[0033] High purity: Reduce cell clump interference through inertial focusing, droplet monodispersity (CV < 5%);

[0034] High activity retention: no labeling, low shear force, bacterial survival rate >90%.

[0035] 2. In a single microfluidic chip, the viscoelastic inertial focusing of bacteria is enhanced through a size-restricted channel, and the downstream is connected with an integrated step structure of flow focusing and step emulsification (the flow rate ratio of the oil phase to the bacterial phase is 1:1 to 1:16), so as to realize the continuous operation of bacterial focusing and droplet encapsulation. The traditional droplet encapsulation technology requires a step-by-step operation of sorting first and then encapsulation, resulting in low efficiency and high risk of cross-contamination. The present invention realizes "one-step" sorting and encapsulation through chip structure innovation.

[0036] 4. Adaptability of viscoelastic fluid formulation: Adjust the viscoelastic fluid concentration (e.g. 50 ppm-400 ppm PEO) according to the bacterial size (1-2 μm) to achieve the best match between rheological properties and inertial migration efficiency.

[0037] 5. Utilizing the dynamic inertial focusing effect of viscoelastic fluids, through the synergistic effect of the fluid elastic modulus and bacterial size, efficient single bacterial sorting can be achieved without fluorescent labeling. Viscoelastic fluids (such as solutions containing high molecular weight polymers) are injected into microfluidic channels. The synergistic effect of their elastic modulus and inertial effect causes bacteria to migrate inertially in the flow field due to size differences, achieving single bacterial focusing.

[0038] 6. Viscoelastic fluids enhance the migration efficiency of small-sized bacteria. Combined with flow focusing droplet generation technology, the throughput can reach 10 3 cells / min, suitable for complex samples (such as soil and fecal extracts), effectively separates low-abundance bacteria (such as <1%) through viscoelastic-inertial focusing, introduces immiscible liquid (fluorinated oil) into the focused bacterial sample flow area through microchannel structure design, and generates single bacteria-encapsulated droplets through shear force. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] To more clearly illustrate the technical solutions in the embodiments of this application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some preferred embodiments of this application, not all embodiments. Regarding the preferred embodiments of this application, it is clear that those skilled in the art can derive other embodiments and drawings based on these embodiments and drawings without inventive effort, and all of them fall within the scope of protection of this application.

[0040] Figure 1 Schematic diagram of the microfluidic chip structure;

[0041] Figure 2 Schematic diagram of the experimental operation, blue dispersed phase, red continuous phase;

[0042] Figure 3 This is a simulation diagram of inertial focusing of viscoelastic fluid;

[0043] Figure 4 The coloring diagram for different λ values;

[0044] Figure 5 Results of single bacterial droplet encapsulation experiments;

[0045] In the figure: 1. Sample inlet, 2. Spiral focusing zone, 3. Droplet generation zone, 4. Continuous phase inlet, 5. Droplet collection port, 6. Shallow spiral channel 1, 7. Shallow spiral channel 2, 8. Continuous phase entry tube, 9. Concave structure. DETAILED DESCRIPTION

[0046] To make the objectives, technical solutions, and advantages of this application more clear, the embodiments of this application will be described in further detail below. It should be understood that the terms "upper," "lower," "top," "bottom," "inner," "outer," and the like indicate positions or location relationships based on the positions or location relationships shown in the accompanying drawings. The foregoing definitions are merely for the purpose of describing this application and simplifying the description, and do not indicate or imply that the structures referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0047] Example 1

[0048] A single bacterial droplet encapsulation method that breaks through the Poisson distribution is prepared in the following steps: (1) preparing a microfluidic chip by soft printing;

[0049] n1. Treat the silicon wafer mold with trimethylchlorosilane vapor for 20 minutes, then mix the PDMS prepolymer and curing agent in a mass ratio of 10:1 to obtain a PDMS mixture for later use; n2. Pour the PDMS mixture onto the mold and place it in a vacuum pump for degassing for 20 minutes; n3. Use an ear bulb to blow away the bubbles on the surface until the bubbles above the channel disappear; n4. Place the mold in an oven and dry and cure at 80°C for 20 minutes; n5. Cut, punch, and thermally bond the chip according to the distance around the structure;

[0050] The purpose of fumigation is to form a hydrophobic layer on the substrate surface through a silanization reaction;

[0051] The curing agent is an organic peroxide (such as benzoyl peroxide) or a platinum catalyst (such as a platinum-vinylsiloxane complex), which is responsible for initiating the cross-linking reaction of the prepolymer to form a three-dimensional network structure;

[0052] (2) Bacterial live or dead staining:

[0053] a. Bacterial culture: Grow bacteria in a suitable liquid medium until the logarithmic growth phase;

[0054] b. Bacterial preparation: Centrifuge the bacterial solution at room temperature for 5 minutes, discard the supernatant, wash the lower layer with physiological saline once, and then adjust the bacterial solution concentration to 10 8 bacteria / mL (OD670≈0.3), and then diluted 100 times with normal saline to make the final bacterial solution concentration 10 6 bacteria / mL;

[0055] c. Dilute DMAO (1000X) and PI (1000X) to 100X using the detection buffer in the bacterial live / dead staining kit. Add 1 μL of DMAO (1000X) and PI (1000X) to 8 μL of detection buffer and mix thoroughly to obtain 10 μL of working solution (100X). For a bacterial suspension containing 5-10 million bacteria, the required amount of bacterial live / dead staining working solution (100X) is 1 μL.

[0056] d. Staining: Add 1 μL of staining working solution (100×) to every 100 μL of bacterial solution, mix thoroughly, and incubate at 37°C in the dark for 20 min.

[0057] (3) Bacterial count

[0058] The stained bacterial solution (OD = 0.3) was aspirated and dropped onto a hemocytometer. The grayscale image and fluorescence image were counted using Image-Pro-Plus software to determine the number of original bacterial solution.

[0059] (4) Adjusting different bacterial densities

[0060] Based on the optimized droplet diameter of 133 μm, the calculated values ​​of λ were 0.5, 0.75, 1, 1.25, 1.5, and 2, and the corresponding numbers of Escherichia coli (E. coli) were 3.3×10 5 / mL, 5×10 5 / mL, 6.67×10 5 / mL, 8.37×10 5 / mL, 1×10 6 / mL, 1.33×10 6 / mL;

[0061] (5) Viscoelastic fluid - Polyethylene oxide (PEO) (6000 kDa) mother liquor preparation (0.05%): Weigh 0.001 g of PEO sample, add ultrapure water to 20 mL and dissolve in a 50 mL round-bottom centrifuge tube. Vortex shaker 3-4 times. Place in a 70°C oven for 45 min. Repeat the above operation three times. After removal, cool to room temperature, filter using a 0.45 μm water filter, and store at -4°C.

[0062] (6) Prepare sample solution

[0063] Bacterial solutions of different densities resuspended in PBS were added to 200 ppm PEO solution (10 μl, 15 μl, 20 μl, 35 μl, 40 μl, 55 μl), and 0.1% glucose aqueous solution was added to maintain the carbon source necessary for bacterial growth, and finally a 1 ml sample solution was prepared;

[0064] (7) Construction of experimental operation platform

[0065] The particle sample suspensions prepared above were loaded into 1mL disposable syringes and connected to silicone tubing (0.5mm ID, 1.5mm OD) via corresponding adapter needles (0.56mm ID, 0.82mm OD). Because the needle's outer diameter is slightly larger than the microtubule's inner diameter, and the microtubule is elastic, a seal is achieved between the needle and the microtubule. The silicone tubing and PDMS chip can be connected using the needle's steel tube, achieving pressure-resistant introduction of the sample liquid. After passing through the microfluidic chip, the sample liquid is introduced into a waste liquid collection bottle through the outlet silicone tubing. The syringe filled with the sample liquid is loaded into a precision syringe pump to provide a stable flow rate set by the sample.

[0066] (8) Observation

[0067] The microfluidic chip was placed on an inverted fluorescence microscope, and the corresponding objective lens and fluorescence excitation module were used to observe the bright field and fluorescence images of the particle movement on the horizontal center plane of the flow channel. The excitation light was focused on the sample, and the fluorescence signal emitted by the sample was collected. Nikon's high-performance objective lens can provide high resolution and high numerical aperture, which helps to obtain clear fluorescence images. The high-speed CCD camera and supporting software are used to record the dynamic movement of the particles and store them as sequence frames or AVI format image videos. The bright field particle real-time motion photos use 4000fps and a 20ms shutter speed, so the movement of single particles can be distinguished. The motion trajectories are then superimposed using Image pro plus 6.0 software to obtain a particle trajectory distribution map. The dark background fluorescence spectrum uses a long exposure time of 600ms.

[0068] Further, such as Figure 1-5 As shown, the microfluidic chip is provided with a spiral focusing area 2, one end of the spiral focusing area 2 is provided with a shallow spiral channel 1 6, the shallow spiral channel 1 6 is provided with a sample inlet 1 connected to a disposable syringe, the other end of the spiral focusing area 2 is provided with a shallow spiral channel 2 7, the shallow spiral channel 2 7 is connected to a droplet collecting tube, the end of the droplet collecting tube is provided with a droplet collecting port 5, the droplet collecting tube is provided with a continuous phase inlet tube 8, and the continuous phase inlet tube 8 is provided with a continuous phase inlet 4.

[0069] Furthermore, a double helix pipe is provided in the spiral focusing area 2, and the double helix pipe is an Archimedean spiral, the angular velocity of the spiral line is constant, the width of the double helix pipe is 0.1 mm, the interval between the double helix pipes is 0.1 mm, the inner radius of the double helix pipe is 1.05 mm, the outer radius of the double helix pipe is 2.25 mm, there are 7 turns in total, and the center of the double helix pipe is S-shaped.

[0070] Furthermore, a concave structure 9 is provided on the outer wall of the double helix pipe, and the width of the concave structure 9 is 1 / 2 of the width of the double helix pipe; the same number of concave structures 9 are evenly provided on each circle of the double helix pipe, and the number of concave structures 9 on a circle of the double helix pipe is 6-10; the S-shaped surface in the center of the double helix pipe is provided with the same number of concave structures 9 as on a circle of the spiral pipe.

[0071] Preferably, Figure 1 As shown, the concave structures on the double helix pipe are on a straight line.

[0072] By providing a concave structure and changing the local flow field, the shear force and elastic force gradient of the Dean flow are enhanced, significantly improving the focusing efficiency.

[0073] Furthermore, one end of the continuous phase inlet tube 8 is connected to the droplet collection tube, and the other end of the continuous phase inlet tube 8 is connected to the droplet collection tube; the two ends of the continuous phase inlet tube 8 are respectively connected to the same horizontal position of the droplet collection tube, and the connection positions correspond.

[0074] The intersection of the continuous phase inlet tube, the droplet collection tube, and the shallow spiral channel 2 forms a droplet generation zone 3;

[0075] Furthermore, the continuous phase inlet 4 is arranged in the middle position of the continuous phase inlet pipe; the continuous phase inlet 4 coincides with the vertical center line of the droplet collection port 5 .

[0076] Furthermore, the flow rate of the bacteria phase is 1 to 4 μL / min; the flow rate of the fluorinated oil phase is 1 to 16 μL / min.

[0077] Furthermore, the height of the shallow spiral channel 2 7 and the continuous phase inlet tube is 10 μm and the width is 100 μm;

[0078] Furthermore, the height 100 of the droplet collecting tube is μm, the droplet collecting tube is provided with a droplet collecting tube contraction opening, and the width of the droplet collecting tube contraction opening is 75 μm.

[0079] The pressure distribution in the spiral microchannel is asymmetric due to the presence of the outer micropillars. The pressure gradient induced by the outer micropillars pushes the secondary flow toward the center of the channel. The micropillars suppress the radial expansion of the vortex through the damping effect, and the secondary flow exhibits a bidirectional alternating deviation. And according to the definition of the Weissenberg number ( where λ is the relaxation time, is the shear rate). As the shear rate increases, the elastic lift also increases, indicating the emergence of the viscoelastic-inertial focusing effect. This also verifies that the minimum shear rate distribution areas of the rectangle are at the center and four corners. By predicting and guiding particle trajectories based on the elastic lift dominated by N1, particles move toward areas of lower shear rate, enabling better integration with inertial lift to achieve a focusing effect.

[0080] The kit used in this application is an existing kit, such as the Biyuntian bacterial dead and live staining kit.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present application, which should be included in the scope of the claims of the present application.

Claims

1. A single bacterial droplet encapsulation method that breaks through the Poisson distribution, characterized in that: The preparation steps are as follows: (1) preparation of microfluidic chip by soft printing method; n1. Treat the silicon wafer mold with trimethylchlorosilane vapor for 20 minutes, then mix the PDMS prepolymer and curing agent in a mass ratio of 5:1-15:1 to obtain a PDMS mixture for later use; n2. Pour the PDMS mixture onto the mold and place it in a vacuum pump for degassing for 20 minutes; n3. Use an ear bulb to blow away the bubbles on the surface until the bubbles above the channel disappear; n4. Place the mold in an oven and dry and cure at 80°C for 20 minutes; n5. Cut, punch, and thermally bond the chip according to the distance around the structure; (2) Bacterial live or dead staining: a. Bacterial culture: Grow bacteria in a suitable liquid medium until the logarithmic growth phase; b. Bacterial preparation: Centrifuge the bacterial solution at room temperature for 5 minutes, discard the supernatant, wash the lower layer with physiological saline once, and then adjust the bacterial solution concentration to 10 8 bacteria / mL (OD670≈0.3), and then diluted 100 times with normal saline to make the final bacterial solution concentration 10 6 bacteria / mL; c. Use the detection buffer in the bacterial live / death staining kit to dilute DMAO (1000X) and PI (1000X) to 100X. Take 1 μL of DMAO (1000X) and PI (1000X) and add them to 8 μL of detection buffer. After mixing, the result is 10 μL of working solution (100X). For a bacterial suspension containing 5-10 million bacteria, the amount of bacterial live / death staining working solution (100X) required is 1 μL. d. Staining: Add 1 μL of staining working solution (100×) to every 100 μL of bacterial solution, mix thoroughly, and incubate at 37°C in the dark for 20 min. (3) Bacterial count The stained bacterial solution (OD=0.3) was aspirated and dropped onto a hemocytometer, and the grayscale image and fluorescence image were counted using Image-Pro-Plus software to determine the amount of the original bacterial solution. (4) Adjusting different bacterial densities Based on the optimized droplet diameter of 133 μm, the values ​​of λ were calculated to be 0.5, 0.75, 1, 1.25, 1.5, and 2, and the corresponding numbers of bacteria (Escherichia coli E. coli) were 3.3×10 5 / mL, 5×10 5 / mL, 6.67×10 5 / mL, 8.37×10 5 / mL, 1×10 6 / mL, 1.33×10 6 pieces / mL. (5) Viscoelastic fluid - Polyethylene oxide (PEO) (6000 kDa) mother liquor preparation (0.05%): Weigh 0.001 g of PEO sample, add ultrapure water to 20 mL and dissolve in a 50 mL round-bottom centrifuge tube. Vortex shaker 3-4 times. Place in a 70°C oven for 45 min. Repeat the above operation three times. After removal, cool to room temperature, filter using a 0.45 μm water filter, and store at -4°C. (6) Prepare sample solution Bacterial solutions of different densities resuspended in PBS were added with 200 ppm PEO solution (10 μl 15 ul 20 μl 35 μl 40 μl 55 μl), and 0.1% glucose aqueous solution was added to maintain the carbon source necessary for bacterial growth, and finally 1 ml sample solution was prepared. (7) Construction of experimental operation platform The particle sample suspensions prepared above were loaded into 1mL disposable syringes and connected to silicone tubing (0.5mm ID, 1.5mm OD) via corresponding adapter needles (0.56mm ID, 0.82mm OD). Because the needle's OD is slightly larger than the microtubule's ID, and the microtubule is elastic, a seal between the needle and microtubule is achieved. The silicone tubing and PDMS chip are connected via the needle's steel tube, enabling pressure-resistant introduction of the sample liquid. After passing through the microfluidic chip, the sample liquid is introduced into a waste liquid collection bottle via the outlet silicone tubing. The syringe filled with the sample liquid is then loaded into a precision syringe pump to provide a stable flow rate set for the sample.

2. A single bacterial droplet encapsulation method that breaks through Poisson distribution as claimed in claim 1, characterized in that: The microfluidic chip is provided with a spiral focusing area, one end of the spiral focusing area is provided with a shallow spiral channel 1, the shallow spiral channel 1 is provided with a sample inlet connected to a disposable syringe, the other end of the spiral focusing area is provided with a shallow spiral channel 2, the shallow spiral channel 2 is connected to a droplet collection tube, the end of the droplet collection tube is provided with a droplet collection port, the droplet collection tube is provided with a continuous phase inlet tube, and the continuous phase inlet tube is provided with a continuous phase inlet.

3. A single bacterial droplet encapsulation method that breaks through Poisson distribution as claimed in claim 2, characterized in that: A double helix pipe is provided in the spiral focusing area. The double helix pipe is an Archimedean spiral. The angular velocity of the spiral line is constant. The width of the double helix pipe is 0.1 mm, the spacing between the double helix pipes is 0.1 mm, the inner radius of the double helix pipe is 1.05 mm, the outer radius of the double helix pipe is 2.25 mm, there are 7 turns in total, and the center of the double helix pipe is S-shaped.

4. A single bacterial droplet encapsulation method that breaks through Poisson distribution as claimed in claim 3, characterized in that: A concave structure is provided on the outer wall of the double helix pipe, and the width of the concave structure is 1 / 2 of the width of the double helix pipe; the same number of concave structures are evenly provided on each circle of the double helix pipe, and the number of concave structures on a circle of the double helix pipe is 6-10; the S-shaped shape in the center of the double helix pipe is provided with the same or different number of concave structures as on a circle of the spiral pipe; preferably, the concave structures on the double helix pipe are in a straight line.

5. The single bacterial droplet encapsulation method that breaks through the Poisson distribution as claimed in claim 2, characterized in that: One end of the continuous phase inlet tube is connected to the droplet collection tube, and the other end of the continuous phase inlet tube is connected to the droplet collection tube; the two ends of the continuous phase inlet tube are respectively connected to the same horizontal position of the droplet collection tube, and the connection positions correspond.

6. A single bacterial droplet encapsulation method that breaks through Poisson distribution as claimed in claim 2, characterized in that: The continuous phase inlet is arranged in the middle of the continuous phase inlet pipe; the continuous phase inlet coincides with the vertical center line of the droplet collecting port.

7. A single bacterial droplet encapsulation method that breaks through Poisson distribution as claimed in claim 2, characterized in that: The flow rate of the bacteria phase is 1 to 4 μL / min; the flow rate of the fluorinated oil phase is 1 to 16 μL / min.

8. The single bacterial droplet encapsulation method that breaks through the Poisson distribution as claimed in claim 2, characterized in that: The height of the shallow spiral channel 2 and the continuous phase entry tube is 10 μm and the width is 100 μm; 9. The single bacterial droplet encapsulation method that breaks through the Poisson distribution as claimed in claim 2, characterized in that: The height 100 of the droplet collecting tube is μm. The droplet collecting tube is provided with a droplet collecting tube contraction opening, and the width of the droplet collecting tube contraction opening is 75 μm.

10. The use of a single bacterial droplet encapsulation method that breaks through Poisson distribution as claimed in claim 2, characterized in that: The microfluidic chip was placed on an inverted fluorescence microscope, and the corresponding objective lens and fluorescence excitation module were used to observe the bright field and fluorescence images of the particle movement on the horizontal center plane of the flow channel. The excitation light was focused on the sample, and the fluorescence signal emitted by the sample was collected. Nikon's high-performance objective lens can provide high resolution and high numerical aperture, which helps to obtain clear fluorescence images. The high-speed CCD camera and supporting software are used to record the dynamic movement of the particles and store them as sequence frames or AVI format image videos. The bright field particle real-time motion photos use 4000fps and a 20ms shutter speed, so the movement of single particles can be distinguished. The motion trajectories are then superimposed using Image pro plus 6.0 software to obtain a particle trajectory distribution map. The dark background fluorescence spectrum uses a long exposure time of 600ms.

Images