Method for screening algae cells

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a method for screening algae cells. The method comprises the following steps: mixing a suspension of algae cells with a lipid dye for dyeing to obtain a dyed algae cell suspension; taking the dyed algae cell suspension as a water phase, and generating liquid drops from the water phase and the oil phase through a liquid drop micro-fluidic technology; and separating the liquid drops with the optical signals higher than the reference value, wherein the liquid drops contain the target algae cells. Through a liquid drop sorting method, a reaction system is improved to picoliter grade, finer control and operation are realized, high-flux and high-precision screening in a microenvironment is facilitated, and the problem that the reaction system is limited is solved. In addition, the screening cost of a single strain is also reduced. By adopting a method of firstly dyeing and then generating the liquid drops, omission possibly occurring in the fusion process of the liquid drops and the dye liquid drops is avoided, and fluorescence detection errors caused by different dye concentrations in the algae cell dyeing and liquid drop sorting process are avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology, and in particular relates to a method for screening algae cells. Background Art

[0002] Microalgae usually refers to microorganisms that contain chlorophyll and can perform photosynthesis, which are widely distributed on land, in the ocean and in lakes. Microalgae can adopt a variety of nutritional modes, including photoautotrophy using light energy and inorganic salts, heterotrophy by directly absorbing organic matter, and can also adopt both for mixotrophy. Microalgae need to absorb a large amount of nutrients such as nitrogen and phosphorus during their growth, and synthesize various organic substances such as proteins, polysaccharides, lipids, etc. through cell metabolism. Therefore, they have great development potential in medicine, food, feed, environmental protection, cosmetics, renewable energy and other aspects.

[0003] The premise of developing microalgae varieties is to obtain high-quality algae strains, such as obtaining microalgae with high lipid content to develop biodiesel. This requires targeted sorting from nature in advance. According to the experience and habits of the cultivators, conventional microalgae sorting methods include micropipette method, water drop method, plate method, solvent extraction, weight measurement method, etc. However, these traditional methods not only have low sorting efficiency, but also have high requirements for operating technology, and are generally suitable for sorting larger algae. Some researchers have tried to apply microfluidics technology to the sorting process of microalgae, such as encapsulating single microalgae cells into oil-in-water emulsion droplets, tracking the growth and division of encapsulated cells over time, and then fluorescently staining the chip. However, the accuracy and sensitivity of fluorescence detection in this method are poor. Summary of the invention

[0004] The invention provides a method for screening algae cells, which can ensure good accuracy and sensitivity during fluorescence detection.

[0005] The first aspect of the present invention provides a method for screening algal cells, comprising:

[0006] Taking the algae cell suspension and mixing it with the lipid dye to dye it, so as to obtain a dyed algae cell suspension;

[0007] The dyed algae cell suspension is used as the aqueous phase and the oil phase to generate droplets through droplet microfluidics technology;

[0008] Droplets with an optical signal higher than a reference value are sorted out, and the droplets contain target algae cells.

[0009] In some embodiments of the present invention, the lipid dye includes at least one of Nile Red, BODIPY, Oil Red O, Sudan, BETA-1, LipidGreen, and PTZ-LD.

[0010] In some embodiments of the invention, the optical signal comprises a fluorescent signal.

[0011] In some embodiments of the present invention, the working concentration of the lipid dye is 1 to 3 μg / mL.

[0012] In some embodiments of the present invention, the algae cell suspension is pre-adjusted to OD before being mixed with the lipid dye for staining. 680 =0.04~0.08.

[0013] In some embodiments of the present invention, the suspension of algal cells is adjusted to OD 680 The cell density after the value is 1×10 6 ~1.5×10 6 Pieces / mL.

[0014] In some embodiments of the present invention, the culture medium in the suspension of algal cells comprises F2 culture medium.

[0015] In some embodiments of the present invention, the diameter of the generated droplets is 40-60 μm.

[0016] In some embodiments of the present invention, the sorting is performed using a high-throughput microbial screening system.

[0017] In some embodiments of the present invention, the droplets are subjected to quality control before sorting, and the quality control includes controlling the integrity and uniformity of the droplets.

[0018] The beneficial effects of the present invention are:

[0019] (1) By introducing microfluidics technology, high-throughput and high-yield lipid microalgae screening was achieved, reaching 10 per day 6 ~10 8 strains, significantly improving the screening efficiency and effectively solving the problem of limited number of well plates in the prior art.

[0020] (2) Through the droplet sorting method, the reaction system is upgraded from the milliliter level to the picoliter level, achieving more precise control and operation. It is helpful to carry out high-throughput and high-precision screening in the microenvironment, solving the problem of limited reaction system. In addition, through picoliter-level screening, the screening cost of a single strain is reduced, which is greatly reduced compared with the milliliter-level cost of existing technologies.

[0021] (3) The method of dyeing first and then generating droplets avoids the omission that may occur during the fusion of droplets and dye droplets, and avoids the fluorescence detection error caused by different dye concentrations during algal cell staining and droplet sorting.

[0022] (4) The process from staining the algae cell suspension to droplet generation and sorting takes no more than 4 hours, thus avoiding the problem of fluorescence detection deviation caused by cell proliferation during the sorting process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a microscopic examination image of algae cells after Nile red staining in an embodiment of the present application, wherein A and B are microscopic examination images of bright field and fluorescent field, respectively.

[0024] Figure 2 This is an instant photo of droplet generation in one embodiment of the present application.

[0025] Figure 3 FIG. 1 is a microscopic image of droplets generated in one embodiment of the present application.

[0026] Figure 4 It is an instant image of a target droplet captured in one embodiment of the present application.

[0027] Figure 5 FIG. 1 is a microscopic image of droplets obtained by sorting in one embodiment of the present application.

[0028] Figure 6 It is the intensity of the fluorescent electrical signal detected at a gain of 700 in one embodiment of the present application. Wherein, A is the fluorescent electrical signal of the Chaetoceros algae droplet at a gain of 700, and B is the fluorescent electrical signal of the Phaeochromis triangularis droplet at a gain of 700; the horizontal and vertical coordinates in the figure are both the detected fluorescence values.

[0029] Figure 7 It is a real-time channel signal of droplet sorting in an embodiment of the present application, wherein the upper side is the signal of Chaetoceros hornii, and the lower side is the signal of Phaeodactylum triangularis.

[0030] Figure 8 This is the fluorescence intensity result after the algae cells are stained with nylon red at different working concentrations in one embodiment of the present application. DETAILED DESCRIPTION

[0031] In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0032] A first aspect of the present invention provides a method for screening algal cells.

[0033] The term "algal cell" generally refers to the cells of microalgae. Microalgae generally refer to microorganisms that contain chlorophyll and can carry out photosynthesis, including one or more of the following: green algae, cyanobacteria, golden algae, diatoms, red algae, cryptophytes, dinoflagellates, brown algae, and yellow algae. Among them, the Chlorophyta includes one or more of the Chlorozoales (such as Botrytis, Chlorella, Paracystis, Unguiculata, Monopterus, Discoideum, Scenedesmus), Volvoxales (such as Chlamydomonas, Volvox, Platyhelminthes, Dunaliella), etc., the Cyanobacteria includes one or more of the Oscillatorales (such as Microcystis), Chrococoacales (such as Microcystis), etc., the Chrysophyta includes one or more of the Chrysophytes (such as Chrysophytes), Rhizochrysophytes (such as Chrysophytes), etc., the Bacillariophyta includes one or more of the Cyclostriales (such as Cyclotella), Bivalviales (such as Pinnata), Tubulariaces (such as Nitzschia, Cylindrotheca), Box-shaped Algae (such as Chaetoceros), and Phaeodactylumales (such as Phaeodactylum triangularis).

[0034] The method includes the following steps S100 to S300.

[0035] S100: taking the algae cell suspension and mixing it with the lipid dye for dyeing to obtain a dyed algae cell suspension.

[0036] The term "suspension" refers to a liquid in which algal cells are dispersed, and the liquid may be one or more of a culture medium and a buffer suitable for the survival of algal cells. The culture medium includes any one of F2 medium, SE medium, BG11 medium, ASW medium, BBM medium, TAP medium, Shuisheng No. 4, Shuisheng 105, Shuisheng Silicon 1, Knop culture medium, Zhu's No. 10, etc., and the buffer includes PBS buffer, etc.

[0037] In some embodiments, the suspension of algal cells is pre-adjusted to OD before being mixed with the lipid dye for staining. 680 is the set value. 680 The setting value is calculated by the following formula:

[0038] OD 680 =λ / (V×σ);

[0039] Among them, λ is the λ of the single-cell droplet number to the total cell droplet number when the Poisson distribution is adopted, the single-cell droplet number is the number of droplets with only one algae cell inside the generated droplets, and the total cell droplet number is the number of droplets with algae cells inside the generated droplets, and the value is 0.1;

[0040] V is the average volume of the droplets;

[0041] σ is the cell density of algae cells in the algae cell suspension, and its value is 2.375×10 7 , unit is mL -1 .

[0042] In some embodiments, the suspension of algal cells is pre-adjusted to OD before being mixed with the lipid dye for staining. 680 It is 0.04 to 0.08, for example, it can be 0.04, 0.05, 0.06, 0.07, or 0.08.

[0043] In some embodiments, the suspension of algal cells is adjusted to OD 680 The cell density was 1×10 6 ~1.5×10 6 / mL, for example, 1×10 6 / mL, 1.1×10 6 / mL, 1.2×10 6 / mL, 1.3×10 6 / mL, 1.4×10 6 / mL, 1.5×10 6 Pieces / mL.

[0044] In some embodiments, the suspension of algae cells is a suspension of algae cells in the logarithmic growth phase. In some embodiments, the suspension of algae cells is a suspension of algae cells at the end of the logarithmic growth phase. In some embodiments, the algae cells at the end of the logarithmic growth phase are obtained by picking the algae cells to be screened and culturing them under light conditions until the end of the logarithmic growth phase. In some embodiments, the algae cells are cultured with a culture medium under light conditions of 10 to 500 rpm for 4 to 10 days to obtain algae cells in the logarithmic growth phase. In some embodiments, the culture medium can be any of the aforementioned culture media, and the specific type is selected according to the type of algae cells. In some embodiments, the rotation speed conditions of the culture can be, for example, 10rpm, 20rpm, 30rpm, 40rpm, 50rpm, 60rpm, 80rpm, 100rpm, 120rpm, 140rpm, 150rpm, 160rpm, 180rpm, 200rpm, 300rpm, 400rpm, 500rpm. In some embodiments, the culture time can be 4, 5, 6, 7, 8, 9, 10 days.

[0045] The term "lipid dye" refers to a dye that can dye lipids, for example, the dye can be combined with lipids in algal cells by one or more effects such as solution effect, adsorption effect, electrostatic interaction, hydration, etc., wherein the lipids include fats, lipids (such as phospholipids, glycolipids, sterols), etc. In some embodiments, the lipid dye includes at least one of Nile Red, BODIPY, Oil Red O, Sudan, BETA-1, LipidGreen, and PTZ-LD, BODIPY dyes include BODIPY 493 / 503, BODIPY-Cholesterol, BODIPY FL Cholesterol, BODIPY505 / 515, BODIPYTR Cadaverine, BODIPY 500 / 510C1, C12, BODIPY 558 / 568C12, BODIPY FL DHPE, etc., LipidGreen includes LipidGreen, LipidGreen2, etc., Sudan dyes include Sudan III, Sudan IV, Sudan Black B, etc. Depending on the different types and principles of lipid dyes, the optical signals used for subsequent sorting may also be different. For example, Oil Red O can dye lipids red, Sudan Black B can dye lipids black, and Nile Red can emit red fluorescence in a lipid-rich environment.

[0046] In some embodiments, the lipid dye is a fluorescent dye, such as Nile Red, BODIPY, BETA-1, LipidGreen, PTZ-LD, etc., and the optical signal based on which the sorting is based includes a fluorescent signal.

[0047] In some embodiments, the working concentration of the lipid dye is 1 to 10 μg / mL, for example, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL. The term "working concentration" refers to the concentration of the lipid dye in the mixed system consisting of the lipid dye and the suspension of algal cells at the beginning of the dyeing process. Since the lipid dye is diluted after mixing, the original concentration of the lipid dye can be 2 times, 3 times, 5 times, 10 times, 20 times, 30 times, 50 times, 100 times, 200 times, 300 times, 500 times, 800 times, 1000 times, 1500 times, 2000 times, 3000 times, 4000 times, 5000 times of the working concentration. In some embodiments, the working concentration of the lipid dye is 1 to 10 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL.

[0048] In some embodiments, the dyeing process includes incubation for 10 to 60 minutes after mixing, for example, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes. In some embodiments, the incubation temperature is 20 to 30 degrees Celsius, for example, 20 degrees Celsius, 21 degrees Celsius, 22 degrees Celsius, 23 degrees Celsius, 24 degrees Celsius, 25 degrees Celsius, 26 degrees Celsius, 27 degrees Celsius, 28 degrees Celsius, 29 degrees Celsius, 30 degrees Celsius. In some embodiments, incubation is carried out under light-proof conditions.

[0049] S200: The dyed algae cell suspension is used as the aqueous phase and the oil phase is used to generate droplets through droplet microfluidics technology.

[0050] The term "droplet microfluidics" is droplet-based microfluidics, which refers to the generation and manipulation of droplets in a microchannel network. Specifically, there are two immiscible liquids in the microchannel as dispersed phase and continuous phase, respectively, and the former is dispersed in the latter in the form of droplets of micro-volume units. The principle of droplet formation is that, with the help of the microchannel structure of the microfluidic chip (which also includes external force manipulation in some embodiments), the continuous phase disperses (e.g., shears) the dispersed phase to form relatively uniform droplets of micro-volume units. In the embodiments of the present application, the purpose is to form droplets containing algae cells, so the stained algae cell suspension is used as the dispersed phase, that is, the water phase, and a liquid that is incompatible with water is provided as the continuous phase, that is, the oil phase, and the oil phase shears the water phase into droplets.

[0051] In some embodiments, the oil phase includes at least one of fluorinated oil, silicone oil, and mineral oil. In some embodiments, the droplet microfluidics system also includes a surfactant, such as a nonionic surfactant, specifically one or more of Triton X-100, Tween 20, Brij 56, Brij 58, etc.

[0052] In some embodiments, the method for generating droplets includes a multiphase flow method, which can be specifically a T-channel method (orthogonal structure method), a flow focusing method, and a coaxial flow method. Accordingly, the microfluidic chip can be a T-channel method microfluidic chip, a flow focusing method microfluidic chip, and a coaxial flow method microfluidic chip. In the T-channel method, the dispersed phase is vertically introduced into the continuous phase, and the dispersed phase is sheared into droplets by the continuous phase at the interface of the two phases; in the flow focusing method, the middle dispersed phase is sheared into droplets under the action of the symmetrical shear force of the continuous phases on both sides; in the coaxial flow method, the dispersed phase and the continuous phase flow coaxially and parallelly in the capillary, and the flow rate of the inner dispersed phase is different from that of the outer continuous phase, which generates fluctuations at the interface of the two phases and causes the dispersed phase to generate droplets. It can be understood that the method for generating droplets can also be any other method known in the art, such as driving the formation of droplets by heat, piezoelectricity, magnetic field, etc.

[0053] In some embodiments, using flow focusing, the pressure of the water phase is 150-250 mbar, for example, it can be 150 mbar, 160 mbar, 170 mbar, 180 mbar, 190 mbar, 200 mbar, 210 mbar, 220 mbar, 230 mbar, 240 mbar, 250 mbar; the pressure of the oil phase is 300-500 mbar, for example, it can be 300 mbar, 320 mbar, 340 mbar, 350 mbar, 360 mbar, 380 mbar, 400 mbar, 420 mbar, 440 mbar, 450 mbar, 460 mbar, 480 mbar, 500 mbar.

[0054] In some embodiments, the diameter of the generated droplets is less than 100 μm. In some embodiments, the diameter of the generated droplets is 40 to 60 μm, for example, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, 47 μm, 48 μm, 49 μm, 50 μm, 51 μm, 52 μm, 53 μm, 54 μm, 55 μm, 56 μm, 57 μm, 58 μm, 59 μm, 60 μm.

[0055] In some embodiments, the generated droplets are subjected to quality control, and the quality control includes controlling the integrity and uniformity of the droplets. In some embodiments, the generated droplets are observed under a microscope for at least one of the integrity of the droplets, the diameter size, and the effective encapsulation of the droplets. In some embodiments, the effective encapsulation of the droplets is calculated using the single spore rate. In some embodiments, the effective encapsulation of the droplets is calculated by counting the cell rate to calculate the single cell rate. In some embodiments, the cell rate = (total number of droplets - number of empty cell droplets) / total number of droplets × 100%, and the single cell rate = number of single cell droplets / total number of droplets. The number of empty cell droplets is the number of droplets that do not contain algae cells inside, and the number of single cell droplets is the number of droplets that only contain 1 algae cell. In some embodiments, the method for calculating the single cell rate is to randomly select a certain number of droplets under a 400× microscope and calculate the single cell rate. In some embodiments, if the quality control results are poor, it is necessary to fine-tune the parameters when the droplets are generated.

[0056] S300: sort out the droplets whose optical signals are higher than the reference value, and the droplets contain target algae cells.

[0057] In some embodiments, the optical signal is a fluorescent signal.

[0058] In some embodiments, the optical signal of the sorted droplets is 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or more of the baseline value.

[0059] In some embodiments, sorting is performed using a high-throughput microbial screening system.

[0060] In some embodiments, sorting is performed using an MSP high-throughput microbial sorter.

[0061] The invention of the present invention mainly includes the following aspects:

[0062] (1) Application of microfluidic technology: Traditional screening methods are limited by manual operation, and the separation and purification steps are cumbersome, and the screening flux is limited. The present invention breaks the limitations of traditional methods for separating and purifying algae strains, and successfully applies microfluidic technology to the process of screening high-lipid-yielding algae strains. Through the automated microfluidic system, it gets rid of the limitations of manual operation and achieves efficient algae strain selection at the single-cell level in a trace system. In addition, microfluidic technology significantly improves the screening flux of high-lipid-yielding algae strains, solves the problem of limited screening flux caused by the number of cell culture plates in traditional screening technologies for high-lipid algae strains, eliminates the limitation of the number of cell culture plates, significantly improves the screening efficiency of algae strains, and ensures the stability of screening results by eliminating the variability in the manual operation process.

[0063] (2) Efficient separation and purification of algae strains: By controlling the algae cell density and droplet generation parameter settings, the reaction system is upgraded from milliliter and microliter levels to picoliter levels, achieving the separation of individual algae cells from complex algae cell samples. Each droplet contains only one algae cell, achieving the purpose of separating and purifying algae strains. Compared with traditional algae screening methods, the procedure is simplified, the cost of consumables is saved, and the fluorescence detection is more precise and sensitive, which improves the reliability of the results. In addition, through picoliter-level screening, the screening cost of a single strain is reduced, which is much lower than the milliliter and microliter levels of existing technologies.

[0064] (3) Introduction of fluorescence screening technology: By combining fluorescence sorting technology with microfluidic high-precision detection, the screening of single cells of high-lipid algae strains was achieved. After staining, the cells are still active and can proliferate in conventional culture medium.

[0065] The present invention successfully applies picoliter-scale microfluidic technology to achieve accurate, fast, efficient, and low-cost screening of high-yield lipid algae strains, achieving the purpose of high-throughput screening at the single-cell level in a micro-droplet system. By operating in a trace system, high-lipid-yielding algae strains can be accurately selected, providing a novel, efficient, and economical solution for the screening of high-yield lipid algae strains. In addition, compared with traditional methods for separating and purifying algae strains, the droplets mentioned in the present invention only wrap one algae cell, achieving a more efficient and convenient method for purifying algae strains, greatly saving time, manpower, and consumables. The screening of high-yield lipid algae strains is upgraded from the milliliter level to the picoliter level, and more sophisticated control and detection are used to improve the accuracy and reliability of the screening.

[0066] The present invention is further described in detail below through specific examples.

[0067] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0068] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0069] Example 1

[0070] 1. Preparation of Algal Cell Suspension

[0071] Two single algae colonies (Chaetoceros muelleri and Phaeodactylum tricornutum Bohlin, from the Laboratory of Marine Microbial Resources Development and Utilization of Hainan University) were picked up on the plate and cultured with F / 2 medium at 180 rpm for 7 days to obtain algae liquid at the end of the logarithmic growth phase. The two algae liquids were mixed in a volume ratio of 1:1 and counted, and then diluted with F / 2 medium to 1-1.2×10 6 cell / mL and OD 680 =0.06 algae cell suspension. Add 1 μL of 2 mg / mL Nile red dye to 1 mL of algae cell suspension to make 2 μg / mL Nile red dye working solution, incubate at room temperature in the dark for 20 minutes to obtain the stained algae cell suspension. Figure 1 , are the results of microscopic examination of algal cells in the algal cell suspension under bright field and fluorescent field after staining.

[0072] Among them, based on previous experimental data, the polyunsaturated fatty acid content of a single Triangular Brown Finger Algae was 31.65fg / pg, and the polyunsaturated fatty acid content of Chaetoceros muelleri was 223.01fg / pg, which were significantly different. Therefore, Chaetoceros muelleri was used as a positive control and Triangular Brown Finger Algae was used as a negative control.

[0073] 2. Generate droplets

[0074] The stained algae cell suspension was used as the aqueous phase, and mineral oil (Zhejiang Dapu Biological Company, droplet generation oil) was used as the oil phase. Droplets with a diameter of about 50 μm were prepared in the matching droplet generation chip using a DG300 droplet encapsulation instrument (Zhejiang Dapu Biological Company). Figure 2 The generated droplets are collected in a Teflon tube. The parameters of the DG300 droplet encapsulation instrument are set as follows: water phase pressure 200mbar, oil phase 400mbar. Figure 3 As shown, the diameter of the generated droplets is about 50±3 μm.

[0075] 3. Droplet microscopy

[0076] The prepared droplets are placed in a droplet observation chip, and the integrity, diameter, and effective droplet encapsulation of the droplets are observed under a microscope. The algae-carrying rate is counted to calculate the single cell rate, and the integrity and uniformity of the droplets are quality controlled.

[0077] Among them, single cell rate = single cell droplet number / total droplet number × 100% = 21.97%;

[0078] The cell rate = (total number of droplets - empty cell droplets) / total number of droplets × 100% = 24.28%. The single cell rate and cell rate conform to the Poisson distribution.

[0079] 4. Droplet sorting

[0080] The uniform and complete droplets after quality control were connected to the MSP droplet sorter (Zhejiang Dapu Biological Company), and signal detection and data collection were performed in the matching sorting chip. Figure 4 , Figure 5 , Figure 6 and Figure 7 shown.

[0081] from Figure 6 It can be seen that under a gain of 700, when a single algae liquid was used to prepare droplets according to the above method for on-machine testing, both algae liquids were overexposed, and the fluorescent electrical signal of the Chaetoceros muelleri droplets was significantly higher than that of the Phaeochromis triangularis.

[0082] Figure 7 Each peak represents the fluorescence of an algal cell. Figure 7 It can be seen that the droplet signal of Chaetoceros muelleri is higher than that of P. triangularis. P. triangularis basically does not exceed 2000 fluorescence units, while Chaetoceros muelleri can reach 3600 fluorescence units. The difference in fluorescent electrical signals detected by the high-throughput microfluidic sorting system of the two algae meets the expected positive and negative control and meets the screening conditions. Collect droplets with specific fluorescent signals to complete the sorting of target algae strains.

[0083] Example 2: Nile red staining concentration screening

[0084] A single colony of Cylindrotheca fusiformis was picked from the plate and cultured with F / 2 medium at 180 rpm for 7 days to obtain algae solution at the end of the logarithmic growth phase. The solution was diluted with F / 2 medium to 1-1.2 × 10 6 cell / mL and OD 680 =0.06 algae cell suspension. Add 1 μL of Nile red dye at concentrations of 0, 0.6, 0.8, 1, 1.2, 1.5, 2 mg / mL to 1 mL of algae cell suspension (the working concentrations of Nile red are 0, 0.6, 0.8, 1, 1.2, 1.5, 2 μg / mL, respectively), incubate at room temperature in the dark for 20 minutes to obtain the stained algae cell suspension. Wash twice, take 200 μL in a 96-well plate and use an enzyme reader to detect the fluorescence intensity. The results are as follows Figure 8 As shown, considering the biological toxicity and fluorescence intensity detection effect of Nile red dye, 2 μg / mL is the optimal staining concentration of Nile red.

[0085] Based on the above embodiments, the method of the present application:

[0086] (1) Microfluidics technology and Nile red dye were used to screen high lipid-producing algae strains at the sub-picoliter level and single-cell level.

[0087] (2) By controlling the algae cell density and droplet generation parameter settings, the separation and purification of algae strains from complex algae cell samples was achieved: ideally, each droplet should contain a single algae cell, but the droplet generation process is random, and the probability of single encapsulation obeys the Poisson distribution. In order to ensure the ideal state of single encapsulated cells to the greatest extent and as few empty droplets as possible. By controlling the algae cell density and droplet generation parameter settings, after microscopic examination and calculation, a cell rate of about 24% and a single cell rate of about 22% were achieved, and the maximum achievable single cell rate was obtained, ensuring that most droplets contained only one algae cell, achieving the purpose of purifying algae strains.

[0088] (3) Combining picoliter-scale microfluidics with Nile red fluorescent dye, the screening of high-lipid-yielding algae strains can be achieved in a high-throughput scenario: Nile red dye has the characteristic of indicating intracellular lipids. After being stained with Nile red, the lipids in the algae cells emit red fluorescence. The microfluidic fluorescent probe captures the intensity of the fluorescent signal emitted from the algae cells and performs diversion screening on the algae cells, thereby achieving the screening of high-lipid-yielding algae strains.

[0089] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for screening algal cells, characterized in that: include: Taking the algae cell suspension and mixing it with the lipid dye to dye it, so as to obtain a dyed algae cell suspension; The dyed algae cell suspension is used as the aqueous phase and the oil phase to generate droplets through droplet microfluidics technology; Droplets with an optical signal higher than a reference value are sorted out, and the droplets contain target algae cells.

2. The method according to claim 1, characterized in that: The lipid dye includes at least one of Nile Red, BODIPY, Oil Red O, Sudan, BETA-1, LipidGreen, and PTZ-LD.

3. The method according to claim 2, characterized in that: The optical signal includes a fluorescent signal.

4. The method according to claim 1, characterized in that: The working concentration of the lipid dye is 1-3 μg / mL.

5. The method according to claim 1, characterized in that: The algae cell suspension was pre-adjusted to OD before being mixed with the lipid dye for staining. 680 =0.04~0.

08.

6. The method according to claim 5, characterized in that: The algal cell suspension was adjusted to OD 680 The cell density after the value is 1×10 6 ~1.5×10 6 Pieces / mL.

7. The method according to claim 5, characterized in that: The culture medium in the algae cell suspension includes F2 culture medium.

8. The method according to claim 1, characterized in that: The diameter of the generated droplets is 40 to 60 μm.

9. The method according to claim 1, characterized in that: The sorting is performed using a high-throughput microbial screening system.

10. The method according to claim 1, characterized in that: The droplets are quality controlled before sorting, and the quality control includes controlling the integrity and uniformity of the droplets.

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