High-flux enzyme directed evolution detection method and magnetic micro-droplet sorting system

Abstract

The invention discloses a high-throughput enzyme directed evolution detection method and a magnetic micro-droplet sorting system, and belongs to the field of biological detection. Aiming at the technical defects of low separation flux, high cost of a separation device and insufficient magnetic separation precision in the existing enzyme directed evolution screening technology, a magnetic micro-droplet technology and a micro-fluidic chip technology are combined, and a micro-fluidic magnetic separation chip system integrating a detection module and a magnetic field separation module is constructed. The system has the technical advantages that the sorting flux and accuracy are remarkably improved, the structure of the sorting device is simplified, and the operation cost is reduced. A high-throughput, high-selectivity, high-universality, economical and efficient screening platform is provided for directed evolution of enzyme molecules.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular, to a high-throughput enzyme directed evolution detection method and a magnetic microdroplet sorting system. Background Art

[0002] Enzymes are highly efficient, highly selective, and environmentally friendly biocatalysts, and are widely used in the fields of biology, medicine, energy, chemical engineering, food, etc. Enzyme directed evolution can screen a very small number of mutants that meet the expected properties from an enzyme mutant library by simulating the Darwinian evolution principle and combining random mutation and high-throughput screening, and optimize and regulate various properties of the enzyme. It is an important tool for optimizing the performance of enzyme molecules.

[0003] Due to the extremely large capacity of random protein mutations (10 6 ~10 9 ), while the library capacity of conventional microplate screening methods is usually only 10 3 -10 5 . The insufficient screening library capacity is the key reason for the failure of directed evolution. Therefore, the development of high-throughput and high-efficiency enzyme activity screening technologies is the key to the success of enzyme directed evolution.

[0004] The enzyme high-throughput screening technology based on microdroplets has the advantages of high throughput (10 8 / day), low sample consumption (reduced by a million times), and high efficiency. However, this technology still has a series of problems: the existing electro-sorting devices are complex and expensive to manufacture, the magnetic sorting throughput and accuracy are insufficient, etc., making it difficult to achieve industrialization and popularization. The microdroplet magnetic sorting system has the advantages of simple production, low cost, long service life, etc., and is an enzyme screening technology with industrialization prospects. However, the incorporation of magnetic materials will bring problems of poor biocompatibility, and the accuracy and throughput are not high, which greatly limits the application of this technology in high-throughput enzyme screening.

[0005] In view of this, the present application is specifically proposed. Summary of the Invention

[0006] The purpose of the present application is to provide a high-throughput enzyme directed evolution detection method and a magnetic microdroplet sorting system to solve the above problems existing in the prior art.

[0007] The present application is implemented as follows:

[0008] In a first aspect, an embodiment of the present application provides a high-throughput enzyme directed evolution detection method. The detection method is based on the integration of magnetic microdroplet technology and microfluidic chip technology, and constructs a microfluidic sorting system integrating a fluorescence detection module and a magnetic field precise sorting module. The method includes: (a) generating amphiphilic magnetic microdroplets containing enzyme mutants through a microfluidic chip; (b) monitoring the dynamic signals of enzyme-catalyzed reactions in the microdroplets using a fluorescence detection module; (c) achieving the directional capture and sorting of target microdroplets based on a magnetic field control module.

[0009] Further, the generation of the magnetic microdroplets in step (a) includes: dispersing magnetic particles with fluorine-containing groups on the surface in an oil phase to prepare a surfactant, and forming single-cell dispersed magnetic microdroplets containing enzyme mutant cells and corresponding substrates through the droplet generation unit of the microfluidic chip;

[0010] The magnetic particles include any one or a combination of more than one of Fe, Fe3O4 nanoparticles, Fe5MnO8 nanoparticles, manganese ferrite, cobalt ferrite magnetic microspheres, or magnetic composite particles coated with silica on the surface;

[0011] The fluorine-containing groups include any one or a combination of more than one of F, CF3, CF2CF3, CHF2;

[0012] The oil phase includes any one or a combination of more than one of HFE 7500, HFE 7100, mineral oil, FC-40;

[0013] The microfluidic chip includes any one of a flow focusing type microfluidic chip, a T-channel method microfluidic chip, or a coaxial flow focusing method microfluidic chip;

[0014] The enzyme mutants include any one or a combination of more than one of oxidoreductase, hydrolase, transferase, lyase, isomerase, synthetase, or horseradish peroxidase mutants with high enzyme activity;

[0015] The mutation types of the enzyme mutants include any one of single point mutation, multiple point mutation, or domain insertion / deletion;

[0016] The host cells of the enzyme mutants include any one or a combination of more than one of Escherichia coli, Pichia pastoris EBY-100 strain, mammalian cell lines, Bacillus subtilis, or Escherichia coli hosts used in combination with M13 filamentous phage;

[0017] The substrates include any one or a combination of more than one of o-phenylenediamine, tetramethylbenzidine, 5-aminosalicylic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, H2O2, amperometric red, chloronaphthol, or aminoethylcarbazole.

[0018] Further, in step (b), a fluorescence dynamic signal is used to collect, convert, and record the enzyme reaction signal;

[0019] The dynamic signal includes any one or a combination of more than one of the following: fluorescence intensity, a curve of fluorescence intensity versus time, enzyme reaction rate, or substrate conversion threshold;

[0020] The time of the enzyme reaction is 1 to 30 minutes;

[0021] The temperature of the enzyme reaction is 4 to 40 °C.

[0022] Further, in step (c), the magnetic field generation unit is integrated into the sorting unit of the sorting chip, and the target droplets move into the collection channel under the action of a micro magnetic field, and the droplet movement rate determines the detection speed;

[0023] The magnetic field generation unit includes one or more of the following: a permanent magnet, an electromagnet, or a superconducting magnet;

[0024] The material of the sorting chip includes any one of polydimethylsiloxane (PDMS), glass, or cycloolefin copolymer (COC);

[0025] The droplet movement rate is 100 to 10,000 per second;

[0026] The detection frequency is 100 to 10,000 times per second.

[0027] In a second aspect, an embodiment of the present application provides a magnetic micro-droplet sorting system applicable to the above high-throughput enzyme directed evolution detection method, including: a microfluidic chip module including a droplet generation unit;

[0028] The material of the microfluidic chip includes any one of polydimethylsiloxane, glass, or cycloolefin copolymer;

[0029] A fluorescence detection module integrated in the detection area of the microfluidic chip, including an excitation light source and an optical sensor;

[0030] The excitation light source is any one of an LED, a laser diode, a mercury lamp, or a xenon lamp;

[0031] The optical sensor is any one of a photomultiplier tube, a high-sensitivity CCD, a CMOS image sensor, or a time-resolved fluorescence detector;

[0032] And the detection wavelength range of the optical sensor is 300 to 800 nm;

[0033] A magnetic field control module including a magnetic field response element and a magnetic droplet sorting chip;

[0034] The magnetic field response element includes: dynamically adjusting any one or more of the direction of the magnetic field acting force, the magnetic field strength gradient, or the pulsed magnetic field acting time;

[0035] The preset screening criterion is to perform real-time analysis on the fluorescence signal through a machine learning algorithm and dynamically adjust the sorting threshold;

[0036] The machine learning algorithm includes: any one or more algorithms of a machine learning algorithm, a threshold sliding window algorithm, or a mathematical model based on enzyme reaction kinetics.

[0037] Furthermore, the microfluidic chip module can adopt a multi-channel parallel structure, including 1 to 20 independent droplet generation units, and the diameter range of the micro-droplets generated by each unit is 20 - 200 μm;

[0038] The arrangement mode of the multi-channel parallel structure is: any one of a linear array, a radial array, or a cross network.

[0039] In a third aspect, the embodiment of the present application provides a detection method for high-throughput enzyme directed evolution, which uses the high-throughput enzyme directed evolution sorting method described in the foregoing embodiment in combination with a high-throughput magnetic micro-droplet sorting system.

[0040] In a fourth aspect, the embodiment of the present application provides a high-throughput enzyme directed evolution integrated working platform, including the high-throughput magnetic micro-droplet sorting system or the detection method implementation unit described in the foregoing embodiment.

[0041] The present application has the following beneficial effects:

[0042] 1. High-throughput screening ability: Through the synergistic effect of the microfluidic multi-channel parallel design and magnetic field sorting, the droplet processing rate reaches 100 - 10,000 per second, and the throughput is increased by 3 orders of magnitude compared with the traditional magnetic sorting technology, meeting the rapid screening requirements of large-scale enzyme mutant libraries.

[0043] 2. High-precision sorting performance: The real-time linkage of the dynamic fluorescence signal and magnetic field sorting, by optimizing the sorting threshold, can distinguish small differences in enzyme activity, and the sorting accuracy is significantly higher than that of the traditional magnetic bead sorting method.

[0044] 3. Good biocompatibility: The proliferation of single bacteria and cells can be completed in magnetic micro-droplets, providing a reliable platform for subsequent enzyme directed evolution.

[0045] 4. Low cost: The integrated microfluidic chip replaces the expensive flow cytometry, and the equipment cost is reduced by more than 80%.

[0046] 5. Wide range of applications: Compatible with the detection of mutants of various enzymes (oxidoreductases, hydrolases, synthases, etc.), and has broad application potential in the fields of analytical chemistry, cell biology, immunology, molecular biology, clinical medicine, etc. Brief Description of the Drawings

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0048] Figure 1 Principle of generating droplets for the microfluidic chip;

[0049] Figure 2 Schematic diagram of the enzymatic reaction of single cells with high fluorescence intensity;

[0050] Figure 3 Schematic diagram of the structure of the controllable magnetic micro-droplet sorting chip;

[0051] Figure 4 Biocompatibility experiment: Schematic diagram and diameter distribution diagram of the growth of single yeast cells in droplets gradually reducing the diameter of the droplets containing yeast cells. The white dashed box is the droplet containing yeast cells, scale bar: 50 μm;

[0052] Figure 5 Reaction time optimization: Schematic diagram and fluorescence intensity distribution diagram of strongly fluorescent droplets and weakly fluorescent droplets at different times, scale bar: 50 μm;

[0053] Figure 6 Amplex Red substrate concentration optimization;

[0054] Figure 7 Reaction temperature optimization;

[0055] Figure 8 Sensitivity verification of the detection method provided in Example 1;

[0056] Figure 9 Specificity verification of the detection method provided in Example 1;

[0057] Figure 10 Comparison of the detection time between the detection method provided in Example 1 and flow cytometry. Detailed Embodiments

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0059] This application is based on the multifunctional magnetic microdroplet technology, combines a microfluidic sorting chip to construct a high-throughput enzyme directed evolution screening system based on fluorescence detection and magnetic field precise sorting, and uses the horseradish peroxidase directed evolution model to test the performance of the system, providing a new detection method for high-throughput enzyme screening. The amphiphilic magnetic microdroplets avoid the problem of poor biocompatibility caused by internal magnetic labeling in conventional droplets; fluorescence detection and magnetic field control are integrated on the microfluidic magnetic sorting chip, which can replace expensive flow cytometers to achieve high-throughput precise screening of magnetic microdroplets, solving the disadvantages of complex and expensive production of electric sorting devices, low magnetic sorting throughput, and insufficient accuracy. The successful implementation of this application provides an important tool for enzyme screening with high throughput, good selectivity, good versatility, and low cost, and promotes the industrial development of magnetic high-throughput enzyme screening.

[0060] Specifically, the embodiments of this application provide a high-throughput enzyme directed evolution detection method. The detection method is based on the integration of magnetic microdroplet technology and microfluidic chip technology, and constructs a microfluidic sorting system integrating a fluorescence detection module and a magnetic field precise sorting module. The method includes: (a) generating amphiphilic magnetic microdroplets containing enzyme mutants through a microfluidic chip; (b) using the fluorescence detection module to monitor the dynamic signals of enzyme-catalyzed reactions in the microdroplets; (c) achieving the directional capture and sorting of target microdroplets based on the magnetic field control module.

[0061] In some embodiments, the generation of the magnetic microdroplets includes: dispersing magnetic particles with fluorine-containing groups on the surface in an oil phase to prepare a surfactant, and forming monodisperse magnetic microdroplets containing enzyme mutant cells and corresponding substrate solutions through the droplet generation unit of the microfluidic chip. The magnetic particles include any one or a combination of more than one of Fe, Fe3O4 nanoparticles, Fe5MnO8 nanoparticles, manganese ferrite, cobalt ferrite magnetic microspheres, or magnetic composite particles coated with silica on the surface.

[0062] In some embodiments, the fluorine-containing groups include any one or a combination of more than one of F, CF3, CF2CF3, and CHF2.

[0063] In some embodiments, the oil phase materials include any one or a combination of more than one of HFE 7500, HFE 7100, mineral oil, and FC-40.

[0064] In some embodiments, the microfluidic chip includes any one of a flow focusing type, a T-channel method, or a coaxial flow focusing method.

[0065] In some embodiments, the enzyme mutant includes any one or a combination of more than one of an oxidoreductase, a hydrolase, a transferase, a lyase, an isomerase, a synthetase, or a highly enzymatically active HRP mutant.

[0066] In some embodiments, the enzyme mutation form includes any one mutation type of a single point mutation, a multi-point mutation, or a domain insertion / deletion.

[0067] In some embodiments, the host cell of the enzyme mutant includes any one or more of Escherichia coli, the Pichia pastoris EBY-100 strain, a mammalian cell line, Bacillus subtilis, or an Escherichia coli host used in combination with an M13 filamentous phage.

[0068] In some embodiments, the substrate includes any one or more of o-phenylenediamine, tetramethylbenzidine, 5-aminosalicylic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, H2O2, amperometric red, chloronaphthol, and aminoethylcarbazole.

[0069] In some embodiments, the fluorescence dynamic signal is used to collect, convert, and record the enzyme reaction signal, and the dynamic signal includes any one or a combination of more than one of fluorescence intensity, a curve of fluorescence intensity versus time, an enzyme reaction rate, or a substrate conversion threshold.

[0070] In some embodiments, the enzyme reaction time is 1 to 30 min, and specifically can be any one or a range between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 min.

[0071] In some embodiments, the enzyme reaction temperature is 4 to 40 °C, and specifically can be any one or a range between any two of 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C.

[0072] In some embodiments, a magnetic field generation unit is integrated in the sorting unit of the sorting chip, and the target droplet moves into the collection channel under the action of a micro magnetic field. The droplet movement rate determines the detection speed, and the magnetic field generation unit is one or more of a permanent magnet, an electromagnet, or a superconducting magnet.

[0073] The material of the sorting chip is any one of polydimethylsiloxane, glass, or cycloolefin copolymer.

[0074] In some embodiments, the droplet moving rate is 100 to 10,000 droplets per second, specifically it can be any one or the range between any two of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 droplets per second.

[0075] In some embodiments, the detection frequency is 100 to 10,000 times per second, specifically it can be any one or the range between any two of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 times per second.

[0076] In some embodiments, the magnetic micro-droplet high-throughput sorting system includes: a microfluidic chip module configured with a droplet generation unit, and the material of the microfluidic chip is any one of polydimethylsiloxane, glass or cycloolefin copolymer.

[0077] In some embodiments, the fluorescence detection module is integrated in the detection area of the microfluidic chip and includes an excitation light source and an optical sensor, and the excitation light source is any one of an LED, a laser diode, a mercury lamp or a xenon lamp.

[0078] In some embodiments, the optical sensor is any one of a photomultiplier tube, a high-sensitivity CCD, a CMOS image sensor, a time-resolved fluorescence detector.

[0079] In some embodiments, and the detection wavelength range of the optical sensor is 300 to 800 nm, specifically it can be any one or the range between any two of 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600, 700, 800 nm.

[0080] In some embodiments, the magnetic field control module includes a magnetic field response element and a magnetic droplet sorting chip, and the magnetic field response element includes any one or more of the ways of dynamically adjusting the direction of the magnetic field force, the magnetic field intensity gradient or the pulsed magnetic field action time.

[0081] In some embodiments, the microfluidic chip module can adopt a multi-channel parallel structure and includes 1 to 20 independent droplet generation units, specifically it can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0082] In some embodiments, the diameter of the microdroplets generated by each unit ranges from 20 to 200 μm, and specifically can be any one of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μm or the range between any two of them.

[0083] In some embodiments, the arrangement of the multi-channel parallel structure is any one of a linear array, a radial array, or a cross network.

[0084] The embodiment of the present application provides a high-throughput detection method for enzyme directed evolution, which uses the high-throughput enzyme directed evolution sorting method described in any of the foregoing embodiments in combination with a high-throughput magnetic microdroplet sorting system.

[0085] The embodiment of the present application also provides a high-throughput enzyme directed evolution integrated work platform, which uses the high-throughput magnetic microdroplet sorting system or the detection method implementation unit described in any of the foregoing embodiments.

[0086] In some embodiments, the steps of the detection include a high-throughput enzyme directed evolution detection method and a magnetic microdroplet sorting system described in any of the foregoing embodiments.

[0087] The features and performance of the present application will be further described in detail below in conjunction with embodiments.

[0088] Example 1

[0089] A high-throughput enzyme directed evolution detection method and a magnetic microdroplet sorting system, which include the following steps.

[0090] (1) Preparation of amphiphilic magnetic microdroplets is as Figure 1 shown. Dissolve 0.06 g of amphiphilic magnetic nanoparticles Fe5MnO8@F-SiO2 in 4 g of HFE7500 to prepare an oil phase of 1.5% (wt%). The aqueous phases are yeast cells containing 10 5 Yeast / mL of high fluorescence intensity HRP mutants and an HRP substrate reagent containing 100 μM Amplex Red and 50 μM H2O2. The oil phase serves as the continuous phase (600 μL / h), and the aqueous phase serves as the dispersed phase (300 μL / h). Monodisperse magnetic microdroplets (diameter 50 ± 5 μm) are generated in a T-shaped microfluidic chip, with a flux of approximately 1000 droplets per second. The droplets react at room temperature in the chip observation slot for 3 min to promote the enzymatic reaction, and the magnetic droplets containing yeast cells with high fluorescence intensity HRP mutants emit strong fluorescence ( Figure 2 ).

[0091] (2) Optical detection module. A 488 nm laser diode is used to excite amperometric red, and a photomultiplier tube (PMT) is combined to collect signals. The signals are processed by an analog signal preprocessing circuit and then reach the A / D converter for signal processing. After signal amplification, data is collected at the data acquisition card ( Figure 3 ). The light spot is focused on the detection area (about 1 mm inside the chip 2 ). When the droplet passes through the detection area, the fluorescence intensity of each droplet is recorded (sampling frequency 1000 Hz). The signals are analyzed in real time by a machine learning algorithm, and droplets with an average fluorescence intensity of 60 ± 5 a.u. or above are determined to be highly active mutant enzymes.

[0092] (3) Electromagnetic control module. The sorting unit integrates an electromagnet array (magnetic field gradient 0.5 T / m). When the target droplet passes through, the electromagnet is instantaneously energized (pulse time 1 ms), driving the droplet to deflect to the collection chip module captured by the magnet, and non-target droplets flow into the waste liquid pool. When the magnet is removed from the capture chip module, the target magnetic droplets can be collected ( Figure 2 , 3). The sorting rate reaches 1000 times per second.

[0093] (4) Verification of sorting efficiency: The permanent magnet is released from the collection chip module, and the sorted droplets are collected. Whether the droplets are yeast cells containing HRP mutants with high fluorescence intensity is verified by a fluorescence microscope.

[0094] Example 2 Good biocompatibility

[0095] Using the oil phase in Example 1 as the continuous phase and 10 5 Yeast / mL yeast cells as the dispersed phase, a certain amount of the dispersed phase and the continuous phase are respectively drawn by a disposable syringe, and the dispersed phase and the continuous phase liquids are pushed into the T-shaped microfluidic chip at a flow rate ratio of 1:2 (300:600 μL / h) by using a micro-injection pump to generate monodisperse magnetic micro-droplets (diameter 50 ± 5 μm), and the generation of droplets is observed in real time under a microscope, and the droplets are collected at the chip outlet. The single cells are proliferated and cultured, and imaging is performed at 0, 6, 12, and 24 hours respectively. The normal growth of a single yeast cell in the droplet will cause the diameter of the droplet containing the yeast cell to gradually decrease. The results are shown in Figure 4 . It can be clearly seen that the droplets in the white dotted box indicate that the yeast cells can grow normally in the magnetic droplets, causing the droplet diameter to decrease, while the diameter of the droplets without yeast cells does not change significantly. This shows that the magnetic micro-droplets used in the embodiments of the present application have good biocompatibility.

[0096] Example 3

[0097] Based on the method provided in Example 1, taking the reaction (step (1)) time as a single-factor variable, multiple experimental groups were set up to verify the effect of different reaction times on the fluorescence intensity of droplets. The detection results are shown in Table 1 and Figure 5 . It was found that yeast cell droplets containing HRP mutants with high fluorescence intensity could be distinguished in 3 minutes.

[0098] Table 1 Detection Results

[0099]

[0100] Example 4

[0101] Based on the method provided in Example 1, taking the Amplex Red concentration in the reaction (step (1)) as a single-factor variable, multiple experimental groups were set up to verify the effect of Amplex Red concentration on the fluorescence intensity of droplets. Among them, the detection results are shown in Table 2 and Figure 6 . It was found that when the Amplex Red concentration was 10 - 100 μM, the average fluorescence intensity of strongly fluorescent droplets increased continuously, and tended to be unchanged after 100 μM.

[0102] Table 2 Detection Results

[0103]

[0104] Example 5

[0105] Based on the method provided in Example 1, taking the reaction temperature in the reaction (step (1)) as a single-factor variable, multiple experimental groups were set up to verify the effect of reaction temperature on the detection effect. Among them, the detection results are shown in Table 3 and Figure 7 . It was found that the average fluorescence of strongly fluorescent droplets at 25 °C was the strongest.

[0106] Table 3 Detection Results

[0107]

[0108]

[0109] Example 6 High sorting sensitivity

[0110] To verify the sorting effect of the detection method provided in Example 1, the sorted droplets were collected, and the proportion of yeast cells with high fluorescence intensity HRP mutants in the droplets was verified by fluorescence microscopy. The detection results are shown in Table 4 and Figure 8 . It can be clearly concluded that the positive rate of the sorted droplets in this application reaches 90.77% - 99%, with high sorting sensitivity.

[0111] Table 4 Detection Results

[0112]

[0113] Example 7 High sorting specificity

[0114] To verify the sorting effect of the detection method provided in Example 1, we collected the droplets in the waste liquid pool after sorting and verified the proportion of yeast cells containing HRP mutants with high fluorescence intensity in the droplets through a fluorescence microscope. The detection results are shown in Table 5 and Figure 9 . It can be clearly seen that the positive rate of the droplets in the waste liquid pool is only 1% - 5.3%, showing high sorting specificity.

[0115] Table 5 Detection results

[0116]

[0117] Example 8 Short detection time

[0118] To verify the detection time of the method of the present application, the detection method of Example 1 of the present application was used to detect yeast cells containing high mutant enzymes. At the same time, a control group was set based on flow cytometer sorting. The yeast cells detected in both groups were 2×10 6 Yeast.

[0119] The time required to complete the detection by the two methods is shown in Table 6 and Figure 10 as shown. It can be clearly seen that the detection time of this method is significantly shorter than that of the flow cytometer.

[0120] Table 6 Detection time

[0121] Detection time min Flow cytometer 120 This method 45

[0122] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high-throughput enzyme directed evolution detection method, characterized in that The detection method is based on the integration of magnetic microdroplet technology and microfluidic chip technology, and a microfluidic sorting system integrating a fluorescence detection module and a magnetic field precise sorting module is constructed. The detection method includes: (a) generating amphiphilic magnetic microdroplets containing enzyme mutants through a microfluidic chip; (b) monitoring the dynamic signals of enzyme-catalyzed reactions in the microdroplets using a fluorescence detection module; (c) achieving the directional capture and sorting of target microdroplets based on a magnetic field control module.

2. The high-throughput enzyme directed evolution detection method according to claim 1, wherein The generation of magnetic microdroplets in step (a) includes: dispersing magnetic particles modified with fluorine-containing groups on the surface in an oil phase to prepare a surfactant, and forming single-cell dispersed magnetic microdroplets containing enzyme mutant cells and corresponding substrates through the droplet generation unit of the microfluidic chip; Preferably, the magnetic particles include any one or a combination of more than one of Fe, Fe3O4 nanoparticles, Fe5MnO8 nanoparticles, manganese ferrite, cobalt ferrite magnetic microspheres, or magnetic composite particles coated with silica on the surface; Preferably, the fluorine-containing groups include any one or a combination of more than one of F, CF3, CF2CF3, CHF2; Preferably, the oil phase includes any one or a combination of more than one of HFE 7500, HFE 7100, mineral oil, FC-40; Preferably, the microfluidic chip includes any one of a flow-focusing microfluidic chip, a T-channel method microfluidic chip, or a coaxial flow-focusing method microfluidic chip; Preferably, the enzyme mutants include any one or a combination of more than one of oxidoreductase, hydrolase, transferase, lyase, isomerase, synthetase, or horseradish peroxidase mutants with high enzyme activity; Preferably, the mutation types of the enzyme mutants include any one of single-point mutation, multi-point mutation, or domain insertion / deletion; Preferably, the host cells of the enzyme mutants include any one or a combination of more than one of Escherichia coli, Pichia pastoris EBY-100 strain, mammalian cell lines, Bacillus subtilis, or Escherichia coli hosts used in combination with M13 filamentous phage; Preferably, the substrates include any one or a combination of more than one of o-phenylenediamine, tetramethylbenzidine, 5-aminosalicylic acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, H2O2, amperometric red, chloronaphthol, or aminoethylcarbazole; 3. The high-throughput enzyme directed evolution detection method according to claim 1, characterized in that, In step (b), fluorescence dynamic signals are used for the acquisition, conversion, and recording of enzyme reaction signals; Preferably, the dynamic signals include any one or a combination of more than one of fluorescence intensity, the change curve of fluorescence intensity over time, enzyme reaction rate, or substrate conversion threshold; Preferably, the time of the enzyme reaction is 1 to 30 minutes; Preferably, the temperature of the enzyme reaction is 4 to 40 °C.

4. The high-throughput enzyme directed evolution detection method according to claim 1, wherein In step (c), a magnetic field generation unit is integrated into the sorting unit of the sorting chip, and target droplets move into the collection channel under the action of a micro-magnetic field. The droplet movement rate determines the detection speed; Preferably, the magnetic field generation unit includes one or more of a permanent magnet, an electromagnet, or a superconducting magnet; Preferably, the material of the sorting chip includes any one of polydimethylsiloxane (PDMS), glass, or cycloolefin copolymer (COC); Preferably, the droplet moving rate is 100 to 10,000 droplets per second; Preferably, the detection frequency is 100 to 10,000 times per second.

5. A magnetic micro-droplet sorting system applicable to the high-throughput enzyme directed evolution detection method according to any one of claims 1 to 4, characterized in that, Comprising: A microfluidic chip module, including a droplet generation unit; Preferably, the material of the microfluidic chip includes any one of polydimethylsiloxane, glass, or cycloolefin copolymer; Preferably, a fluorescence detection module, integrated in the detection area of the microfluidic chip, comprising an excitation light source and an optical sensor; Preferably, the excitation light source is any one of an LED, a laser diode, a mercury lamp, or a xenon lamp; Preferably, the optical sensor is any one of a photomultiplier tube, a high-sensitivity CCD, a CMOS image sensor, or a time-resolved fluorescence detector; Preferably, the detection wavelength range of the optical sensor is 300 to 800 nm; Preferably, a magnetic field control module, comprising a magnetic field response element and a magnetic droplet sorting chip; Preferably, the magnetic field response element includes any one or more of dynamically adjusting the direction of the magnetic field force, the magnetic field intensity gradient, or the pulsed magnetic field action time.

6. The magnetic micro-droplet sorting system according to claim 5, wherein The microfluidic chip module can adopt a multi-channel parallel structure, including 1 to 20 independent droplet generation units, and the diameter range of the micro-droplets generated by each unit is 20 - 200 μm; Preferably, the arrangement mode of the multi-channel parallel structure is any one of a linear array, a radial array, or a cross network.

7. An integrated high-throughput enzymatic directed evolution work platform, characterized in that, Comprising: The magnetic micro-droplet sorting system according to claims 5 - 6.

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