SERS (Surface Enhanced Raman Scattering) optical fiber probe, micro-droplet high-throughput screening system and preparation, detection and screening methods

Abstract

The invention belongs to the technical field of bioengineering, and discloses an SERS (Surface Enhanced Raman Scattering) optical fiber probe, a micro-droplet high-throughput screening system and preparation, detection and screening methods, which are used for realizing high-speed, accurate and lossless droplet sorting based on SERS. The high-throughput screening system comprises: a droplet generation module, which is used for wrapping a single cell into micro-droplets; the liquid drop incubation module is used for incubation and fermentation of strains; and the SERS liquid drop sorting module is used for high-throughput screening of target strains. The system can achieve the screening accuracy of 98% and the screening flux of 180 pieces per second, solves the problem that high-flux sorting is difficult to carry out originally, and has the advantages of being simple, feasible, wide in application range, high in expandability and the like.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a SERS optical fiber probe and a micro-droplet high-throughput screening system and a preparation, detection and screening method. Background Art

[0002] Industrial strains are the "chips" of green biomanufacturing. The development of high-performance industrial strains is inseparable from efficient and automated strain selection technology and equipment. Traditional screening methods mostly use shake flasks or plates, combined with sequencing, PCR or specific response mechanisms to screen strains. The entire system is time-consuming, the instruments are complex, the reagents are high, and the screening efficiency is low.

[0003] In the past few years, with the diversification of screening needs, high-throughput screening of microdroplets has shown great prospects. The core is to encapsulate a single strain in a microfluidic droplet. Each droplet is equivalent to a microreactor to analyze a single specific strain independently without cross-contamination. At present, fluorescence is the most commonly used microdroplet high-throughput screening strategy, but it has key limitations for the determination of metabolite content in cells that cannot be fluorescently labeled. For wild-type strains and engineered strains that cannot be fluorescently labeled, although there are some cases of screening strains through Raman spectroscopy, it is limited to lower-throughput screening, up to 4.5 / second. Moreover, the existing Raman microscopy technology has defects when used for sample analysis. Take the measurement of single microbial cells as an example: the signal intensity of single-cell Raman spectroscopy is weak, especially when the cells are suspended in liquid, usually only 10 6-8 One in ten photons passes through Raman scattering, resulting in a long spectral scanning time to obtain a complete and reliable Raman spectral signal. When analyzing a large number of samples, full spectrum identification of each cell will greatly increase the analysis time of the samples, resulting in a low collection throughput, which cannot fully meet actual needs. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a SERS optical fiber probe and a micro-droplet high-throughput screening system and a preparation, detection and screening method to achieve high-speed, accurate and non-destructive micro-droplet sorting based on SERS. When sorting micro-droplets, at least one of the following requirements can be met:

[0005] a) Low requirements on strain type;

[0006] b) High requirements for detection and screening sensitivity;

[0007] c) High requirements for screening throughput and accuracy;

[0008] d) No physiological damage is caused to the contents of the sorted droplets, such as single cells.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a SERS fiber optic probe, comprising a tapered fiber optic probe, one end of the tapered fiber optic probe is connected to a tapered structure, and a layer of metal nanoparticles is deposited on the surface of the tapered structure as a SERS substrate.

[0011] Preferably, the metal nanoparticles are gold nanoparticles, silver nanoparticles or a composite of gold nanoparticles and silver nanoparticles.

[0012] In a second aspect of the present invention, a method for preparing the SERS optical fiber probe as described above is provided, comprising:

[0013] The tapered optical fiber probe is etched by hydrofluoric acid to form a tapered structure at one end of the tapered optical fiber probe.

[0014] A layer of metal nanoparticles is deposited on the surface of the cone-shaped structure as a SERS substrate.

[0015] In a third aspect, the present invention provides a detection method based on the SERS optical fiber probe as described above, comprising:

[0016] Compare the Raman spectrum of the target compound with the SERS spectrum of the standard solution simulating microdroplets to determine the Raman characteristic peaks that match the detection signal;

[0017] Standard solutions of target compounds of different concentrations are made into microdroplets, and the corresponding relationship between the characteristic peak intensity and the concentration of the target compound is determined by using the linear relationship between the relative Raman intensity of the Raman characteristic peak of the Raman spectrum of the microdroplets of each concentration and the concentration. The characteristic peak intensity is the relative Raman intensity of the Raman characteristic peak of the Raman spectrum.

[0018] In a fourth aspect, the present invention provides a micro-droplet high-throughput screening system, comprising:

[0019] A droplet generation module, used for encapsulating a single cell in a bacterial solution into a first micro-droplet;

[0020] A droplet incubation module, used for incubating and fermenting the first micro-droplet to obtain a second micro-droplet;

[0021] A SERS droplet sorting module is used for high-throughput screening of target strains; the SERS droplet sorting module comprises a micro-droplet sorting chip, a Raman system and the SERS optical fiber probe as described above; the micro-droplet sorting chip is provided with a liquid flow channel, a first oil phase channel, a second oil phase channel, a sorting channel, a target channel and a waste channel; the liquid flow channel is used to access the second micro-droplet, the first oil phase channel and the second oil phase channel are both used to access the oil phase, the two ends of the sorting channel are respectively a first end and a second end, the liquid flow channel, the first oil phase channel, the second oil phase channel The channels are all arranged at the first end to pass the second micro-droplets and the oil phase into the sorting channel, the target channel and the waste channel are both arranged at the second end, the SERS optical fiber probe is arranged in the sorting channel to monitor the second micro-droplets in the sorting channel to obtain a SERS spectrum, the Raman system is arranged corresponding to the position of the sorting channel to obtain the Raman spectrum of the second micro-droplets in the sorting channel, and a sorting power unit is arranged corresponding to the second end, and the sorting power unit is used to control the flow direction of the second micro-droplets in the sorting channel.

[0022] Preferably, the SERS droplet sorting module further comprises a control module, the signal output ends of the Raman system and the SERS optical fiber probe are connected to the signal input end of the control module, the signal output end of the control module is connected to the signal input end of the sorting power unit, and the control module is used to:

[0023] Comparing the SERS spectra and the Raman spectra fed by the Raman system and the SERS optical fiber probe to determine the Raman characteristic peaks;

[0024] Determining the concentration of the second micro-droplet in the sorting channel based on the corresponding relationship between the characteristic peak intensity and the concentration of the target compound determined by the detection method as described above;

[0025] Based on the set concentration threshold, when the concentration of the second micro-droplets is greater than or equal to the concentration threshold, the second micro-droplets are controlled to flow to the target channel by the sorting power unit; when the concentration of the second micro-droplets is less than the concentration threshold, the second micro-droplets are controlled to flow to the waste channel by the sorting power unit.

[0026] Preferably, the droplet generation module includes a droplet preparation chip, on which an internal phase flow channel, a first external phase flow channel, a second external phase flow channel and a mixed flow channel are arranged, the internal phase flow channel, the first external phase flow channel and the second external phase flow channel are all arranged at one end of the mixed flow channel, and the other end of the mixed flow channel is connected to the droplet incubation module; wherein the internal phase flow channel is used to introduce bacterial liquid into the mixed flow channel, and the first external phase flow channel and the second external phase flow channel are used to introduce oil phase into the mixed flow channel.

[0027] Preferably, the device further comprises a target droplet collecting unit and a waste droplet collecting unit, wherein the target droplet collecting unit is used to collect droplets flowing out of the target channel, and the waste droplet collecting unit is used to collect droplets flowing out of the waste channel.

[0028] In a fifth aspect, the present invention provides a microdroplet high-throughput screening method, based on the microdroplet high-throughput screening system as described above, the method comprises:

[0029] Establish a microbial mutant library;

[0030] Inputting the bacterial liquid in the microbial mutant library into a droplet generation module to prepare a plurality of first micro droplets;

[0031] Incubate and ferment the plurality of first micro-droplets in a droplet incubation module to obtain second micro-droplets;

[0032] The second micro-droplet is then injected into the SERS droplet sorting module for microfluidic sorting.

[0033] Preferably, the oil phase used in the droplet generation module is HFE-7500 fluorinated fluid containing 2.0%-5% Pico-Surf surfactant.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention ensures that the strains are independent of each other and do not affect each other during the culture and fermentation process by encapsulating the microdroplets first and then incubating them.

[0036] 2. The present invention realizes high-throughput screening of micro-droplets based on SERS, with a throughput of up to 180 per second, which is more than two orders of magnitude higher than other existing single-cell sorting technologies based on Raman spectroscopy.

[0037] 3. The present invention improves the throughput while still ensuring the sorting accuracy (>98%).

[0038] 4. The screening system and method of the present invention adopts a droplet microfluidic system, which greatly reduces damage to cells and facilitates downstream processing after sorting.

[0039] In summary, the present invention provides a SERS-based micro-droplet high-throughput screening system and a corresponding sorting method, which solves the problem of high-throughput sorting that was previously difficult to perform, and has the advantages of being simple and feasible, having a wide range of applications, and having strong scalability. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0041] Figure 1 4 is a structural diagram of a SERS optical fiber probe according to an embodiment of the present invention.

[0042] Figure 2 The figure is a flow chart of preparing a SERS optical fiber probe according to an embodiment of the present invention.

[0043] Figure 3 4 is a structural diagram of a micro-droplet high-throughput screening system according to an embodiment of the present invention.

[0044] Figure 4 4 is a structural diagram of a SERS droplet sorting module according to an embodiment of the present invention. DETAILED DESCRIPTION

[0045] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0046] In the description of the present invention, unless otherwise specified, "plurality" means two or more than two; the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right", "inner", "outer", "front end", "rear end", "head", "tail", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0047] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0048] The present invention will now be further described with reference to the accompanying drawings.

[0049] Embodiment 1:

[0050] Figure 1 FIG. 1 is a structural diagram of a SERS optical fiber probe according to an embodiment of the present invention. The present invention provides a SERS optical fiber probe, such as Figure 1 As shown, the SERS fiber optic probe 100 includes a tapered fiber optic probe 101 , one end of which is connected to a tapered structure 102 , and a layer of metal nanoparticles 103 is deposited on the surface of the tapered structure 102 as a SERS substrate.

[0051] In some embodiments, the metal nanoparticles are gold nanoparticles, silver nanoparticles, or a composite of gold nanoparticles and silver nanoparticles.

[0052] In some embodiments, the SERS substrate is formed by self-assembly of metal nanoparticles.

[0053] In some embodiments, the tapered optical fiber probe 101 is etched by hydrofluoric acid to form a tapered structure 102 at one end of the tapered optical fiber probe 101 .

[0054] Embodiment 2:

[0055] Figure 2 FIG. 4 is a flow chart of preparing a SERS optical fiber probe according to an embodiment of the present invention. Figure 2 As shown, the embodiment of the present invention provides a preparation method as described in any embodiment of embodiment 1, which specifically comprises the following steps:

[0056] S201. Fiber pretreatment: First, use a stripping tool to cut the optical fiber into multiple sections of 15 cm in length, and use Miller pliers to strip the coating layer of about 5 cm at one end of each section of optical fiber. Then, use a cotton ball dipped in anhydrous ethanol to clean the end of the optical fiber from which the protective layer has been stripped to remove residual impurities. After cleaning, store the optical fiber in a clean and dust-free container for subsequent use.

[0057] S202, preparation of tapered optical fiber: The tapered optical fiber is prepared by chemical etching. Add 40wt% hydrofluoric acid solution and dimethyl silicone oil (an isolation layer to prevent hydrofluoric acid from volatilizing) into a plastic beaker. After the solution is layered, install the pretreated optical fiber on an iron stand and keep it vertically inserted into the solution in the beaker. After 20 minutes, take out the tapered optical fiber, rinse it with ethanol and deionized water several times, and carefully store it for later use.

[0058] S203, silanization of the optical fiber end face: prepare 5uL / mL 3-aminopropyltriethoxysilane (3-APTS) aqueous solution, fully immerse the treated optical fiber probe end in the 3-APTS aqueous solution, let it stand for 30 minutes and then take it out to obtain the optical fiber modified with the silane coupling agent.

[0059] S204, assembly of optical fiber and metal nanomaterials: After the end face of the optical fiber is silanized, it is immediately inserted into the silver nanoparticle dispersion and left to stand for 12 hours. Do not take it out during this process to avoid contact with air that affects the assembly effect. Through chemical coupling, the silver nanoparticles will be firmly attached to the surface of the optical fiber. After the assembly process is completed, the optical fiber is thoroughly cleaned with anhydrous ethanol to remove unbound nanoparticles, and then the optical fiber is dried. The obtained SERS optical fiber probe should be carefully stored for later use.

[0060] Embodiment 3:

[0061] The embodiment of the present invention provides a detection method based on the SERS optical fiber probe as described in Embodiment 1, the detection method comprising:

[0062] S301, comparing the Raman spectrum of the target compound with the SERS spectrum of the standard solution simulating the microdroplets, and determining the Raman characteristic peaks that match the detection signal.

[0063] Exemplarily, the Raman laser signal comparison method used in SERS detection is selected from one or more of the following combinations: single peak comparison, multi-peak comparison, and full spectrum comparison.

[0064] It should be noted that the Raman spectrum of the target compound is the standard Raman spectrum of the target compound solution, and the data comes from the published Raman spectrum database. The "standard solution simulating microdroplets" is to prepare the standard solution of the target compound into microdroplets, which are input into the sorting system for early Raman detection and data analysis. The "Raman characteristic peak that conforms to the detection signal" refers to the Raman characteristic peak of the target compound structure, and the relative Raman intensity of the characteristic peak conforms to the concentration-intensity correspondence, and data analysis can be performed. Taking the Raman spectrum of succinic acid solution as an example, the Raman characteristic peak of succinic acid is 1285cm -1The carbon-oxygen double bond absorption peak near the succinic acid has the largest relative intensity in the entire spectrum, and there is a corresponding concentration-Raman intensity linear relationship between the peak intensity and the succinic acid concentration, which facilitates the conversion of the characteristic Raman absorption peak intensity of the target compound succinic acid into the succinic acid concentration for analysis and calculation.

[0065] S302, making micro-droplets of target compound standard solutions of different concentrations, and determining the corresponding relationship between the characteristic peak intensity and the target compound concentration by using the linear relationship between the relative Raman intensity of the Raman characteristic peak of the Raman spectrum of the micro-droplets of each concentration and the concentration, wherein the characteristic peak intensity is the relative Raman intensity of the Raman characteristic peak of the Raman spectrum.

[0066] In this embodiment, the correspondence between the determined characteristic peak intensity and the concentration of the target compound can be applied in the subsequent detection of the concentration of the target compound in other solutions. For example, a certain micro-droplet A contains a target compound, which is detected using a SERS fiber optic probe and a Raman system. The SERS fiber optic probe can obtain a SERS spectrum, and the Raman system can obtain a Raman spectrum. Based on the SERS spectrum and the Raman spectrum, the Raman characteristic peak can be determined, and then the characteristic peak intensity can be obtained. Under the known characteristic peak intensity, the concentration of the target compound in the micro-droplet A can be determined based on the correspondence between the characteristic peak intensity and the target compound concentration.

[0067] Embodiment 4:

[0068] Figure 3 FIG. 1 is a structural diagram of a micro-droplet high-throughput screening system according to an embodiment of the present invention. The embodiment of the present invention provides a micro-droplet high-throughput screening system, such as Figure 1 As shown, the microdroplet high-throughput screening system includes:

[0069] A droplet generation module 401 is used to encapsulate a single cell in a bacterial solution into a first micro-droplet;

[0070] A droplet incubation module 402 is used for incubating and fermenting the first micro-droplet to obtain a second micro-droplet;

[0071] The SERS droplet sorting module 403 is used for high-throughput screening of target strains.

[0072] Figure 4 The structure diagram of the SERS droplet sorting module according to the embodiment of the present invention is shown in FIG. Figure 3 and Figure 4As shown, the SERS droplet sorting module 403 includes a micro-droplet sorting chip 4031, a Raman system 4032 and the SERS optical fiber probe 100 as described in Example 1, the micro-droplet sorting chip 4031 is provided with a liquid flow channel 4033, a first oil phase channel 4034, a second oil phase channel 4035, a sorting channel 4036, a target channel 4037 and a waste channel 4038, the liquid flow channel 4033 is used to access the second micro-droplet, the first oil phase channel 4034 and the second oil phase channel 4035 are both used to access the oil phase, the two ends of the sorting channel 4036 are respectively a first end and a second end, and the liquid flow channel 4033, the first oil phase channel 4034, the second oil phase channel 4035 are connected to the oil phase. The channels 4035 are all arranged at the first end to pass the second micro-droplets and the oil phase into the sorting channel 4036. The target channel 4037 and the waste channel 4038 are all arranged at the second end. The SERS fiber optic probe 100 is arranged in the sorting channel 4036 to monitor the second micro-droplets in the sorting channel 4036 to obtain a SERS spectrum. The Raman system 4032 is arranged corresponding to the position of the sorting channel 4036 to obtain the Raman spectrum of the second micro-droplets in the sorting channel 4036. A sorting power unit 4039 is arranged corresponding to the second end, and the sorting power unit 4039 is used to control the flow direction of the second micro-droplets in the sorting channel 4036.

[0073] It should be noted that the oil phase introduced through the first oil phase channel 4034 and the second oil phase channel 4035 is the same as the oil phase introduced into the droplet generation module 401, and its function is to control the speed of the droplets in the sorting module passing through the SERS optical fiber probe to ensure that the droplets pass the detection completely independently. The oil phase introduced into the droplet generation module 401 will be specifically mentioned in the subsequent embodiments.

[0074] In some embodiments, the SERS droplet sorting module 403 also includes a control module, and the signal output ends of the Raman system 4032 and the SERS optical fiber probe 100 are connected to the signal input end of the control module, and the signal output end of the control module is connected to the signal input end of the sorting power unit 4039. The control module is used to: compare the SERS spectrum and the Raman spectrum fed by the Raman system and the SERS optical fiber probe to determine the Raman characteristic peak; determine the concentration of the second micro-droplets in the sorting channel based on the correspondence between the characteristic peak intensity and the target compound concentration determined by the detection method described in Example 3; based on the set concentration threshold, when the concentration of the second micro-droplets is greater than or equal to the concentration threshold, control the second micro-droplets to flow to the target channel through the sorting power unit, and when the concentration of the second micro-droplets is less than the concentration threshold, control the second micro-droplets to flow to the waste channel through the sorting power unit.

[0075] It should be noted that the control module is an existing device capable of data processing, such as a single chip microcomputer. Considering that the built-in chip in the existing Raman system enables it to have a data analysis function, the chip in the Raman system can be used as the control module. Of course, the control module can also be installed in the Raman system, that is, a control chip is added to the Raman system, and the control chip is used to execute the flow control process described above.

[0076] In some embodiments, Figure 3 As shown, the droplet generation module 401 includes a droplet preparation chip 4011, on which an internal phase flow channel 4012, a first external phase flow channel 4013, a second external phase flow channel 4014 and a mixed flow channel 4015 are arranged, and the internal phase flow channel 4012, the first external phase flow channel 4013, and the second external phase flow channel 4014 are all arranged at one end of the mixed flow channel 4015, and the other end of the mixed flow channel 4015 is connected to the droplet incubation module 402; wherein, the internal phase flow channel 4012 is used to pass the bacterial liquid into the mixed flow channel 4015, and the first external phase flow channel 4013 and the second external phase flow channel 4014 are used to pass the oil phase into the mixed flow channel 4014.

[0077] The droplet generation module 401 is used to generate a first droplet. The droplet generation module 401 may also include a fluid injection component, wherein three fluid injection components are provided, respectively connected to the inner phase flow channel 4012, the first outer phase flow channel 4013, and the second outer phase flow channel 4014, so as to input bacterial liquid into the inner phase flow channel 4012 and inject the carrier external phase into the first outer phase flow channel 4013 and the second outer phase flow channel 4014. As an example only, the fluid injection component includes a syringe pump and a liquid inlet syringe, wherein the liquid inlet syringe is used to absorb the liquid to be input. Taking the input of bacterial liquid as an example, the liquid inlet syringe first absorbs a certain amount of bacterial liquid, and then the liquid inlet syringe containing the bacterial liquid is connected to the syringe pump through a pipeline, and the syringe pump is connected to the inner phase flow channel 4012, and the working parameters (such as the rotation speed) of the syringe pump are controlled to make the bacterial liquid in the liquid inlet syringe flow into the mixed flow channel 4015 at a set flow rate. The method of injecting the carrier external phase into the mixing channel 4015 through the first external phase flow channel 4013 and the second external phase flow channel 4014 using the liquid injection assembly is the same as the method of injecting the bacterial solution, which will not be repeated here.

[0078] In one embodiment, the droplet generation module 401 also includes a microscope. During the droplet generation process, the droplet preparation chip 4011 can be placed on the operating table of the microscope to observe the droplet generation state, and the flow rate of the internal phase (such as bacterial liquid) and the carrier external phase can be controlled to ensure that the generated micro droplets meet the requirements.

[0079] In this embodiment, the microscope can be a WMJ-9590 metallographic microscope with a high-precision CCD, which can accurately collect image information of micro-droplets.

[0080] In one embodiment, the carrier oil phase used in the droplet generation module 401 is HFE-7500 fluorinated fluid (3M) containing 2.0% (v / v) Pico-Surf surfactant (5% in HFE-7500, Sphere Fluidics).

[0081] In one embodiment, the oil phase flow rate used in the droplet generation module 401 is 8-12 μL / min, and the water phase bacterial liquid flow rate is 3-5 μL / min. Under this flow rate control, the water-in-oil droplets generated by the droplet generation module contain no more than one single cell / particle per droplet on average.

[0082] In one embodiment, a microbial mutant library is constructed to provide bacterial liquid to the droplet generation module 401 , and the aqueous phase used in the droplet generation module 401 may be a diluted microbial mutant library, wherein the liquid used for dilution includes a microbial culture medium, a fermentation substrate, and an inducer.

[0083] The microorganisms used in the microbial mutant library may be yeast and / or Escherichia coli, and more preferably organic acid-producing Escherichia coli. The microdroplet high-throughput screening system obtains an engineered strain of Escherichia coli with high organic acid production by screening signals.

[0084] In one embodiment, the oil phase flow rate used in the droplet generation module 401 is 10 μL / min, and the water phase bacterial liquid flow rate is 4 μL / min. Under this flow rate control, the oil-in-water droplets generated by the droplet generation module contain an average of 0.5 single cells / particles in each droplet.

[0085] In one embodiment, the diameter of the water-in-oil droplets generated by the droplet generation module 401 is 50 μm.

[0086] In some embodiments, Figure 4 As shown, the micro-droplet high-throughput screening system also includes a target droplet collection unit 404 and a waste droplet collection unit 405 . The target droplet collection unit 404 is used to collect droplets flowing out of the target channel 4037 , and the waste droplet collection unit 405 is used to collect droplets flowing out of the waste channel 4038 .

[0087] In some embodiments, the sorting power unit 4039 performs droplet sorting by any of the following methods: dielectrophoresis, ultrasound, electric field, magnetic field, pressure squeezing or pressure suction.

[0088] Exemplarily, the separation power unit 4039 includes at least two dielectric electrodes to separate droplets by dielectrophoresis. Of course, the separation power unit 4039 can also adopt other structures, such as using a solenoid valve as the separation power unit 4039, and the solenoid valve can be, for example, a solenoid three-way valve, etc., which is not specifically limited here.

[0089] In some embodiments, the droplet incubation module 402 uses a constant temperature static incubation, and the incubation conditions are 30-37° C. for 12-24 hours. Figure 4 The container shown in Figure 4 Two droplet incubation modules 402 are marked in order to show the changes of micro-droplets in the droplet incubation module 402 before and after incubation. The droplet generation module 401 generates micro-droplets, which are then incubated using the droplet incubation module 402. This ensures that the strains are independent of each other and do not affect each other during the culture and fermentation process.

[0090] Embodiment 5:

[0091] The embodiment of the present invention provides a microdroplet high-throughput screening method, based on the microdroplet high-throughput screening system as described in any embodiment of embodiment 4, the method comprises the following steps:

[0092] S1. Strain culture: Escherichia coli DC1515 is a strain in which the genes of lactate dehydrogenase (ldhA), pyruvate formate lyase (plfA), and glucose transporter (ptsG) in wild-type Escherichia coli are knocked out. It has the potential to produce succinic acid by fermentation and was donated by Professor P. Clark of Southern Illinois University. After culturing DC1515 in LB liquid medium for 48 hours, the OD 600 =0.6 was subjected to atmospheric pressure room temperature plasma (ARTP) mutagenesis. Specifically, the bacterial solution was mixed with a 5% glycerol solution, 10 μL was applied on an iron sheet, the ARTP mutagenizer power was set to 120 W, the airflow was 10 SLM, and the mutagenesis was performed for 40 s. After the mutagenesis was completed, the induced mutant strain was inoculated into a fresh LB liquid culture medium, and cultured at a temperature of 37° C. for 10 h to obtain a seed solution of a succinic acid-producing strain mutant library, and diluted with a buffer to OD 600 =0.05-0.1 to obtain the culture solution of the mutant library of succinic acid-producing strains. Buffer composition: fresh LB medium, 100ug / mL ampicillin sodium, 15g / L glucose, 0.5mM IPTG, 15g / L NaHCO3.

[0093] S2, select fluorinated oil with HFE-7500 added (the content of Pico-Surf surfactant in the fluorinated oil is 5wt%) as the external phase, and use the culture solution obtained in step S1 as the internal phase; keep the droplet preparation chip at a constant temperature of 35°C, use a syringe to draw 1mL of the internal phase and the external phase respectively, the syringe of the external phase is installed on the external phase peristaltic pump, and is connected to the two oil phase injection ports of the droplet preparation chip through a polyethylene tube, and the external phase is input into the chip at a flow rate of 10μL / min; the syringe of the internal phase is installed on the internal phase peristaltic pump, and is connected to a water phase injection port of the droplet preparation chip through a polyethylene tube, and the internal phase is input into the chip at a flow rate of 4μL / min. When the external phase meets the internal phase fluid, due to the combined effect of shear force and surface tension, the internal phase fluid is stretched and eventually broken to form monodisperse emulsion microdroplets.

[0094] S3. The generated microdroplets were collected in anaerobic tubes and cultured anaerobically in a vertical incubator at 37°C for 12 h without stirring.

[0095] S4. Pump the microdroplets into the SERS droplet sorting module for microfluidic sorting. When the droplets flow through, the optical fiber automatically penetrates into the microdroplets for measurement. One microdroplet is measured at a time, and the Raman spectrum information of each droplet is collected. Voltage is applied to sort the microdroplets with the highest relative Raman intensity of the characteristic peak, so that they flow into the collection channel.

[0096] A batch of micro-droplets with high signal intensity were screened out, and shake flask fermentation was performed to screen out high-yield succinic acid strains. The highest succinic acid yield among the screened high-yield succinic acid strains was 13.16 g / L, which was 34% higher than the starting strain (the original strain was 9.82 g / L). It was verified that the accuracy of strain screening by the SERS droplet sorting module in this embodiment can reach more than 98%, and the screening speed is about 180 per second.

[0097] Embodiment 6:

[0098] High-throughput screening of high-yielding succinate strains was performed according to the method of Example 5, except that the ARTP mutagenesis used in step S1 was replaced by error-prone PCR random mutagenesis: phosphoenolpyruvate carboxylase, a key enzyme in the succinate biosynthesis process, was used as the mutation target, and a gene mutant library was constructed using error-prone PCR, which was then introduced into the host bacterium DC1515 to obtain a strain mutant library.

[0099] A batch of microdroplets with higher signal intensity were screened out, and shake flask fermentation was performed to screen out high-yielding succinic acid strains. The highest succinic acid yield among the screened high-yielding succinic acid strains was 32.06 g / L, which was 226% higher than that of the starting strain.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and should all be included in the scope of the claims and specification of the present invention.

Claims

1. A SERS optical fiber probe, characterized in that: The method comprises a tapered optical fiber probe, one end of which is connected to a tapered structure, and a layer of metal nanoparticles is deposited on the surface of the tapered structure as a SERS substrate.

2. The SERS optical fiber probe according to claim 1, characterized in that: The metal nanoparticles are gold nanoparticles, silver nanoparticles or a composite of gold nanoparticles and silver nanoparticles.

3. A method for preparing a SERS optical fiber probe as claimed in any one of claims 1 to 2, characterized in that: include: The tapered optical fiber probe is etched with hydrofluoric acid to form a tapered structure at one end of the tapered optical fiber probe, and a layer of metal nanoparticles is deposited on the surface of the tapered structure as a SERS substrate.

4. A detection method based on the SERS optical fiber probe according to any one of claims 1 to 2, characterized in that: include: Compare the Raman spectrum of the target compound with the SERS spectrum of the standard solution simulating microdroplets to determine the Raman characteristic peaks that match the detection signal; Standard solutions of target compounds of different concentrations are made into microdroplets, and the corresponding relationship between the characteristic peak intensity and the concentration of the target compound is determined by using the linear relationship between the relative Raman intensity of the Raman characteristic peak of the Raman spectrum of the microdroplets of each concentration and the concentration. The characteristic peak intensity is the relative Raman intensity of the Raman characteristic peak of the Raman spectrum.

5. A microdroplet high-throughput screening system, characterized in that: include: A droplet generation module, used for encapsulating a single cell in a bacterial solution into a first micro-droplet; A droplet incubation module, used for incubating and fermenting the first micro-droplet to obtain a second micro-droplet; A SERS droplet sorting module is used for high-throughput screening of target strains; the SERS droplet sorting module comprises a micro-droplet sorting chip, a Raman system and a SERS optical fiber probe as described in any one of claims 1 to 2, the micro-droplet sorting chip is provided with a liquid flow channel, a first oil phase channel, a second oil phase channel, a sorting channel, a target channel and a waste channel, the liquid flow channel is used to access the second micro-droplet, the first oil phase channel and the second oil phase channel are both used to access the oil phase, the two ends of the sorting channel are respectively a first end and a second end, the liquid flow channel, the first oil phase channel , and the second oil phase channel are both arranged at the first end to pass the second micro-droplets and the oil phase into the sorting channel. The target channel and the waste channel are both arranged at the second end. The SERS optical fiber probe is arranged in the sorting channel to monitor the second micro-droplets in the sorting channel to obtain a SERS spectrum. The Raman system is arranged corresponding to the position of the sorting channel to obtain the Raman spectrum of the second micro-droplets in the sorting channel. A sorting power unit is arranged corresponding to the second end, and the sorting power unit is used to control the flow direction of the second micro-droplets in the sorting channel.

6. The microdroplet high-throughput screening system according to claim 5, characterized in that: The SERS droplet sorting module further comprises a control module, the signal output ends of the Raman system and the SERS optical fiber probe are connected to the signal input end of the control module, the signal output end of the control module is connected to the signal input end of the sorting power unit, and the control module is used to: Comparing the SERS spectra and the Raman spectra fed by the Raman system and the SERS optical fiber probe to determine the Raman characteristic peaks; Determining the concentration of the second micro-droplet in the sorting channel based on the corresponding relationship between the characteristic peak intensity and the concentration of the target compound determined by the detection method as claimed in claim 4; Based on the set concentration threshold, when the concentration of the second micro-droplets is greater than or equal to the concentration threshold, the second micro-droplets are controlled to flow to the target channel by the sorting power unit; when the concentration of the second micro-droplets is less than the concentration threshold, the second micro-droplets are controlled to flow to the waste channel by the sorting power unit.

7. The microdroplet high-throughput screening system according to claim 5, characterized in that: The droplet generation module includes a droplet preparation chip, on which an inner phase flow channel, a first outer phase flow channel, a second outer phase flow channel and a mixed flow channel are arranged, the inner phase flow channel, the first outer phase flow channel and the second outer phase flow channel are all arranged at one end of the mixed flow channel, and the other end of the mixed flow channel is connected to the droplet incubation module; wherein the inner phase flow channel is used to introduce bacterial liquid into the mixed flow channel, and the first outer phase flow channel and the second outer phase flow channel are used to introduce oil phase into the mixed flow channel.

8. The micro-droplet high-throughput screening system according to claim 5, characterized in that: It also includes a target droplet collection unit and a waste droplet collection unit. The target droplet collection unit is used to collect droplets flowing out of the target channel, and the waste droplet collection unit is used to collect droplets flowing out of the waste channel.

9. A microdroplet high-throughput screening method, characterized in that: Based on the microdroplet high-throughput screening system according to any one of claims 5 to 8, the method comprises: Establish a microbial mutant library; Inputting the bacterial liquid in the microbial mutant library into a droplet generation module to prepare a plurality of first micro droplets; Incubate and ferment the plurality of first micro-droplets in a droplet incubation module to obtain second micro-droplets; The second micro-droplet is then injected into the SERS droplet sorting module for microfluidic sorting.

10. The microdroplet high-throughput screening method according to claim 9, characterized in that: The oil phase used in the droplet generation module is HFE-7500 fluorinated fluid containing 2.0%-5% Pico-Surf surfactant.

Images