Micro-organism in-situ sorting device and method for deep sea, equipment and medium

Abstract

The invention provides a miniature organism in-situ sorting device and method for deep sea, equipment and a medium. The device comprises a dry cabin module, a wet cabin module, a transition block module and a pressure buffer, the dry cabin module is used for detecting, collecting, analyzing, processing and controlling optical signals; the wet cabin module is used for collecting seawater samples, focusing sound waves and standing waves and sorting target microorganisms; the transition block module is used for transmitting electric signals and optical signals of the dry cabin and the wet cabin; the pressure buffer is used for compensating the pressure in the wet cabin, so that the pressure in the wet cabin is equal to the in-situ pressure of the deep sea. The problems that in-situ microbiological analysis and sorting are difficult to carry out due to the severe deep sea environment and the in-situ analysis and sorting flux is low can be solved; based on the structural design that the dry cabin and the wet cabin are separated and the wet cabin pressure compensation technology, the sorting device is suitable for a deep-sea in-situ high hydrostatic pressure environment, and based on the design of a flow control cell sorting chip of an acoustic wave electrophoresis or acoustic wave standing wave method, in-situ high-throughput analysis and sorting of deep-sea microorganisms are effectively achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of micro-organism sampling, identification and sorting, and in particular to an in-situ micro-organism sorting device and method, equipment and medium for deep-sea micro-organisms. Background Art

[0002] The deep sea usually refers to an area with a water depth of more than 1,000 meters, which has extreme environmental characteristics such as darkness, high hydrostatic pressure, high salt, and low temperature. At the same time, the deep sea also has a very complex ecological environment and rich biodiversity. The study of deep-sea microorganisms can not only reveal the physiological metabolism and dynamic changes of various deep-sea microorganisms, but also contribute to the development and utilization of deep-sea biological resources. Its unique physiological mechanisms and metabolites have great application prospects in the fields of pharmaceutical development, gene therapy, food processing, chemical industry, and environmental protection. Traditional sampling methods such as water sample bottles or in-situ pressure maintenance will have a great impact on deep-sea microbial samples, such as causing rapid growth or stress changes in microorganisms, and even leading to the loss of in-situ gene expression information. These changes limit the study of the life process of microorganisms and the distribution of their biological populations in extreme deep-sea environments. In addition, traditional sampling methods can only obtain observation data at a certain depth and a certain time point in the ocean, which cannot meet the research on the dynamic laws of deep-sea ecosystems. Therefore, the research on in-situ detection methods or device development of deep-sea microorganisms has become one of the most cutting-edge fields in the current deep-sea ecosystem research.

[0003] The in-situ sorting instrument for microorganisms in deep-sea environments based on the principle of Raman spectroscopy proposed in relevant patents or literature can be used for in-situ identification and sorting of deep-sea microorganisms. However, due to the limitations of Raman spectroscopy detection and the principle of "optical tweezers", the device has a low throughput for identification or sorting of microorganisms, usually at the level of 1 microorganism per minute, or even lower, which makes it difficult to meet the needs of detecting or sorting a large number of microorganisms in the deep sea. In addition, the method or device has very high requirements for the stability of water flow in the deep-sea environment. External fluid disturbances will seriously affect the identification or sorting performance of the instrument, making it difficult to apply to complex hot springs or cold springs in the deep-sea environment and their nearby locations. Summary of the invention

[0004] In order to achieve the above-mentioned purpose and other advantages of the present invention, the first purpose of the present invention is to provide a micro-organism in-situ sorting device for deep sea, comprising a dry tank module, a wet tank module, a transition block module and a pressure buffer; wherein,

[0005] The dry cabin module is used for optical signal detection, collection, analysis, processing and control;

[0006] The wet tank module is used for collecting seawater samples, focusing acoustic standing waves and sorting target microorganisms;

[0007] The transition block module is used for transmitting electrical signals and optical signals between the dry tank module and the wet tank module;

[0008] The pressure buffer is used to compensate for the pressure in the wet tank module so that the pressure in the wet tank module is equal to the pressure in the deep sea.

[0009] Furthermore, the dry cabin module includes an electronics system, an optical system and a first cabin penetration joint;

[0010] The first penetration connector includes an input power line and a communication line for realizing power management and communication of the device;

[0011] The electronics system is used to collect and process signals in the optical system, and to control the wet tank module through the transition block module.

[0012] Furthermore, the transition block module includes a second through-cabin joint, a third through-cabin joint and an optical glass slide. The electronics system is connected to the wet cabin module through the second through-cabin joint and the third through-cabin joint to achieve control of the wet cabin module. The optical glass slide is used to transmit the optical signal of the optical system.

[0013] Furthermore, the electronic system includes a power control board, a main control board, a data acquisition board, a signal generator, and a liquid flow control board;

[0014] The power control board is connected to the input power line of the first penetration connector and is used for power management and power distribution of the device;

[0015] The main control board performs data exchange with the data acquisition board, the signal generator, and the liquid flow control board, and is used for control, data analysis, and processing of the data acquisition board, the signal generator, and the liquid flow control board;

[0016] The main control board is connected to the communication line of the first through-cabin connector for communication and control of the device by the outside world;

[0017] The data acquisition board is used to collect and process the detection signal of the optical system;

[0018] The signal generator is used to control each transducer on the microfluidic chip in the wet tank module through the third tank penetration connector;

[0019] The liquid flow control panel is used to control the fluid system in the wet tank module through the second tank penetration joint.

[0020] Further, the optical system includes an excitation light path and a first collection light path;

[0021] The excitation light path is used to shape the excitation light and transmit it to the detection position of the microfluidic chip in the wet chamber module to perform fluorescence excitation on the microorganisms;

[0022] The first collecting light path is used for focusing and imaging the excitation light of the microorganism;

[0023] The optical system comprises an excitation light source, a first mirror group, a first dichroic mirror, an objective lens, a light-transmitting sheet, a second dichroic mirror, a second mirror group, a third mirror group, a first detector, and a second detector;

[0024] The excitation light source, the first mirror group, the first dichroic mirror, the objective lens, and the light-transmitting sheet form an excitation light path; the objective lens is used to focus and shape the excitation light source in the excitation light path;

[0025] The light-transmitting sheet, the objective lens, the first dichroic mirror, the second dichroic mirror, the second lens group, the third lens group, the first detector, and the second detector form a first light collecting path; the objective lens is used to focus and image the excitation light of the microorganisms in the first light collecting path, and the data acquisition board is connected to the first detector and the second detector in the optical system to collect and process the signals of the first detector and the second detector.

[0026] Furthermore, the wet cabin module includes a microfluidic chip, a focusing / sorting amplifier board, a fluid system, a fourth through-cabin connector and a fifth through-cabin connector;

[0027] The signal generator is connected to the focusing / sorting power amplifier board through the third through-chamber connector to control each transducer on the microfluidic chip;

[0028] The liquid flow control panel is connected to each pump and solenoid valve of the fluid system through the second through-tank joint to control the fluid system;

[0029] The pressure buffer is connected to the fourth penetration joint to compensate for the pressure in the wet tank;

[0030] The microfluidic chip interacts with the outside world through the fifth through-chamber connector.

[0031] Furthermore, the microfluidic chip adopts a glass-to-glass bonding method to form a microchannel for sample flow; wherein the microchannel includes a sample inlet, a first outlet, a second outlet and a third outlet, and the sample inlet and each outlet interact with the outside world through the fifth through-chamber joint.

[0032] Furthermore, the microfluidic chip is glued with a first transducer, a second transducer, and a third transducer 3, and each transducer adopts a piezoelectric ceramic material and an ultrasonic standing wave method.

[0033] Furthermore, the focusing / sorting power amplifier board includes a first power amplifier board, a second power amplifier board, and a third power amplifier board; wherein, the first transducer and the second transducer respectively realize three-dimensional focusing of particles in the sample flow in the microchannel under the action of the first power amplifier board and the second power amplifier board; the third transducer realizes the displacement of target particles in the sample flow and flows them into the first outlet or the second outlet under the action of the third power amplifier board.

[0034] Furthermore, the fluid system includes a pump body, a solenoid valve body, a fluid bag and a fluid pipeline; wherein the pump body includes a first pump, a second pump and a third pump, and the first pump, the second pump and the third pump are respectively used for extracting or delivering external sample flows, reagents and mixed samples; the fluid bag includes a sample bag and a reagent bag, the sample bag is used to store a mixed solution of an external sample flow and a reagent, and the reagent bag is used to store a staining reagent; the fluid pipeline is used for delivering sample flows or reagents; and the solenoid valve body is used to control the flow direction of sample flows or reagents.

[0035] Furthermore, the wet chamber module also includes a second collecting light path, which includes a fourth lens group and a third detector. The fourth lens group is used to focus the forward scattered light, and the third detector is used to collect fluorescence signals. The data acquisition board is connected to the third detector and is used to collect and process the signal of the third detector.

[0036] Furthermore, the first cabin penetration joint, the second cabin penetration joint and the third cabin penetration joint are all pressure-bearing multi-core wire joints, and the fourth cabin penetration joint and the fifth cabin penetration joint are all pressure-bearing single-core or multi-core fluid joints.

[0037] Furthermore, the optical glass slide is sealed and fixed on the end face of the transition block module facing the wet tank module, and is made of a transparent material that can withstand hydrostatic high pressure, and is used to deliver the excitation light in the dry tank module to the detection area of ​​the microfluidic chip in the wet tank module, and at the same time deliver the stimulated light of the microparticles in the microchannel of the microfluidic chip in the wet tank module to the optical system of the dry tank module.

[0038] The second object of the present invention is to provide a method for in-situ sorting of deep-sea microorganisms, based on the above-mentioned device, comprising the following steps:

[0039] After the main control board of the dry cabin module is powered on, the parameters of each component in the device are initialized and set;

[0040] When executing the automatic sorting stage, the main control board sends an enable instruction to the signal generator of the dry chamber module, so that the signal generator transmits the pre-set pulse signals of different resonant frequencies and voltages to the first power amplifier board and the second power amplifier board respectively, so as to drive the first transducer and the second transducer on the microfluidic chip to realize three-dimensional focusing of the sample flow in the microchannel;

[0041] By judging whether the intensity of the FSC pulse signal obtained when the particles arranged in a single cell stream pass through the laser spot is higher than a threshold, the data of each channel of the particles can be automatically stored or the collected data can be discarded;

[0042] By analyzing whether the fluorescence channel pulse signal intensity of the particle is within the gate size range, it is determined whether to execute the sorting trigger instruction;

[0043] If the sorting trigger instruction is executed, the sorting enable of the signal generator is controlled according to the set sorting delay and sorting pulse width, so that the signal generator sends a pulse signal of a specific duration to the third power amplifier board at a specified time, which is used to drive the third transducer on the microfluidic chip to achieve the sorting of target particles.

[0044] A third object of the present invention is to provide a computer device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the steps of the above method when executing the computer program.

[0045] A fourth object of the present invention is to provide a computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the steps of the above method when executed by a processor.

[0046] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0047] The present invention provides a fully automated deep-sea microorganism in-situ sorting device and method based on microfluidic technology, which can overcome the problem of the existing technology that the deep-sea environment is harsh and it is difficult to carry out in-situ microbial analysis and sorting, as well as the problem of low in-situ analysis and sorting throughput. The present invention is based on the structural design of separation of dry and wet tanks, and the wet tank pressure compensation technology, which is conducive to the sorting device being suitable for the high hydrostatic pressure environment in the deep sea, and the design of the flow control cell sorting chip based on the acoustic electrophoresis or acoustic standing wave method, effectively realizing the in-situ high-throughput analysis and sorting of deep-sea microorganisms.

[0048] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0050] Figure 1 This is a functional framework diagram of a micro-organism in-situ sorting device for deep sea;

[0051] Figure 2 This is a functional schematic diagram of a micro-organism in-situ sorting device for deep sea use;

[0052] Figure 3 This is a structural diagram of the shell of a micro-organism in-situ sorting device used in the deep sea;

[0053] Figure 4 This is a cross-sectional view of a micro-organism in-situ sorting device used in the deep sea;

[0054] Figure 5 This is a diagram of the internal structure of a micro-organism in-situ sorting device used in the deep sea;

[0055] Figure 6 Flow chart of the in situ sorting method for deep-sea microorganisms;

[0056] Figure 7 It is a schematic diagram of computer equipment;

[0057] Figure 8 A schematic diagram of a computer-readable storage medium.

[0058] In the figure: 1. Dry tank module; 2. Wet tank module; 3. Transition block; 4. Dry tank shell; 5. Dry tank end cover; 6. Wet tank shell; 7. Wet tank end cover; 8. Soft gasket. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0060] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0061] The figure numbers in this application are only used to distinguish the various steps in the scheme, and are not used to limit the execution order of the various steps. The specific execution order is subject to the description in the specification.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0063] Example 1

[0064] A micro-organism in-situ sorting device for deep sea, such as Figure 1 As shown, it includes a dry tank module, a wet tank module, a transition block module and a pressure buffer; wherein,

[0065] The dry cabin module is used for optical signal detection, collection, analysis, processing and control;

[0066] The wet tank module is used for collecting seawater samples, focusing acoustic standing waves and sorting target microorganisms;

[0067] The transition block module is used for transmitting electrical signals and optical signals between the dry tank module and the wet tank module;

[0068] The pressure buffer is used to compensate for the pressure in the wet tank module so that the pressure in the wet tank module is equal to the pressure in the deep sea.

[0069] In some embodiments, the dry tank module includes an electronics system, an optical system and a first penetration connector (i.e. Figure 1 Middle penetration joint 1);

[0070] The first penetration connector includes an input power line and a communication line for realizing power management and communication of the device;

[0071] The electronics system is used to collect and process signals in the optical system, and to control the wet tank module through the transition block module.

[0072] In some embodiments, the transition block module includes a second penetration joint (ie Figure 1 Middle penetration joint 4), the third penetration joint (i.e. Figure 1 The electronic system is connected to the wet cabin module through the second and third through-cabin joints to realize the control of the wet cabin module, and the optical glass is used to transmit the optical signal of the optical system.

[0073] In some embodiments, the electronics system includes a power control board, a main control board, a data acquisition board, a signal generator, and a liquid flow control board;

[0074] The power control board is connected to the input power line of the first penetration connector and is used for power management and power distribution of the device;

[0075] The main control board performs data exchange with the data acquisition board, the signal generator, and the liquid flow control board, and is used for control, data analysis, and processing of the data acquisition board, the signal generator, and the liquid flow control board;

[0076] The main control board is connected to the communication line of the first through-cabin connector for communication and control of the device by the outside world;

[0077] The data acquisition board is used to collect and process the detection signals of the optical system; specifically, the data acquisition board is connected to each detector in the optical system, and after collecting and processing the signals of these detectors, it exchanges data with the main control board;

[0078] The signal generator is controlled by the main control board, and controls each transducer on the microfluidic chip in the wet tank module through the third through-tank connector; specifically, the signal generator is connected to the focusing / sorting amplifier board in the wet tank module through the third through-tank connector, and is used to control each transducer on the microfluidic chip.

[0079] The liquid flow control board is used to control the fluid system in the wet tank module through the second tank penetration joint; specifically, the liquid flow control board is connected to each pump and solenoid valve of the fluid system in the wet tank module through the second tank penetration joint to control the fluid system.

[0080] In some embodiments, the optical system includes an excitation light path and a first collection light path;

[0081] The excitation light path is used to shape the excitation light and transmit it to the detection position of the microfluidic chip in the wet chamber module to perform fluorescence excitation on the microorganisms;

[0082] The first collecting light path is used for focusing and imaging the excitation light of the micro-organisms.

[0083] like Figure 2 As shown, the optical system includes an excitation light source, a first mirror group ( Figure 2 Middle mirror group 4), first dichroic mirror ( Figure 2 Middle dichroic mirror 1), objective lens, light-transmitting sheet, second dichroic mirror ( Figure 2 Middle two-color mirror 2), the second mirror group ( Figure 2 Middle lens group 1), third lens group ( Figure 2 Middle mirror group 3), the first detector ( Figure 2 Middle detector 1), second detector ( Figure 2 Middle detector 3);

[0084] The excitation light source, the first mirror group, the first dichroic mirror, the objective lens, and the light-transmitting sheet form an excitation light path; the objective lens is used to focus and shape the excitation light source in the excitation light path;

[0085] The excitation light source is preferably a laser, and the wavelength of the laser is selected according to the dye used to stain the microorganism. For example, when SYBR Green dye is used to stain the microorganism, a laser with a wavelength of 488 nm can be used.

[0086] The light-transmitting sheet, the objective lens, the first dichroic mirror, the second dichroic mirror, the second lens group, the third lens group, the first detector, and the second detector form a first light collecting path; the objective lens is used to focus and image the excitation light of the microorganisms in the first light collecting path, and the data acquisition board is connected to the first detector and the second detector in the optical system to collect and process the signals of the first detector and the second detector.

[0087] The second lens group and the third lens group both use a combination of lenses and filters; wherein the lenses are used to focus excitation light of different wavelengths; and the filters are used to prevent excitation light of non-specific wavelengths from entering the first detector and the second detector. The first detector and the second detector both use photomultiplier tubes PMT or avalanche photodiodes APD to collect fluorescence signals.

[0088] In some embodiments, the wet cabin module includes a microfluidic chip, a focusing / sorting amplifier board, a fluid system, a fourth through-cabin connector (i.e. Figure 1 Middle penetration joint 2) and the fifth penetration joint (i.e. Figure 1 Middle penetration joint 3);

[0089] The signal generator is connected to the focusing / sorting power amplifier board through the third through-chamber connector to control each transducer on the microfluidic chip;

[0090] The liquid flow control panel is connected to each pump and solenoid valve of the fluid system through the second through-tank joint to control the fluid system;

[0091] The pressure buffer is connected to the fourth penetration joint to compensate for the pressure in the wet tank so that the pressure in the wet tank is equal to the original pressure in the deep sea;

[0092] The microfluidic chip interacts with the outside world through the fifth through-chamber connector.

[0093] like Figure 2 As shown, the microfluidic chip adopts a glass-to-glass bonding method to form a microchannel for sample flow; wherein the microchannel includes a sample inlet, a first outlet ( Figure 2Middle Exit 1), Second Exit ( Figure 2 Middle Exit 2) and the third exit ( Figure 2 The sample inlet and each outlet interact with the outside world through the fifth through-cabin joint.

[0094] The microfluidic chip is glued with a first transducer ( Figure 2 Middle transducer 1), second transducer ( Figure 2 Middle transducer 2), third transducer ( Figure 2 In the transducer 3), each transducer adopts piezoelectric ceramic material and ultrasonic standing wave method.

[0095] The focusing / sorting power amplifier board includes a first power amplifier board ( Figure 2 Middle power amplifier board 1), second power amplifier board ( Figure 2 Middle power amplifier board 2), third power amplifier board ( Figure 2 wherein, the first transducer and the second transducer respectively realize three-dimensional focusing of particles in the sample flow in the microchannel under the action of the first power amplifier board and the second power amplifier board; the third transducer realizes the displacement of target particles in the sample flow and flows them into the first outlet or the second outlet under the action of the third power amplifier board.

[0096] The fluid system includes a pump body, a solenoid valve body, a fluid bag and a fluid pipeline; wherein the pump body includes a first pump ( Figure 2 Middle pump 1), second pump ( Figure 2 Middle pump 2) and third pump ( Figure 2 The middle pump 3) may adopt a peristaltic pump or a plunger pump, the first pump, the second pump and the third pump are respectively used for extracting or delivering external sample flow, reagents and mixed samples; the fluid bags are all sterile soft plastic bags, including sample bags and reagent bags, the sample bags are used to store the mixed solution of external sample flow and reagents, and the reagent bags are used to store staining reagents; the fluid pipeline is a thick-walled hard pipe or a hose, which is used for delivering sample flow or reagents; the solenoid valve body is a plurality of dispersed solenoid valves or an integrated solenoid valve, which is used to control the flow direction of sample flow or reagents.

[0097] The wet cabin module also includes a second light collecting path, and the second light collecting path includes a fourth lens group ( Figure 2 Middle mirror group 2), the third detector ( Figure 2 The fourth mirror group is used to focus the forward scattered light, the third detector is used to collect the fluorescence signal, and the data acquisition board is connected to the third detector to collect the signal of the third detector and process it.

[0098] The fourth lens group is a combination of a lens and a filter; the lens is used to focus the forward scattered light; the filter is used to prevent the forward scattered light of non-specific wavelength band from entering the third detector. The third detector is a photodiode PD.

[0099] like Figure 3 , Figure 4 , Figure 5 As shown, the wet tank module 1 is fixed in the dry tank shell 4, and the wet tank module 2 is fixed in the wet tank shell 6. The first through-tank joint is fixed on the dry tank end cover 5, the second through-tank joint and the third through-tank joint are both fixed on the transition block 3, and the fourth through-tank joint and the fifth through-tank joint are both fixed on the wet tank end cover 7. Among them, the first through-tank joint, the second through-tank joint and the third through-tank joint are all pressure-bearing multi-core wire joints, and the fourth through-tank joint and the fifth through-tank joint are all pressure-bearing single-core or multi-core fluid joints.

[0100] The transition block 3 is made of titanium alloy. The dry tank end cover 5, dry tank shell 4, wet tank shell 6 and wet tank end cover 7 are made of aluminum alloy or titanium alloy. All shells in contact with seawater are sprayed with anti-corrosion coating.

[0101] The structural assembly process of the micro-organism in-situ sorting device for deep sea is as follows:

[0102] The dry tank module 1 and the wet tank module 2 are respectively fixed on two sides of the transition block 3;

[0103] The dry tank shell 4 is assembled with the transition block 3, and then the dry tank end cover 5 is assembled with the dry tank shell 4;

[0104] The wet tank shell 6 is assembled with the transition block 3 , and then the wet tank end cover 7 is assembled with the wet tank shell 6 .

[0105] Axial and radial soft gaskets 8 are installed on the dry tank end cover 5, the two side surfaces of the transition block 3 and the wet tank end cover 7.

[0106] The optical glass slide is sealed and fixed on the end face of the transition block 3 facing the wet chamber. It is made of a transparent material that can withstand hydrostatic high pressure, such as sapphire, and is used to deliver the excitation light in the dry chamber to the detection area of ​​the microfluidic chip in the wet chamber, and at the same time deliver the stimulated light of the particles in the microchannel of the microfluidic chip in the wet chamber to the optical system of the dry chamber.

[0107] The in situ sorting methods for deep-sea microorganisms are based on flow cytometry, e.g. Figure 2As shown, however, unlike the laboratory desktop flow detection platform, considering the in-situ working conditions of deep-sea detection, the microfluidic chip is installed in the wet chamber of the instrument, and the inlet and outlet of the chip are both located in the deep sea. The detection sample inside the chip is balanced with the hydrostatic pressure of the deep sea in situ; at the same time, the inside of the instrument wet chamber is filled with an insulating liquid medium, such as silicone oil, and a pressure buffer is connected to the end cover of the instrument wet chamber, so that the external pressure of all devices inside the wet chamber is balanced with the hydrostatic pressure of the deep sea in situ; based on the acoustic electrophoresis method, transducers 1 and 2 on the microfluidic chip are used to realize the focusing and "single-cell flow" arrangement of particles in the chip microchannel in three-dimensional space; then, the particles arranged in a single row will pass through the laser detection area in turn to identify and sort the target microorganisms; finally, the target microorganisms are screened and enriched under the ultrasonic standing wave of the piezoelectric transducer 3.

[0108] like Figure 3 As shown, the automatic sorting process of the sorting device is:

[0109] After the main control board is powered on, the initialization settings of the gain, FSC threshold, gate size, sorting delay, sorting pulse width and other parameters of PMTs and PD are completed. When executing the automatic sorting stage, the main control board sends an enable command to the signal generator. Then, the signal generator transmits the pre-set pulse signals of different resonant frequencies and voltages to the power amplifier board 1 and the power amplifier board 2 respectively to drive the piezoelectric transducer 1 and the transducer 2 on the microchip to achieve three-dimensional focusing of the sample flow in the microchannel.

[0110] When particles arranged in a "single cell stream" pass through the laser spot, FSC and fluorescence channel pulse signals are obtained at the same time. When the intensity of the acquired FSC pulse signal is higher than the threshold, the data of each channel of the particle will be automatically stored, otherwise, the collected data will be discarded.

[0111] The main control board will further analyze whether the intensity of the fluorescence channel pulse signal of the particle is within the range of the gate size to decide whether to execute the sorting trigger instruction. If sorting is executed, the main control board controls the sorting enable of the signal generator according to the set sorting delay and sorting pulse width. Then, the signal generator sends a pulse signal of a specific duration to the sorting amplifier board at a specified time to drive the piezoelectric transducer in the sorting area on the microchip, and finally achieve the sorting of the target particles.

[0112] This embodiment provides a micro-organism in-situ sorting device for deep-sea environment based on flow cytometry, dry and wet compartment separation structure design, acoustic standing wave focusing and sorting technology, and microfluidic technology, which can overcome the problem of the existing technology that the deep-sea environment is harsh and it is difficult to carry out in-situ microbial analysis and sorting, as well as the problem of low in-situ analysis and sorting throughput. The present invention is based on the structural design of the separation of the dry and wet compartments, and the wet compartment pressure compensation technology, which is conducive to the sorting device being suitable for the high hydrostatic pressure environment in the deep sea, and the design of the flow cell sorting chip based on the acoustic electrophoresis or acoustic standing wave method effectively realizes the in-situ high-throughput analysis and sorting of deep-sea microorganisms.

[0113] Example 2

[0114] A method for in-situ sorting of deep-sea microorganisms is provided, based on the above-mentioned device. For a detailed description of the device, reference can be made to the corresponding description in the above-mentioned device embodiment, which will not be repeated here. Figure 6 As shown, the method comprises the following steps:

[0115] After the main control board of the dry chamber module is powered on, the parameters of each component in the device are initialized; specifically, after the main control board is powered on, the initialization settings of the gain, FSC threshold, gate size, sorting delay, sorting pulse width and other parameters of the PMTs and PD are completed.

[0116] When executing the automatic sorting stage, the main control board sends an enable instruction to the signal generator of the dry chamber module, so that the signal generator transmits the pre-set pulse signals of different resonant frequencies and voltages to the first power amplifier board and the second power amplifier board respectively, so as to drive the first transducer and the second transducer on the microfluidic chip to realize three-dimensional focusing of the sample flow in the microchannel;

[0117] When particles arranged in a "single cell stream" pass through the laser spot, FSC and fluorescence channel pulse signals are acquired simultaneously.

[0118] By judging whether the intensity of the FSC pulse signal obtained when the particles arranged in a single-cell stream pass through the laser spot is higher than a threshold, the data of each channel of the particle can be automatically stored or the collected data can be discarded; specifically, when the intensity of the FSC pulse signal obtained is higher than the threshold, the data of each channel of the particle can be automatically stored, otherwise, the collected data will be discarded.

[0119] By analyzing whether the fluorescence channel pulse signal intensity of the particle is within the gate size range, it is determined whether to execute the sorting trigger instruction;

[0120] If the sorting trigger instruction is executed, the sorting enable of the signal generator is controlled according to the set sorting delay and sorting pulse width, so that the signal generator sends a pulse signal of a specific duration to the third power amplifier board at a specified time, which is used to drive the third transducer on the microfluidic chip to achieve the sorting of target particles.

[0121] This embodiment provides an in-situ sorting method for deep-sea microorganisms, which can overcome the problems of the prior art that the harsh deep-sea environment makes it difficult to carry out in-situ microbial analysis and sorting, as well as the problem of low in-situ analysis and sorting throughput. The present invention is based on a structural design of separation of dry and wet tanks, and a wet tank pressure compensation technology, which is conducive to the sorting device being suitable for the high hydrostatic pressure environment in the deep sea, and a flow control cell sorting chip design based on acoustic electrophoresis or acoustic standing wave method, which effectively realizes in-situ high-throughput analysis and sorting of deep-sea microorganisms.

[0122] Example 3

[0123] A computer device 900, such as Figure 7 As shown, it includes a memory 910, a processor 920, and a computer program 930 stored in the memory and executable on the processor. When the processor executes the computer program, the steps of a method for in-situ sorting of deep-sea microorganisms are implemented. For a detailed description of the method, reference may be made to the corresponding description in the above method embodiment, which will not be repeated here.

[0124] Example 4

[0125] A computer readable storage medium such as Figure 8 As shown, a computer program is stored thereon, and when the computer program is executed by the processor, the steps of a method for in-situ sorting of deep-sea microorganisms are implemented. For a detailed description of the method, reference may be made to the corresponding description in the above method embodiment, and no further description is given here.

[0126] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0127] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

[0128] The apparatus, computer device, non-volatile computer storage medium and method provided in the embodiments of this specification correspond to each other, and therefore, the apparatus, computer device and non-volatile computer storage medium also have similar beneficial technical effects as the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding apparatus, computer device and non-volatile computer storage medium will not be repeated here.

[0129] Those skilled in the art also know that, in addition to implementing the controller in a purely computer-readable program code, the controller can be made to implement the same function in the form of logic gates, switches, application-specific integrated circuits, programmable logic controllers, and embedded microcontrollers by logically programming the method steps. Therefore, such a controller can be considered as a hardware component, and the devices for implementing various functions included therein can also be considered as structures within the hardware component. Or even, the devices for implementing various functions can be considered as both software units for implementing the method and structures within the hardware component.

[0130] The systems, devices or units described in the above embodiments can be implemented by computer chips or entities, or by products with certain functions. For the convenience of description, the above devices are described separately by functions in various units. Of course, when implementing one or more embodiments of this specification, the functions of each unit can be implemented in the same or more software and / or hardware.

[0131] Those skilled in the art will appreciate that the embodiments of this specification may be provided as methods, systems, or computer program products. Therefore, the embodiments of this specification may be in the form of complete hardware embodiments, complete software embodiments, or embodiments in combination with software and hardware. Moreover, the embodiments of this specification may be in the form of a computer program product implemented in one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) that contain computer-usable program code.

[0132] This specification is described with reference to the flowcharts and / or block diagrams of the methods, devices (systems), and computer program products according to the embodiments of this specification. It should be understood that each process and / or box in the flowchart and / or block diagram, as well as the combination of the processes and / or boxes in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 A process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0133] These computer program instructions may also be stored in a computer-readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer-readable memory produce an article of manufacture comprising an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 A function specified in one or more boxes.

[0134] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operating steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for implementing the process. Figure 1 A process or multiple processes and / or boxes Figure 1 A step that specifies a function in one or more boxes.

[0135] It should also be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, commodity or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, commodity or device. In the absence of more restrictions, the elements defined by the sentence "comprises a ..." do not exclude the existence of other identical elements in the process, method, commodity or device including the elements.

[0136] The specification may be described in the general context of computer-executable instructions executed by a computer, such as program units. Generally, program units include routines, programs, objects, components, data structures, etc. that perform specific tasks or implement specific abstract data types. The specification may also be practiced in distributed computing environments where tasks are performed by remote processing devices connected via a communications network. In a distributed computing environment, program units may be located in local and remote computer storage media, including storage devices.

[0137] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0138] The above description is only an embodiment of this specification and is not intended to limit one or more embodiments of this specification. For those skilled in the art, one or more embodiments of this specification may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of one or more embodiments of this specification shall be included in the scope of the claims of one or more embodiments of this specification.

Claims

1. A device for in-situ sorting of microorganisms in the deep sea, characterized by: It includes a dry tank module, a wet tank module, a transition block module and a pressure buffer; among which, The dry cabin module is used for optical signal detection, collection, analysis, processing and control; The wet tank module is used for collecting seawater samples, focusing acoustic standing waves and sorting target microorganisms; The transition block module is used for transmitting electrical signals and optical signals between the dry tank module and the wet tank module; The pressure buffer is used to compensate for the pressure in the wet tank module so that the pressure in the wet tank module is equal to the pressure in the deep sea.

2. The in-situ microorganism sorting device for deep sea as claimed in claim 1, characterized in that: The dry cabin module includes an electronics system, an optical system and a first cabin penetration joint; The first penetration connector includes an input power line and a communication line for realizing power management and communication of the device; The electronics system is used to collect and process signals in the optical system, and to control the wet tank module through the transition block module.

3. The in-situ microorganism sorting device for deep sea as claimed in claim 2, characterized in that: The transition block module includes a second through-cabin joint, a third through-cabin joint and an optical glass slide. The electronics system is connected to the wet cabin module through the second through-cabin joint and the third through-cabin joint to achieve control of the wet cabin module. The optical glass slide is used to transmit the optical signal of the optical system.

4. The in-situ microorganism sorting device for deep sea as claimed in claim 3, characterized in that: The electronic system includes a power control board, a main control board, a data acquisition board, a signal generator, and a liquid flow control board; The power control board is connected to the input power line of the first penetration connector and is used for power management and power distribution of the device; The main control board performs data exchange with the data acquisition board, the signal generator, and the liquid flow control board, and is used for control, data analysis, and processing of the data acquisition board, the signal generator, and the liquid flow control board; The main control board is connected to the communication line of the first through-cabin connector for communication and control of the device by the outside world; The data acquisition board is used to collect and process the detection signal of the optical system; The signal generator is used to control each transducer on the microfluidic chip in the wet tank module through the third tank penetration connector; The liquid flow control panel is used to control the fluid system in the wet tank module through the second tank penetration joint.

5. The in-situ microorganism sorting device for deep sea as claimed in claim 4, characterized in that: The optical system comprises an excitation light path and a first collection light path; The excitation light path is used to shape the excitation light and transmit it to the detection position of the microfluidic chip in the wet chamber module to perform fluorescence excitation on the microorganisms; The first collecting light path is used for focusing and imaging the excitation light of the microorganism; The optical system comprises an excitation light source, a first mirror group, a first dichroic mirror, an objective lens, a light-transmitting sheet, a second dichroic mirror, a second mirror group, a third mirror group, a first detector, and a second detector; The excitation light source, the first mirror group, the first dichroic mirror, the objective lens, and the light-transmitting sheet form an excitation light path; the objective lens is used to focus and shape the excitation light source in the excitation light path; The light-transmitting sheet, the objective lens, the first dichroic mirror, the second dichroic mirror, the second lens group, the third lens group, the first detector, and the second detector form a first light collecting path; the objective lens is used to focus and image the excitation light of the microorganisms in the first light collecting path, and the data acquisition board is connected to the first detector and the second detector in the optical system to collect and process the signals of the first detector and the second detector.

6. The in-situ microorganism sorting device for deep sea as claimed in claim 5, characterized in that: The wet cabin module includes a microfluidic chip, a focusing / sorting amplifier board, a fluid system, a fourth through-cabin connector and a fifth through-cabin connector; The signal generator is connected to the focusing / sorting power amplifier board through the third through-chamber connector to control each transducer on the microfluidic chip; The liquid flow control panel is connected to each pump and solenoid valve of the fluid system through the second through-tank joint to control the fluid system; The pressure buffer is connected to the fourth penetration joint to compensate for the pressure in the wet tank; The microfluidic chip interacts with the outside world through the fifth through-chamber connector.

7. The in-situ microorganism sorting device for deep sea as claimed in claim 6, characterized in that: The microfluidic chip adopts a glass-to-glass bonding method to form a microchannel for sample flow; wherein the microchannel includes a sample inlet, a first outlet, a second outlet and a third outlet, and the sample inlet and each outlet interact with the outside world through the fifth through-chamber joint.

8. The in-situ microorganism sorting device for deep sea as claimed in claim 6, characterized in that: The microfluidic chip is glued with a first transducer, a second transducer and a third transducer, and each transducer adopts a piezoelectric ceramic material and an ultrasonic standing wave method.

9. The deep-sea microorganism in-situ sorting device according to claim 8, characterized in that: The focusing / sorting power amplifier board includes a first power amplifier board, a second power amplifier board, and a third power amplifier board; wherein, the first transducer and the second transducer respectively realize three-dimensional focusing of particles in the sample flow in the microchannel under the action of the first power amplifier board and the second power amplifier board; the third transducer realizes the deviation of target particles in the sample flow and flows them into the first outlet or the second outlet under the action of the third power amplifier board.

10. The in-situ microorganism sorting device for deep sea as claimed in claim 6, characterized in that: The fluid system includes a pump body, a solenoid valve body, a fluid bag and a fluid pipeline; wherein the pump body includes a first pump, a second pump and a third pump, and the first pump, the second pump and the third pump are respectively used for extracting or delivering external sample flows, reagents and mixed samples; the fluid bag includes a sample bag and a reagent bag, the sample bag is used to store a mixed solution of an external sample flow and a reagent, and the reagent bag is used to store a staining reagent; the fluid pipeline is used for delivering sample flows or reagents; and the solenoid valve body is used to control the flow direction of sample flows or reagents.

11. The deep-sea microorganism in-situ sorting device according to claim 6, characterized in that: The wet chamber module also includes a second light collecting path, which includes a fourth lens group and a third detector. The fourth lens group is used to focus the forward scattered light, and the third detector is used to collect fluorescence signals. The data acquisition board is connected to the third detector and is used to collect and process the signal of the third detector.

12. The deep-sea microorganism in-situ sorting device according to claim 6, characterized in that: The first, second and third cabin penetration joints are all pressure-bearing multi-core wire joints, and the fourth and fifth cabin penetration joints are all pressure-bearing single-core or multi-core fluid joints.

13. The in-situ microorganism sorting device for deep sea as claimed in claim 7, characterized in that: The optical glass slide is sealed and fixed on the end face of the transition block module facing the wet chamber module. It is made of a transparent material that can withstand hydrostatic high pressure and is used to deliver the excitation light in the dry chamber module to the detection area of ​​the microfluidic chip in the wet chamber module, and at the same time deliver the excited light of the microparticles in the microchannel of the microfluidic chip in the wet chamber module to the optical system of the dry chamber module.

14. A method for in-situ sorting of microorganisms in the deep sea, based on the device according to any one of claims 1 to 13, characterized in that: The following steps are involved: After the main control board of the dry cabin module is powered on, the parameters of each component in the device are initialized and set; When executing the automatic sorting stage, the main control board sends an enable instruction to the signal generator of the dry chamber module, so that the signal generator transmits the pre-set pulse signals of different resonant frequencies and voltages to the first power amplifier board and the second power amplifier board respectively, so as to drive the first transducer and the second transducer on the microfluidic chip to realize three-dimensional focusing of the sample flow in the microchannel; By judging whether the intensity of the FSC pulse signal obtained when the particles arranged in a single cell stream pass through the laser spot is higher than a threshold, the data of each channel of the particles can be automatically stored or the collected data can be discarded; By analyzing whether the fluorescence channel pulse signal intensity of the particle is within the gate size range, it is determined whether to execute the sorting trigger instruction; If the sorting trigger instruction is executed, the sorting enable of the signal generator is controlled according to the set sorting delay and sorting pulse width, so that the signal generator sends a pulse signal of a specific duration to the third power amplifier board at a specified time, which is used to drive the third transducer on the microfluidic chip to achieve the sorting of target particles.

15. A computer device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the steps of the method according to claim 14 are implemented.

16. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the method according to claim 14 are implemented.

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