Miniature in-situ biological sorting devices, methods, equipment, and media for deep-sea applications

Abstract

This invention provides a microscopic in-situ biological sorting device, method, equipment, and medium for deep-sea applications. The device includes a dry chamber module, a wet chamber module, a transition block module, and a pressure buffer. The dry chamber module is used for optical signal detection, acquisition, analysis, processing, and control. The wet chamber module is used for seawater sample collection, acoustic standing wave focusing, and target microorganism sorting. The transition block module is used for the transmission of electrical and optical signals between the dry and wet chambers. The pressure buffer is used to compensate for the pressure in the wet chamber, ensuring that the pressure in the wet chamber is equal to the in-situ pressure in the deep sea. This invention overcomes the problems of the harsh deep-sea environment making in-situ microbial analysis and sorting difficult, as well as the low throughput of in-situ analysis and sorting. Based on the structural design of separating the dry and wet chambers and the wet chamber pressure compensation technology, the sorting device is suitable for the high hydrostatic pressure environment in deep-sea in-situ. The flow-controlled cell sorting chip design based on acoustic electrophoresis or acoustic standing wave methods effectively realizes high-throughput in-situ analysis and sorting of deep-sea microorganisms.

Description

Technical Field

[0001] This invention relates to the field of microbial sampling, identification and sorting technology, and particularly to microbial in-situ sorting devices, methods, equipment and media for deep-sea applications. Background Technology

[0002] The deep sea typically refers to areas with depths exceeding 1000 meters, characterized by extreme environments such as darkness, high hydrostatic pressure, high salinity, and low temperatures. Simultaneously, the deep sea possesses an extremely complex ecosystem and rich biodiversity. Research on 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. Their unique physiological mechanisms and metabolites have significant application prospects in fields such as pharmaceutical development, gene therapy, food processing, chemical industry, and environmental protection. Traditional sampling methods, such as water sample bottles or in-situ pressure holding, can significantly impact deep-sea microbial samples, causing rapid proliferation or stress-induced changes, and even leading to the loss of in-situ gene expression information. These changes limit research on the life processes and population distribution of microorganisms in the extreme environment of the deep sea. Furthermore, traditional sampling methods can only obtain observational data at a specific depth and time point in the ocean, failing to meet the needs of studying the dynamic laws of deep-sea ecosystems. Therefore, research on in-situ detection methods or the development of devices for deep-sea microorganisms has become one of the most cutting-edge areas in current deep-sea ecosystem research.

[0003] In-situ microbial sorting instruments based on Raman spectroscopy, proposed in relevant patents or literature, can be used for in-situ identification and sorting of deep-sea microorganisms. However, limited by Raman spectroscopy detection and the "optical tweezers" principle, the throughput of these devices for identifying or sorting microorganisms is low, typically on the order of one microorganism per minute or even lower, making it difficult to meet the needs of detecting or sorting and enriching large numbers of microorganisms in the deep sea. Furthermore, these methods or devices require extremely high water flow stability in the deep-sea environment; external fluid disturbances can severely affect the instrument's identification or sorting performance, making them unsuitable for the complex environments of hydrothermal vents or cold seeps and their vicinity. Summary of the Invention

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

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

[0006] The wet chamber module is used for seawater sample collection, acoustic standing wave focusing, and sorting of target microorganisms;

[0007] The transition block module is used for the transmission of electrical and optical signals between the dry chamber module and the wet chamber module;

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

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

[0010] The first through-cabin connector includes an input power line and a communication line, used to realize power management and communication of the device;

[0011] The electronics system is used to acquire and process signals from the optical system, and to control the wet chamber module via the transition block module.

[0012] Furthermore, the transition block module includes a second chamber penetration connector, a third chamber penetration connector, and an optical glass plate. The electronic system is connected to the wet chamber module through the second and third chamber penetration connectors to control the wet chamber module. The optical glass plate is used to transmit the optical signals 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 fluid flow control board;

[0014] The power control board is connected to the input power line of the first through-cabin connector and is used for power management and power distribution of the device.

[0015] The main control board interacts with the data acquisition board, the signal generator, and the fluid flow control board to control, analyze, and process data from the data acquisition board, the signal generator, and the fluid flow control board.

[0016] The main control board is connected to the communication line of the first through-cabin joint for external communication and control of the device;

[0017] The data acquisition board is used to acquire and process the detection signals of the optical system.

[0018] The signal generator is used to control each transducer on the microfluidic chip in the wet chamber module through the third transforaminal connector.

[0019] The fluid control board is used to control the fluid system in the wet chamber module via the second transom connector.

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

[0021] The excitation optical 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 excite microorganisms with fluorescence.

[0022] The first optical path is used to focus and image the excitation light of the microorganism;

[0023] The optical system includes an excitation light source, a first mirror group, a first dichroic mirror, an objective lens, a light-transmitting plate, 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 plate form the excitation light path; the objective lens is used in the excitation light path to focus and shape the excitation light source.

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

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

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

[0028] The fluid control board is connected to each pump and solenoid valve of the fluid system via the second through-chamber connector to control the fluid system;

[0029] The pressure buffer is connected to the fourth transom connector to compensate for the pressure in the wet chamber;

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

[0031] Furthermore, the microfluidic chip employs 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 transconducting connector.

[0032] Furthermore, the microfluidic chip is bonded with a first transducer, a second transducer, and a third transducer 3, each of which is made of piezoelectric ceramic material and 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, under the action of the first power amplifier board and the second power amplifier board respectively, realize the three-dimensional focusing of particles in the sample flow in the microchannel; the third transducer, under the action of the third power amplifier board, realizes the deflection of target particles in the sample flow and flows into the first outlet or the second outlet.

[0034] Furthermore, the fluid system includes a pump body, a solenoid valve body, a fluid bag, and fluid tubing; wherein, the pump body includes a first pump, a second pump, and a third pump, which are respectively used for the extraction or delivery of external sample streams, reagents, and mixed samples; the fluid bag includes a sample bag and a reagent bag, the sample bag being used to store the mixture of external sample streams and reagents, and the reagent bag being used to store staining reagents; the fluid tubing is used for the delivery of sample streams or reagents; and the solenoid valve body is used to control the flow direction of sample streams or reagents.

[0035] Furthermore, the wet chamber module also includes a second optical collection path, which includes a fourth mirror group and a third detector. The fourth mirror group is used to focus the forward dispersive 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 signals from the third detector.

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

[0037] Furthermore, the optical glass slide is sealed and fixed on the end face of the transition block module facing the wet chamber module, and is made of a transparent material that can withstand hydrostatic pressure. It 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 particles in the microchannel of the microfluidic chip in the wet chamber module to the optical system of the dry chamber module.

[0038] A second objective of this invention is to provide a method for in-situ sorting of microorganisms in the deep sea, based on the aforementioned apparatus, comprising the following steps:

[0039] After powering on the main control board of the dry compartment module, the parameters of each component in the device are initialized.

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

[0041] By determining whether the intensity of the FSC pulse signal obtained when a particle arranged in a single-cell flow passes through a 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.

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

[0043] If the sorting trigger command 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 to drive the third transducer on the microfluidic chip and realize the sorting of target particles.

[0044] A third objective of the present invention is to provide a computer device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps of the above-described method.

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

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

[0047] This invention provides a fully automated, microfluidic-controlled in-situ sorting device and method for deep-sea microorganisms, overcoming the problems of harsh deep-sea environments hindering in-situ microbial analysis and sorting, as well as low throughput in in-situ analysis and sorting. Based on a separate dry and wet chamber structure and wet chamber pressure compensation technology, this invention facilitates the sorting device's applicability to the high hydrostatic pressure environment of deep-sea in-situ. Furthermore, the fluid-controlled cell sorting chip design based on acoustic electrophoresis or acoustic standing wave methods effectively enables high-throughput in-situ analysis and sorting of deep-sea microorganisms.

[0048] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[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 Functional framework diagram of a micro-organism in-situ sorting device for deep-sea applications;

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

[0052] Figure 3 This is a structural diagram of the outer shell of a miniature in-situ biological sorting device for deep-sea applications.

[0053] Figure 4 Cross-sectional view of a miniature in-situ biological sorting device for deep-sea applications;

[0054] Figure 5 This is a diagram of the internal structure of a miniature in-situ biological sorting device for deep-sea applications.

[0055] Figure 6 Flowchart of an in-situ sorting method for microorganisms used in the deep sea;

[0056] Figure 7 This is a schematic diagram of a computer device.

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

[0058] In the diagram: 1. Dry compartment module; 2. Wet compartment module; 3. Transition block; 4. Dry compartment shell; 5. Dry compartment end cover; 6. Wet compartment shell; 7. Wet compartment end cover; 8. Soft gasket. Detailed Implementation

[0059] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0060] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0061] The drawing numbers in this application are only used to distinguish the steps in the scheme and are not used to limit the execution order of the steps. The specific execution order is as described 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 this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0063] Example 1

[0064] A miniature in-situ biological sorting device for deep-sea applications, such as... Figure 1 As shown, it includes a dry compartment module, a wet compartment module, a transition block module, and a pressure buffer; among which,

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

[0066] The wet chamber module is used for seawater sample collection, acoustic standing wave focusing, and sorting of target microorganisms;

[0067] The transition block module is used for the transmission of electrical and optical signals between the dry chamber module and the wet chamber module;

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

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

[0070] The first through-cabin connector includes an input power line and a communication line, used to realize power management and communication of the device;

[0071] The electronics system is used to acquire and process signals from the optical system, and to control the wet chamber module via the transition block module.

[0072] In some embodiments, the transition block module includes a second through-cabin connector (i.e. Figure 1 4) Third compartment joint (i.e. Figure 1 The electronic system is connected to the wet chamber module through the second and third transom connectors to control the wet chamber module. The optical glass is used to transmit the optical signals of the optical system.

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

[0074] The power control board is connected to the input power line of the first through-cabin connector and is used for power management and power distribution of the device.

[0075] The main control board interacts with the data acquisition board, the signal generator, and the fluid flow control board to control, analyze, and process data from the data acquisition board, the signal generator, and the fluid flow control board.

[0076] The main control board is connected to the communication line of the first through-cabin joint for external communication and control of the device;

[0077] The data acquisition board is used to acquire and process the detection signals of the optical system; specifically, the data acquisition board is connected to each detector in the optical system, acquires and processes the signals from these detectors, and then interacts 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 chamber module through the third transducer connector; specifically, the third transducer connector is connected to the focusing / sorting power amplifier board in the wet chamber module to control each transducer on the microfluidic chip.

[0079] The fluid control board is used to control the fluid system in the wet chamber module through the second through-chamber connector; specifically, the fluid control board is connected to each pump and solenoid valve of the fluid system in the wet chamber module through the second through-chamber connector to control the fluid system.

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

[0081] The excitation optical 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 excite microorganisms with fluorescence.

[0082] The first optical path is used to focus and image the excitation light of the microorganism.

[0083] like Figure 2 As shown, the optical system includes an excitation light source and a first mirror group ( Figure 2 Intermediate lens group 4), first dichroic lens ( Figure 2 Dichroic lens 1), objective lens, lens, second dichroic lens ( Figure 2 Medium dichroic mirror 2), second group of mirrors ( Figure 2 Intermediate lens group 1), Third lens group ( Figure 2 Mid-mirror group 3), first detector ( Figure 2 Detector 1), Second Detector ( Figure 2 Detector 3);

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

[0085] The excitation source laser is selected based on the wavelength of the dye used for staining microorganisms. For example, when using SYBR Green dye to stain microorganisms, a 488nm wavelength laser can be used.

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

[0087] Both the second and third mirror groups employ a combination of lenses and filters. The lenses focus excitation light of different wavelengths, while the filters prevent excitation light of non-specific wavelengths from entering the first and second detectors. Both the first and second detectors utilize photomultiplier tubes (PMTs) or avalanche photodiodes (APDs) to acquire fluorescence signals.

[0088] In some embodiments, the wet chamber module includes a microfluidic chip, a focusing / sorting power amplifier board, a fluid system, and a fourth trans-chamber connector (i.e., Figure 1 The middle compartment joint 2) and the fifth compartment joint (i.e. Figure 1 3) Intermediate-section connector;

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

[0090] The fluid control board is connected to each pump and solenoid valve of the fluid system via the second through-chamber connector to control the fluid system;

[0091] The pressure buffer is connected to the fourth transom connector to compensate for the pressure in the wet chamber, so that the pressure in the wet chamber is equal to the pressure in the deep sea in situ.

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

[0093] like Figure 2 As shown, the microfluidic chip uses a glass-to-glass bonding method to form a microchannel for sample flow; wherein, the microchannel includes a sample inlet and a first outlet ( Figure 2China's export 1) Second export ( Figure 2 China's exports 2) and third exports ( Figure 2 (3) The sample inlet and each outlet are connected to the outside world through the fifth transom connector.

[0094] A first transducer is glued onto the microfluidic chip. Figure 2 Middle transducer 1), Second transducer ( Figure 2 Middle transducer 2), third transducer ( Figure 2 Transducer 3), each transducer uses piezoelectric ceramic material and ultrasonic standing wave method.

[0095] The focusing / sorting power amplifier board includes a first power amplifier board ( Figure 2 First power amplifier board 1), Second power amplifier board ( Figure 2 2) Middle power amplifier board, 3rd power amplifier board Figure 2 The first and second transducers, under the action of the first and second power amplifier boards respectively, realize the three-dimensional focusing of particles in the sample flow in the microchannel; the third transducer, under the action of the third power amplifier board, realizes the deflection of target particles in the sample flow and flows into the first or second outlet.

[0096] The fluid system includes a pump body, a solenoid valve body, a fluid bag, and fluid piping; wherein, the pump body includes a first pump ( Figure 2 Medium pump 1), Second pump ( Figure 2 Medium pump 2) and third pump ( Figure 2 The pump 3) can be a peristaltic pump or a plunger pump. The first pump, the second pump, and the third pump are used for the extraction or delivery of external sample streams, reagents, and mixed samples, respectively. The fluid bags are all sterile soft plastic bags, including sample bags and reagent bags. The sample bags are used to store the mixture of external sample streams and reagents, and the reagent bags are used to store staining reagents. The fluid lines are thick-walled rigid tubes or flexible tubes, used for the delivery of sample streams or reagents. The solenoid valve body is a plurality of dispersed solenoid valves or an integrated solenoid valve, used to control the flow direction of sample streams or reagents.

[0097] The wet chamber module also includes a second optical collection path, which includes a fourth mirror group. Figure 2 Mid-mirror group 2), third detector ( Figure 2 The fourth mirror group is used to focus the forward-scattering light, the third detector is used to collect fluorescence signals, and the data acquisition board is connected to the third detector to collect and process the signals from the third detector.

[0098] The fourth mirror group employs a combination of lenses and filters; the lenses are used to focus forward-scattered light; and the filters are used to prevent forward-scattered light of non-specific wavelength bands from entering the third detector. The third detector is a photodiode (PD).

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

[0100] The transition block 3 is made of titanium alloy. The dry tank end cap 5, dry tank hull 4, wet tank hull 6, and wet tank end cap 7 are made of aluminum alloy or titanium alloy. All hull surfaces in contact with seawater are coated with an anti-corrosion coating.

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

[0102] Dry chamber module 1 and wet chamber module 2 are respectively fixed on both sides of transition block 3;

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

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

[0105] Axial and radial soft gaskets 8 are installed on both sides of the dry compartment end cover 5, the transition block 3, and the wet compartment 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 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, while delivering the excited light of the particles in the microchannel of the microfluidic chip in the wet chamber to the optical system of the dry chamber.

[0107] In-situ sorting methods for deep-sea microorganisms are based on flow cytometry, such as... Figure 2As shown, but unlike the laboratory desktop flow cytometry platform, considering the in-situ working conditions of deep-sea detection, the microfluidic chip is installed in the instrument's wet chamber. The chip's inlet and outlet are both located in the deep sea, and the sample inside the chip is kept in equilibrium with the hydrostatic pressure of the in-situ deep sea. At the same time, the instrument's wet chamber is filled with an insulating liquid medium, such as silicone oil, and a pressure buffer is connected to the end cap of the instrument's wet chamber, so that the external pressure of all devices inside the wet chamber is kept in equilibrium with the hydrostatic pressure of the in-situ deep sea. Based on the acoustic electrophoresis method, transducers 1 and 2 on the microfluidic chip are used to focus the particles in the chip's microchannels in three-dimensional space and arrange them in a "single-cell flow". Then, the single-column arranged particles will pass through the laser detection area in sequence 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 this sorting device is as follows:

[0109] After the main control board is powered on, it initializes the parameters such as the gain, FSC threshold, gate size, sorting delay, and sorting pulse width of the PMTs and PDs. During the automatic sorting stage, the main control board sends an enable command to the signal generator. The signal generator then transmits pre-set pulse signals with different resonant frequencies and voltages to power amplifier boards 1 and 2, respectively, to drive piezoelectric transducers 1 and 2 on the microchip, achieving three-dimensional focusing of the sample flow in the microchannel.

[0110] When particles arranged in a "single-cell stream" pass through a laser spot, both FSC and fluorescence channel pulse signals are simultaneously acquired. When the intensity of the acquired FSC pulse signal is higher than a threshold, the data for each channel of the particle is automatically stored; otherwise, the acquired data is discarded.

[0111] The main control board further analyzes whether the intensity of the fluorescence channel pulse signal of the particle is within the range of the gate size to determine whether to execute the sorting trigger command. 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 power amplifier board at a specified time to drive the piezoelectric transducer in the sorting area on the microchip, ultimately achieving the sorting of the target particles.

[0112] This embodiment provides a microscopic in-situ biological sorting device for deep-sea environments, integrating flow cytometry, a dry and wet chamber separation structure design, acoustic standing wave focusing and sorting technology, and microfluidic technology. It overcomes the problems of existing technologies, such as the harsh deep-sea environment making in-situ microbial analysis and sorting difficult, and the low throughput of in-situ analysis and sorting. The invention's dry and wet chamber separation structure design, along with wet chamber pressure compensation technology, facilitates the sorting device's applicability to the high hydrostatic pressure environment of deep-sea in-situ environments. Furthermore, the flow-controlled cell sorting chip design based on acoustic electrophoresis or acoustic standing wave methods effectively achieves high-throughput in-situ analysis and sorting of deep-sea microorganisms.

[0113] Example 2

[0114] A method for in-situ sorting of microorganisms in the deep sea is based on the aforementioned device. For a detailed description of the device, please refer to the corresponding description in the above-described device embodiments; it will not be repeated here. Figure 6 As shown, the method includes the following steps:

[0115] After the main control board of the dry compartment 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 parameters such as the gain of PMTs and PDs, FSC threshold, gate size, sorting delay, and sorting pulse width are completed.

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

[0117] When particles arranged in a "single-cell flow" pass through a laser spot, they simultaneously acquire FSC and fluorescence channel pulse signals.

[0118] By determining whether the intensity of the FSC pulse signal obtained when a particle arranged in a single-cell flow passes through a laser spot is higher than a threshold, the data of each channel of the particle is automatically stored or the collected data is discarded. Specifically, when the intensity of the FSC pulse signal is higher than the threshold, the data of each channel of the particle is automatically stored; otherwise, the collected data is discarded.

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

[0120] If the sorting trigger command 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 to drive the third transducer on the microfluidic chip and realize the sorting of target particles.

[0121] This embodiment provides a method for in-situ sorting of microorganisms in the deep sea, overcoming the problems of the harsh deep-sea environment making in-situ microbial analysis and sorting difficult, as well as the low throughput of in-situ analysis and sorting. Based on a structural design separating dry and wet chambers and wet chamber pressure compensation technology, this invention facilitates the sorting device's applicability to the high hydrostatic pressure environment of the deep sea. Furthermore, the flow-controlled cell sorting chip design based on acoustic electrophoresis or acoustic standing wave methods effectively achieves high-throughput in-situ analysis and sorting of deep-sea microorganisms.

[0122] Example 3

[0123] A computer device 900, such as Figure 7 As shown, the system 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, it implements the steps of a method for in-situ sorting of microorganisms in the deep sea. For a detailed description of the method, please refer to the corresponding description in the above method embodiments; it 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, which, when executed by a processor, implements the steps of a method for in-situ sorting of microorganisms in the deep sea. For a detailed description of the method, please refer to the corresponding description in the above method embodiments, which will not be repeated here.

[0126] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0127] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. 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 illustrations shown and described herein.

[0128] The apparatus, computer device, and non-volatile computer storage medium and method provided in the embodiments of this specification are corresponding. 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 will also know that, besides implementing the controller in the form of purely computer-readable program code, the same functions can be achieved by logically programming the method steps, making the controller take the form of logic gates, switches, application-specific integrated circuits (ASICs), programmable logic controllers (PLCs), and embedded microcontrollers. Therefore, such a controller can be considered a hardware component, and the devices included within it for implementing various functions can also be considered structures within that hardware component. Alternatively, the devices for implementing various functions can be considered as both software units implementing the method and structures within a hardware component.

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

[0131] Those skilled in the art will understand that the embodiments of this specification can be provided as methods, systems, or computer program products. Therefore, the embodiments of this specification can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the embodiments of this specification can take the form of a computer program product implemented on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0132] This specification is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this specification. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create a machine for implementing the flowchart illustrations and / or block diagrams. Figure 1 a process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0133] These computer program instructions may also be stored in a computer readable memory that can direct a computer or other programmable data processing device to work 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 The 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 operational steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing the instructions executed on the computer or other programmable device 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 "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

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

[0137] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0138] The above description is merely an embodiment of this specification and is not intended to limit the scope of one or more embodiments of this specification. Various modifications and variations can be made to one or more embodiments of this specification by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of one or more embodiments of this specification should be included within the scope of the claims of one or more embodiments of this specification.

Claims

1. A micro-organism in-situ sorting device for deep-sea organisms, characterized in that: It includes a dry compartment module, a wet compartment module, a transition block module, and a pressure buffer; among which, The dry chamber module is used for optical signal detection, acquisition, analysis, processing, and control; The wet chamber module is used for seawater sample collection, acoustic standing wave focusing, and sorting of target microorganisms; The transition block module is used for the transmission of electrical and optical signals between the dry chamber module and the wet chamber module; The pressure buffer is used to compensate for the pressure in the wet chamber module, so that the pressure in the wet chamber module is equal to the pressure in the deep sea in situ; The dry cabin module includes an electronics system, an optical system, and a first cabin penetration connector; The first through-cabin connector includes an input power line and a communication line, used to realize power management and communication of the device; The electronics system is used to acquire and process signals from the optical system, and to control the wet chamber module via the transition block module; The transition block module includes a second chamber connector, a third chamber connector, and an optical glass plate. The electronic system is connected to the wet chamber module through the second chamber connector and the third chamber connector to control the wet chamber module. The optical glass plate is used to transmit the optical signals of the optical system. The electronic system includes a power control board, a main control board, a data acquisition board, a signal generator, and a fluid flow control board; The power control board is connected to the input power line of the first through-cabin connector and is used for power management and power distribution of the device. The main control board interacts with the data acquisition board, the signal generator, and the fluid flow control board to control, analyze, and process data from the data acquisition board, the signal generator, and the fluid flow control board. The main control board is connected to the communication line of the first through-cabin joint for external communication and control of the device; The data acquisition board is used to acquire and process the detection signals of the optical system. The signal generator is used to control each transducer on the microfluidic chip in the wet chamber module through the third transforaminal connector. The fluid control board is used to control the fluid system in the wet chamber module via the second trans-chamber joint; The optical system includes an excitation optical path and a first collection optical path; The excitation optical 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 excite microorganisms with fluorescence. The first optical path is used to focus and image the excitation light of the microorganism; The optical system includes an excitation light source, a first mirror group, a first dichroic mirror, an objective lens, a light-transmitting plate, 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 plate form the excitation light path; the objective lens is used in the excitation light path to focus and shape the excitation light source. The light-transmitting sheet, the objective lens, the first dichroic mirror, the second dichroic mirror, the second mirror group, the third mirror group, the first detector, and the second detector form a first light-collecting path; the objective lens is used in the first light-collecting path to focus and image the excitation light of the microorganism; the data acquisition board is connected to the first detector and the second detector in the optical system to collect and process the signals from the first detector and the second detector. The wet chamber module includes a microfluidic chip, a focusing / sorting power amplifier board, a fluid system, a fourth chamber penetration connector, and a fifth chamber penetration connector. The signal generator is connected to the focusing / sorting power amplifier board via the third through-chamber connector to control each transducer on the microfluidic chip; The fluid control board is connected to each pump and solenoid valve of the fluid system via the second through-chamber connector to control the fluid system; The pressure buffer is connected to the fourth transom connector to compensate for the pressure in the wet chamber; The microfluidic chip interacts with the outside world through the fifth trans-chamber connector; The microfluidic chip uses 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 transconducting connector; The microfluidic chip has a first transducer, a second transducer, and a third transducer bonded to it. Each transducer is made of piezoelectric ceramic material and ultrasonic standing wave method. 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, under the action of the first power amplifier board and the second power amplifier board respectively, realize the three-dimensional focusing of particles in the sample flow in the microchannel; the third transducer, under the action of the third power amplifier board, realizes the deflection of target particles in the sample flow and flows into the first outlet or the second outlet. The optical 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 pressure. It 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 particles in the microchannel of the microfluidic chip in the wet chamber module to the optical system of the dry chamber module.

2. The micro-organism in-situ sorting device for deep-sea organisms as described in claim 1, characterized in that: The fluid system includes a pump body, a solenoid valve body, a fluid bag, and fluid tubing; wherein, the pump body includes a first pump, a second pump, and a third pump, which are used for the extraction or delivery of external sample streams, reagents, and mixed samples, respectively; the fluid bag includes a sample bag and a reagent bag, the sample bag being used to store the mixture of external sample streams and reagents, and the reagent bag being used to store staining reagents; the fluid tubing is used for the delivery of sample streams or reagents; and the solenoid valve body is used to control the flow direction of sample streams or reagents.

3. The micro-organism in-situ sorting device for deep-sea organisms as described in claim 1, characterized in that: The wet chamber module also includes a second optical collection path, which includes a fourth mirror group and a third detector. The fourth mirror group is used to focus 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 signals from the third detector.

4. The micro-organism in-situ sorting device for deep-sea organisms as described in claim 1, characterized in that: The first, second, and third through-cabin connectors are all pressure-bearing multi-core wire connectors, while the fourth and fifth through-cabin connectors are all pressure-bearing single-core or multi-core fluid connectors.

5. A method for in-situ sorting of microorganisms in the deep sea, based on the apparatus described in any one of claims 1 to 4, characterized in that, Includes the following steps: After powering on the main control board of the dry compartment module, the parameters of each component in the device are initialized. During the automatic sorting stage, the main control board sends an enable command to the signal generator of the dry chamber module, so that the signal generator transmits pulse signals with different pre-set resonant frequencies and voltages to the first power amplifier board and the second power amplifier board respectively, which are used to drive the first transducer and the second transducer on the microfluidic chip to realize the three-dimensional focusing of the sample flow in the microchannel. By determining whether the intensity of the FSC pulse signal obtained when a particle arranged in a single-cell flow passes through a 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. By analyzing whether the intensity of the fluorescence channel pulse signal of the particles is within the range of the gate size, it can be determined whether to execute the sorting trigger command; If the sorting trigger command 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 to drive the third transducer on the microfluidic chip and realize the sorting of target particles.

6. 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, it implements the steps of the method as described in claim 5.

7. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the steps of the method as described in claim 5.

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