A high-throughput single-cell sorting device and method for marine in-situ environment

Abstract

This invention proposes a high-throughput single-cell sorting device for marine in-situ environments, comprising a pressure-resistant visual sorting chamber, an optical recognition system, and an automatic sorting system. The device involves injecting a microbial-containing bacterial solution into the pressure-resistant visual sorting chamber; a carrier chip placed within the chamber disperses enriched microorganisms through a channel; as the enriched microorganisms pass through the carrier chip, the optical recognition system observes and identifies them; and the automatic sorting system automatically sorts the microorganisms based on the identification results. This invention also proposes a high-throughput single-cell sorting method for marine in-situ environments. After single-cell dispersion of microorganisms using a carrier chip, the method involves observation and identification via an optical recognition system, followed by intelligent sorting by an automatic sorting system. This achieves high-throughput single-cell identification and sorting of marine microorganisms under high pressure and extreme temperature environments through optical and spectral detection, effectively improving the culturability of marine microorganisms.

Description

Technical Field

[0001] This invention relates to the field of marine microbial technology, and in particular to a high-throughput sorting device and method for single cells in marine in situ. Background Technology

[0002] The vast ocean is the cradle of life on Earth, teeming with abundant and diverse microorganisms. Marine microorganisms are vital marine biological resources. The metabolic products of marine bacteria, fungi, actinomycetes, and archaea found in seawater and marine sediments contain a wealth of bioactive substances, with significant application prospects in energy, materials, environment, and medicine. For example, autotrophic microorganisms capable of producing bioenergy have been discovered in deep-sea cold seeps and hydrothermal environments; marine microorganisms capable of degrading plastics have been identified; scientists have isolated effective antibiotics from marine bacteria and actinomycetes; and marine methanogens and other archaea possess strong methane metabolism capabilities, serving as primary producers in extreme marine ecosystems, providing crucial carbon and energy sources to metazoans through chemosynthesis and symbiosis. Therefore, marine microorganisms are important biological resources with significant development and utilization value.

[0003] Isolation and culture are crucial prerequisites for the development and utilization of marine microorganisms. Currently, most marine microorganism isolation methods involve streak plating or single-cell sorting instruments under normal pressure. However, the number of isolated marine microorganisms remains less than 1%. Furthermore, the physiological, biogeochemical, and ecological mechanisms and characteristics of microorganisms are not easily obtained directly from nature. Isolating microorganisms from their natural environment and establishing pure cultures is a fundamental step in studying their gene sequences, morphological characteristics, physiological features, and ecological characteristics. However, because many marine microorganisms live in extreme environments—for example, barophilic microorganisms are almost impossible to isolate and culture under normal pressure—this limits our understanding of marine microorganisms and their potential for development and utilization. Therefore, there is an urgent need to develop effective identification and sorting technologies for microorganisms living under high-pressure marine environments.

[0004] Existing technology discloses a method for detecting aerobic anaerobic photosynthetic bacteria based on single-cell Raman spectroscopy, enabling single-cell detection of aerobic anaerobic photosynthetic bacteria in environmental water. Furthermore, the Raman spectroscopy detection method is non-destructive, and the detected aerobic anaerobic photosynthetic bacteria can be used for single-cell sorting and sequencing. However, due to the unique environment in which deep-sea microorganisms live, the above method is not suitable for the identification and sorting of deep-sea microorganisms. Summary of the Invention

[0005] In order to overcome at least one of the above-mentioned technical defects, the present invention provides a high-throughput single-cell sorting device and method for marine in-situ environment. Under high pressure, the device achieves high-throughput single-cell identification and sorting of marine microorganisms through optical and spectral detection, thereby improving the culturability of marine microorganisms.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] A high-throughput single-cell sorting device for marine in-situ environments includes a microbial enrichment injection system, a pressure-resistant visual sorting chamber, an annular wall temperature control system, a pressurization system, an annular pressure control system, an optical recognition system, an automatic sorting system, and a data acquisition and processing system. The microbial enrichment injection system is used to cultivate and inject a microbial-containing bacterial solution into the pressure-resistant visual sorting chamber. A carrier chip, made of a visible material, is embedded in the pressure-resistant visual sorting chamber, allowing the enriched microorganisms to disperse and pass through the channels. As the enriched microorganisms pass through the carrier chip, the optical recognition system observes and identifies the microorganisms. The outlet of the pressure-resistant visual sorting chamber is connected to the automatic sorting system. The system is interconnected, and the automatic sorting system automatically sorts microorganisms based on the results of microbial identification by the optical recognition system. The annular temperature control system ensures a consistent internal temperature within the pressure-resistant visual sorting chamber. The pressurization system ensures that the internal pressure of the pressure-resistant visual sorting chamber matches the internal pressure of the microbial enrichment injection system. The annular pressure control system maintains a consistent internal pressure within the pressure-resistant visual sorting chamber based on pressure changes within the automatic sorting system, preventing deformation or damage to the carrier chip due to pressure differential. The microbial enrichment injection system, annular temperature control system, pressurization system, annular pressure control system, optical recognition system, and automatic sorting system are all electrically connected to the data acquisition and processing system.

[0008] In the above scheme, the carrier chip is equipped with micro-inlet and outlet channels. The inlet channels are mainly for pumping in bacterial liquid containing microorganisms from the microbial enrichment injection system, and for injecting gas and liquid for pressurization from the pressurization system.

[0009] The above scheme proposes a carrier chip that enables the dispersion of microorganisms. After observation and identification by an optical recognition system, the microorganisms are intelligently sorted by an automatic sorting system. This achieves high-throughput single-cell identification and sorting of marine microorganisms under high pressure through optical and spectral detection, effectively improving the culturability of marine microorganisms.

[0010] This solution addresses the challenge of isolating marine microorganisms by proposing a device and technology for high-throughput single-cell sorting under high-pressure environments. Compared to existing atmospheric pressure isolation and culture methods, it enables the enrichment and isolation of microorganisms in situ under high pressure in the deep sea, solving the problems of deep-sea in-situ barophilic bacteria failing to survive or exhibiting differential expression under atmospheric pressure. Furthermore, compared to conventional enrichment and streak plating techniques, this solution provides a high-throughput screening approach and method based on specific morphological and metabolic characteristics. It not only solves the difficulties in enriching, isolating, and culturing microorganisms outside of high-pressure environments but also enables high-throughput identification and screening of single-cell-scale marine microorganisms under high pressure, thus improving isolation efficiency.

[0011] The microbial enrichment injection system includes a microfluidic pump, a high-pressure microbial enrichment culture chamber, and an inlet pressure detection device. The microfluidic pump control terminal is electrically connected to the data acquisition and processing system. The microfluidic pump input terminal is connected to the outlet terminal of the high-pressure microbial enrichment culture chamber, and its output terminal is connected to the inlet terminal of the pressure-resistant visual sorting chamber via the inlet pressure detection device. The high-pressure microbial enrichment culture chamber is used to cultivate a microbial-containing bacterial solution, which is then injected into the pressure-resistant visual sorting chamber via the microfluidic pump.

[0012] The pressure-resistant visual sorting chamber further includes a pressure-resistant visual cavity, an annular cooling / heating cavity, and an annular high-pressure cavity. The pressure-resistant visual cavity is made of pressure-resistant and corrosion-resistant metal material, with pressure-resistant visual material inlaid on its front and back sides. It can withstand pressure up to 5000 meters deep and is connected to a pressurization system. The sample-carrying chip is located in the center of the pressure-resistant visual cavity. The microfluidic channel inlet of the sample-carrying chip is connected to the enrichment microorganism injection system, and its outlet is the outlet end of the pressure-resistant visual sorting chamber, connected to the automatic sorting system. The annular high-pressure cavity is located on the outer ring of the pressure-resistant visual cavity to protect the sample-carrying chip from damage within the cavity. The annular high-pressure cavity is connected to the pressurization system and the annular pressure control system. The annular cooling / heating cavity surrounds the outer wall of the pressure-resistant visual cavity, is used to load cooling / heating fluid, and is connected to the annular temperature control system via the cooling / heating fluid.

[0013] In the above scheme, the pressure-resistant viewing cavity is equipped with a vent valve, the output of which is electrically connected to the data acquisition and processing system for convenient pressure regulation within the cavity. To protect the chip from damage within the pressure-resistant viewing cavity, a high-pressure chamber is provided around the annular wall. This chamber simultaneously pressurizes the outer ring of the pressure-resistant viewing cavity and is equipped with an annular pressure control system. This system automatically increases or decreases the pressure in the high-pressure chamber based on changes in the pressure within the pressure-resistant viewing cavity, achieving pressure balance between the pressure-resistant viewing cavity and the high-pressure chamber. This ensures the chip withstands minimal pressure difference without being damaged.

[0014] The annular wall temperature control system employs a circulating cooling / heating device and a temperature sensor. The control terminal of the circulating cooling / heating device is electrically connected to the data acquisition and processing system, and is used for cooling / heating and circulating the cooling / heating fluid within the annular wall cooling / heating cavity. The temperature sensor probe is installed inside the pressure-resistant visible cavity, and its signal output terminal is electrically connected to the data acquisition and processing system.

[0015] In the above scheme, the temperature of the pressure-resistant visual cavity is mainly maintained by injecting a cooling / heating fluid into the annular wall cooling / heating cavity, and by circulating the fluid for cooling or heating to ensure that the fluid in the annular wall cooling / heating cavity is in a low-temperature or high-temperature state. Then, the low-temperature or high-temperature state in the pressure-resistant visual cavity is maintained by heat exchange between the cooling / heating fluid and the pressure-resistant visual cavity.

[0016] The pressurization system includes an air compressor, a booster pump, an air tank, a pressure regulating valve, and a pressure sensor. The air compressor, booster pump, air tank, and pressure regulating valve are connected in sequence and then connected to the pressure-resistant visual cavity and the annular high-pressure cavity, respectively. The pressure sensor probe is installed inside the pressure-resistant visual cavity, and its signal output terminal is electrically connected to the data acquisition and processing system.

[0017] In the above scheme, the temperature sensor and pressure sensor are used to measure and monitor the temperature and pressure of the pressure-resistant visual cavity throughout the entire microbial sorting process.

[0018] The ring pressure control system includes a ring pressure detection device, a first back pressure tracking pump, a back pressure detection device, a back pressure valve, a buffer tank, and a second back pressure tracking pump. Specifically: the probe of the ring pressure detection device is disposed within the high-pressure chamber of the ring wall, and its output end is electrically connected to the data acquisition and processing system; the back pressure tracking pump is connected to the high-pressure chamber of the ring wall, and its control end is electrically connected to the data acquisition and processing system; the detection end of the back pressure detection device is connected to the pressure-resistant visual chamber, and its signal output end is electrically connected to the data acquisition and processing system; one end of the back pressure valve is connected to the automatic sorting system, and the other end is connected to the second back pressure tracking pump via the buffer tank; the control end of the second back pressure tracking pump is electrically connected to the data acquisition and processing system.

[0019] The optical recognition system employs a spectral / optical observation module. When enriched microorganisms pass through the carrier chip, the spectral / optical observation module observes and identifies the microorganisms and sends the identification results to the data acquisition and processing system.

[0020] During the passage of enriched microorganisms through a microarray, a spectroscopic / optical observation module is used to observe and identify them. High-resolution optical microscopy allows for the identification of single-cell morphology above the chip, while Raman spectroscopy identifies intracellular marker biocompounds. By combining optical and spectroscopic identification signals, it can be determined whether the microorganisms on the chip are the target microorganisms desired by the researchers.

[0021] The automatic sorting system includes an intelligent control three-way module, a target microorganism storage module, and a non-target microorganism storage module. The target microorganism storage module and the non-target microorganism storage module are respectively connected to two connection terminals of the intelligent control three-way module, and the other connection terminal of the intelligent control three-way module is connected to the outlet terminal of the pressure-resistant visual sorting chamber. The control terminal of the intelligent control three-way module is electrically connected to the data acquisition and processing system.

[0022] In the above scheme, an automatic sorting system is installed at the outlet of the pressure-resistant visual cavity to directionally sort the identified microorganisms. The automatic sorting system is mainly controlled by an intelligent three-way control module. This module is an automatically opening and closing three-way valve. When an identified single cell is determined to be a target microorganism, the valve of the target microorganism storage module opens, allowing the single cell to enter the target microorganism storage module. When an identified single cell is determined to be a non-target microorganism, the target microorganism storage module opens, allowing the cell to enter the target microorganism storage module, thus achieving high-throughput single-cell sorting. The target microorganism storage module can be selected as either an atmospheric pressure container or a high-pressure container, depending on experimental needs. Both containers contain the corresponding culture medium to meet the requirements for further cultivation of the sorted microorganisms.

[0023] This solution also provides a high-throughput sorting method for single cells in marine in-situ environments, implemented using a high-throughput sorting device for single cells in marine in-situ environments, specifically including the following steps:

[0024] S1: Determine the pressure value inside the pressure-resistant visual sorting chamber based on the pressure value of the enriched microorganism injection system; inject gas into the pressure-resistant visual chamber through the pressurization system so that the pressure value inside the pressure-resistant visual chamber is consistent with that of the enriched microorganism injection system;

[0025] S2: Activate the ring pressure control system to make the pressure of the pressure-resistant visual cavity and the high-pressure cavity of the ring wall consistent;

[0026] S3: Determine the temperature value inside the pressure-resistant visual cavity based on the temperature value inside the enriched microorganism injection system, and make the temperature value inside the ring wall cooling / heating cavity consistent with the temperature value inside the pressure-resistant visual cavity by activating the ring wall temperature control system.

[0027] S4: Adjust the optical recognition system so that it can clearly observe the situation inside the chip.

[0028] S5: Inject the microbial-containing bacterial solution from the microbial enrichment injection system into the pressure-resistant visual cavity via a microfluidic pump, allowing the bacterial solution to slowly pass through the carrier chip, enabling it to pass through the etching channel in the form of single cells; turn on the ring wall temperature control system to keep the pressure at the outlet of the pressure-resistant visual cavity constant when the liquid flows out of the pressure-resistant visual cavity;

[0029] S6: During the process of bacterial culture passing through the carrier chip, the morphology of the cells and the spectroscopic information of intracellular and extracellular metabolic compounds are fully observed and collected through the optical recognition system to determine whether the cell is the target microorganism and send the recognition result to the data acquisition and processing system.

[0030] S7: The automatic sorting system intelligently activates the intelligent control three-way module based on the identification results, sending the target microorganisms to the target microorganism storage module and the non-target microorganisms to the non-target microorganism storage module;

[0031] S8: Once the number of target microorganisms in the target microorganism storage module meets the requirements, the sorting process ends.

[0032] Before performing step S1, the marine in-situ single-cell high-throughput sorting device needs to be pretreated. Specifically, the outlet end of the pressure-resistant visual cavity is opened, and the pressure-resistant visual cavity is repeatedly cleaned by pumping in deionized water. After rinsing, 75% alcohol is pumped in. After the pressure-resistant visual cavity is completely filled with alcohol, the pressure-resistant visual cavity is closed, left to stand for 24 hours, and then the alcohol in the pressure-resistant visual cavity is emptied to complete the pretreatment.

[0033] Throughout the sorting process, the pressure and temperature within the pressure-resistant visual cavity are maintained consistent with the pressure and temperature environment of the enriched microorganism injection system where the microorganisms were initially located, allowing the microorganisms to be sorted under in-situ high pressure conditions. During the sorting process, the ring pressure control system is activated, and the pressure value within the high-pressure chamber of the ring wall is maintained consistent with the pressure value within the pressure-resistant visual cavity, ensuring that the carrier chip does not experience pressure differentials and does not deform or break.

[0034] This proposed solution utilizes a high-throughput single-cell sorting chip and technology for marine microorganisms under high-pressure environments. This enables the identification and sorting of microorganisms under high-pressure conditions, meeting the needs of subsequent purification and culture. Compared to traditional techniques for enriching and separating marine microorganisms under normal pressure, this solution effectively addresses the challenges of low survival rates of pressure-tolerant and barophilic bacteria under normal pressure, and the inability to effectively express deep-sea native characteristics under normal pressure. It also solves the problems of low culture density and difficulty in culturing pure cultures of marine microorganisms. Furthermore, this solution achieves high-throughput identification and automated sorting at the single-cell scale under high pressure, significantly improving the efficiency of microbial culture and purification compared to conventional microbial isolation and culture techniques.

[0035] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0036] This invention proposes a high-throughput single-cell sorting device and method for marine in-situ environment, and proposes a carrier chip to enable the dispersion of microorganisms. After observation and identification by an optical recognition system, the microorganisms are intelligently sorted by an automatic sorting system. This invention realizes the high-throughput single-cell identification and sorting process of marine microorganisms under high pressure environment through optical and spectral detection, effectively improving the culturability of marine microorganisms. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of the marine in-situ environment single-cell high-throughput sorting device of the present invention;

[0038] Figure 2 This is a schematic diagram showing the connection between the data acquisition and processing system and its various system modules in this invention;

[0039] Figure 3 This is a flowchart illustrating the high-throughput sorting method for single cells in the marine in-situ environment according to the present invention.

[0040] The system includes: 1. Microbial enrichment injection system; 11. Microfluidic pump; 12. High-pressure microbial enrichment culture chamber; 13. Imported pressure detection device; 2. Pressure-resistant visual sorting chamber; 21. Carrier chip; 22. Annular wall cooling / heating chamber; 23. Vent valve; 3. Annular wall temperature control system; 31. Circulating cooling / heating device; 32. Temperature sensor; 4. Pressurization system; 41. Air compressor; 42. Pressurization pump; 43. Air storage tank; 44. Pressure regulating valve; 45. Pressure sensor; 5. Annular pressure control system; 51. Annular pressure detection device; 52. First back pressure tracking pump; 53. Back pressure detection device; 54. Back pressure valve; 55. Buffer tank; 56. Second back pressure tracking pump; 6. Optical recognition system; 7. Automatic sorting system; 71. Intelligent control three-way module; 72. Target microbial storage module; 73. Non-target microbial storage module; 8. Data acquisition and processing system. Detailed Implementation

[0041] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent.

[0042] This embodiment is a complete usage example with rich content.

[0043] To better illustrate this embodiment, some parts in the accompanying drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions;

[0044] It will be understood by those skilled in the art that certain well-known structures and their descriptions may be omitted in the accompanying drawings.

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0046] Example 1

[0047] like Figure 1 , Figure 2 As shown, this embodiment proposes a marine in-situ single-cell high-throughput sorting device, including a microbial enrichment injection system 1, a pressure-resistant visual sorting chamber 2, an annular wall temperature control system 3, a pressurization system 4, an annular pressure control system 5, an optical recognition system 6, an automatic sorting system 7, and a data acquisition and processing system 8; wherein: the microbial enrichment injection system 1 is used to cultivate and inject microbial-containing bacterial solutions into the pressure-resistant visual sorting chamber 2; the pressure-resistant visual sorting chamber 2 is equipped with a carrier chip 21, which is made of a visible material and has embedded etched microfluidic channels for dispersing and passing through the channels; when the enriched microbial microbes pass through the carrier chip 21, the microbes are observed and identified by the optical recognition system 6; the outlet end of the pressure-resistant visual sorting chamber 2 is connected to... The automatic sorting system 7 is connected and automatically sorts microorganisms based on the microbial identification results of the optical recognition system 6. The annular temperature control system 3 is used to ensure that the internal temperature of the pressure-resistant visual sorting chamber 2 is consistent. The pressurization system 4 is used to make the internal pressure of the pressure-resistant visual sorting chamber 2 consistent with the internal pressure of the enriched microorganism injection system 1. The annular pressure control system 5 is used to maintain the internal pressure of the pressure-resistant visual sorting chamber 2 consistent with the pressure value of the automatic sorting system 7 according to the pressure value change, so as to avoid deformation or damage to the carrier chip 21 due to pressure difference. The enriched microorganism injection system 1, the annular temperature control system 3, the pressurization system 4, the annular pressure control system 5, the optical recognition system 6, and the automatic sorting system 7 are all electrically connected to the data acquisition and processing system 8.

[0048] In the specific implementation process, the carrier chip 21 is provided with micro-inlet and outlet channels. The inlet channels are mainly for pumping in bacterial liquid containing microorganisms from the microbial enrichment injection system 1, and for injecting gas and liquid pressurization from the pressurization system 4.

[0049] In the specific implementation process, this embodiment proposes a carrier chip 21 to enable the dispersion of microorganisms. After observation and identification by the optical recognition system 6, the microorganisms are intelligently sorted by the automatic sorting system 7. This realizes a high-throughput single-cell identification and sorting process of marine microorganisms through optical and spectral detection under high pressure, effectively improving the culturability of marine microorganisms.

[0050] This embodiment addresses the challenge of isolating marine microorganisms by proposing a device and technology for high-throughput single-cell sorting under high-pressure environments. Compared to existing atmospheric pressure isolation and culture methods, it enables the enrichment and isolation of microorganisms in situ under high pressure in the deep sea, solving the problems of deep-sea in-situ barophilic bacteria failing to survive or exhibiting differential expression under atmospheric pressure. Furthermore, compared to conventional enrichment and streak plating techniques, this embodiment provides a method for high-throughput screening of microorganisms based on specific morphological and metabolic characteristics. This not only solves the difficulties in enriching, isolating, and culturing microorganisms outside of high-pressure environments but also enables high-throughput identification and screening of single-cell-scale marine microorganisms under high pressure, thus improving isolation efficiency.

[0051] More specifically, the microbial enrichment injection system 1 includes a microfluidic pump 11, a high-pressure microbial enrichment culture chamber 12, and an inlet pressure detection device 13; wherein: the control end of the microfluidic pump 11 is electrically connected to the data acquisition and processing system 8; the input end of the microfluidic pump 11 is connected to the outlet end of the high-pressure microbial enrichment culture chamber 12, and the output end is connected to the inlet end of the pressure-resistant visual sorting chamber 2 through the inlet pressure detection device 13; the high-pressure microbial enrichment culture chamber 12 is used to cultivate bacterial solutions containing microorganisms, and injects them into the pressure-resistant visual sorting chamber 2 through the microfluidic pump 11.

[0052] More specifically, the pressure-resistant visual sorting chamber 2 further includes a pressure-resistant visual cavity, an annular wall cooling / heating cavity 22, and an annular wall high-pressure cavity; wherein: the pressure-resistant visual cavity is made of pressure-resistant and corrosion-resistant metal material, with pressure-resistant visual material inlaid on its front and back, and the entire cavity can withstand the pressure of a water depth of 5000 meters, and it is connected to the pressurization system 4; the carrier chip 21 is located in the center of the pressure-resistant visual cavity; the inlet of the microfluidic channel of the carrier chip 21 is connected to the enrichment microorganism injection system 1, and its outlet is the outlet end of the pressure-resistant visual sorting chamber 2, which is connected to the automatic sorting system 7; the annular wall high-pressure cavity is located on the outer ring of the pressure-resistant visual cavity to protect the carrier chip 21 from damage in the pressure-resistant visual cavity; the annular wall high-pressure cavity is connected to the pressurization system 4 and the annular pressure control system 5; the annular wall cooling / heating cavity 22 is wrapped around the outer wall of the pressure-resistant visual cavity, used to fill the cooling / heating fluid, and is connected to the annular wall temperature control system 3 through the cooling / heating fluid.

[0053] In the specific implementation process, the pressure-resistant visual cavity is equipped with a vent valve 23, the output of which is electrically connected to the data acquisition and processing system 8 to facilitate pressure adjustment within the cavity. To protect the carrier chip 21 from damage within the pressure-resistant visual cavity, an annular high-pressure chamber is provided. Pressure is simultaneously increased in the outer ring of the pressure-resistant visual cavity, and an annular pressure control system 5 is installed to automatically increase or decrease the pressure in the annular high-pressure chamber according to pressure changes in the pressure-resistant visual cavity, achieving pressure balance between the pressure-resistant visual cavity and the annular high-pressure chamber. This ensures that the carrier chip 21 withstands minimal pressure difference without being damaged.

[0054] In the specific implementation process, the annular wall temperature control system 3 adopts a circulating cooling / heating device 31 and a temperature sensor 32; the control terminal of the circulating cooling / heating device 31 is electrically connected to the data acquisition and processing system 8, and is used for cooling / heating and circulating the cooling / heating fluid in the annular wall cooling / heating cavity 22; the probe of the temperature sensor 32 is set in the pressure-resistant visible cavity, and its signal output terminal is electrically connected to the data acquisition and processing system 8.

[0055] In the specific implementation process, the temperature of the pressure-resistant visual cavity is mainly maintained by injecting a cooling / heating fluid into the annular wall cooling / heating cavity 22, and by circulating the fluid for cooling or heating to ensure that the fluid in the annular wall cooling / heating cavity 22 is in a low-temperature or high-temperature state. Then, the low-temperature or high-temperature state in the pressure-resistant visual cavity is maintained by heat exchange between the cooling / heating fluid and the pressure-resistant visual cavity.

[0056] More specifically, the pressurization system 4 includes an air compressor 41, a booster pump 42, an air tank 43, a pressure regulating valve 44, and a pressure sensor 45; the air compressor 41, booster pump 42, air tank 43, and pressure regulating valve 44 are connected in sequence and then connected to the pressure-resistant visual cavity and the annular high-pressure cavity respectively; the probe of the pressure sensor 45 is set in the pressure-resistant visual cavity, and its signal output terminal is electrically connected to the data acquisition and processing system 8.

[0057] In the specific implementation process, the temperature sensor 32 and the pressure sensor 45 are set up to measure and monitor the temperature and pressure of the pressure-resistant visual cavity throughout the entire microbial sorting process.

[0058] More specifically, the ring pressure control system 5 includes a ring pressure detection device 51, a first back pressure tracking pump 52, a back pressure detection device 53, a back pressure valve 54, a buffer tank 55, and a second back pressure tracking pump 56; wherein: the probe of the ring pressure detection device 51 is disposed in the high-pressure chamber of the ring wall, and its output end is electrically connected to the data acquisition and processing system 8; the back pressure tracking pump is connected to the high-pressure chamber of the ring wall, and its control end is electrically connected to the data acquisition and processing system 8; the detection end of the back pressure detection device 53 is connected to the pressure-resistant visual chamber, and its signal output end is electrically connected to the data acquisition and processing system 8; one end of the back pressure valve 54 is connected to the automatic sorting system 7, and the other end is connected to the second back pressure tracking pump 56 through the buffer tank 55; the control end of the second back pressure tracking pump 56 is electrically connected to the data acquisition and processing system 8.

[0059] More specifically, the optical recognition system 6 employs a spectral / optical observation module. When enriched microorganisms pass through the carrier chip 21, the spectral / optical observation module observes and identifies the microorganisms and sends the identification results to the data acquisition and processing system 8.

[0060] During the passage of enriched microorganisms through the carrier chip 21, the microorganisms are observed and identified using a spectroscopic / optical observation module. The morphology of single cells can be identified by observation above the chip using a high-resolution optical microscope, and intracellular marker biocompounds can be identified using Raman spectroscopy. By combining optical and spectroscopic identification signals, it can be determined whether the microorganisms in the chip are the target microorganisms desired by the researchers.

[0061] More specifically, the automatic sorting system 7 includes an intelligent control three-way module 71, a target microorganism storage module 72, and a non-target microorganism storage module 7372; wherein, the target microorganism storage module 72 and the non-target microorganism storage module 7372 are respectively connected to two connection ends of the intelligent control three-way module 71, and the other connection end of the intelligent control three-way module 71 is connected to the outlet end of the pressure-resistant visual sorting chamber 2; the control end of the intelligent control three-way module 71 is electrically connected to the data acquisition and processing system 8.

[0062] In the specific implementation process, an automatic sorting system 7 is installed at the outlet end of the pressure-resistant visual cavity to perform directional sorting of identified microorganisms. The automatic sorting system 7 is mainly controlled by an intelligent control three-way module 71. This module is an automatically opening and closing three-way valve. When an identified single cell is determined to be a target microorganism, the valve of the target microorganism storage module 72 opens, allowing the single cell to enter. When an identified single cell is determined to be a non-target microorganism, the channel of the target microorganism storage module 72 is opened, allowing the cell to enter, thus achieving high-throughput single-cell sorting. The target microorganism storage module 72 can be selected as either a normal pressure container or a high-pressure container, depending on experimental needs. Both containers contain the corresponding culture medium to meet the requirements for further cultivation of the sorted microorganisms.

[0063] Example 2

[0064] More specifically, such as Figure 3 As shown, this solution also provides a high-throughput sorting method for single cells in the marine in-situ environment, which is implemented using a high-throughput sorting device for single cells in the marine in-situ environment, specifically including the following steps:

[0065] S1: Determine the pressure value inside the pressure-resistant visual sorting chamber 2 based on the pressure value of the enriched microorganism injection system 1; inject gas into the pressure-resistant visual chamber through the pressurization system 4 so that the pressure value inside the pressure-resistant visual chamber is consistent with that of the enriched microorganism injection system 1;

[0066] S2: Open the ring pressure control system 5 to make the pressure of the pressure-resistant visible cavity and the high-pressure cavity of the ring wall consistent;

[0067] S3: Determine the temperature value inside the pressure-resistant visual cavity based on the temperature value inside the enriched microorganism injection system 1, and make the temperature value inside the annular wall temperature control system 3 consistent with the temperature value inside the pressure-resistant visual cavity by activating the annular wall cooling / heating cavity 22.

[0068] S4: Adjust the optical recognition system 6 so that it can clearly observe the situation inside the object carrier chip 21;

[0069] S5: Inject the microbial-containing bacterial solution from the microbial enrichment injection system 1 into the pressure-resistant visual cavity through the microfluidic pump 11, so that the bacterial solution slowly passes through the carrier chip 21, allowing it to pass through the etching channel in the form of single cells; turn on the ring wall temperature control system 3 to keep the pressure at the outlet of the pressure-resistant visual cavity constant when the liquid flows out of the pressure-resistant visual cavity;

[0070] S6: During the process of the bacterial solution passing through the carrier chip 21, the optical recognition system 6 fully observes and collects the morphology of the cells, as well as the spectroscopic information of intracellular and extracellular metabolic compounds, to determine whether the cell is the target microorganism, and sends the recognition result to the data acquisition and processing system 8;

[0071] S7: The automatic sorting system 7 intelligently opens the intelligent control three-way module 71 according to the identification result, sending the target microorganism to the target microorganism storage module 72 and the non-target microorganism to the non-target microorganism storage module 7372;

[0072] S8: Once the number of target microorganisms in the target microorganism storage module 72 meets the requirements, the sorting process ends.

[0073] More specifically, before performing step S1, the marine in-situ single-cell high-throughput sorting device needs to be pre-treated. Specifically, the outlet end of the pressure-resistant visual cavity is opened, and the pressure-resistant visual cavity is repeatedly cleaned by pumping in deionized water. After rinsing, 75% alcohol is pumped in. After the pressure-resistant visual cavity is completely filled with alcohol, the pressure-resistant visual cavity is closed, left to stand for 24 hours, and then the alcohol in the pressure-resistant visual cavity is emptied to complete the pre-treatment.

[0074] Throughout the sorting process, the pressure and temperature within the pressure-resistant visual cavity are maintained consistent with the pressure and temperature environment of the enriched microorganism injection system 1 where the microorganisms were initially located, allowing the microorganisms to be sorted under in-situ high pressure conditions. During the sorting process, the ring pressure control system 5 is activated, and the pressure value within the high-pressure chamber of the ring wall is maintained consistent with the pressure value within the pressure-resistant visual cavity, ensuring that the carrier chip 21 does not experience pressure differentials and does not deform or break.

[0075] The high-throughput single-cell sorting chip and sorting technology for marine microorganisms under high-pressure environments proposed in this embodiment can achieve the identification and sorting of microorganisms under high-pressure marine conditions, meeting the needs of subsequent purification and culture. Compared with the current traditional techniques for enriching and separating marine microorganisms under normal pressure environments, this method effectively solves the problems of low survival rates of marine pressure-tolerant and barophilic bacteria under normal pressure, and the inability to effectively express deep-sea native characteristics under normal pressure. It also addresses the current issues of low culture density and difficulty in culturing pure bacteria in marine microorganisms. Furthermore, this solution enables high-throughput identification and automatic sorting at the single-cell scale under high pressure, effectively improving the efficiency of microbial culture and purification compared to conventional microbial isolation and culture techniques.

[0076] Example 3

[0077] More specifically, to further illustrate the technical implementation process and effects of this solution, the high-throughput single-cell sorting microfluidic chip for deep-sea methanogens involved in this embodiment can achieve high-throughput single-cell sorting of enriched deep-sea methanogens under in-situ high pressure, meeting the requirements for subsequent culture and functional determination. The core of this example is the pressure-resistant and visually oriented pressure-resistant sorting chamber 2. Other components mainly include a pressurization system 4, an annular pressure control system 5, an optical recognition system 6, an automatic sorting system 7, and a data acquisition and processing system 8.

[0078] The core component, the pressure-resistant visual sorting chamber 2, mainly includes a pressure-resistant visual cavity, a carrier chip 21, an annular cooling / heating cavity 22, and an annular high-pressure cavity. The pressure-resistant visual cavity is made of pressure-resistant and corrosion-resistant titanium alloy, with pressure-resistant sapphire material inlaid on the front and back. The entire cavity can withstand pressure at a depth of 5000 meters. The carrier chip 21 is located in the center of the pressure-resistant visual cavity. Microfluidic channels are provided on the carrier chip 21, allowing microbial-containing bacterial solutions to be injected from the high-pressure microbial enrichment culture chamber 12 into the pressure-resistant visual cavity via a microfluidic pump 11. This allows the bacterial solutions to slowly pass through the carrier chip 21, and methane is injected from the pressurization system 4. An inlet pressure detection device 13 is installed between the enrichment microbial injection system 1 and the pressure-resistant visual sorting chamber 2. The outlet of the pressure-resistant visual cavity is mainly used for the fluid containing the deep-sea methanophilic bacteria enrichment solution after sorting to exit the pressure-resistant visual cavity and enter the automatic sorting system 7. A backpressure control system 5 is installed at the outlet end to control backpressure. This system mainly includes a backpressure detection device 53, a backpressure valve 54, a buffer tank 55, and a second backpressure tracking pump 56. This ensures that the fluid containing microorganisms flows out of the sorting system under a set pressure condition, maintaining a constant pressure within the pressure-resistant visual cavity throughout the sorting process. The pressure-resistant visual cavity is equipped with a vent valve 23 for easy pressure adjustment. A temperature sensor 32 and a pressure sensor 45 are also installed in the pressure-resistant visual cavity to measure and monitor the temperature and pressure within the cavity during the sorting of deep-sea methanogens. The carrier chip 21 is made of a visible material and has embedded etched microfluidic channels. This allows the enriched deep-sea methanogen solution to enter the pressure-resistant visual sorting cavity and pass through the chip at a relatively low flow rate, enabling single cells to disperse within the channels. To protect the carrier chip 21 from damage within the pressure-resistant viewing cavity, a high-pressure chamber is provided around the annular wall. Pressure is simultaneously increased in the outer ring of the pressure-resistant viewing cavity, and a ring pressure control system 5 is installed. This system automatically increases or decreases the pressure in the high-pressure chamber based on changes in the pressure within the pressure-resistant viewing cavity, achieving pressure balance between the pressure-resistant viewing cavity and the high-pressure chamber. This ensures that the carrier chip 21 withstands minimal pressure difference without being damaged. The temperature of the pressure-resistant viewing cavity is maintained primarily by injecting a cooling / heating fluid, such as a refrigeration solution containing ethylene glycol, into the annular wall cooling / heating chamber 22. The fluid is circulated through a cooling / heating device 31 to maintain a low temperature of 4°C within the annular wall chamber. Heat exchange between the cooling fluid and the pressure-resistant viewing cavity further maintains this low temperature.

[0079] During the process of enriched microorganisms passing through the sorting chip, the microorganisms are observed and identified using a spectral / optical observation module. For example, the morphology of single cells can be identified by observing above the chip using a high-resolution optical microscope, and intracellular marker biocompounds can be identified using Raman spectroscopy. Combining optical and spectroscopic identification signals, it can be determined whether the microorganisms in the chip are deep-sea methanogens. An automatic sorting system 7 is set at the outlet of the pressure-resistant visual cavity to perform directional sorting of the identified microorganisms. The automatic sorting system is equipped with an intelligent control three-way module 71, which is an automatically opening and closing three-way valve. When the identified single cell is determined to be a deep-sea methanogen, the valve of the target microorganism storage module 72 is opened, and the single cell enters the target microorganism storage module 72. When the identified single cell is determined to be a non-deep-sea methanogen, the collection channel of the non-target microorganism storage module 73 is opened, and the cell enters the non-target microorganism storage module 73, thereby achieving the purpose of high-throughput single-cell sorting. The target microbial storage module 72 can be an atmospheric pressure container or a high pressure container to meet the needs of the sorted deep-sea methanophiles to continue to be cultured in a high-pressure environment.

[0080] This example demonstrates a high-throughput single-cell sorting technology for marine microorganisms under high pressure, primarily requiring the creation of a high-pressure environment within a pressure-resistant visible cavity that mimics the living conditions of deep-sea methanogens. First, the pressure-resistant visible cavity is cleaned by opening the inlet and outlet and pumping in deionized water for repeated rinsing. After thorough rinsing, 75% alcohol is pumped in until the cavity is completely filled with alcohol. The cavity is then closed and allowed to stand for 24 hours before being vented. Next, the pressure within the visible cavity is determined based on the initial pressure of 12 MPa in the enrichment microorganism injection system 1. CH4 gas is injected into the cavity via the pressurization system 4 to increase the pressure to 12 MPa. The annular pressure control system 5 is then activated to ensure that the pressure in the annular high-pressure chamber matches that of the visible cavity. During the sorting process, if pressure changes occur within the visible cavity, gas is injected into the annular high-pressure chamber via the first backpressure tracking pump 52, or a valve is opened to release pressure, ensuring that the pressure in the annular high-pressure chamber remains consistent with that of the visible cavity. Then, based on the temperature value of 4°C within the enriched microbial injection system 1, the temperature value within the pressure-resistant visual cavity is determined. By activating the annular wall temperature control system 3, the temperature value within the annular wall cooling / heating cavity is made consistent with the temperature within the pressure-resistant visual cavity. Next, the spectral / optical observation module is adjusted to clearly observe the conditions within the carrier chip 21. Then, the bacterial solution containing deep-sea methanophores is injected into the pressure-resistant visual cavity from the enriched microbial injection system 1 via the microfluidic pump 11, and the outlet backpressure is opened, setting the outlet pressure to 11.5 MPa. This allows the bacterial solution to slowly pass through the carrier chip 21, enabling it to pass through the etched channel in the form of single cells. During the passage of the bacterial solution through the carrier chip 21, the spectral / optical observation module is activated to fully observe and collect the morphology of the cells and the spectroscopic information of the single-celled microorganisms. It is determined whether the cell is a methanophore. If it is a methanophore, the valve of the automatic sorting system 7 is opened, allowing it to enter the methanophore collection module; otherwise, it enters the non-target bacteria collection module. Once all the fluid within the enriched microorganism injection system 1 has been sorted and identified, the sorting process ends. Throughout the sorting process, the pressure and temperature within the pressure-resistant visual cavity are maintained consistent with the pressure and temperature environment of the enriched microorganism injection system 1 where the deep-sea methanogens were initially located, allowing the microorganisms to be sorted under in-situ high pressure conditions. During the sorting process, the annular pressure control system 5 is activated, and the pressure value within the high-pressure chamber of the annular wall is maintained consistent with the pressure value within the pressure-resistant visual cavity, ensuring that the carrier chip 21 does not experience pressure differentials and does not deform or break.

[0081] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A high-throughput single-cell sorting device for marine in-situ environment, characterized in that, It includes a microbial enrichment injection system (1), a pressure-resistant visual sorting chamber (2), an annular wall temperature control system (3), a pressurization system (4), an annular pressure control system (5), an optical recognition system (6), an automatic sorting system (7), and a data acquisition and processing system (8); among which: The enriched microbial injection system (1) is used to cultivate and inject microbial-containing bacterial solutions into the pressure-resistant visual sorting chamber (2); The pressure-resistant visual sorting chamber (2) is equipped with a carrier chip (21), which is made of a visible material and has embedded etched microfluidic channels to enable the enriched microorganisms to disperse and pass through the channels; when the enriched microorganisms pass through the carrier chip (21), the microorganisms are observed and identified by an optical recognition system (6); The outlet end of the pressure-resistant visual sorting chamber (2) is connected to the automatic sorting system (7), and the automatic sorting system (7) automatically sorts microorganisms according to the microorganism identification results of the optical recognition system (6); The annular wall temperature control system (3) is used to ensure that the internal temperature of the pressure-resistant visual sorting chamber (2) is consistent; The pressurization system (4) is used to make the internal pressure of the pressure-resistant visual sorting chamber (2) consistent with the internal pressure of the enriched microorganism injection system (1); The ring pressure control system (5) is used to maintain the pressure inside the pressure-resistant visual sorting chamber (2) consistent with the pressure value change in the automatic sorting system (7), so as to avoid deformation or damage to the loaded chip (21) due to pressure difference. The enrichment microbial injection system (1), the ring wall temperature control system (3), the pressurization system (4), the ring pressure control system (5), the optical recognition system (6), and the automatic sorting system (7) are all electrically connected to the data acquisition and processing system (8). The pressure-resistant visual sorting chamber (2) also includes a pressure-resistant visual cavity, an annular wall cooling / heating cavity (22) and an annular wall high-pressure cavity; wherein: the pressure-resistant visual cavity is made of pressure-resistant and corrosion-resistant metal material, and its front and back are inlaid with pressure-resistant visual material, and it is connected to the pressurization system (4); The carrier chip (21) is disposed in the center of the pressure-resistant visual cavity; the inlet of the microfluidic channel of the carrier chip (21) is connected to the enriched microorganism injection system (1), and its outlet is the outlet end of the pressure-resistant visual sorting chamber (2), which is connected to the automatic sorting system (7). The high-pressure chamber is located on the outer ring of the pressure-resistant visible cavity to protect the chip (21) from damage in the pressure-resistant visible cavity; the high-pressure chamber is connected to the pressurization system (4) and the ring pressure control system (5); The annular wall cooling / heating cavity (22) is wrapped around the outer wall of the pressure-resistant visible cavity and is used to load the cooling / heating fluid, and is connected to the annular wall temperature control system (3) through the cooling / heating fluid; The ring pressure control system (5) includes a ring pressure detection device (51), a first back pressure tracking pump (52), a back pressure detection device (53), a back pressure valve (54), a buffer tank (55), and a second back pressure tracking pump (56); wherein: The probe of the ring pressure detection device (51) is set inside the high-pressure chamber of the ring wall, and its output end is electrically connected to the data acquisition and processing system (8); the back pressure tracking pump (52) is connected to the high-pressure chamber of the ring wall, and its control end is electrically connected to the data acquisition and processing system (8); The back pressure detection device (53) has its detection end connected to the pressure-resistant visual cavity, and its signal output end is electrically connected to the data acquisition and processing system (8). One end of the back pressure valve (54) is connected to the automatic sorting system (7), and the other end is connected to the second back pressure tracking pump (56) through the buffer tank (55); The control terminal of the second back pressure tracking pump (56) is electrically connected to the data acquisition and processing system (8); The automatic sorting system (7) includes an intelligent control three-way module (71), a target microorganism storage module (72), and a non-target microorganism storage module (73); wherein the target microorganism storage module (72) and the non-target microorganism storage module (73) are respectively connected to two connection ends of the intelligent control three-way module (71), and the other connection end of the intelligent control three-way module (71) is connected to the outlet end of the pressure-resistant visual sorting chamber (2); the control end of the intelligent control three-way module (71) is electrically connected to the data acquisition and processing system (8).

2. The marine in-situ single-cell high-throughput sorting device according to claim 1, characterized in that, The enriched microbial injection system (1) includes a microfluidic pump (11), a high-pressure microbial enrichment culture chamber (12), and an inlet pressure detection device (13); wherein: The control terminal of the microfluidic pump (11) is electrically connected to the data acquisition and processing system (8); The input end of the microfluidic pump (11) is connected to the liquid outlet of the high-pressure microbial enrichment culture chamber (12), and the output end is connected to the inlet end of the pressure-resistant visual sorting chamber (2) through the inlet pressure detection device (13). The high-pressure microbial enrichment culture chamber (12) is used to cultivate bacterial solutions containing microorganisms, which are then injected into the pressure-resistant visual sorting chamber (2) via the microfluidic pump (11).

3. The marine in-situ single-cell high-throughput sorting device according to claim 1, characterized in that, The annular wall temperature control system (3) employs a circulating cooling / heating device (31) and a temperature sensor (32); the control terminal of the circulating cooling / heating device (31) is electrically connected to the data acquisition and processing system (8) for cooling / heating and circulating the cooling / heating fluid in the annular wall cooling / heating cavity (22); the probe of the temperature sensor (32) is set in the pressure-resistant visible cavity, and its signal output terminal is electrically connected to the data acquisition and processing system (8).

4. The marine in-situ single-cell high-throughput sorting device according to claim 1, characterized in that, The pressurization system (4) includes an air compressor (41), a booster pump (42), an air tank (43), a pressure regulating valve (44), and a pressure sensor (45). The air compressor (41), booster pump (42), air tank (43), and pressure regulating valve (44) are connected in sequence and then connected to the pressure-resistant visual cavity and the annular high-pressure cavity, respectively. The probe of the pressure sensor (45) is set in the pressure-resistant visual cavity, and its signal output terminal is electrically connected to the data acquisition and processing system (8).

5. A high-throughput single-cell sorting device for marine in-situ environment according to claim 1, characterized in that, The optical recognition system (6) uses a spectral / optical observation module. When enriched microorganisms pass through the carrier chip (21), the spectral / optical observation module observes and identifies the microorganisms and sends the identification results to the data acquisition and processing system (8).

6. A high-throughput sorting method for single cells in situ in marine environments, characterized in that, The method employs the marine in-situ single-cell high-throughput sorting device as described in claim 1, specifically including the following steps: S1: Determine the pressure value inside the pressure-resistant visual sorting chamber (2) based on the pressure value of the enriched microorganism injection system (1); inject gas into the pressure-resistant visual chamber through the pressurization system (4) so ​​that the pressure value inside the pressure-resistant visual chamber is consistent with or has a slight pressure difference with the enriched microorganism injection system (1); S2: Open the ring pressure control system (5) to make the pressure of the pressure-resistant visible cavity and the high-pressure cavity of the ring wall consistent; S3: Determine the temperature value inside the pressure-resistant visual cavity based on the temperature value inside the enriched microorganism injection system (1), and make the temperature value inside the ring wall cooling / heating cavity (22) consistent with the temperature value inside the pressure-resistant visual cavity by turning on the ring wall temperature control system (3); S4: Adjust the optical recognition system (6) so that it can clearly observe the situation inside the carrier chip (21); S5: Inject the microbial-containing bacterial solution from the microbial enrichment injection system (1) into the pressure-resistant visual cavity through the microfluidic pump (11), so that the bacterial solution slowly passes through the carrier chip (21) and can pass through the etching channel in the form of single cells; turn on the ring wall temperature control system (3) to keep the pressure at the outlet of the pressure-resistant visual cavity constant when the liquid flows out of the pressure-resistant visual cavity; S6: During the process of the bacterial solution passing through the carrier chip (21), the morphology of the collected cells and the spectroscopic information of the single-celled microorganisms are fully observed and collected through the optical recognition system (6) to determine whether the cell is the target microorganism and send the recognition result to the data acquisition and processing system (8). S7: The automatic sorting system (7) intelligently opens the intelligent control three-way module (71) according to the identification result, sends the target microorganism to the target microorganism storage module (72), and sends the non-target microorganism to the non-target microorganism storage module (73); S8: When the number of target microorganisms in the target microorganism storage module (72) meets the requirements, the sorting process ends.

7. The method for high-throughput sorting of single cells in situ in marine environments according to claim 6, characterized in that, Before performing step S1, the marine in-situ single-cell high-throughput sorting device needs to undergo pretreatment, specifically: Open the outlet of the pressure-resistant visual cavity and repeatedly clean it with deionized water by pumping in deionized water. After rinsing, pump in 75% alcohol. After the pressure-resistant visual cavity is completely filled with alcohol, close the pressure-resistant visual cavity, let it stand for 24 hours, and then drain the alcohol from the pressure-resistant visual cavity to complete the pretreatment.

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