An in-situ detection device and method for detecting microbial fluorescence activity and regulating carbon dioxide microenvironment underwater

Abstract

The application relates to the technical field of underwater in-situ detection and microbial activity analysis, and particularly discloses a device and a method for underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation, which comprises the following: an underwater sampling module, which is used for obtaining water samples containing microorganisms in a target water body; an AF2400 gas permeable membrane, which is used for realizing the transmission of carbon dioxide in the water body or a microfluidic chip to a gas detection optical cell or a gas regulation area; and a microfluidic chip, which is used for transporting, capturing, enriching and positioning the microorganisms entering the chip. The device couples the dense non-microporous gas permeable membrane and the microfluidic chip, realizes the transmembrane transmission and gas-liquid isolation of carbon dioxide in the underwater in-situ environment, completes the transfer of gas molecules from the water phase to the gas phase through the dissolution, diffusion and desorption mechanisms, can effectively block the liquid water and dissolved ions from entering the gas phase detection area, and significantly improves the stability and reliability of the underwater long-term continuous detection.

Description

Technical Field

[0001] This invention relates to the field of underwater in-situ detection and microbial activity analysis technology, and in particular to an underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device and method. Background Technology

[0002] In underwater environments, carbon dioxide is a key gaseous component of microbial metabolic activities and the global carbon cycle. Its concentration changes directly or indirectly reflect the physiological metabolic intensity and ecological function of microbial communities. As an important driver of underwater ecosystems, the activity state of microorganisms is closely related to the surrounding carbon dioxide microenvironment. Traditional underwater microbial detection methods mostly rely on analysis after sample collection on land, making it difficult to simultaneously obtain dynamic information on microbial activity and the carbon dioxide concentration in the real underwater environment. Fluorescence detection can characterize the physiological state of microorganisms, while accurate measurement of carbon dioxide concentration usually requires the use of gas sensors.

[0003] In existing technologies, traditional underwater microbial detection relies on offline sampling, making it difficult to simultaneously acquire information on the in-situ physiological activity of microorganisms and the carbon dioxide concentration of their environment on the same platform. Moreover, conventional gas detection methods require introducing aqueous samples into the gas phase detection area, but liquid water can interfere with spectral detection and is difficult to achieve gas-liquid isolation. At the same time, existing systems lack the ability to actively change the carbon dioxide gas environment around microorganisms, making it difficult to further observe the dynamic response of microbial activity to changes in carbon dioxide concentration. Therefore, how to simultaneously solve the three progressive problems of microbial activity characterization, carbon dioxide concentration detection, and active regulation of the gas microenvironment on the same in-situ underwater platform is the problem that this invention aims to solve. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides an underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device and method, which can effectively solve the problems involved in the prior art.

[0005] The objective of this invention can be achieved through the following technical solution: Firstly, this invention provides an underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device, comprising: The underwater sampling module is used to obtain water samples containing microorganisms in the target water body, realizing in-situ acquisition of microbial samples and avoiding the activity distortion caused by in vitro sampling; The AF2400 gas permeable membrane is used to enable the transfer of carbon dioxide from water or microfluidic chips to the gas detection optical cell or gas control region, achieving non-destructive cross-membrane transfer of carbon dioxide under gas-liquid isolation and avoiding interference of liquid water with spectral detection. Microfluidic chips are used to transport, capture, enrich, and locate microorganisms that enter the chip, enabling automated localization and controllable observation of microorganisms, reducing manual intervention and sample consumption; The fluorescence detection module is used to collect fluorescence signals of microorganisms in the microfluidic chip and determine the physiological activity status of microorganisms based on changes in fluorescence intensity, thereby realizing real-time in-situ characterization of microbial activity status and evaluating their physiological response to environmental changes. The mid-infrared TDLAS detection module for carbon dioxide is used to determine the concentration of carbon dioxide after transmission through the AF2400 gas permeation membrane based on 2.6μm mid-infrared tunable semiconductor laser absorption spectroscopy technology, thereby achieving accurate spectral determination of carbon dioxide concentration and adapting to the complex environment of underwater in-situ detection. The carbon dioxide microenvironment control module is used to inject, release, exchange, or adjust the concentration gradient of carbon dioxide gas environment within the microfluidic chip or around microorganisms, enabling artificial controllable adjustment of the carbon dioxide environment around microorganisms and supporting the study of dynamic response patterns. The temperature and pressure environment monitoring module is used to collect temperature and pressure parameters in the underwater environment, gas detection optical cell and microfluidic chip, to achieve simultaneous monitoring and compensation of temperature and pressure at multiple locations, and to ensure the accuracy of concentration inversion and activity discrimination. The data processing and control module is used to perform fusion analysis on microbial fluorescence intensity, carbon dioxide concentration, temperature, pressure and gas regulation parameters, and evaluate the changes in physiological activity of microorganisms under different carbon dioxide gas environments based on the analysis results. It realizes multi-parameter fusion and closed-loop regulation decision-making, and outputs a quantitative evaluation of the microbial response to the gas environment.

[0006] Preferably, the AF2400 gas permeable membrane is a dense non-microporous membrane structure. The AF2400 gas permeable membrane is disposed between the aqueous phase region and the gas phase region to block liquid water from entering the gas detection optical cell, while allowing carbon dioxide molecules dissolved in the water to be transported to the gas detection optical cell through the membrane material. The microfluidic chip includes an injection channel, a microbial capture area, a fluorescence observation area, a gas exchange area, and a drainage channel; Among them, the underwater sampling module obtains microbial-containing water samples in situ in the target water body, and the water samples are introduced into the microfluidic chip injection channel using an isothermal and isobaric flow path. At the same time, the temperature and pressure environment of the water samples are maintained to be consistent with the in situ water body to avoid temperature and pressure changes interfering with microbial activity and to ensure that the detection truly reflects the in situ state. Within a microfluidic chip, fluid manipulation structures are used to transport, capture, enrich, and locate microorganisms in water samples, enabling microorganisms to be positioned in a controllable micro-region between the fluorescence observation area and the gas exchange area, thus achieving automated microorganism enrichment and fixed-point observation without human intervention. By using the AF2400 gas permeable membrane placed between the aqueous and gas phases, CO2 molecules are dissolved, diffused, and desorbed into the gas detection optical cell or gas control channel based on the dissolution-diffusion-desorption cascade mechanism, thus blocking interference from liquid water and ion spectra and achieving poreless transmembrane transport under gas-liquid isolation.

[0007] Preferably, the fluorescence detection module includes an excitation light source, a fluorescence excitation filter unit, a fluorescence collection optical unit, a fluorescence filter unit, an imaging detector, and a fluorescence intensity analysis unit. The fluorescence detection module obtains the fluorescence intensity of the microorganism through one or more of the following: microbial autofluorescence, fluorescence staining signal, or fluorescence probe signal. It then determines the physiological activity state of the microorganism based on the fluorescence intensity, fluorescence intensity change rate, or normalized fluorescence intensity, thereby achieving in-situ non-destructive discrimination of the microbial activity state. Specifically, the excitation light source generates a specific wavelength of excitation light through the filter unit, which irradiates the pre-positioned microbial area within the microfluidic chip, stimulating its own fluorescence, staining signal, or fluorescent probe signal, ensuring the high efficiency and specificity of fluorescence excitation. Microbial fluorescence images are acquired by using a fluorescence collection optical unit and an imaging detector. The fluorescence intensity, fluorescence distribution area, or integral fluorescence value of a single microorganism within the observation area are extracted to obtain multi-dimensional quantitative information on microbial activity and distribution. The normalized fluorescence change rate is calculated based on the ratio of fluorescence intensity to initial fluorescence intensity to determine whether the physiological activity of microorganisms is in an enhanced, weakened, or stable state, thus eliminating batch differences and enabling comparable judgment of activity change trends.

[0008] Preferably, the fluorescence detection module further includes: Microbial fluorescence images were acquired multiple times in a continuous time series, and the integrated fluorescence intensity at each moment was extracted to form a dynamic curve of fluorescence intensity changing over time, thereby tracking the continuous evolution of microbial activity in real time. The dynamic curve is processed by moving average filtering, and the instantaneous rate of change of fluorescence intensity and the first derivative of the rate of change are calculated to identify the rapid rise, decay or plateau of fluorescence signal, and to accurately identify the stage change characteristics of fluorescence signal. When the normalized fluorescence change rate exceeds the preset threshold range and continues to exceed the set time window, it is determined that the microorganism has entered an abnormal activity state, and carbon dioxide microenvironment regulation or data labeling is triggered to realize automatic identification and closed-loop response of abnormal activity.

[0009] Preferably, the mid-infrared TDLAS detection module for carbon dioxide includes a 2.6μm mid-infrared tunable laser, a collimating optical element, a gas detection optical cell, a mid-infrared detector, a laser driving and temperature control unit, a signal acquisition unit, and a concentration inversion unit, effectively integrating dedicated optical components to achieve highly selective underwater carbon dioxide detection; In this process, carbon dioxide gas, which is transmitted to the gas detection optical cell through the AF2400 gas permeation membrane, is placed in a closed optical path after temperature and pressure correction. A 2.6μm mid-infrared tunable laser emits a laser using a sinusoidal scanning and sawtooth superposition modulation method. By using a closed optical path and modulated laser, the spectral signal quality and detection stability are improved. The absorption signal of carbon dioxide at the characteristic absorption line of 2.6 μm is acquired by direct absorption spectroscopy or wavelength modulation spectroscopy, and the second harmonic signal is extracted in wavelength modulation mode. When wavelength modulation spectroscopy is used, the carbon dioxide concentration is inverted by the first harmonic signal, the second harmonic signal or the normalized second harmonic signal. The use of multiple harmonic signals for inversion enhances the flexibility and anti-interference ability of concentration measurement. The concentration inversion unit calculates the absolute concentration of carbon dioxide in the gas detection optical cell based on the absorption signal amplitude, the peak intensity of the second harmonic, and the real-time temperature and pressure compensation coefficient. It then performs the inversion by integrating temperature and pressure compensation to ensure the authenticity and accuracy of the concentration output.

[0010] Preferably, the carbon dioxide microenvironment control module specifically includes: Based on the fusion analysis results of the current carbon dioxide concentration and the fluorescence activity status of microorganisms, it is determined whether it is necessary to change the carbon dioxide gas environment around the microorganisms in the microfluidic chip, so as to realize intelligent control decision based on real-time detection results. When adjustment is needed, the carbon dioxide microenvironment control module is activated, and a set concentration of carbon dioxide gas or mixed carrier gas is introduced into the gas exchange area through the gas injection channel to achieve active and precise intervention in the gas environment around microorganisms. Carbon dioxide molecules are transported across the membrane from the gas phase control channel to the aqueous phase region of the microfluidic chip through the AF2400 gas permeation membrane, enabling the raising, lowering, or maintaining of carbon dioxide concentration around microorganisms, and completing gas concentration regulation under bubble-free and undisturbed conditions.

[0011] Preferably, the carbon dioxide microenvironment control module further includes: By introducing carbon dioxide gas of different concentrations into different spatial locations of the gas exchange zone through multiple independent gas inlets, the AF2400 membrane exhibits differentiated gas phase carbon dioxide partial pressure in different regions, thereby achieving a stable and controllable spatial partial pressure distribution pattern on the gas phase side of the membrane. By utilizing the fluid isolation structure within the microfluidic chip to maintain the carbon dioxide concentration differences in different regions of the aqueous phase, a stable carbon dioxide concentration gradient or periodic fluctuation environment is formed in the microbial capture zone, enabling parallel observation of microbial communities simultaneously exposed to multiple concentration environments. Based on real-time fluorescence response feedback, the flow rate ratio or gas switching frequency of each gas inlet is dynamically adjusted to make the carbon dioxide concentration around the microorganisms change according to a set curve. This is used to analyze the sensitive response range of microorganisms to the gas environment, and to achieve accurate analysis and quantitative characterization of the sensitivity of microbial activity to the gas environment.

[0012] Preferably, the temperature and pressure environment monitoring module specifically includes: Temperature and absolute pressure sensors calibrated underwater were deployed at three independent locations: the inlet of the underwater sampling module, inside the gas detection optical cell, and in the fluorescence observation area of ​​the microfluidic chip. This eliminated the temperature and pressure transfer delay between different media interfaces and avoided spectral inversion deviations caused by temperature and pressure lag at the gas-liquid interface. Three temperature and pressure data points are collected synchronously at a sampling frequency of no less than 1Hz. The temperature and pressure data in the gas detection optical cell are time-domain aligned and abnormal transient fluctuations are removed. The data is then transmitted to the data processing and control module in real time to form a temperature and pressure time series synchronized with environmental conditions, effectively suppressing occasional noise interference and improving data processing stability. When performing concentration inversion, the carbon dioxide mid-infrared TDLAS detection module reads the temperature and pressure values ​​of the current gas detection optical cell, performs numerical compensation on the concentration inverted by the absorption signal according to the ideal gas law, corrects the concentration inverted by the absorption signal point by point, eliminates measurement drift caused by changes in underwater depth or thermal disturbances, and ensures the comparability of detection results under different underwater environmental conditions.

[0013] Preferably, the data processing control module specifically includes: The normalized fluorescence intensity of microorganisms, absolute carbon dioxide concentration, temperature, pressure and gas regulation parameters within the same time window are constructed into a multi-source heterogeneous feature matrix with timestamps. The time window is divided according to the trigger time of the regulation event, effectively establishing the time sequence correspondence of multiple parameters and ensuring that the regulation response process is clearly segmented. Using a sliding window cross-correlation analysis method, the time delay correlation coefficient and peak response time between the carbon dioxide concentration change sequence and the fluorescence intensity change rate sequence were calculated. The lag response time and response sensitivity of microorganisms to changes in the gas environment were extracted, the microbial response delay was accurately quantified, and the temporal association between activity changes and gas stimulation was revealed. Based on the dynamic correlation curves between carbon dioxide concentration and fluorescence intensity under different regulatory stages, the quantitative evaluation results of microbial physiological activity as a function of carbon dioxide microenvironment are output, intuitively presenting the dependence of activity on gas concentration, and realizing quantitative assessment of environmental effects.

[0014] Secondly, the present invention provides a method for detecting the fluorescence activity of underwater microorganisms and regulating the carbon dioxide microenvironment, based on the aforementioned device for detecting the fluorescence activity of underwater microorganisms and regulating the carbon dioxide microenvironment, comprising the following steps: S1: In the target water body, underwater in-situ sampling is carried out to obtain water samples containing microorganisms. The water samples are introduced into the microfluidic chip through an isothermal and isobaric flow path to maintain the water sample environment consistent with the in-situ environment and ensure that the microbial activity truly reflects the in-situ state. S2: Microorganisms are transported, captured, enriched and located within a microfluidic chip, stabilizing them in a controllable micro-region between the fluorescence observation area and the gas exchange area, thus achieving precise microbial positioning and long-term stable observation. S3: Use the fluorescence detection module to acquire microbial fluorescence images, extract integrated fluorescence intensity, calculate normalized fluorescence change rate, determine the physiological activity status of microorganisms, and realize real-time quantitative discrimination of microbial activity status; S4: Enables carbon dioxide molecules in the water sample to dissolve, diffuse, and desorb through the dense non-microporous membrane AF2400, and then transport them across the membrane to the gas detection optical cell, achieving gas-liquid isolation and avoiding interference from liquid water on spectral detection; S5: The carbon dioxide concentration is measured using 2.6μm mid-infrared TDLAS technology, and temperature and pressure parameters are collected simultaneously for correction to obtain an accurate carbon dioxide concentration value, ensuring the accuracy and long-term stability of carbon dioxide concentration measurement. S6: Integrates fluorescence activity and carbon dioxide concentration data, activates the regulation module as needed to change the gaseous environment around microorganisms, obtains the response characteristics of activity to changes in carbon dioxide, and realizes the linkage analysis of gaseous environment changes and microbial responses.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device and method, by coupling a dense non-microporous gas permeable membrane with a microfluidic chip, realizes the transmembrane transport and gas-liquid isolation of carbon dioxide in the underwater in-situ environment. The membrane structure does not rely on perforated micropores, and completes the transfer of gas molecules from the aqueous phase to the gas phase through dissolution, diffusion and desorption mechanisms. It can effectively block liquid water and its dissolved ions from entering the gas phase detection area, significantly improving the stability and reliability of long-term continuous underwater detection.

[0016] 2. This underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device and method integrates microbial fluorescence activity detection and carbon dioxide spectral detection on the same underwater in-situ platform. It can simultaneously acquire microbial fluorescence activity signals and carbon dioxide concentration information in the microenvironment while maintaining the original environmental state of the microorganisms. Fluorescence activity is used to determine the physiological state of the microorganisms, and spectral detection enables quantitative determination of gas concentration. The two are correlated and analyzed on a unified time axis, overcoming the information fragmentation problem of traditional offline sampling and single-parameter detection.

[0017] 3. This underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device and method has the ability to actively regulate the carbon dioxide microenvironment around microorganisms. It can perform gas concentration increase, decrease or gradient construction operations based on real-time fusion analysis results. During the regulation process, gas molecules are transported across the dense membrane without bubbles, which will not cause bubble impact or flow field disturbance to the located microorganisms. Combined with continuous fluorescence response monitoring, a closed-loop linkage between environmental changes and activity response is formed, providing technical support for studying the dynamic adaptation behavior of microorganisms to the gas environment. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the workflow of the underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device and method of the present invention. Figure 2 This is a schematic diagram of the process flow for the underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation method of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0020] Example 1, please refer to Figure 1 , Figure 2 This invention provides a technical solution: an underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device, comprising: The underwater sampling module is used to obtain water samples containing microorganisms in the target water body, realizing in-situ acquisition of microbial samples and avoiding the activity distortion caused by in vitro sampling; The AF2400 gas permeable membrane is used to enable the transfer of carbon dioxide from water or microfluidic chips to the gas detection optical cell or gas control region, achieving non-destructive cross-membrane transfer of carbon dioxide under gas-liquid isolation and avoiding interference of liquid water with spectral detection. Microfluidic chips are used to transport, capture, enrich, and locate microorganisms that enter the chip, enabling automated localization and controllable observation of microorganisms, reducing manual intervention and sample consumption; Furthermore, the AF2400 gas permeable membrane is a dense, non-microporous membrane structure. It does not rely on interconnected micropores for gas transport; instead, it achieves carbon dioxide transport from the aqueous phase to the gas phase detection region through the dissolution, diffusion, and desorption of carbon dioxide molecules within the AF2400 membrane material. Positioned between the aqueous and gas phase regions, the AF2400 membrane prevents liquid water from entering the gas detection optical cell while allowing dissolved carbon dioxide molecules to pass through the membrane material. The AF2400 membrane is not used for microbial filtration, cell sieving, or particulate matter retention; its primary function is the transmembrane transport of carbon dioxide molecules between the aqueous and gas phase detection regions, preventing pore blockage and liquid infiltration, and improving the reliability of long-term in-situ detection. The microfluidic chip includes an inlet channel, a microbial capture zone, a fluorescence observation zone, a gas exchange zone, and a drain channel. The gas exchange zone connects to the carbon dioxide mid-infrared TDLAS detection module or the carbon dioxide microenvironment adjustment module via the AF2400 gas permeable membrane. The control module is coupled to achieve integrated precise microbial localization and gas-liquid interface functional zoning. Specifically, an underwater sampling module acquires microbial-containing water samples in situ from the target water body. An isothermal and isobaric flow path introduces the water sample into the microfluidic chip's sampling channel, maintaining the water sample's temperature and pressure environment consistent with the in-situ water body. This avoids temperature and pressure fluctuations that could interfere with microbial activity, ensuring the detection accurately reflects the in-situ state. Within the microfluidic chip, a fluid manipulation structure transports, captures, enriches, and locates microorganisms in the water sample, placing them in a controllable micro-region between the fluorescence observation area and the gas exchange area. This achieves automated microbial enrichment and fixed-point observation without human intervention. An AF2400 gas permeation membrane, positioned between the aqueous and gas phases, allows CO2 molecules to dissolve, diffuse, and desorb into the gas detection optical cell or gas control channel based on a dissolution-diffusion-desorption cascade mechanism. This blocks interference from liquid water and ion spectra, achieving poreless cross-membrane transport under gas-liquid isolation, eliminating interference from liquid water on spectral detection, and improving the accuracy of carbon dioxide measurement. It should be noted that the underwater sampling module is deployed at a predetermined depth in the target water body. This module integrates temperature and pressure sensors at the sampling inlet to monitor the environmental parameters of the in-situ water body in real time. Once the sampling process is initiated, the underwater sampling module extracts the water sample containing microorganisms from the target water body through an isothermal and isobaric flow path and directly introduces it into the microfluidic chip's injection channel. Throughout the sampling and introduction process, the flow path system compares the in-situ temperature and pressure at the sampling inlet with those at the microfluidic chip inlet in real time, and dynamically compensates for this through an active temperature control element and a pressure balancing chamber. This ensures that the temperature and pressure of the water sample entering the microfluidic chip remain consistent with the in-situ water body. Maintaining isothermal and isobaric conditions prevents non-experimental changes in microbial physiological activity caused by sudden temperature or pressure changes during water sample transfer, ensuring that the microbial state reflected in subsequent fluorescence detection truly represents its activity level in the original underwater environment. Simultaneously, it avoids the impact of bubble precipitation or water sample degassing on subsequent carbon dioxide transmembrane transport. Interference during transport; After the water sample enters the microfluidic chip, the fluid control program is activated. The microfluidic chip is equipped with an inlet channel, a microbial capture zone, a fluorescence observation zone, a gas exchange zone, and a drain channel. The fluid control structure controls the flow direction and velocity of the water sample inside the chip through a precision injection pump or electroosmosis drive. The water sample first enters the microbial capture zone along the inlet channel. This zone is designed with microcolumn arrays, flow channel contraction structures, or dielectrophoretic capture electrodes of appropriate size to transport, capture, enrich, and locate the microorganisms in the water sample. After a set time of capture and enrichment, the microorganisms are confined to a controllable micro-region between the fluorescence observation zone and the gas exchange zone. This micro-region simultaneously meets the optical pathway requirements for fluorescence detection and the distance requirements for carbon dioxide transmembrane exchange. The drain channel is used to discharge the captured water sample or excess liquid from the chip, preventing the flowing liquid from scouring and displacing the located microorganisms. The entire transport and positioning process is completed within a closed microfluidic system without relying on external human intervention.After the microorganisms are stably positioned between the fluorescence observation area and the gas exchange area, the carbon dioxide transmembrane transport process is initiated. The AF2400 gas permeable membrane is positioned between the aqueous and gas phase regions. One side is the aqueous phase channel of the microfluidic chip containing the positioned microorganisms, and the other side is the gas detection optical cell or gas control channel. The AF2400 membrane is a dense, non-microporous structure that does not rely on perforated micropores for gas exchange. Dissolved carbon dioxide molecules in the aqueous phase first migrate to the aqueous phase interface of the AF2400 membrane, then dissolve in the membrane material and diffuse within the membrane under the drive of the concentration gradient. Finally, they desorb at the gas phase interface and enter the gas detection optical cell or gas control channel. This dissolution-diffusion-desorption cascade mechanism realizes the non-porous transmembrane transport of carbon dioxide from the aqueous phase to the gas phase. At the same time, the AF2400 membrane effectively blocks liquid water and its dissolved ions from entering the gas phase region, avoiding direct absorption interference of liquid water on mid-infrared spectral detection and contamination of the optical window by ion crystallization. Under gas-liquid isolation conditions, in-situ sampling and detection of carbon dioxide underwater are completed. The fluorescence detection module is used to collect fluorescence signals from microorganisms within the microfluidic chip and determine the physiological activity state of the microorganisms based on changes in fluorescence intensity. This enables real-time in-situ characterization of microbial activity and assessment of their physiological responses to environmental changes. The fluorescence detection module includes an excitation source, a fluorescence excitation filter unit, a fluorescence collection optics unit, a fluorescence filter unit, an imaging detector, and a fluorescence intensity analysis unit. The module acquires the fluorescence intensity of the microorganisms through one or more of the following: microbial autofluorescence, fluorescent staining signals, or fluorescent probe signals. It then determines the physiological activity state of the microorganisms based on the fluorescence intensity, the rate of change of fluorescence intensity, or the normalized fluorescence intensity, thus achieving in-situ characterization of microbial activity. Non-destructive discrimination involves generating specific wavelength excitation light from an excitation source through a filter unit, illuminating the pre-positioned microbial region within the microfluidic chip, and exciting its own fluorescence, staining signal, or fluorescent probe signal to ensure the high efficiency and specificity of fluorescence excitation. Microbial fluorescence images are acquired through a fluorescence collection optical unit and an imaging detector, and the fluorescence intensity, fluorescence distribution area, or integral fluorescence value of a single microorganism within the observation area are extracted to obtain multi-dimensional quantitative information on microbial activity and distribution. The normalized fluorescence change rate is calculated based on the ratio of fluorescence intensity to initial fluorescence intensity to determine whether the physiological activity of the microorganism is in an enhanced, weakened, or stable state, eliminating batch differences and achieving comparable judgment of activity change trends. It should be noted that after the microorganism is stably located in the fluorescence observation area of ​​the microfluidic chip, the fluorescence detection module is activated. The light beam emitted by the excitation source is converted into narrowband excitation light with a specific wavelength that matches the excitation wavelength of the fluorescent probe or autofluorescence of the microorganism. This excitation light is focused on the area where the microorganism is located through a microscopic optical path, effectively exciting the fluorescent substances in the microorganism and its surrounding medium. After absorbing the excitation light energy, the fluorescent substances transition to the excited state and emit a longer-wavelength fluorescence signal during the return to the ground state. To eliminate the influence of residual excitation light on detection, a fluorescence filter unit is installed before the fluorescence collection optical unit, allowing only the target fluorescence band to pass through and enter the imaging detector. Quantitative analysis is performed on the acquired fluorescence images to extract the integrated fluorescence intensity, fluorescence distribution area, or integrated fluorescence value of a single microorganism within the observation area. The integrated fluorescence intensity reflects the total amount of fluorescence signal in the observation area and is related to the microorganism's fluorescence intensity. The fluorescence expression level of individual microorganisms is related to the quantity of microorganisms; the fluorescence distribution area can be used to assess the diffusion or aggregation of microbial communities; the integrated fluorescence value of individual microorganisms is used to characterize the activity differences between different microbial individuals. The above parameters can be used to distinguish microbial regions from background regions through image segmentation algorithms, and quantitative fluorescence data can be obtained after removing background noise; the initial fluorescence intensity is defined as the fluorescence intensity of microorganisms at the start of detection or the first measurement, serving as a benchmark value for comparison. The normalized fluorescence change rate is the difference between the current fluorescence intensity and the initial fluorescence intensity divided by the initial fluorescence intensity, eliminating the uncertainty caused by the difference in absolute fluorescence intensity between different batches or different observation areas. When the normalized fluorescence change rate is positive and exceeds the set threshold, it is judged that the physiological activity of microorganisms is enhanced; when it is negative and below the set negative threshold, it is judged that the physiological activity of microorganisms is weakened; when the absolute value of the change rate is continuously within the threshold range, it is judged that the activity of microorganisms is stable. Furthermore, the fluorescence detection module also includes: acquiring microbial fluorescence images multiple times in a continuous time series, extracting the integrated fluorescence intensity at each moment, forming a dynamic curve of fluorescence intensity changing with time, tracking the continuous evolution of microbial activity in real time, performing moving average filtering on the dynamic curve, calculating the instantaneous rate of change of fluorescence intensity and the first derivative of the rate of change, which is used to identify the rapid rise, decay or plateau phase of fluorescence signal, accurately identify the stage change characteristics of fluorescence signal, and when the normalized fluorescence rate of change exceeds the preset threshold range and continues to exceed the set time window, it is determined that the microorganism has entered an abnormal activity state, and triggers carbon dioxide microenvironment regulation or data labeling, realizing automatic identification and closed-loop response of abnormal activity; It should be noted that, within the continuous time series framework, the fluorescence detection module acquires microbial fluorescence images multiple times according to a set sampling frequency. After background subtraction and region segmentation, the integrated fluorescence intensity within the observation area is extracted from each frame. The integrated fluorescence intensity at each sampling time is then arranged chronologically to form a dynamic curve of fluorescence intensity changing over time. This dynamic curve comprehensively records the continuous evolution of the fluorescence signal of microorganisms before, during, and after the regulation of the carbon dioxide microenvironment, covering the initial stable phase, the regulatory response phase, and the possible recovery phase. The vertical axis of the dynamic curve represents the integrated fluorescence intensity, and the horizontal axis represents the sampling time point. Its overall trend directly reflects the persistence of microbial physiological activity under the disturbance of the external gas environment. To further analyze the changing patterns and facilitate subsequent rate of change calculations and activity state identification, the dynamic curve was aligned along the time axis and outliers were removed to ensure high reliability and repeatability of the obtained fluorescence intensity time series. The fluorescence intensity dynamic curve serves as the foundational data for subsequent rate of change analysis, response time extraction, and trigger determination. A moving average filter was applied to the fluorescence intensity dynamic curve to suppress the adverse effects of high-frequency noise and random detection errors on the rate of change calculation. The moving average filter uses a fixed-width time window, and the arithmetic mean of the integrated fluorescence intensity within the window is taken as the smoothed fluorescence intensity value at the center of the window. Then, the smoothed fluorescence intensity is differentiated with respect to time to obtain the instantaneous change in fluorescence intensity. The rate of change is further differentiated again to obtain the first derivative of the rate of change. The instantaneous rate of change is used to characterize the speed at which the fluorescence intensity increases or decreases at the current moment, while the first derivative of the rate of change is used to identify the acceleration characteristics of the fluorescence intensity change trend, i.e., the enhancement, weakening, or zero-crossing state of the rate of change itself. Through the joint analysis of the instantaneous rate of change and the first derivative, the rapid rise phase, decay phase, and plateau phase where the rate of change approaches zero of the fluorescence signal can be accurately distinguished, providing a quantitative basis for the precise identification of the microbial activity state. A normal fluctuation threshold range for the normalized fluorescence rate of change is preset, including an upper threshold and a lower threshold. When the real-time calculated normalized fluorescence rate of change exceeds this threshold range and continues to do so... When the time exceeds the preset time window length, the microorganism is determined to have entered an abnormal activity state. Abnormal activity states include excessive fluorescence response caused by excessive activity enhancement and low fluorescence response caused by activity inhibition or damage. Once an abnormal activity state is confirmed, the data processing control module automatically triggers corresponding operations according to the abnormality type: if it is a positive over-threshold abnormality, the carbon dioxide microenvironment regulation module can be activated to reduce the carbon dioxide concentration around the microorganism or change the gas composition to alleviate high activity stress; if it is a negative over-threshold abnormality, data marking is triggered and the current gas environment is maintained, or the carbon dioxide concentration is actively adjusted according to the set regulation strategy to try to restore microbial activity, thereby realizing automatic identification and closed-loop response of abnormal microbial activity states. The carbon dioxide mid-infrared TDLAS detection module is used to determine the concentration of carbon dioxide after transmission through an AF2400 gas-permeable membrane based on 2.6μm mid-infrared tunable semiconductor laser absorption spectroscopy. This achieves accurate spectral determination of carbon dioxide concentration and is suitable for complex underwater in-situ detection environments. The module includes a 2.6μm mid-infrared tunable laser, collimating optical elements, a gas detection optical cell, a mid-infrared detector, a laser drive and temperature control unit, a signal acquisition unit, and a concentration inversion unit. It effectively integrates dedicated optical components to achieve highly selective underwater carbon dioxide detection. Specifically, the carbon dioxide gas transmitted through the AF2400 gas-permeable membrane to the gas detection optical cell is placed in a closed optical path after temperature and pressure correction, and then detected by the 2.6μm mid-infrared tunable laser. The instrument emits a laser using a sinusoidal scanning and sawtooth superposition modulation method. By sealing the optical path and modulating the laser, the quality of the spectral signal and the stability of detection are improved. The absorption signal of carbon dioxide at the characteristic absorption line of 2.6μm is collected by direct absorption spectroscopy or wavelength modulation spectroscopy. The second harmonic signal is extracted in wavelength modulation mode. When wavelength modulation spectroscopy is used, the carbon dioxide concentration is inverted by the first harmonic signal, the second harmonic signal, or the normalized second harmonic signal. The use of multiple harmonic signals for inversion enhances the flexibility and anti-interference ability of concentration measurement. The concentration inversion unit calculates the absolute concentration value of carbon dioxide in the gas detection optical cell based on the absorption signal amplitude, the peak intensity of the second harmonic, and the real-time temperature and pressure compensation coefficient. The inversion is performed by integrating temperature and pressure compensation to ensure the authenticity and accuracy of the concentration output. It should be noted that carbon dioxide gas, transmitted through the AF2400 gas permeation membrane to the gas detection optical cell, is introduced into the closed optical path system as the detection target. The gas detection optical cell adopts a pressure-resistant sealed structure, with its inlet directly coupled to the gas phase-side outlet of the AF2400 membrane to shorten the gas transmission path and reduce dead volume. Before detection begins, the temperature and pressure monitoring module reads the internal temperature and pressure values ​​of the optical cell in real time and performs temperature and pressure correction on the optical path system to ensure that the absorption spectral parameters match the current environmental conditions. The 2.6μm mid-infrared tunable laser outputs laser light under the control of the laser driver and temperature control unit. The laser uses a sinusoidal scanning... The modulation method, combining scanning and sawtooth patterns, allows the output wavelength to periodically scan past the characteristic absorption peak of carbon dioxide. A collimating optical element shapes the laser beam into parallel light before guiding it into the gas detection optical cell. Within the cell, the beam undergoes multiple reflections or direct transmission before being received by a mid-infrared detector, forming a complete closed optical path detection link. During the laser wavelength scanning process, carbon dioxide molecules selectively absorb laser energy at the 2.6 μm characteristic absorption line, with the absorption intensity proportional to the gas concentration. The mid-infrared detector acquires the laser intensity signal transmitted through the gas detection optical cell in real time. The signal acquisition unit amplifies, filters, and performs analog-to-digital conversion on the detector output before sending it to the... The concentration inversion unit selects either direct absorption spectroscopy or wavelength modulation spectroscopy based on application requirements. When using wavelength modulation spectroscopy, the laser drive signal operates under high-frequency modulation, demodulating the first and second harmonic signals from the detection signal. The second harmonic signal exhibits a peak response at the center of the absorption peak and is insensitive to background interference. The concentration inversion unit extracts the peak intensity of the second harmonic as the main characteristic quantity for concentration inversion. If necessary, it combines the first harmonic signal for normalization to suppress the impact of light intensity fluctuations and non-absorption losses on detection accuracy. During the inversion process, the unit reads data provided in real-time by the temperature and pressure environment monitoring module. The current optical cell temperature and pressure data are used to compensate for the inversion results based on the ideal gas law and the temperature dependence of absorption line intensity. Temperature changes affect the line intensity and linewidth of the absorption line, while pressure changes affect the collision broadening of the line. Both need to be included in the concentration inversion model. The compensation coefficient is obtained by the response curve calibrated in advance under a standard gas of known concentration, and is dynamically adjusted according to the real-time temperature and pressure values ​​during actual detection. The final output absolute carbon dioxide concentration value is corrected by temperature and pressure to eliminate the measurement drift caused by pressure fluctuations caused by changes in underwater depth and environmental heat flow disturbances, ensuring the accuracy and comparability of concentration data in long-term in-situ detection. The carbon dioxide microenvironment control module is used to inject, release, exchange, or adjust the concentration gradient of carbon dioxide gas environment within the microfluidic chip or around microorganisms, enabling artificial controllable adjustment of the carbon dioxide environment around microorganisms and supporting the study of dynamic response patterns. The temperature and pressure environment monitoring module is used to collect temperature and pressure parameters in the underwater environment, gas detection optical cell and microfluidic chip, to achieve simultaneous monitoring and compensation of temperature and pressure at multiple locations, and to ensure the accuracy of concentration inversion and activity discrimination. The data processing and control module is used to perform fusion analysis on microbial fluorescence intensity, carbon dioxide concentration, temperature, pressure and gas regulation parameters, and evaluate the changes in physiological activity of microorganisms under different carbon dioxide gas environments based on the analysis results. It realizes multi-parameter fusion and closed-loop regulation decision-making, and outputs a quantitative evaluation of the microbial response to the gas environment.

[0021] Example 2, as Figure 1 , Figure 2 As shown, based on Example 1, the present invention provides a technical solution: the carbon dioxide microenvironment control module specifically includes: determining whether it is necessary to change the carbon dioxide gas environment around the microorganisms in the microfluidic chip based on the fusion analysis results of the current carbon dioxide concentration and the fluorescence activity state of the microorganisms, realizing intelligent control decision based on real-time detection results; when adjustment is required, the carbon dioxide microenvironment control module is activated, and a set concentration of carbon dioxide gas or mixed carrier gas is introduced into the gas exchange area through the gas injection channel to realize active and precise intervention on the gas environment around the microorganisms; carbon dioxide molecules are transported across the membrane from the gas phase control channel to the aqueous phase region of the microfluidic chip through the AF2400 gas permeation membrane to realize the operation of increasing, decreasing or maintaining the carbon dioxide concentration around the microorganisms, and completing the gas concentration control under bubble-free and undisturbed conditions; It should be noted that the data processing and control module receives the normalized fluorescence change rate from the fluorescence detection module and the absolute concentration value from the carbon dioxide mid-infrared TDLAS detection module in real time. After aligning these two values ​​with a timestamp, a fused feature vector is constructed. The system internally presets carbon dioxide concentration threshold ranges and normal fluctuation ranges of fluorescence activity for different microbial groups or experimental scenarios. When the fusion analysis results indicate that the current carbon dioxide concentration deviates from the set threshold range, or the fluorescence change rate shows a continuous trend exceeding the normal fluctuation range, or both show abnormal fluctuations simultaneously, it is determined that active intervention in the gaseous environment surrounding the microorganisms is required. This judgment logic simultaneously considers the degree of gas concentration deviation, the direction of fluorescence activity response, and the temporal correlation between the two, avoiding false triggers caused by instantaneous fluctuations of a single parameter, and ensuring the accuracy and reliability of the control decision. The carbon dioxide microenvironment control module integrates multiple independent gas pathways, respectively connected to a high-purity carbon dioxide source, a carrier gas source, and a dynamic gas mixing unit for dilution ratio control. Based on the fusion analysis results, the target control direction and control amplitude are determined. If it is necessary to increase the carbon dioxide concentration, the volume fraction of carbon dioxide gas in the mixed gas is increased; if... To reduce carbon dioxide concentration, the carrier gas ratio is increased or the pure carrier gas purge channel is switched. The dynamic gas distribution unit monitors the output gas flow rate and concentration in real time, and ensures that the gas parameters entering the gas exchange zone are stable at the set value through closed-loop feedback control. The outlet of the gas injection channel is close to the gas phase side surface of the AF2400 gas permeation membrane, so that the control gas reaches the membrane interface in a uniform distribution. In this process, the AF2400 dense non-microporous membrane acts as a functional interface between the gas phase and the aqueous phase. Carbon dioxide molecules dissolve into the membrane material on the gas phase side of the membrane, diffuse through the membrane layer under the drive of the concentration gradient, and then diffuse into the aqueous phase side. The desorbed water sample medium is introduced into the microfluidic chip. Since the membrane structure itself does not rely on the through-pores, the overall flow of the control gas will not directly penetrate the membrane layer into the aqueous phase, avoiding the physical impact of bubble generation or flow field disturbance on the located microorganisms. The rate of change of dissolved carbon dioxide concentration in the aqueous phase is jointly determined by the partial pressure of carbon dioxide on the gas phase side, the membrane area, and the concentration gradient on both sides of the membrane. By rationally selecting the effective working area of ​​the membrane and the gas phase flow configuration in the design, the control response speed can be matched with the time resolution of microbial activity detection, realizing the synchronous observation and closed-loop control of gas environment changes and fluorescence response signals. The carbon dioxide microenvironment control module also includes: introducing carbon dioxide gas of different concentrations to different spatial locations in the gas exchange zone through multiple independent gas inlets, so that different regions of the AF2400 membrane exhibit differentiated gas phase carbon dioxide partial pressures, achieving a stable and controllable spatial partial pressure distribution pattern on the gas phase side of the membrane; maintaining the carbon dioxide concentration difference in different regions of the aqueous phase using the fluid isolation structure within the microfluidic chip, forming a stable carbon dioxide concentration gradient or periodic fluctuation environment in the microbial capture zone, enabling parallel observation of microbial communities simultaneously exposed to multiple concentration environments; and dynamically adjusting the flow rate ratio or gas switching frequency of each gas inlet based on real-time fluorescence response feedback, so that the carbon dioxide concentration around the microorganisms changes according to a set curve, which is used to analyze the sensitive response range of microorganisms to the gas environment, and to achieve accurate analysis and quantitative characterization of the sensitivity of microbial activity to the gas environment. It should be noted that in the carbon dioxide microenvironment control module, carbon dioxide gas of different concentrations is introduced into different spatial locations of the gas exchange zone through multiple independent gas inlets. Each gas inlet forms a local coupling region with the gas phase side surface of the AF2400 gas permeation membrane. Each inlet is equipped with an independent dynamic gas distribution unit and mass flow controller, which can independently adjust the carbon dioxide volume fraction and flow rate of the gas output from each inlet according to control commands. Due to the difference in gas concentration at different inlets, a differentiated carbon dioxide partial pressure distribution is formed on the gas phase side of the AF2400 membrane at different spatial locations. The membrane material has a dense, non-microporous structure, and carbon dioxide molecules... Diffusion within the membrane is controlled by local partial pressure on the gas phase side. The pressure differences between different regions do not rapidly homogenize due to lateral diffusion within the membrane, thus forming a stable spatial partial pressure distribution pattern on the gas phase side. This provides controllable gas partial pressure boundary conditions for establishing the subsequent concentration gradient on the aqueous phase side. The microfluidic chip incorporates a fluid isolation structure between the gas exchange region and the microbial capture region, including multiple parallel microchannels, fluid distribution ridges, or local flow resistance adjustment structures. This maintains fluid independence between different regions of the aqueous phase. When the AF2400 membrane exhibits differentiated carbon dioxide partial pressures on the gas phase side, carbon dioxide molecules are transported across the membrane into the corresponding aqueous microfluidic regions. The system ensures that the concentration of dissolved carbon dioxide in the aqueous phase continuously varies along the spatial distribution direction of the chip. Because the fluid isolation structure restricts lateral convection mixing between different regions of the aqueous phase, the established concentration differences are stably maintained, thus forming a carbon dioxide concentration gradient with a predetermined spatial distribution characteristic within the microbial capture zone. This concentration gradient can remain stable over time and can also be dynamically adjusted by changing the concentration and flow rate ratios of each gas inlet. The data processing and control module receives the normalized fluorescence change rate and its spatial distribution information from the fluorescence detection module in real time and performs correlation analysis with the current control parameters of each gas inlet. When it is necessary to analyze the effect of microorganisms on... When the carbon dioxide gas environment is in a sensitive response range, the flow rate ratio of each gas inlet or the gas switching frequency is changed according to the preset dynamic control curve. This causes the partial pressure of different regions on the gas phase side of the AF2400 membrane to fluctuate periodically, rise, fall, or change stepwise over time. This drives the carbon dioxide concentration in the microbial capture zone to change according to a set trajectory. The fluorescence detection module records the fluorescence response behavior of microorganisms under different gas concentrations. The data processing and control module calculates the time delay correlation coefficient between the concentration change sequence and the fluorescence response sequence to extract the sensitive concentration range, response threshold, and recovery characteristic parameters of microbial activity, providing a quantitative basis for the assessment of microbial ecological response. The temperature and pressure environment monitoring module specifically includes: underwater-calibrated temperature and absolute pressure sensors deployed at three independent locations: the inlet of the underwater sampling module, inside the gas detection optical cell, and in the fluorescence observation area of ​​the microfluidic chip. This eliminates the temperature and pressure transmission delay between different media interfaces, avoids spectral inversion deviations caused by temperature and pressure lag at the gas-liquid interface, and synchronously collects temperature and pressure data from the three locations at a sampling frequency of no less than 1Hz. The temperature and pressure data in the gas detection optical cell are time-domain aligned and abnormal transient fluctuations are removed. The data is transmitted in real time to the data processing and control module to form a temperature and pressure time series synchronized with environmental conditions, effectively suppressing occasional noise interference and improving data processing stability. When performing concentration inversion, the carbon dioxide mid-infrared TDLAS detection module reads the current temperature and pressure values ​​of the gas detection optical cell, performs numerical compensation on the absorption signal inversion concentration according to the ideal gas law, corrects the absorption signal inversion concentration point by point, eliminates measurement drift caused by changes in underwater depth or thermal disturbances, and ensures the comparability of detection results under different underwater environmental conditions. It should be noted that underwater-calibrated temperature and absolute pressure sensors are deployed at three independent locations: the inlet of the underwater sampling module, inside the gas detection optical cell, and in the fluorescence observation area of ​​the microfluidic chip. The sensor at the inlet of the underwater sampling module acquires the baseline temperature and pressure values ​​of the target water body's original environment. This value is compared with temperature and pressure changes in subsequent processes, providing closed-loop feedback for isothermal and isobaric sampling. The sensor inside the gas detection optical cell is directly installed in the gas region of the closed optical path to collect real-time gas temperature and pressure parameters involved in absorption spectral inversion, avoiding heat transfer and delay errors introduced by external indirect calculations. The sensor in the fluorescence observation area of ​​the microfluidic chip monitors the temperature and pressure status of the aqueous phase region where the microorganisms reside, ensuring that environmental conditions are known and controllable during fluorescence activity data acquisition. The three sensors synchronously acquire temperature and pressure data at a sampling frequency of no less than 1 Hz, eliminating temperature and pressure transmission delays caused by thermal conduction lag between the aqueous and gas phases and differences in the response speed of different media interfaces. The obtained temperature and pressure time series is transmitted in real-time to the data processing and control module, aligned with the fluorescence intensity and carbon dioxide concentration data on a unified time axis. After receiving data from three temperature and pressure sensors simultaneously, the block first performs time-domain alignment on the temperature and pressure sequences in the gas detection optical cell to eliminate micro-delays introduced by differences in sensor physical location and signal transmission paths. The aligned temperature and pressure data is then processed using a sliding window method to remove abnormal transient fluctuations. When a temperature or pressure value at a sampling point changes abruptly beyond the physical possible range or statistical threshold within an adjacent time window, it is judged as instantaneous interference from the sensor or occasional signal noise, and is replaced by linear interpolation or the average value of the adjacent window to avoid significant deviations in subsequent concentration inversion caused by single-point anomalies. After anomaly removal, the three temperature and pressure data form continuous and smooth environmental change time series. The temperature and pressure sequence at the inlet of the underwater sampling module is used to characterize the changing trend of the external water environment, the temperature and pressure sequence in the fluorescence observation area of ​​the microfluidic chip is used to correct the possible temperature and pressure dependence of microbial activity, and the temperature and pressure sequence of the gas detection optical cell is directly input into the carbon dioxide concentration inversion model. The data processing and control module compares the differences between the three temperature and pressure data in real time. When the difference exceeds the set range, it automatically marks an abnormal state and prompts the system to check the integrity of the sampling flow path or sealing structure.The data processing and control module reads the temperature and pressure values ​​of the gas detection optical cell at the current moment. The concentration inversion unit establishes the relationship between the absorption signal and the number density of gas molecules based on the ideal gas law. Temperature changes directly affect the line intensity and Doppler broadening of the absorption spectrum, while pressure changes determine the width and line shape distribution of the Lorentz collision broadening. During the inversion process, the theoretical line intensity of the carbon dioxide characteristic absorption spectrum at the current temperature is first calculated by looking up a table or interpolating based on the real-time temperature value. Then, the collision broadening half-width of the spectrum is calculated based on the real-time pressure value. Subsequently, a standard absorption line shape function under the current temperature and pressure conditions is constructed. Then, the measured absorption signal is fitted with this standard line shape, and the absorption signal is corrected point by point to invert the concentration. This eliminates the measurement drift caused by external pressure fluctuations due to changes in underwater depth and internal temperature changes due to heat flow disturbances. After temperature and pressure compensation, the output absolute carbon dioxide concentration value has consistent comparability under different underwater depths and different ambient temperatures, ensuring the accuracy and stability of concentration data in long-term in-situ continuous detection. This allows the gas detection results to truly reflect the actual carbon dioxide microenvironment conditions experienced by microorganisms within the microfluidic chip. The data processing and control module specifically includes: constructing a multi-source heterogeneous feature matrix with timestamps from the normalized fluorescence intensity, absolute carbon dioxide concentration, temperature, pressure, and gas regulation parameters of microorganisms within the same time window; dividing the time window according to the trigger time of the regulation event to effectively establish the time-series correspondence of multiple parameters, ensuring clear segmentation of the regulation response process; using the sliding window cross-correlation analysis method to calculate the time delay correlation coefficient and peak response time between the carbon dioxide concentration change sequence and the fluorescence intensity change rate sequence; extracting the lag response duration and response sensitivity of microorganisms to changes in the gas environment; accurately quantifying the microbial response delay; revealing the time-series correlation between activity changes and gas stimulation; and outputting quantitative evaluation results of microbial physiological activity with changes in the carbon dioxide microenvironment based on the dynamic correlation curve between carbon dioxide concentration and fluorescence intensity under different regulation stages, intuitively presenting the dependence of activity on gas concentration, and realizing quantitative assessment of environmental effects. It should be noted that, within the continuous time series framework, the data processing and control module constructs a timestamped multi-source heterogeneous feature matrix by combining the normalized fluorescence intensity of microorganisms, absolute carbon dioxide concentration, temperature, pressure, and gas regulation parameters acquired within the same time window. This matrix is ​​then divided into time windows based on the trigger time of each regulation event, ensuring that each time window corresponds to a complete regulation response process, including the pre-regulation baseline stage, the response stage during regulation, and the post-regulation recovery stage. This feature matrix construction method ensures that the temporal correspondence between different physical quantities is accurately maintained. The length of the time window segment is determined based on the fluorescence response rate. The rate and regulation response time are adaptively adjusted to ensure that each window contains a sufficient number of sampling points to support the reliability of subsequent statistical analysis, while avoiding feature ambiguity caused by cross-window information aliasing. A sliding window cross-correlation analysis method is used. The data processing control module calculates the time delay correlation coefficient between the carbon dioxide concentration change sequence and the fluorescence intensity change rate sequence segment by segment along the time dimension of the feature matrix. The optimal lag response time of microbial activity to gas concentration changes is determined by searching for the peak of the correlation coefficient. The width of the sliding window is set to three to five times the characteristic time constant of the regulation response, and the sliding step size is set to an integer multiple of the sampling interval to ensure information continuity between adjacent windows. For each time window, the peak value of the normalized cross-correlation function and its corresponding delay time are extracted. The peak value reflects the linear correlation strength between fluorescence response and concentration change, and the delay time characterizes the time required for microorganisms to perceive changes in the gas environment and exhibit detectable activity. The trends in peak value and delay time changes between different windows are used to determine whether microorganisms have undergone adaptive changes due to continuous regulation. The data processing control module outputs quantitative evaluation results of microbial physiological activity with changes in the carbon dioxide microenvironment. The dynamic correlation curve is plotted with carbon dioxide concentration as the horizontal axis and normalized fluorescence intensity as the vertical axis. Plotting the curve on the vertical axis, the slope, inflection point, and hysteresis area are used to characterize the sensitivity of microorganisms to changes in the gaseous environment, the concentration threshold for activity switching, and the reversibility of the response process, respectively. When the correlation curves show different slopes in different concentration ranges, the sensitive concentration range and the optimal concentration range for microbial activity are identified. When there is a hysteresis between the positive regulation curve and the negative recovery curve, the hysteresis area is quantitatively calculated to evaluate the integrity of the response recovery. The final quantitative evaluation results include the upper and lower limits of the sensitive concentration range, the distribution of response hysteresis time, and the activity recovery coefficient, which directly serve the underwater microbial ecological monitoring and carbon cycle response assessment. The rate of change of fluorescence intensity can be expressed as ,in The initial fluorescence intensity, for fluorescence intensity at any time To normalize the fluorescence change rate, by The size and trend of changes can be used to determine the activity response of microorganisms under different carbon dioxide environments.

[0022] Example 3, as Figure 1 , Figure 2 As shown, based on Examples 1 and 2, the present invention also provides a method for underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation, which is implemented based on the above-mentioned underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device, and includes the following steps: S1: In the target water body, underwater in-situ sampling is carried out to obtain water samples containing microorganisms. The water samples are introduced into the microfluidic chip through an isothermal and isobaric flow path to maintain the water sample environment consistent with the in-situ environment and ensure that the microbial activity truly reflects the in-situ state. S2: Microorganisms are transported, captured, enriched and located within a microfluidic chip, stabilizing them in a controllable micro-region between the fluorescence observation area and the gas exchange area, thus achieving precise microbial positioning and long-term stable observation. S3: Use the fluorescence detection module to acquire microbial fluorescence images, extract integrated fluorescence intensity, calculate normalized fluorescence change rate, determine the physiological activity status of microorganisms, and realize real-time quantitative discrimination of microbial activity status; S4: Enables carbon dioxide molecules in the water sample to dissolve, diffuse, and desorb through the dense non-microporous membrane AF2400, and then transport them across the membrane to the gas detection optical cell, achieving gas-liquid isolation and avoiding interference from liquid water on spectral detection; S5: The carbon dioxide concentration is measured using 2.6μm mid-infrared TDLAS technology, and temperature and pressure parameters are collected simultaneously for correction to obtain an accurate carbon dioxide concentration value, ensuring the accuracy and long-term stability of carbon dioxide concentration measurement. S6: Integrates fluorescence activity and carbon dioxide concentration data, activates the regulation module as needed to change the gaseous environment around microorganisms, obtains the response characteristics of activity to changes in carbon dioxide, and realizes the linkage analysis of gaseous environment changes and microbial responses.

[0023] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. The scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device, characterized in that, include: The underwater sampling module is used to obtain water samples containing microorganisms from the target water body; The AF2400 gas-permeable membrane is used to enable the transfer of carbon dioxide from water or microfluidic chips to the gas detection optical cell or gas control region. Microfluidic chips are used to transport, capture, enrich, and locate microorganisms that enter the chip; The fluorescence detection module is used to collect fluorescence signals from microorganisms within the microfluidic chip and to determine the physiological activity status of the microorganisms based on changes in fluorescence intensity. A mid-infrared TDLAS detection module for carbon dioxide is used to determine the concentration of carbon dioxide after transmission through the AF2400 gas permeation membrane based on 2.6μm mid-infrared tunable semiconductor laser absorption spectroscopy. The carbon dioxide microenvironment control module is used to inject, release, exchange, or regulate the concentration gradient of carbon dioxide gas environment within the microfluidic chip or around microorganisms. The temperature and pressure environment monitoring module is used to collect temperature and pressure parameters in the underwater environment, gas detection optical cell, and microfluidic chip. The data processing and control module is used to perform fusion analysis on microbial fluorescence intensity, carbon dioxide concentration, temperature, pressure and gas regulation parameters, and evaluate the changes in the physiological activity of microorganisms under different carbon dioxide gas environments based on the analysis results.

2. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device according to claim 1, characterized in that: The AF2400 gas permeable membrane is a dense non-microporous membrane structure. The AF2400 gas permeable membrane is disposed between the aqueous phase region and the gas phase region to block liquid water from entering the gas detection optical cell, while allowing carbon dioxide molecules dissolved in the water to be transported to the gas detection optical cell through the membrane material. The microfluidic chip includes an injection channel, a microbial capture area, a fluorescence observation area, a gas exchange area, and a drainage channel; Among them, the underwater sampling module obtains microbial water samples in situ in the target water body, and the water samples are introduced into the microfluidic chip sampling channel using an isothermal and isobaric flow path, while maintaining the water sample temperature and pressure environment consistent with the in situ water body. Within a microfluidic chip, fluid manipulation structures are used to transport, capture, enrich, and locate microorganisms in a water sample, enabling the microorganisms to be positioned in a controllable micro-region between the fluorescence observation area and the gas exchange area. By using the AF2400 gas permeable membrane placed between the aqueous and gas phases, CO2 molecules are dissolved, diffused, and desorbed into the gas detection optical cell or gas control channel through a dissolution-diffusion-desorption cascade mechanism, thus blocking interference from liquid water and ion spectra.

3. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device according to claim 1, characterized in that: The fluorescence detection module includes an excitation light source, a fluorescence excitation filter unit, a fluorescence collection optical unit, a fluorescence filter unit, an imaging detector, and a fluorescence intensity analysis unit. The fluorescence detection module obtains the fluorescence intensity of microorganisms through one or more of the following: microbial autofluorescence, fluorescence staining signal, or fluorescence probe signal, and determines the physiological activity state of microorganisms based on fluorescence intensity, fluorescence intensity change rate, or normalized fluorescence intensity. Specifically, a specific wavelength of excitation light is generated by the excitation source through the filter unit, which irradiates the pre-positioned microbial area within the microfluidic chip, stimulating its own fluorescence, staining signal, or fluorescent probe signal. Microbial fluorescence images are acquired using a fluorescence collection optical unit and an imaging detector, and the fluorescence intensity, fluorescence distribution area, or integrated fluorescence value of a single microorganism within the observation area are extracted. The normalized fluorescence change rate is calculated based on the ratio of fluorescence intensity to initial fluorescence intensity to determine whether the physiological activity of microorganisms is enhanced, weakened, or stable.

4. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device according to claim 3, characterized in that: The fluorescence detection module further includes: Microbial fluorescence images were acquired multiple times in a continuous time series, and the integrated fluorescence intensity at each moment was extracted to form a dynamic curve of fluorescence intensity changing over time. The dynamic curve is processed by moving average filtering, and the instantaneous rate of change of fluorescence intensity and the first derivative of the rate of change are calculated to identify the rapid rise, decay or plateau of fluorescence signal. When the normalized fluorescence change rate exceeds the preset threshold range and continues to exceed the set time window, it is determined that the microorganism has entered an abnormal activity state, and carbon dioxide microenvironment regulation or data labeling is triggered.

5. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device according to claim 1, characterized in that: The carbon dioxide mid-infrared TDLAS detection module includes a 2.6μm mid-infrared tunable laser, collimating optical elements, a gas detection optical cell, a mid-infrared detector, a laser driving and temperature control unit, a signal acquisition unit, and a concentration inversion unit; Among them, carbon dioxide gas, which is transmitted to the gas detection optical cell through the AF2400 gas permeation membrane, is placed in a closed optical path after temperature and pressure correction, and a laser is emitted by a 2.6μm mid-infrared tunable laser using a sinusoidal scanning and sawtooth superposition modulation method. The absorption signal of carbon dioxide at the characteristic absorption line of 2.6 μm was acquired by direct absorption spectroscopy or wavelength modulation spectroscopy, and the second harmonic signal was extracted in wavelength modulation mode. When wavelength modulation spectroscopy was used, the carbon dioxide concentration was inverted by the first harmonic signal, the second harmonic signal or the normalized second harmonic signal. The concentration inversion unit calculates the absolute concentration of carbon dioxide in the gas detection optical cell based on the absorption signal amplitude, the second harmonic peak intensity, and the real-time temperature and pressure compensation coefficient.

6. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device according to claim 1, characterized in that: The carbon dioxide microenvironment regulation module specifically includes: Based on the fusion analysis results of the current carbon dioxide concentration and the fluorescence activity status of microorganisms, it is determined whether it is necessary to change the carbon dioxide gas environment around the microorganisms in the microfluidic chip. When adjustment is required, the carbon dioxide microenvironment control module is activated, and a set concentration of carbon dioxide gas or mixed carrier gas is introduced into the gas exchange area through the gas injection channel; Carbon dioxide molecules are transported across the membrane from the gas phase control channel to the aqueous phase region of the microfluidic chip through the AF2400 gas permeation membrane, enabling the increase, decrease, or maintenance of carbon dioxide concentration around microorganisms.

7. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment regulation device according to claim 6, characterized in that: The carbon dioxide microenvironment control module also includes: Different concentrations of carbon dioxide gas are introduced into different spatial locations of the gas exchange zone through multiple independent gas inlets, so that different regions of the AF2400 membrane exhibit differentiated gas phase carbon dioxide partial pressures. By utilizing the fluid isolation structure within the microfluidic chip, the carbon dioxide concentration differences in different regions of the aqueous phase are maintained, thereby creating a stable carbon dioxide concentration gradient or a periodically fluctuating environment in the microbial capture zone. Based on real-time fluorescence response feedback, the flow rate ratio or gas switching frequency of each gas inlet is dynamically adjusted to make the carbon dioxide concentration around the microorganisms change according to a set curve, which is used to analyze the sensitive response range of microorganisms to the gas environment.

8. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device according to claim 1, characterized in that: The temperature and pressure environment monitoring module specifically includes: A temperature sensor and an absolute pressure sensor, both calibrated underwater, were deployed at three independent locations: the inlet of the underwater sampling module, the inside of the gas detection optical pool, and the fluorescence observation area of ​​the microfluidic chip, to eliminate the temperature and pressure transmission delay between different media interfaces. Three temperature and pressure data points are collected synchronously at a sampling frequency of not less than 1Hz. The temperature and pressure data in the gas detection optical cell are time-domain aligned and abnormal transient fluctuations are removed. The data is then transmitted to the data processing and control module in real time to form a temperature and pressure time series synchronized with the environmental conditions. When performing concentration inversion, the carbon dioxide infrared TDLAS detection module reads the temperature and pressure values ​​of the current gas detection optical cell, performs numerical compensation on the concentration inverted by the absorption signal according to the ideal gas law, and corrects the concentration inverted by the absorption signal point by point.

9. The underwater in-situ microbial fluorescence activity detection and carbon dioxide microenvironment control device according to claim 1, characterized in that: The data processing control module specifically includes: The normalized fluorescence intensity of microorganisms, absolute carbon dioxide concentration, temperature, pressure and gas regulation parameters within the same time window are constructed into a multi-source heterogeneous feature matrix with timestamps, and the time window is segmented according to the trigger time of the regulation event. The sliding window cross-correlation analysis method was used to calculate the time delay correlation coefficient and peak response time between the carbon dioxide concentration change sequence and the fluorescence intensity change rate sequence, and to extract the hysteresis response time and response sensitivity of microorganisms to changes in the gas environment. Based on the dynamic correlation curves between carbon dioxide concentration and fluorescence intensity under different regulatory stages, quantitative evaluation results of microbial physiological activity as a function of the carbon dioxide microenvironment are output.

10. A method for detecting the fluorescence activity of underwater microorganisms and regulating the carbon dioxide microenvironment, implemented based on the device for detecting the fluorescence activity of underwater microorganisms and regulating the carbon dioxide microenvironment as described in any one of claims 1-9, characterized in that, Includes the following steps: S1: Perform underwater in-situ sampling in the target water body to obtain water samples containing microorganisms, and introduce the water samples into the microfluidic chip through an isothermal and isobaric flow path to maintain the water sample environment consistent with the in-situ environment; S2: Transporting, capturing, enriching, and localizing microorganisms within a microfluidic chip, stabilizing them in a controllable micro-region between the fluorescence observation area and the gas exchange area; S3: Use the fluorescence detection module to acquire microbial fluorescence images, extract integrated fluorescence intensity, calculate normalized fluorescence change rate, and determine the physiological activity status of microorganisms; S4: Enables carbon dioxide molecules in the water sample to dissolve, diffuse, and desorb through the AF2400 dense non-microporous membrane, and then transport them across the membrane to the gas detection optical cell, thus achieving gas-liquid isolation; S5: The carbon dioxide concentration is determined using 2.6μm mid-infrared TDLAS technology, and temperature and pressure parameters are collected simultaneously for correction to obtain an accurate carbon dioxide concentration value; S6: Integrates fluorescence activity and carbon dioxide concentration data, activates the regulation module as needed to change the gaseous environment around the microorganism, and obtains the response characteristics of activity to changes in carbon dioxide.

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