Method and device for detecting algal cell density and living cell density based on microfluidic microfluorescence

Abstract

The present invention provides a method and device for detecting algal cell density and live cell density based on microfluidic microfluorescence. This method allows individual algal cells in a microfluidic channel to pass through the microscopic field of view at a constant speed. The number of fluorescence peaks exhibited by the cell fluorescence intensity as it changes with position is detected to accurately count floating algae cells. Cell activity is then determined by detecting the variable fluorescence inversion of the algal cells, enabling accurate counting of live algal cells. The algal cell density and live cell density in a sample are calculated by recording the number of algal cells and live cells in a specific volume of sample. Based on this, a microfluidic microfluorescence-based algal cell density device was designed. This device utilizes microfluidics for quantitative sample injection and employs an episcopic microfluorescence optical path combined with an integral amplifier circuit and a high-speed acquisition circuit to measure single algal cell fluorescence and variable fluorescence. Combined with the algal cell density and live cell density detection methods proposed in the invention, algal cell density and live cell density detection can be achieved.

Description

Technical Field

[0001] The present invention belongs to the fields of botany and environmental science, and particularly relates to a method and device for detecting algae cell density and living cell density based on microfluidic microfluorescence. Background Art

[0002] Phytoplankton is an important primary producer in aquatic ecosystems and an indicator of water health. Measuring phytoplankton cell density, particularly viable cell density, directly and effectively reflects its growth status, providing important scientific and practical applications for water quality monitoring and early warning of algal blooms.

[0003] Currently, methods for measuring phytoplankton cell density can be divided into direct and indirect methods. Indirect methods primarily include spectrophotometry, high-performance liquid chromatography, and fluorescence detection. These methods all infer algal cell density by measuring chlorophyll a concentration. However, scholars both domestically and internationally have demonstrated that chlorophyll a concentration is susceptible to factors such as algal cell growth state, species, and size, making it inaccurately reflecting algal cell density. Direct methods primarily include microscopy, Coulter counting, and flow fluorescence cytometry. Microscopy involves manual observation and recording under a microscope. To determine algal cell viability, staining is required to determine the number of viable algal cells. This is labor-intensive, inefficient, and subject to significant human influence, resulting in large errors. The Coulter counting method involves passing a suspended algae solution through a small aperture. Electrodes are placed on either side of the aperture, and the change in resistance is detected as algal cells pass through the aperture to measure and count particle size. This method cannot determine algal cell viability and is susceptible to interference from other suspended matter, resulting in low accuracy in actual algal cell counts. Flow fluorescence cytometry uses laser-induced fluorescence from algal cells, which is then detected and counted within a microchannel tube. This method is highly efficient and has the advantages of being fast and accurate. However, to improve the accuracy of detection, traditional flow cytometry uses a sheath fluid-focused injection method to ensure that individual cells pass through the detection window. This method first requires the suspension to be tested to be pressurized, then injected into the sheath fluid through a nozzle, and finally the pressurized sheath fluid drives the algal cells to flow through the detection window. This makes the injection structure very complex and requires the consumption of a large amount of sheath fluid. At the same time, to obtain multiple parameters, flow cytometry fluorescence usually uses an orthogonal structure for the optical path. This structure must ensure the perpendicularity of the excitation light path and the fluorescence collection light path. Before use, it is necessary to use fluorescent beads for tedious focusing, which is not suitable for miniaturized on-site rapid measurements. In addition, when counting planktonic living cells, a complex pre-treatment process for staining is required, and staining has the risk of environmental pollution, which is not currently suitable for miniaturized on-site rapid measurements. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method and device for detecting the cell density and living cell density of phytoplankton based on microfluidic microfluorescence. Algal cells pass through the microscopic field of view at a uniform speed in a microfluidic channel. By measuring and analyzing the fluorescence and variable fluorescence of individual algal cells, the number of phytoplankton cells and the number of living cells can be quickly and accurately detected. By recording the number of algal cells and the number of living cells in a specific volume of sample, the algal cell density and the number of living cells in the sample can be calculated.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for detecting phytoplankton cell density based on microfluidic microfluorescence adopts an epi-illumination excitation-emission optical structure. Single algae cells in the microfluidic channel pass through the microscopic field of view at a uniform speed. The fluorescence intensity of the stimulated emission of individual algae cells changes with their position and presents a fluorescence peak shape. The phytoplankton cells are accurately counted by detecting the number of fluorescence peaks, and the algae cell density in the sample is calculated by recording the number of algae cells in a specific volume of sample.

[0007] Furthermore, the precise counting of phytoplankton cells comprises the following steps:

[0008] Step 1: When algal cells enter the edge of the microscopic field of view, a weak fluorescence signal begins to appear; as the algal cells flow toward the center of the microscopic field of view, the intensity of the fluorescence signal gradually increases;

[0009] Step ② When the algae cells reach the center of the microscopic field of view, the fluorescence signal intensity reaches its peak;

[0010] Step 3: When the algae cells pass through the center and leave the microscope field of view, the fluorescence signal gradually weakens until it disappears;

[0011] Step ④ accurately counts the phytoplankton cells by detecting the number of fluorescence peaks presented by the changes in cell fluorescence intensity with the position, and calculates the algae cell density in the sample by recording the number of algae cells in a specific volume of sample.

[0012] The present invention also provides a method for detecting the density of living algae cells based on microfluidic microscopic variable fluorescence. The method adopts an epi-illumination excitation emission optical structure. The algae cells in the microfluidic channel pass through the microscopic field of view at a uniform speed. By measuring the variable fluorescence curve generated by a single algae cell under the excitation of a strong light pulse, the activity parameter F of the fluorescent cell is inverted. V / F M , the activity state of algae cells is judged according to their size, and the accurate counting of algae living cells is achieved. The density of algae living cells in the sample is calculated by recording the number of algae living cells in a specific volume of sample; Among them, F M is the maximum fluorescence, F v is the maximum fluorescence F M The difference from the initial fluorescence F0;.

[0013] Furthermore, the accurate counting of living algae cells comprises the following steps:

[0014] Step 1: Weak light indicator pulses are used to identify algae cells reaching the detection window: Algae living cells flow at a constant speed in the microfluidic channel. To reduce measurement errors, weak light with a 1 kHz pulse width of 5 μs is used to continuously illuminate the microfluidic channel. The integral amplifier circuit detects weak fluorescence. If the fluorescence is greater than the set threshold, the algae living cells have reached the edge of the detection window.

[0015] Step 2: Dark adaptation to promote QA-oxidation: Turn off the excitation light source to promote QA-oxidation and wait for the living algae cells to move from the edge of the detection window to the center of the detection window;

[0016] Step 3: Measure variable fluorescence with strong light pulses: Move the living algae cells to the center of the detection window, use a pulse width of 160 μs, and a light intensity of 10,000 μmol quanta / m 2 / s strong light pulses stimulate living algae cells, and an oversampling rate circuit is used to measure the variable fluorescence curve;

[0017] Step 4: Determine whether algae cells are alive or dead: Use the variable fluorescence curve inversion to obtain the initial fluorescence F0 and the maximum fluorescence F m , calculate the maximum photochemical quantum efficiency F v / F m , where F v =F m -F0, with F v / F m >0.2 is the standard for judging living cells, which enables the counting of living algae cells. The density of living algae cells in the sample is calculated by recording the number of living algae cells in a specific volume of sample.

[0018] The present invention also provides a device for measuring the cell density of phytoplankton and living cells based on microfluidic microfluorescence, which includes an excitation and emission optical structure, a microfluidic sampling module, an excitation light source driving module, a fluorescence detection module and a main control module;

[0019] The excitation emission optical structure uses a high-brightness LED chip as the excitation light source, and a BP455 bandpass filter is placed at the front end to preliminarily eliminate the influence of the light source on the fluorescence. The excitation light source is then focused by a lens combination, and after focusing, it passes through a D470 dichroic mirror and vertically enters the objective lens. The excitation light is refocused by the objective lens and irradiates the microfluidic chip. The fluorescence generated by the excited algae cells passes through the objective lens, passes through the D470 dichroic mirror and the filter combination, and is filtered out stray light before being focused by the lens onto the aperture. The aperture filters the stray light twice, and then the light is received by a photomultiplier tube and detected by a fluorescence detection module.

[0020] The microfluidic injection module consists of a sample pool, a microfluidic chip, a two-way valve, a high-precision injection pump and a waste liquid pool;

[0021] The excitation light source driving module is composed of a DAC driver, a constant voltage driving unit, and a high-power MOS switch circuit. Under the control of the 16-bit digital-to-analog converter of the main control module, a variable amplitude voltage is generated. After being driven by the DAC driver, the voltage of the constant voltage driving unit is adjusted to achieve control of the excitation light intensity. The PWM of the main control module generates an electrical pulse signal to control the high-power MOS switch circuit to drive the excitation light source to generate variable light pulses.

[0022] The fluorescence detection module includes a dual-channel analog switch, a fast fluorescence detection channel, and a weak fluorescence detection channel;

[0023] The main control module is based on the Cortex-M8 processor, combined with RAM and Flash memory, a touch liquid crystal display and peripheral circuits to realize excitation light source control, fluorescence detection module data acquisition, data analysis and processing, as well as input and output control of the entire device.

[0024] Furthermore, the filter combination includes a BP675 narrowband filter and a BP655 material filter.

[0025] Furthermore, the microfluidic chip is designed according to the particle size characteristics of phytoplankton, has a channel cross-sectional size of 200 μm*50 μm, is made of quartz glass, and uses lateral sampling as the sampling method.

[0026] Furthermore, the flow path system of the microfluidic injection module is divided into two processes: injection and discharge;

[0027] During sampling, channel A of the two-way valve is opened and channel B is closed. The algae solution in the sample pool flows into the microfluidic chip under the action of a high-precision syringe pump. In the microfluidic chip, single algae cells continuously pass through the optical detection window to achieve the excitation and collection of algae cell fluorescence. After measurement, the algae solution flows out of the microfluidic chip and is extracted into the high-precision syringe pump through the two-way valve, completing the entire sampling process.

[0028] During sample discharge, the B channel of the two-way valve is opened and the A channel is closed, and the high-precision injection pump pushes the waste sample liquid into the waste liquid pool to complete the reset.

[0029] Furthermore, the fast fluorescence detection channel, the weak fluorescence detection channel and the PMT detector are switched through a dual-channel analog switch; the PMT detector signal is pre-amplified and collected by a high-speed data acquisition circuit, and the collected data is output to the main control module through DMA. This channel can achieve accurate measurement of fast-changing fluorescence processes within 150us, and distinguish the life and death of algae cells, thereby achieving accurate measurement of the density of living algae cells; when measuring the density of algae cells, the weak fluorescence detection channel is switched through the dual-channel analog switch, and the PMT detector signal is implemented through an integral amplifier circuit to achieve a flow integration process, which is equivalent to summing the current signal within a certain time, and the signal-to-noise ratio after integration is 1 / 4 of the original signal-to-noise ratio. times, and the output signal is collected by the analog / digital converter of the main control module. This channel can achieve high-sensitivity detection of fluorescence at a specific frequency, thereby achieving accurate measurement of algae cell density; the acquisition rate of the high-speed data acquisition circuit reaches more than 50Mbps.

[0030] Beneficial effects:

[0031] This method utilizes chlorophyll fluorescence in the characteristic wavelength band of stimulated emission from algal cells, combining a microfluidic chip with microfluorescence detection technology. By detecting the fluorescence and variable fluorescence of single algal cells within a specific volume, this method overcomes the interference of suspended particles in the water and enables rapid and accurate detection of both floating and living algae cell density. The device has a simple structure, a rapid measurement process, and requires no reagents, providing a new approach for the rapid and accurate detection of algal cell density, particularly that of living algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the method for counting phytoplankton cells and living cells based on microfluidic microfluorescence;

[0033] Figure 2 This is a structural diagram of the device for measuring phytoplankton cell density and living cell density based on microfluidic microfluorescence of the present invention;

[0034] Figure 3a , Figure 3b , Figure 3c , Figure 3d This is a comparison chart of algae cell density under microscope and density detected by microfluidic microfluorescence, where: Figure 3a It is Dunaliella salina; Figure 3b It is Heterosigma akashiwo; Figure 3c For cryptoalgae; Figure 3d For Chromococcus;

[0035] Figure 4 The density of living algae cells under different volume ratios is shown. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0037] The present invention provides a method and device for detecting phytoplankton cell density and living cell density based on microfluidic microfluorescence. The entire operation process does not require pretreatment and can be used for on-site rapid investigation of phytoplankton in wild water bodies.

[0038] The present invention proposes a method for detecting the density of phytoplankton cells based on microfluidic microfluorescence. In this method, a single algae cell in a microfluidic channel passes through the microscopic field of view at a uniform speed. The intensity of the stimulated fluorescence emission of the cell changes with its position and presents a fluorescence peak. The phytoplankton cells are accurately counted by detecting the number of fluorescence peaks, and the algae cell density in the sample is calculated by recording the number of algae cells in a specific volume of sample.

[0039] The present invention also proposes a method for detecting the density of living algae cells based on microfluidic microscopic variable fluorescence. The algae cells in the microfluidic channel pass through the microscopic field of view at a uniform speed. By measuring the variable fluorescence curve generated by a single algae cell under the excitation of a strong light pulse, the activity parameter F of the fluorescent cell is inverted. V / F M The activity state of algae cells can be judged according to their size, and the accurate counting of living algae cells can be achieved. By recording the number of living algae cells in a specific volume of sample, the density of living algae cells in the sample can be calculated.

[0040] The present invention adopts a combination of a microfluidic chip, a syringe pump, and a solenoid valve to realize algae cell sampling, an epi-illumination microfluorescence optical path to realize algae cell fluorescence excitation and collection, and a dual-channel fluorescence detection circuit to realize algae cell weak fluorescence and variable fluorescence measurement. The present invention provides a device for measuring the cell density of floating algae and the density of living cells based on microfluidic microfluorescence. The device has a simple sampling structure and optical structure, high measurement accuracy, and is suitable for miniaturized on-site rapid measurement.

[0041] like Figure 1 As shown in , the method for detecting phytoplankton cell density based on microfluidic microfluorescence of the present invention includes:

[0042] Using an epi-illumination excitation emission optical structure 10, a single algae cell in a microfluidic channel 60 passes through the microscopic field of view at a constant speed. The fluorescence intensity of the stimulated emission of a single algae cell depends on the excitation light intensity and fluorescence collection efficiency at the location of the algae cell. The fluorescence peak shape changes with the location. By detecting the number of fluorescence peaks, the floating algae cells can be accurately counted 70, such as Figure 1The precise counting of phytoplankton cells can be divided into three steps:

[0043] Step ① When algae cells enter the edge of the microscopic field of view, the fluorescence signal begins to be generated but is relatively weak; as the algae cells flow toward the center of the microscopic field of view, the fluorescence signal intensity gradually increases, such as Figure 1 The precise count of phytoplankton cells is shown in A at 70;

[0044] Step ② When the algae cells reach the center of the microscopic field of view, the fluorescence signal intensity reaches a peak, such as Figure 1 The precise count of phytoplankton cells is shown in B at 70;

[0045] Step ③ When the algae cells pass through the center and leave the microscope field of view, the fluorescence signal gradually weakens until it disappears. Figure 1 The precise count of phytoplankton cells at 70°C is shown.

[0046] Therefore, each algae cell flowing through the microscopic field of view will correspond to a fluorescence peak. By detecting the number of fluorescence peaks, the floating algae cells can be accurately counted, and the algae cell density in the sample can be calculated by recording the number of cells in a specific volume of sample.

[0047] like Figure 2 As shown, the method for detecting the density of living phytoplankton cells based on microfluidic microscopic variable fluorescence of the present invention includes:

[0048] When living algae cells are stimulated by strong light, the fluorescence yield of the living algae cells shows a fluorescence change curve that first rises rapidly and then flattens. However, after the death of dead algae cells, under strong light stimulation, they show a stable fluorescence intensity instantly, and there is no fluorescence rise process. Based on this, using an epi-microscopic optical structure, the living algae cells in the microfluidic channel pass through the microscopic field of view at a uniform speed. By measuring the variable fluorescence curve of a single living algae cell excited by a strong light pulse, the activity of the living algae cells is judged based on the activity parameter FV / FM, and the living algae cell count is achieved. Figure 1 The counting of 80 viable algal cells can be divided into three steps:

[0049] Step 1: Weak light indicator pulses are used to identify algae cells reaching the detection window. Algae living cells flow at a constant speed in the microfluidic channel. To reduce measurement errors, weak light with a pulse width of 5μs at 1kHz is used to continuously illuminate the microfluidic channel. The integral amplifier circuit detects weak fluorescence. If the fluorescence is greater than the set threshold, the algae living cells have reached the edge of the detection window. Figure 1 The count of viable algal cells was 80 as shown;

[0050] Step ② Dark adaptation promotes QA oxidation. Turn off the excitation light source to promote QA oxidation and wait for the algae living cells to move from the edge of the detection window to the center of the detection window. Figure 1The count of viable algal cells is 80 as shown in B;

[0051] Step 3: Measure variable fluorescence using strong light pulses. The living algae cells are moved to the center of the detection window, and a pulse width of 160 μs and a light intensity of 10,000 μmol quanta / m 2 / s strong light pulses stimulate algae cells, and at the same time, an oversampling rate circuit is used to measure the variable fluorescence curve, such as Figure 1 The count of living algal cells is shown in 80C;

[0052] Step 4: Determine whether algae cells are alive or dead. Use the variable fluorescence curve inversion to obtain the initial fluorescence F0 and the maximum fluorescence F m , calculate the maximum photochemical quantum efficiency F v / F m (where F v =F m -F0), with F v / F m >0.2 is the standard for judging living cells, which enables the counting of living algae cells. By recording the number of living cells in a specific volume of sample, the density of living algae cells in the sample can be calculated.

[0053] like Figure 2 As described above, the phytoplankton cell density and living cell density measuring device based on microfluidic microfluorescence of the present invention is mainly composed of an excitation emission optical structure 10, a microfluidic sampling module 20, an excitation light source driving module 30, a fluorescence detection module 40 and a main control module 50.

[0054] The excitation emission optical structure 10 uses a high-brightness LED chip as the excitation light source 1. The central wavelength of the excitation light source 1 is 469nm (which can be selected based on the absorption characteristics of phytoplankton). A BP455 bandpass filter 2 is placed at the front end to initially eliminate the influence of the light source on fluorescence. The excitation light source 1 is then focused by a lens combination 3. After focusing, it passes through a D470 dichroic mirror 4 and vertically enters an objective lens 5 (magnification 40, numerical aperture 0.64). After secondary focusing by the objective lens 5, the excitation light is irradiated onto the microfluidic chip 22. The fluorescence generated by the excited algae cells passes through the objective lens 5, passes through the D470 dichroic mirror 4 and the filter combination 9, and is then focused by the lens 7 onto the aperture 8. After the aperture filters the stray light twice, the light is received by the photomultiplier tube (PMT) and detected by the fluorescence detection module 40. The filter combination 9 includes a BP675 narrowband filter and a BP655 material filter. This optical path design has the following advantages: ① The epi-illumination optical path, the filter system at the excitation and emission ends, and the aperture 8 effectively overcome the interference of scattered light from the light source on fluorescence detection. ② The excitation and collection optical paths are coaxial, improving excitation and collection efficiency while reducing the tedious focusing process.

[0055] The microfluidic injection module 20 consists of a sample reservoir 21, a microfluidic chip 22, a two-way valve 23, a high-precision syringe pump 24, and a waste liquid reservoir 25. The microfluidic chip 22 is a quartz glass chip designed based on the particle size characteristics of phytoplankton. It has a channel cross-section of 200 μm by 50 μm and uses lateral injection. The flow system of the microfluidic injection module 20 can be divided into two processes: injection and discharge.

[0056] During sampling, channel A of the two-way valve 23 is opened and channel B is closed. The algae solution in the sample pool 21 flows into the microfluidic chip 22 under the action of the high-precision injection pump 24. In the microfluidic chip 22, single algae cells continuously pass through the optical detection window to achieve the excitation and collection of algae cell fluorescence. After measurement, the algae solution flows out of the microfluidic chip 22 and is extracted into the high-precision injection pump 24 through the two-way valve 23, completing the entire sampling process.

[0057] During sample discharge, the two-way valve B channel is opened and the A channel is closed, and the high-precision injection pump 24 pushes the waste sample liquid into the waste liquid pool 25 to complete the reset.

[0058] The excitation light source driver module 30 consists of a DAC driver 31, a constant voltage driver unit 32, and a high-power MOS switch circuit 33. Under the control of the 16-bit digital-to-analog converter (DAC) of the main control module 50, a variable amplitude voltage is generated. After being driven by the DAC driver 31, the constant voltage driver unit 32 is regulated to achieve control of the excitation light intensity. The PWM of the main control module 50 generates an electrical pulse signal to control the high-power MOS switch circuit 33 to drive the excitation light source 1 to generate variable light pulses.

[0059] The fluorescence detection module 40 is mainly composed of a dual-channel analog switch 41, a fast fluorescence detection channel 42, and a weak fluorescence detection channel 43. When measuring the density of living algae cells, the dual-channel analog switch 41 is used to switch to the weak fluorescence detection channel 42. The PMT detector signal is pre-amplified and collected by a high-speed data acquisition circuit (the acquisition rate reaches more than 50Mbps). The collected data is output to the main control module 50 through DMA. This channel can accurately measure the fast-changing fluorescence process within 150us and distinguish the life and death of algae cells, thereby achieving accurate measurement of the density of living algae cells; when measuring the density of algae cells, the dual-channel analog switch 41 is used to switch to the weak fluorescence detection channel 43. The PMT detector signal is used to implement a flow integration process through an integration amplifier circuit, which is equivalent to summing the current signal within a certain time. The signal-to-noise ratio after integration is 1 / 4 of the original signal-to-noise ratio. times, and the output signal is collected by the analog / digital converter (ADC) of the main control module 50. This channel can achieve high-sensitivity detection of fluorescence at a specific frequency, thereby achieving accurate measurement of algae cell density;

[0060] The main control module 50 is based on the Cortex-M8 processor, combined with RAM and Flash memory, touch LCD and other peripheral circuits to realize excitation light source control, fluorescence detection module data collection, data analysis and processing, as well as input and output control of the entire device.

[0061] Specifically, the algae cell density measurement results are:

[0062] Four algae species were selected for the experiment: Dunaliella salina, Chromococcus, Cryptoalgae, and Heterosigma akashiwo. These four algae species vary widely in cell size, phylum, and chlorophyll content. All algae species were obtained from the freshwater algae collection of the Institute of Hydrobiology, Chinese Academy of Sciences. Detailed information is shown in Table 1. The experiment was carried out by gradual gradient dilution at a 2-fold ratio, with the dilution to the lowest algae density of 1.5×105cells / L as the benchmark, and obtained 5 Dunaliella salina, 5 Cryptomonas, 4 Chromococcus, and 4 Red tide flexible algae with different density gradient samples. The microfluidic microfluorescence system was used at an injection speed of 50μL / min to calculate the algal cell density of each sample by recording the fluorescence peak information. Each sample was measured 10 times and the average value was taken. The algal cell density number under microscope was used as the standard, and the microscopic density and the density measured by this method were used as X and Y to draw a dot-line graph, respectively, and the absolute value of the relative error was also plotted. The results are shown in Figure 2. Figure 3a , Figure 3b , Figure 3c , Figure 3d shown.

[0063] Table 1 Experimental algae species information

[0064]

[0065] The test results of the four algae samples were highly consistent with the standard curves, with correlation coefficients R2 greater than 0.996. The absolute values of the relative errors of different types of algae cells were all less than 3.96% within 1.3×106Cells / L. The accuracy of the test results was not affected by the size or type of algae.

[0066] Specifically, the measurement results of live algae cell density are:

[0067] Coscinodiscus granii was selected as the experimental object, and the microscopic staining method was used to count the Coscinodiscus granii. A certain amount of original algae solution was taken for inactivation, and the inactivated algae solution was tested according to the microscopic vital staining method to ensure that there were no living algae cells in the inactivated algae solution. The uninactivated algae solution and the inactivated algae solution were mixed in volume ratios of 1:1, 2:1, 3:1, and 4:1. A microfluidic microfluorescence system was used to measure 1 mL of each sample at an injection speed of 50 μL / min. The algae cell density and the number of living algae cells detected by microscope were used as standards to calculate the theoretical value of the living algae cell density under each volume ratio. The Y-axis of the bar graph was plotted with the theoretical living algae cell density, algae cell density, living algae cell density measured by the system, and algae cell density under each volume ratio and the mixing of each volume ratio. The relative error under each mixing ratio was also marked. The results are shown as follows Figure 4 shown.

[0068] Depend on Figure 4 It can be seen that the relative errors of the algae cell density detection results and microscope detection under various volume ratios are both lower than 3.38%, and the relative errors of the live algae cell density detection results and theoretical values are both lower than 7.37%. The test results show that this technology has good accuracy in different ratios of live algae.

[0069] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A device for measuring phytoplankton cell density and living cell density based on microfluidic microfluorescence, characterized in that: It includes excitation and emission optical structure, microfluidic sampling module, excitation light source driving module, fluorescence detection module and main control module; The excitation emission optical structure uses a high-brightness LED chip as the excitation light source, and a BP455 bandpass filter is placed at the front end to preliminarily eliminate the influence of the light source on the fluorescence. The excitation light source is then focused by a lens combination, and after focusing, it passes through a D470 dichroic mirror and vertically enters the objective lens. The excitation light is refocused by the objective lens and irradiates the microfluidic chip. The fluorescence generated by the excited algae cells passes through the objective lens, passes through the D470 dichroic mirror and the filter combination, and is filtered out stray light before being focused by the lens onto the aperture. The aperture filters the stray light twice, and then the light is received by a photomultiplier tube and detected by a fluorescence detection module. The microfluidic injection module consists of a sample pool, a microfluidic chip, a two-way valve, a high-precision injection pump and a waste liquid pool; The excitation light source driving module is composed of a DAC driver, a constant voltage driving unit, and a high-power MOS switch circuit. Under the control of the 16-bit digital-to-analog converter of the main control module, a variable amplitude voltage is generated. After being driven by the DAC driver, the voltage of the constant voltage driving unit is adjusted to achieve control of the excitation light intensity. The PWM of the main control module generates an electrical pulse signal to control the high-power MOS switch circuit to drive the excitation light source to generate variable light pulses. The fluorescence detection module includes a dual-channel analog switch, a fast fluorescence detection channel, and a weak fluorescence detection channel; The main control module is based on the Cortex-M8 processor, combined with RAM and Flash memory, a touch LCD and peripheral circuits to achieve excitation light source control, fluorescence detection module data acquisition, data analysis and processing, as well as input and output control of the entire device; The flow path system of the microfluidic injection module is divided into two processes: injection and discharge; During sampling, channel A of the two-way valve is opened and channel B is closed. The algae solution in the sample pool flows into the microfluidic chip under the action of a high-precision syringe pump. In the microfluidic chip, single algae cells continuously pass through the optical detection window to achieve the excitation and collection of algae cell fluorescence. After measurement, the algae solution flows out of the microfluidic chip and is extracted into the high-precision syringe pump through the two-way valve, completing the entire sampling process. When discharging samples, the B channel of the two-way valve is opened, the A channel is closed, and the high-precision syringe pump pushes the waste sample liquid into the waste liquid pool to complete the reset; The fast fluorescence detection channel, the weak fluorescence detection channel and the PMT detector are switched through a dual-channel analog switch; the PMT detector signal is pre-amplified and collected by a high-speed data acquisition circuit, and the collected data is output to the main control module through DMA. This channel can accurately measure the fast-changing fluorescence process within 150 microseconds, distinguish the life and death of algae cells, and thus achieve accurate measurement of the density of living algae cells; when measuring the density of algae cells, the dual-channel analog switch is switched to the weak fluorescence detection channel, and the PMT detector signal is implemented through an integral amplifier circuit to implement a flow integration process, which is equivalent to summing the current signal within a certain time. The signal-to-noise ratio after integration is 1 / 4 of the original signal-to-noise ratio. times, and the output signal is collected by the analog / digital converter of the main control module. This channel can achieve high-sensitivity detection of fluorescence at a specific frequency, thereby achieving accurate measurement of algae cell density; the acquisition rate of the high-speed data acquisition circuit reaches more than 50Mbps.

2. The device for measuring phytoplankton cell density and living cell density based on microfluidic microfluorescence according to claim 1, characterized in that: The filter combination includes a BP675 narrowband filter and a BP655 narrowband filter.

3. The device for measuring phytoplankton cell density and living cell density based on microfluidic microfluorescence according to claim 1, characterized in that: The microfluidic chip is designed based on the particle size characteristics of phytoplankton, with a channel cross-section size of 200 μm × 50 μm, a material of quartz glass, and a lateral sampling method.

4. The method for measuring phytoplankton cell density and living cell density based on a microfluidic microfluorescence device according to any one of claims 1 to 3, characterized in that: Using an epi-illumination excitation-emission optical structure, the algae cells in the microfluidic channel pass through the microscopic field of view at a uniform speed. By measuring the variable fluorescence curve produced by a single algae cell under the excitation of a strong light pulse, the activity parameter F of the fluorescent cell is inverted. V / F M , the activity state of algae cells is judged according to their size, and the accurate counting of algae living cells is achieved. The density of algae living cells in the sample is calculated by recording the number of algae living cells in a specific volume of sample; Among them, F M is the maximum fluorescence, F v is the maximum fluorescence F M The difference from the initial fluorescence F0.

5. The method for detecting the density of living phytoplankton cells based on microfluidic microfluorescence according to claim 4, characterized in that: The accurate counting of living algae cells comprises the following steps: Step 1: Weak light indicator pulses are used to identify algae cells reaching the detection window: Algae living cells flow at a constant speed in the microfluidic channel. To reduce measurement errors, weak light with a 1 kHz pulse width of 5 μs is used to continuously illuminate the microfluidic channel. The integral amplifier circuit detects weak fluorescence. If the fluorescence is greater than the set threshold, the algae living cells have reached the edge of the detection window. Step ② Dark adaptation promotes QA - Oxidation: Turn off the excitation light source to promote QA - Oxidation, waiting for the living algae cells to move from the edge of the detection window to the center of the detection window; Step 3: Measure variable fluorescence with strong light pulses: Move the living algae cells to the center of the detection window, use a pulse width of 160 μs, and a light intensity of 10,000 μmol quanta / m 2 / s strong light pulses stimulate living algae cells, and an oversampling rate circuit is used to measure the variable fluorescence curve; Step 4: Determine whether algae cells are alive or dead: Use the variable fluorescence curve inversion to obtain the initial fluorescence F0 and the maximum fluorescence F m , calculate the maximum photochemical quantum efficiency F v / F m , where F v =F m -F0, with F v / F m >0.2 is the standard for judging living cells, which enables the counting of living algae cells. The density of living algae cells in the sample is calculated by recording the number of living algae cells in a specific volume of sample.

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