Photoelectric microfluidic automatic control method for screening microalgae growth conditions

Abstract

The invention discloses a photoelectric microfluidic automatic control method for screening microalgae growth conditions, which is characterized by comprising the following steps: preparing different types of culture medium solutions, respectively introducing the culture medium solutions into a photoelectric wetting chip, and generating different types of culture medium drops on the photoelectric wetting chip; automatically obtaining the current positions and IDs of a plurality of culture medium drops, and automatically obtaining the current positions and IDs of algae drops; according to the current positions and the IDs of the plurality of culture medium drops and the current positions and the IDs of the algae drops, respectively planning moving paths of the plurality of culture medium drops to the corresponding algae drops; respectively driving the plurality of culture medium drops to move to the corresponding algae drops in parallel according to the moving path through the plurality of light spots for fusion; and culturing the fused liquid drops in an illumination incubator so as to screen out a culture medium solution which is most suitable for the growth of the algae drops.

Description

Technical Field

[0001] The invention relates to the technical field of microfluidic chips, and in particular to a photoelectric microfluidic automatic control method for screening microalgae growth conditions. Background Art

[0002] With global warming and the depletion of fossil energy, biodiesel has attracted widespread attention from the industry and academia as a renewable and sustainable new energy source. Microalgae, as a type of single-cell organism, does not compete for arable land. It can produce a variety of substances such as lipids, vitamins, pigments, fatty acids, etc. through photosynthesis, and its growth rate and lipid content are higher than those of traditional crops. Therefore, it is considered to be a promising raw material for biodiesel production. Recent studies have shown that the growth conditions of microalgae will affect its yield as a biological product. Factors affecting the growth of microalgae include light, temperature, pH value, nitrogen and phosphorus elements, titanium dioxide concentration, cell density, salinity, pressure, etc. Therefore, in order to ensure the economic feasibility of microalgae as biofuel, it is necessary to have a deep understanding of the effects of culture conditions on microalgae growth and lipid accumulation. The key lies in developing an efficient screening method to optimize its growth conditions and maximize the yield of algae-derived products.

[0003] Traditionally, research on the growth environment of microalgae has relied on large-scale experimental techniques, including fluorescence activated cell sorting, hyperspectral imaging, mass spectrometry, and nuclear magnetic resonance. Although these methods can provide rich data information, they are costly, and it is difficult to optimize the optimal growth conditions of microalgae in conventional flask experiments, mainly due to the heterogeneity of cells. The limitations of these traditional technologies have made it challenging for researchers to fine-tune the optimization of microalgae culture conditions. Microalgae cell culture conditions rely on the development of tools and equipment. Current screening methods usually use components such as syringes to inject microalgae-containing liquids into culture media for cultivation, but they have the problems of low efficiency and insufficient precision. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a photoelectric microfluidic automatic control method for screening the growth conditions of microalgae, which mainly solves the technical problems existing in the above-mentioned background technology.

[0005] To achieve the above object, the technical solution of the embodiment of the present invention is implemented as follows:

[0006] The first aspect of the present invention discloses a photoelectric microfluidic automatic control method for screening microalgae growth conditions, the method comprising the following steps:

[0007] Different types of culture medium solutions are configured and introduced into the photoelectric wetting chip respectively, so as to generate different types of culture medium droplets on the photoelectric wetting chip;

[0008] Automatically obtain the current position and ID of multiple culture medium drops, and automatically obtain the current position and ID of algae drops;

[0009] According to the current positions and IDs of the multiple culture medium droplets and the current positions and IDs of the algae droplets, respectively planning the moving paths of the multiple culture medium droplets to the corresponding algae droplets;

[0010] Using multiple light spots to drive multiple culture medium droplets to move in parallel along the moving path to the corresponding algae droplets for fusion;

[0011] The fused droplets are placed in a light incubator for culture, thereby screening out the culture medium solution most suitable for the growth of algal droplets.

[0012] Optionally, different types of culture medium solutions include: standard culture medium solution, nitrogen-deficient culture medium solution, phosphorus-deficient culture medium solution, nitrogen-phosphorus-deficient culture medium solution, nitrogen-excess culture medium solution, phosphorus-excess culture medium solution, nitrogen-phosphorus-excess culture medium solution, nitrogen-excess phosphorus-deficient culture medium solution, and phosphorus-excess nitrogen-deficient culture medium solution.

[0013] Optionally, the current positions and IDs of multiple culture medium droplets and the current position and ID of the algae droplet are obtained through a target tracking algorithm, wherein the target tracking algorithm is composed of a ByteTrack tracker and a YOLOv5s detector, wherein the YOLOv5s detector is used to realize target recognition, and the ByteTrack tracker is used to add a bounding box and ID to the target recognition result, and the ByteTrack tracker includes a Kalman filter part and an IoU matching part.

[0014] Optionally, the current position and ID of different culture medium droplets and the current position and ID of the algae droplet are obtained respectively through a target tracking algorithm, specifically including: acquiring a target image on the photoelectric wetting chip, using a YOLOv5s detector to identify the target image, and obtaining an identification result, wherein the identification result includes the current position and ID of different culture medium droplets and the current position and ID of the algae droplet, and the current position includes the bounding box coordinates.

[0015] Optionally, when multiple culture medium droplets are driven by multiple light spots to move in parallel along the moving path to the corresponding algae droplets for fusion, the current position of each culture medium droplet is continuously updated through the target tracking algorithm, and the corresponding light spot is adjusted according to the deviation between the updated current position of the culture medium droplet and the current position of the algae droplet, so that the corresponding culture medium droplets can move in parallel along the moving path until all culture medium droplets reach the current position of the algae droplet.

[0016] Optionally, the current position of each culture medium droplet is continuously updated by a target tracking algorithm, including:

[0017] Identify multiple culture medium drops in the target image of the previous frame by using a target tracking algorithm, and obtain the bounding box coordinates and bounding box ID name of each culture medium drop in the previous frame;

[0018] The bounding box coordinates of the plurality of culture medium drops in one frame are simultaneously input into the Kalman filter, and the positions where the plurality of culture medium drops appear in the next frame are predicted respectively according to the speed and position of the corresponding culture medium drops;

[0019] Identify multiple culture medium drops in the target image of the current frame by using a target tracking algorithm to obtain the bounding box coordinates of each culture medium drop in the current frame;

[0020] For the bounding box coordinates of the current frame with high confidence, the IoU calculation is performed with the corresponding predicted position of the Kalman filter. If the IoU is greater than the set threshold, it is considered that the bounding box coordinates of the current frame of the culture medium droplet match the predicted position, and the bounding box ID name of the previous frame corresponding to the culture medium droplet is assigned to the bounding box of the current frame;

[0021] For the bounding box coordinates of the current frame with low confidence, the IoU is calculated with the predicted positions that have not been matched, and the bounding box of the current frame with low confidence is assigned a bounding box ID name.

[0022] Optionally, if any bounding box of the current frame cannot be assigned a corresponding bounding box ID of the previous frame, a new bounding box ID is assigned to the bounding box.

[0023] Optionally, the corresponding light spot is adjusted according to the deviation between the updated current position of the culture medium droplet and the current position of the algae droplet, specifically including: adjusting the position or shape of the corresponding light spot pattern according to the deviation, so that the corresponding culture medium droplets can move in parallel along the moving path until all culture medium droplets reach the current position of the algae droplet.

[0024] The second aspect of the present invention discloses a photoelectric microfluidic automatic control system for screening microalgae growth conditions, wherein the control system comprises a visual acquisition module, a droplet tracking module, an interaction module, and a light spot generation module.

[0025] The visual acquisition module is configured to acquire a real-time image on the photoelectric wetting chip as a target image;

[0026] The droplet tracking module is configured to obtain the current position and ID of the culture medium droplet, obtain the current position and ID of the algae droplet, and continuously update the current position of each culture medium droplet during the movement of each culture medium droplet;

[0027] The interaction module is used to receive input parameters from the user and adjust parameters including the motion trajectory and speed of the culture medium droplet based on the input parameters;

[0028] The light spot generation module is configured to generate a plurality of light spots based on the tracking results of the droplet tracking module, and respectively guide the corresponding plurality of culture medium droplets to move in parallel along the moving path to the corresponding algae droplets for fusion.

[0029] The beneficial effects of the present invention are as follows: through the proposed optoelectronic microfluidic automatic control method for screening microalgae growth conditions, the microalgae culture environment can be efficiently and accurately controlled, the automated mixing operation of the culture medium and algae droplets can be realized, and the growth of microalgae under different culture conditions can be monitored and analyzed in real time, thereby screening out the optimal microalgae growth conditions and maximizing the yield of microalgae-derived products. Secondly, the photoelectric wetting technology ensures high-precision control of the droplet generation and mixing process, reduces human errors, and avoids the risks of cross-contamination and external contamination. Efficient automated operation: The combination of photoelectric wetting technology and control software realizes the automated mixing operation of the culture medium and algae droplets, greatly reducing manual operation time and workload, and can process multiple samples at the same time, improving experimental efficiency. In addition, the target tracking algorithm is used to detect and monitor the position and type of the culture medium in real time to achieve high-throughput screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the structure of the photoelectrowetting microfluidic chip in the embodiment of the present application;

[0031] Figure 2 is a schematic flow chart of the optoelectronic microfluidic automatic control method for screening microalgae growth conditions in an embodiment of the present application;

[0032] Figure 3 is a schematic diagram of the microalgae droplet screening results provided by an embodiment of the present invention;

[0033] Figure 4 2 is a schematic diagram comparing the culture medium results with and without the pigment culture medium according to an embodiment of the present invention;

[0034] Figure 5 It is a module block diagram of the optoelectronic microfluidic automatic control system for screening microalgae growth conditions in the embodiment of the present application;

[0035] Figure 6 It is a principle block diagram of the optoelectronic microfluidic automatic control system for screening microalgae growth conditions in the embodiment of the present application.

[0036] Description of Figure Numbers:

[0037] 1. Visual acquisition module; 2. Droplet tracking module; 3. Interaction module; 4. Light spot halo generation module; 11. Upper glass base; 12. Upper indium tin oxide film; 13. Upper hydrophobic layer; 14. Droplet layer; 15. Lower hydrophobic layer; 16. Dielectric layer; 17. Photoconductive layer; 18. Lower indium tin oxide film; 19. Lower glass base. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further elaborated in detail below in conjunction with the drawings and specific embodiments of the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In the following description, the expression "some embodiments" is related to a subset of all possible embodiments, but it should be understood that "some embodiments" can be the same subset or different subsets of all possible embodiments, and can be combined with each other without conflict.

[0039] In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is apparent to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0040] It should be understood that the present invention can be implemented in different forms and should not be interpreted as being limited to the embodiments proposed herein. On the contrary, providing these embodiments will make the disclosure thorough and complete, and the scope of the present invention will be fully conveyed to those skilled in the art. And the purpose of the terms used herein is only to describe specific embodiments and is not intended to be a limitation of the present invention. When used herein, the singular forms of "one", "one" and "said / the" are also intended to include plural forms, unless the context clearly indicates another way. It should also be understood that the terms "compose" and / or "include" when used in this specification determine the presence of the features, integers, steps, operations, elements and / or parts, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts and / or groups. When used herein, the term "and / or" includes any and all combinations of the relevant listed items.

[0041] It should also be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "inside", "outside", "left", "right" and similar expressions used herein are for illustrative purposes only and are not intended to be the only implementation method.

[0042] In order to fully understand the present invention, a detailed structure will be proposed in the following description to illustrate the technical solution proposed by the present invention. The optional embodiments of the present invention are described in detail as follows, but in addition to these detailed descriptions, the present invention may also have other implementations.

[0043] Please refer to the attached Figure 1 The photoelectric wetting chip described in the embodiment of the present application comprises, from top to bottom, an upper glass substrate 11 (700 micrometers thick), an upper indium tin oxide film 12 (200 nanometers thick), an upper hydrophobic layer 13 (50 nanometers thick), a droplet layer 14 (130 micrometers thick), a lower hydrophobic layer (50 nanometers thick), a dielectric layer 16 (1 micrometer thick), a photoconductive layer 17 (1 micrometer thick), a lower indium tin oxide film 18 (200 nanometers thick) and a lower glass substrate 19 (700 micrometers thick). The upper indium tin oxide film 12 and the lower indium tin oxide film 18 serve as electrodes. When in use, an AC bias is applied between the two transparent indium tin oxide electrodes. In the absence of light, the externally applied voltage mainly exists in the high-resistance photoconductive layer 17. Under light, the conductivity of the photoconductive layer 17 increases by several orders of magnitude, which causes the externally applied voltage drop to be mainly in the dielectric layer, forming a strong electric field region near the light spot. In order to conveniently, efficiently and accurately screen the optimal growth conditions of microalgae, thereby maximizing the yield of microalgae-derived products, the first aspect of the present invention discloses an optoelectronic microfluidic automatic control method for screening microalgae growth conditions, see Figure 2 , the method comprises the following steps:

[0044] S1. Prepare different types of culture medium solutions and pass them into the photoelectric wetting chip respectively, so as to generate different types of culture medium droplets on the photoelectric wetting chip;

[0045] S2, automatically obtaining the current position and ID of multiple culture medium droplets, and automatically obtaining the current position and ID of algae droplets;

[0046] S3, planning the movement paths of the multiple culture medium droplets to the corresponding algae droplets according to the current positions and IDs of the multiple culture medium droplets and the current positions and IDs of the algae droplets;

[0047] S4, using a plurality of light spots to drive a plurality of culture medium droplets to move in parallel along the moving path to the corresponding algae droplets for fusion;

[0048] S5. Place the fused droplets in a light incubator for cultivation, thereby selecting the culture medium solution most suitable for the growth of algae droplets.

[0049] In step S1, the present application customizes the BG11 culture medium and sets different nitrogen and phosphorus concentrations in the customized culture medium, wherein the concentrations of various components in the customized culture medium are: MgSO4 (36.6 mg / L), CaCl 2 ·2H 2 O (27.2 mg / L), Na 2 CO 3 (20mg / L) 、 Citric acid (6mg / L), EDTA (1mg / L), Ferric ammonium citrate (6mg / L), MnCl 2 ·H 2 O (1.81mg / L), ZnSO4·7H2O (0.22mg / L), CuSO 4 ·5H 2 O (0.08 mg / L), Na 2 MoO 4 ·2H 2 O (0.39 mg / L), CoCl 2 6H 2 O(0.0409mg / L), H 3 BO 3 (2.86mg / L)

[0050] Furthermore, according to the requirements of the culture medium environment, the nitrogen and phosphorus conditions can be set, and the concentrations of nitrogen and phosphorus elements can be increased or decreased based on the standard level.

[0051] The standard content of sodium nitrate in the standard culture medium solution is 1500 mg / L, and the standard content of potassium hydrogen phosphate trihydrate is 30.5 mg / L, which is recorded as 1Nx1P;

[0052] The sodium nitrate content in the nitrogen-deficient medium solution is zero, and the potassium phosphate trihydrate is 30.5 mg / L, recorded as 0Nx1P;

[0053] The content of sodium nitrate in the phosphorus-deficient medium solution was 1500 mg / L, and the content of potassium hydrogen phosphate trihydrate was zero, which was recorded as 1Nx0P;

[0054] The contents of sodium nitrate and dipotassium hydrogen phosphate trihydrate in the culture medium solution lacking both nitrogen and phosphorus are both zero, recorded as 0Nx0P;

[0055] The content of sodium nitrate in the nitrogen excess medium solution is 2250 mg / L, and the content of potassium phosphate trihydrate is 30.5 mg / L, which is recorded as 1.5Nx1P;

[0056] The content of sodium nitrate in the phosphorus excess medium solution is 1500 mg / L, and the content of potassium hydrogen phosphate trihydrate is 45.75 mg / L, which is recorded as 1Nx1.5P;

[0057] The sodium nitrate content of the nitrogen and phosphorus excess culture medium solution is 2250 mg / L, and the potassium phosphate trihydrate content is 45.75 mg / L, which is recorded as 1.5Nx1.5P;

[0058] The sodium nitrate content in the nitrogen excess and phosphorus deficiency culture medium solution is 2250 mg / L, and the content of potassium hydrogen phosphate trihydrate is zero, which is recorded as 1.5Nx0P;

[0059] The content of sodium nitrate in the phosphorus excess and nitrogen deficiency culture medium solution is zero, and the content of potassium hydrogen phosphate trihydrate is 45.75 mg / L, recorded as 0Nx1.5P;

[0060] It should be noted that the above ratios are only partial ratios. In fact, more ratios of culture medium ratios can be selected according to the needs of the culture environment.

[0061] Furthermore, since the chip contains a large number of algae droplets and culture media, the requirements for the target detection algorithm are relatively high, so food coloring is added to the culture media with different nitrogen and phosphorus concentrations for differentiation.

[0062] In one possible implementation, the current positions and IDs of multiple culture medium droplets and the current position and ID of algae droplets are obtained through a target tracking algorithm, wherein the target tracking algorithm is composed of a ByteTrack tracker and a YOLOv5s detector, wherein the YOLOv5s detector is used to realize target recognition, and the ByteTrack tracker is used to add a bounding box and an ID to the target recognition result, and the ByteTrack tracker includes a Kalman filter part and an IoU matching part.

[0063] YOLOv5s (You Only Look Once version 5 small) is a lightweight target detector. While maintaining high detection accuracy, YOLOv5s has a faster inference speed and a smaller model size, making it very suitable for real-time target detection on devices with limited resources.

[0064] ByteTrack is an efficient multi-target tracking framework that uses a Kalman filter to predict the location of the target and a Hungarian algorithm to determine the best match between the detection result and the prediction result. It can achieve real-time target detection and tracking. The algorithm is particularly suitable for processing large-scale video data and performs well in multiple scenarios, including but not limited to traffic monitoring, security monitoring, sports analysis and other fields. It has the following main features: 1. Efficiency. ByteTrack can achieve real-time performance and maintain a high frame rate even on low-power devices. It uses a lightweight detection model and tracking strategy to reduce computational complexity. 2. Robustness. ByteTrack can stably track targets in complex environments and maintain good tracking performance even in cases of occlusion, lighting changes, etc. It can handle the sudden appearance and disappearance of targets. 3. Flexibility: It supports multiple detection models and can select the appropriate detection model according to the application scenario; it can be easily integrated into existing video analysis systems.

[0065] ByteTrack trackers typically use a Kalman filter to predict the location of an object and match detections and predictions using the Hungarian Algorithm. For each frame, the algorithm attempts to associate the detected object with the tracked object in the previous frame.

[0066] Furthermore, the current positions and IDs of different culture medium droplets and the current position and ID of algae droplets are obtained respectively through the target tracking algorithm, specifically including: acquiring the target image on the photoelectric wetting chip, using the YOLOv5s detector and the ByteTrack tracker to identify the target image, and obtaining the recognition result, wherein the recognition result includes the current position and ID of different culture medium droplets and the current position and ID of the algae droplet, and the current position includes the bounding box coordinates.

[0067] Furthermore, when multiple culture medium droplets are driven by multiple light spots to move in parallel along the moving path to the corresponding algae droplets for fusion, the current position of each culture medium droplet is continuously updated through the target tracking algorithm, and the corresponding light spot is adjusted according to the deviation between the updated current position of the culture medium droplet and the current position of the algae droplet, so that the corresponding culture medium droplets can move in parallel along the moving path until all culture medium droplets reach the current position of the algae droplet.

[0068] In one possible implementation, the current position of each culture medium droplet is continuously updated by a target tracking algorithm, specifically including:

[0069] Identify multiple culture medium drops in the target image of the previous frame by using a target tracking algorithm, and obtain the bounding box coordinates and bounding box ID name of each culture medium drop in the previous frame;

[0070] The bounding box coordinates of the plurality of culture medium drops in one frame are simultaneously input into the Kalman filter, and the positions where the plurality of culture medium drops appear in the next frame are predicted respectively according to the speed and position of the corresponding culture medium drops;

[0071] Identify multiple culture medium drops in the target image of the current frame by using a target tracking algorithm to obtain the bounding box coordinates of each culture medium drop in the current frame;

[0072] For the bounding box coordinates of the current frame with high confidence, the IoU calculation is performed with the corresponding predicted position of the Kalman filter. If the IoU is greater than the set threshold, it is considered that the bounding box coordinates of the current frame of the culture medium droplet match the predicted position, and the bounding box ID name of the previous frame corresponding to the culture medium droplet is assigned to the bounding box of the current frame;

[0073] For the bounding box coordinates of the current frame with low confidence, the IoU is calculated with the predicted positions that have not been matched, and the bounding box of the current frame with low confidence is assigned a bounding box ID name.

[0074] Specifically, when performing motion detection, first obtain the target image of the previous frame in a static state, and recognize the target image of the previous frame through a recognition algorithm to obtain the bounding box coordinates of each culture medium drop in the previous frame, and assign a corresponding bounding box ID name to each bounding box.

[0075] The obtained bounding box coordinates are input into the Kalman filter, which predicts the possible position of each culture medium drop in the current frame. Then, the target image of the current frame in a moving state is obtained. The target image of the current frame is identified through the target tracking algorithm to obtain the bounding box coordinates of each culture medium drop in the current frame, where the bounding box of the current frame has two types of situations: high confidence and low confidence.

[0076] For the identified bounding box of the current frame, ID matching needs to be performed. The ID matching adopts two-step matching, in which the first matching is for the bounding box coordinates of the current frame with high confidence, and the IoU calculation is performed between it and the corresponding predicted position of the Kalman filter. If the IoU is greater than the set threshold, it is considered that the bounding box coordinates of the current frame of the culture medium droplet match the predicted position, and the bounding box ID name of the previous frame corresponding to the culture medium droplet is assigned to the bounding box of the current frame;

[0077] The second match is for the low-confidence bounding box that has not been matched yet, and the IoU calculation is performed again. If the IoU between the low-confidence bounding box and the predicted position is greater than the threshold, the bounding box ID name of the previous frame corresponding to the culture medium droplet is assigned to the bounding box of the current frame.

[0078] In a possible implementation, if any bounding box of the current frame cannot be assigned a corresponding bounding box ID of the previous frame, a new bounding box ID is assigned to the bounding box.

[0079] In an optional embodiment, the corresponding light spot is adjusted according to the deviation between the updated current position of the culture medium droplet and the current position of the algae droplet, specifically including: according to the deviation, adjusting the position or shape of the corresponding light spot pattern so that the corresponding culture medium droplets can move in parallel along the moving path until all culture medium droplets reach the current position of the algae droplet.

[0080] Exemplarily, for each culture medium droplet, the deviation between its current position and the nearest point on the target path is calculated, and the nearest point can be determined by calculating the distance from the current position of the culture medium droplet to all points on the target path and then selecting the point with the smallest distance. If the deviation exceeds a threshold, it is considered that the culture medium droplet has deviated from the predetermined path.

[0081] If the culture medium drop deviates from the predetermined path, the culture medium drop is guided back to the predetermined path by adjusting the position or shape of the light spot pattern. For example, if the culture medium drop deviates to the left, the light spot pattern on the right can be strengthened to guide the culture medium drop to move to the right.

[0082] Furthermore, the shape of the light spot pattern can be rectangular, circular, annular or other geometric shapes, and different shapes can be used to guide the culture medium droplet to move in a specific direction. Changing the shape of the light spot pattern can change the distribution of the electric field around the culture medium droplet, thereby affecting the moving direction of the culture medium droplet.

[0083] In order to verify the method proposed in the embodiment of the present invention, two types of culture medium environments are taken as examples, namely, the standard content of sodium nitrate in the standard culture medium solution is 1500 mg / L, the standard content of dipotassium hydrogen phosphate trihydrate is 30.5 mg / L, denoted as 1Nx1P, and the content of sodium nitrate and dipotassium hydrogen phosphate trihydrate in the nitrogen and phosphorus deficient culture medium solution is zero, denoted as 0Nx0P.

[0084] During the implementation process, the culture medium solution needs to be prepared first. First, 1633 mg of BG11 culture medium is weighed and dissolved in 1000 mL of deionized water to prepare two bottles of BG11 culture medium solution. According to the above two experimental conditions, the corresponding amount of sodium nitrate and dipotassium hydrogen phosphate trihydrate samples are weighed respectively, and the pH is adjusted to 7.1 with dilute hydrochloric acid. The culture medium solution is sterilized at high temperature and high pressure at 121°C in a water bath for 15 minutes. After sterilization, the culture medium solution is cooled to room temperature, and part of the culture medium solution is used for cell culture, and food coloring is added to the two culture media.

[0085] Chlorella vulgaris was selected as the microalgae, and the Chlorella cell solution and two BG11 culture medium solutions were respectively passed through a polytetrafluoroethylene hose and a syringe pump to push the syringe into the photoelectric wetting chip at a flow rate of 1 μL / min to generate a number of culture medium droplets and algae droplets.

[0086] like Figure 3 As shown, the upper area of ​​the chip is the fusion operation area, and the lower area is the storage area of ​​the microalgae cells. The target in the photoelectric wetting chip is detected and ID assigned by the target tracking algorithm, and the algae droplets algae_1 and algae_2, culture medium N(1)_P(1)_3 and N(0)_P(0)_4 are identified. Then, according to the ID sequence, the movement routes of the algae droplets and culture medium are planned respectively, and the appropriate size of the light ring is generated at the four detection targets, driving the culture medium droplets containing different nitrogen and phosphorus concentrations to merge with the algae droplets. After fusion, according to the different pigments in the droplets, N(1)_P(1)_algae_1 and N(0)_P(0)_algae_2, i.e., droplets with sufficient nitrogen and phosphorus and droplets with insufficient nitrogen and phosphorus, are detected respectively. The above two droplets are then placed in a light incubator for culture.

[0087] The incubation environment of the light incubator was set as follows: light intensity of 4000 lm, temperature of 25 °C, light-dark switching time of 12h:12h, no additional CO 2 The culture was continued for one cycle (7 days), and the algal cells in the droplets were counted daily using ImageJ.

[0088] Three sets of repeated experiments were set up on a 96-well plate under the same experimental conditions, using the same culture medium as in the chip. The volume of the algae culture solution in each set of experiments was about 300 μL.

[0089] The cells were placed in a continuous culture incubator and cultured continuously for one cycle (7 days), and the number of Chlorella cells was observed and counted using a hemocytometer every day;

[0090] To verify the feasibility of screening the optimal growth environment of algae cells in the photoelectrowetting digital microfluidic chip, the number and growth rate of Chlorella cells cultured on chip and traditional off-chip were compared. Figure 4As shown in the figure, in the culture medium with standard nitrogen and phosphorus, the number of cells cultured in the 96-well plate and on the chip were 7183.33 cells / μL and 6214.68 cells / μL, respectively; in the culture medium with nitrogen and phosphorus deficiency, the number of cells cultured in the 96-well plate and on the chip were 4902.22 cells / μL and 4361.74 cells / μL, respectively; in the culture medium with nitrogen and phosphorus deficiency, the maximum growth rates inside and outside the chip were 0.20412 / d and 0.15261 / d, respectively; in the culture medium with sufficient nitrogen and phosphorus, the maximum growth rates inside and outside the chip were 0.24737 / d and 0. 19909 / d; in the nitrogen and phosphorus standard experiment, the daily growth rates were 0.3914 / d and 0.2778 / d respectively; in the nitrogen and phosphorus deficiency experiment, due to the lack of nitrogen and phosphorus elements, the growth rates on the well plate and chip were lower than the values ​​under the nitrogen and phosphorus sufficient experimental conditions, which were 0.1877 / d and 0.1231 / d respectively; in the nitrogen and phosphorus deficient culture medium environment, the average growth rates of algae cells inside and outside the chip were 0.07562 / d and 0.07136 / d respectively; in the nitrogen and phosphorus sufficient culture medium environment, the maximum growth rates inside and outside the chip were 0.24737 / d and 0.19909 / d respectively.

[0091] The above data show that within a growth cycle, the growth rate v of the two types of culture experiments of Chlorella cells has the same trend of change, and the values ​​of both the average growth rate and the maximum growth rate are very similar.

[0092] These trends demonstrate the feasibility of this system in the field of screening growth conditions for microalgae strains.

[0093] See also Figures 5 and 6 The second aspect of the present invention discloses a photoelectric microfluidic automatic control system for screening microalgae growth conditions, the control system comprises a visual acquisition module 1, a droplet tracking module 2, an interaction module 3, and a light spot generation module 4.

[0094] The visual acquisition module 1 is configured to acquire a real-time image on the photoelectric wetting chip as a target image;

[0095] The droplet tracking module 2 is configured to obtain the current position and ID of the culture medium droplet, obtain the current position and ID of the algae droplet, and continuously update the current position of each culture medium droplet during the movement of each culture medium droplet;

[0096] The interaction module 3 is used to receive input parameters from the user and adjust parameters including the motion trajectory and speed of the culture medium droplet based on the input parameters;

[0097] The light spot generating module 4 is configured to generate a plurality of light spots based on the tracking result of the droplet tracking module, and respectively guide the corresponding plurality of culture medium droplets to move in parallel along the moving path to the corresponding algae droplets for fusion.

[0098] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. The protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A photoelectric microfluidic automatic control method for screening microalgae growth conditions, characterized in that: The method comprises the following steps: Different types of culture medium solutions are configured and introduced into the photoelectric wetting chip respectively, so as to generate different types of culture medium droplets on the photoelectric wetting chip; Automatically obtain the current position and ID of multiple culture medium drops, and automatically obtain the current position and ID of algae drops; According to the current positions and IDs of the multiple culture medium droplets and the current positions and IDs of the algae droplets, respectively planning the moving paths of the multiple culture medium droplets to the corresponding algae droplets; Using multiple light spots to drive multiple culture medium droplets to move in parallel along the moving path to the corresponding algae droplets for fusion; The fused droplets are placed in a light incubator for culture, thereby screening out the culture medium solution most suitable for the growth of algal droplets.

2. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 1, characterized in that: Different types of culture medium solutions include: standard culture medium solution, nitrogen-deficient culture medium solution, phosphorus-deficient culture medium solution, nitrogen-phosphorus-deficient culture medium solution, nitrogen-excess culture medium solution, phosphorus-excess culture medium solution, nitrogen-phosphorus-excess culture medium solution, nitrogen-excess phosphorus-deficient culture medium solution, and phosphorus-excess nitrogen-deficient culture medium solution.

3. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 1, characterized in that: The current positions and IDs of multiple culture medium droplets and the current positions and IDs of algae droplets are obtained through a target tracking algorithm. The target tracking algorithm consists of a ByteTrack tracker and a YOLOv5s detector, wherein the YOLOv5s detector is used to realize target recognition, and the ByteTrack tracker is used to add a bounding box and an ID to the target recognition result. The ByteTrack tracker includes a Kalman filter part and an IoU matching part.

4. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 3, characterized in that: The current positions and IDs of different culture medium droplets and the current position and ID of algae droplets are obtained respectively through the target tracking algorithm, specifically including: obtaining the target image on the photoelectric wetting chip, using the YOLOv5s detector and ByteTrack tracker to identify the target image, and obtaining the recognition result, which includes the current position and ID of different culture medium droplets and the current position and ID of the algae droplet, and the current position includes the bounding box coordinates.

5. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 4, characterized in that: When multiple culture medium droplets are driven by multiple light spots to move in parallel along the moving path to the corresponding algae droplets for fusion, the current position of each culture medium droplet is continuously updated through the target tracking algorithm, and the corresponding light spot is adjusted according to the deviation between the updated current position of the culture medium droplet and the current position of the algae droplet, so that the corresponding culture medium droplets can move in parallel along the moving path until all culture medium droplets reach the current position of the algae droplet.

6. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 5, characterized in that: The current position of each culture medium droplet is continuously updated through a target tracking algorithm, including: Using the YOLOv5s detector and ByteTrack tracker, multiple culture medium drops in the target image of the previous frame are identified and assigned, and the bounding box coordinates and bounding box ID name of each culture medium drop in the previous frame are obtained; The bounding box coordinates of the plurality of culture medium drops in one frame are simultaneously input into the Kalman filter, and the positions where the plurality of culture medium drops appear in the next frame are predicted respectively according to the speed and position of the corresponding culture medium drops; Identify multiple culture medium drops in the target image of the current frame by using a target tracking algorithm to obtain the bounding box coordinates of each culture medium drop in the current frame; For the bounding box coordinates of the current frame with high confidence, the IoU calculation is performed with the corresponding predicted position of the Kalman filter. If the IoU is greater than the set threshold, it is considered that the bounding box coordinates of the current frame of the culture medium droplet match the predicted position, and the bounding box ID name of the previous frame corresponding to the culture medium droplet is assigned to the bounding box of the current frame; For the bounding box coordinates of the current frame with low confidence, the IoU is calculated with the predicted positions that have not been matched, and the bounding box of the current frame with low confidence is assigned a bounding box ID name.

7. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 6, characterized in that: If any bounding box of the current frame cannot be assigned a corresponding bounding box ID of the previous frame, a new bounding box ID is assigned to the bounding box.

8. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to claim 7, characterized in that: Adjusting the corresponding light spot according to the deviation between the updated current position of the culture medium droplet and the current position of the algae droplet specifically includes: adjusting the position or shape of the corresponding light spot pattern according to the deviation so that the corresponding culture medium droplets can move in parallel along the moving path until all culture medium droplets reach the current position of the algae droplet.

9. The optoelectronic microfluidic automatic control method for screening microalgae growth conditions according to any one of claims 1 to 8, characterized in that: The method is applied to an optoelectronic microfluidic automatic control system for screening microalgae growth conditions, wherein the control system comprises a visual acquisition module, a droplet detection and tracking module, an automatic control module, and a light spot generation module. The visual acquisition module is configured to acquire a real-time image on the photoelectric wetting chip as a target image; The droplet detection and tracking module is configured to obtain the current position and ID of the culture medium droplet, obtain the current position and ID of the algae droplet, and continuously update the current position of each culture medium droplet during the movement of each culture medium droplet; The automatic control module is used to receive input parameters from the user and adjust parameters including the motion trajectory and speed of the culture medium droplet based on the input parameters; The light spot generation module is configured to generate a plurality of light spots based on the tracking results of the droplet detection and tracking module, and guide the corresponding plurality of culture medium droplets to move in parallel along the moving path to the corresponding algae droplets for fusion.

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