Photoelectric microfluidic automatic control method for screening microalgae growth conditions

Abstract

The application discloses a photoelectric microfluidic automatic control method for screening microalgae growth conditions, and is characterized in that the method comprises the following steps: configuring different types of culture medium solutions and respectively inputting the culture medium solutions into a photoelectric wetting chip to generate culture medium drops belonging to different types on the photoelectric wetting chip; automatically obtaining current positions and IDs of the multiple culture medium drops, and automatically obtaining current positions and IDs of algal drops; respectively planning moving paths of the multiple culture medium drops to the corresponding algal drops according to the current positions and IDs of the multiple culture medium drops and the current positions and IDs of the algal drops; driving the multiple culture medium drops to move to the corresponding algal drops in parallel according to the moving paths by multiple light spots to perform fusion; and placing the fused drops in a light culture box to perform culture, so that the culture medium solution most suitable for growth of the algal drops is screened out.

Description

Technical Field

[0001] The present 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, as a renewable and sustainable energy source, has attracted widespread attention from both industry and academia. Microalgae, as single-celled organisms that do not compete for arable land, can produce a variety of substances such as lipids, vitamins, pigments, and fatty acids through photosynthesis. Their growth rate and lipid content are both higher than those of traditional crops, making them considered a promising feedstock for biodiesel production. Recent studies have shown that microalgae growth conditions influence the yield of bioproducts. Factors influencing microalgae growth include light, temperature, pH, nitrogen and phosphorus, titanium dioxide concentration, cell density, salinity, and pressure. Therefore, to ensure the economic viability of microalgae as biofuels, a deeper understanding of the effects of culture conditions on microalgae growth and lipid accumulation is necessary. The key lies in developing efficient screening methods to optimize 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 optimizing the optimal growth conditions of microalgae in conventional flask experiments is difficult, 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 syringes and other components to inject microalgae-containing liquid into the culture medium 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 microalgae growth conditions, which mainly solves the technical problems existing in the above-mentioned background technology.

[0005] To achieve the above-mentioned purpose, 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 prepared and introduced into the photoelectrowetting chip respectively, so as to generate different types of culture medium droplets on the photoelectrowetting chip;

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

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

[0010] The plurality of culture medium droplets are driven by the plurality of light spots to move in parallel along the movement 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- and 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 position and ID of multiple culture medium droplets and the current position and ID of the algae droplet 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 ID to the target recognition result. 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 the target tracking algorithm, specifically including: obtaining the target image on the photoelectric wetting chip, using the YOLOv5s detector to identify the target image, and obtaining the recognition result, the recognition result including the current position and ID of different culture medium droplets and the current position and ID of the algae droplet, and the current position including 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 using a target tracking algorithm, specifically including:

[0017] Identify multiple culture medium drops in the previous frame of the target image 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 multiple culture medium drops in one frame are simultaneously input into the Kalman filter, and the positions of the multiple culture medium drops in the next frame are predicted based on the speed and position of the corresponding culture medium drops;

[0019] Identify multiple culture medium droplets in the target image of the current frame using a target tracking algorithm to obtain the bounding box coordinates of each culture medium droplet 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, the bounding box coordinates of the current frame of the culture droplet are considered to match the predicted position, and the bounding box ID name of the previous frame corresponding to the culture 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 are not 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 droplet can move in parallel according to 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, the control system includes 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 configured to receive input parameters from a user and adjust parameters including the motion trajectory and speed of the culture medium droplet based on the input parameters;

[0028] The light spot generating module 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.

[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 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 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 culture medium in real time to achieve high-throughput screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the structure of the photoelectrowetting microfluidic chip in the embodiment of this application;

[0031] Figure 2 Schematic diagram of the process of the optoelectronic microfluidic automatic control method for screening microalgae growth conditions in the embodiment of the present application;

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

[0033] Figure 4 2. It 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 This is a module block diagram of the optoelectronic microfluidic automatic control system for screening microalgae growth conditions in an embodiment of the present application;

[0035] Figure 6 This is a principle block diagram of the optoelectronic microfluidic automatic control system for screening microalgae growth conditions in the embodiments 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. Light guide 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 those skilled in the art to which the present invention belongs. 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, reference is made to "some embodiments", which describes 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, numerous specific details are provided to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these details. In other instances, certain technical features well known in the art are not described to avoid confusion with the present invention.

[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 will fully convey the scope of the present invention 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 limit the present invention. When used herein, the singular forms "one", "an" 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 "comprising" and / or "comprising" 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 an intermediate element. When an element is referred to as being "connected to" another element, it may be directly connected to the other element or there may be an intermediate element. The terms "vertical," "horizontal," "inner," "outer," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only implementation methods.

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

[0043] Please refer to the attached Figure 1 The photoelectrowetting chip described in the embodiment of the present application comprises, from top to bottom, an upper glass substrate 11 (700 microns thick), an upper indium tin oxide film 12 (200 nanometers thick), an upper hydrophobic layer 13 (50 nanometers thick), a droplet layer 14 (130 microns thick), a lower hydrophobic layer (50 nanometers thick), a dielectric layer 16 (1 micron thick), a photoconductive layer 17 (1 micron thick), a lower indium tin oxide film 18 (200 nanometers thick), and a lower glass substrate 19 (700 microns 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 mainly occur 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 a photoelectric 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 introduce them into the photoelectrowetting chip respectively, generating different types of culture medium droplets on the photoelectrowetting chip;

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

[0046] S3, planning 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 multiple light spots to drive multiple culture medium droplets to move in parallel along the movement path to the corresponding algae droplets for fusion;

[0048] S5. Place the fused droplets in a light incubator for cultivation, thereby screening out 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), CaCl2·2H2O (27.2 mg / L), Na2CO3 (20 mg / L) 、 Citric acid (6mg / L), EDTA (1mg / L), Ferric ammonium citrate (6mg / L), MnCl2·H2O (1.81mg / L), ZnSO4·7H2O (0.22mg / L), CuSO4·5H2O (0.08mg / L), Na2MoO4·2H2O (0.39mg / L), CoCl2·6H2O (0.0409mg / L), H3BO3 (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 levels.

[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 hydrogen phosphate trihydrate content is 30.5 mg / L, which is recorded as 0Nx1P;

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

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

[0055] The nitrogen excess medium solution contained 2250 mg / L of sodium nitrate and 30.5 mg / L of potassium hydrogen phosphate trihydrate, which was recorded as 1.5Nx1P;

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

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

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

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

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

[0061] Further, since the chip contains many algae droplets and culture medium, the requirements for the target detection algorithm are relatively high, and therefore edible pigments are added to the culture medium with different nitrogen and phosphorus concentrations for differentiation.

[0062] In a possible implementation, the current positions and IDs of the plurality of culture medium droplets and the current positions and IDs of the algae droplets are obtained by a target tracking algorithm, the target tracking algorithm is composed of a ByteTrack tracker and a YOLOv5s detector, the YOLOv5s detector is used to realize target recognition, 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. YOLOv5s has a faster inference speed and a smaller model size while maintaining high detection accuracy, and is 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 target's position and the Hungarian algorithm to determine the best match between the detection result and the prediction result. It enables real-time target detection and tracking. This algorithm is particularly well-suited for processing large-scale video data and excels in a variety of scenarios, including but not limited to traffic monitoring, security surveillance, and sports analysis. Its key features include: 1. Efficiency: ByteTrack achieves real-time performance, maintaining high frame rates even on low-power devices. It employs a lightweight detection model and tracking strategy, reducing computational complexity. 2. Robustness: ByteTrack can stably track targets in complex environments, maintaining good tracking performance even in situations such as occlusion and lighting changes, and can handle the sudden appearance and disappearance of targets. 3. Flexibility: ByteTrack supports multiple detection models, allowing you to select the appropriate detection model based on the application scenario, and 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 position and ID of different culture medium droplets and the current position and ID of the algae droplet 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.

[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 previous frame of the target image 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 boundary box coordinates of multiple culture medium drops in a frame are input into the Kalman filter at the same time, and the positions of the multiple culture medium drops in the next frame are predicted according to the speed and position of the corresponding culture medium drops.

[0071] The multiple culture medium drops in the target image of the current frame are identified by the target tracking algorithm, and the boundary box coordinates of each culture medium drop in the current frame are obtained.

[0072] For the high-confidence boundary box coordinates of the current frame, IoU calculation is performed between the boundary box coordinates and the corresponding predicted position of the Kalman filter, and if the IoU is greater than a set threshold, it is considered that the boundary box coordinates of the culture medium drop in the current frame match the predicted position, and the boundary box ID name of the previous frame of the corresponding culture medium drop is assigned to the boundary box of the current frame.

[0073] For the low-confidence boundary box coordinates of the current frame, IoU calculation is sequentially performed between the boundary box coordinates and the predicted positions that have not been matched, and the boundary box ID name is assigned to the low-confidence boundary box of the current frame.

[0074] Specifically, when mobile detection is performed, the target image of the previous frame in a stationary state is first obtained, and the target image of the previous frame is identified by an identification algorithm to obtain the boundary box coordinates of each culture medium drop in the previous frame, and a corresponding boundary box ID name is assigned to each boundary box.

[0075] The obtained boundary box coordinates are input into the Kalman filter, and the possible positions of each culture medium drop in the current frame are predicted by the Kalman filter, and then the target image of the current frame in a moving state is obtained, and the target image of the current frame is identified by a target tracking algorithm to obtain the boundary box coordinates of each culture medium drop in the current frame, wherein the boundary box of the current frame has high confidence and low confidence.

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

[0077] The second matching is for the low-confidence boundary box that has not been matched, and IoU calculation is performed again, and if the IoU of the low-confidence boundary box and the predicted position is greater than the threshold, the boundary box ID name of the previous frame of the corresponding culture medium drop is assigned to the boundary 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: adjusting the position or shape of the corresponding light spot pattern according to the deviation, so that the corresponding culture medium droplet can move in parallel according to the moving path until all culture medium droplets reach the current position of the algae droplet.

[0080] For example, for each culture medium droplet, the deviation between its current position and the nearest point on the target path is calculated. 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, the culture medium droplet is considered to have deviated from the predetermined path.

[0081] If the culture medium droplet deviates from the predetermined path, the position or shape of the light spot pattern can be adjusted to guide the culture medium droplet back to the predetermined path. For example, if the culture medium droplet deviates to the left, the light spot pattern on the right can be strengthened to guide the culture medium droplet 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 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 droplet, thereby affecting the movement direction of the culture 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, and the standard content of potassium hydrogen phosphate trihydrate is 30.5 mg / L, which is recorded as 1Nx1P, and the content of sodium nitrate and potassium hydrogen phosphate trihydrate in the nitrogen and phosphorus deficient culture medium solution is zero, which is recorded 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. Heat in a water bath at 121°C and sterilize under high temperature and high pressure for 15 minutes. After sterilization is completed, wait for the culture medium solution to cool to room temperature, take part of the culture medium solution for cell culture, and add food coloring 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 was pushed by a syringe pump at a flow rate of 1 μL / min into the photoelectric wetting chip to generate several 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 targets in the photoelectric wetting chip are detected and ID assigned by the target tracking algorithm, and the algae droplets algae_1 and algae_2, and the culture media 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 the culture media are planned respectively. At the four detection targets, a light ring of appropriate size is generated to drive the culture media 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 culture environment of the light incubator was set as follows: light intensity of 4000 lm, temperature of 25 °C, light-dark switching time of 12 h:12 h, and no additional CO2 supply. The culture was continued for one cycle (7 days), and the algal cells in the droplets were counted daily using ImageJ.

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

[0089] Place the cells in a continuous culture incubator and culture them for one cycle (7 days). Observe and count the number of Chlorella cells daily using a hemocytometer.

[0090] To verify the feasibility of screening the optimal growth environment for algae cells in the photoelectrowetting digital microfluidic chip, the number and growth rate of Chlorella cells cultured on the chip and traditional off-chip were compared. Figure 4As shown in the figure, in the medium with standard nitrogen and phosphorus, the number of cells cultured in the 96-well plate and on the chip was 7183.33 cells / μL and 6214.68 cells / μL, respectively; in the medium with nitrogen and phosphorus deficiency, the number of cells cultured in the 96-well plate and on the chip was 4902.22 cells / μL and 4361.74 cells / μL, respectively; in the 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 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 standard nitrogen and phosphorus 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, the growth rates on the well plate and chip were lower than those 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 algal 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 average growth rate and maximum growth rate values ​​are very similar.

[0092] These trends demonstrate the feasibility of this system in screening the growth conditions of 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 includes 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 configured to receive input parameters from a 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 merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. The scope of protection of the present invention should be based on the scope of protection 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 prepared and introduced into the photoelectrowetting chip respectively, so as to generate different types of culture medium droplets on the photoelectrowetting chip; Automatically obtain the current position and ID of multiple culture medium droplets, and automatically obtain the current position and ID of 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, the moving paths of the multiple culture medium droplets to the corresponding algae droplets are planned respectively; The plurality of culture medium droplets are driven by the plurality of light spots to move in parallel along the movement 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- and phosphorus-deficient culture medium solution, nitrogen-excess culture medium solution, phosphorus-excess culture medium solution, nitrogen- and phosphorus-excess culture medium solution, nitrogen-excess and phosphorus-deficient culture medium solution, and phosphorus-excess and 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 position and ID of multiple culture medium droplets and the current position and ID of algae droplets are obtained through the 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 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 position and ID 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 a target image on the photoelectric wetting chip, using the YOLOv5s detector and ByteTrack tracker to identify the target image, and obtaining an identification result. 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.

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, which includes: Use the YOLOv5s detector and ByteTrack tracker to identify and assign multiple culture medium droplets in the previous frame target image, and obtain the bounding box coordinates and bounding box ID name of each culture medium droplet in the previous frame; The bounding box coordinates of multiple culture medium drops in one frame are simultaneously input into the Kalman filter, and the positions of the multiple culture medium drops in the next frame are predicted based on the speed and position of the corresponding culture medium drops; Identify multiple culture medium droplets in the target image of the current frame using a target tracking algorithm to obtain the bounding box coordinates of each culture medium droplet 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, the bounding box coordinates of the current frame of the culture droplet are considered to match the predicted position, and the bounding box ID name of the previous frame corresponding to the culture 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 are not 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 droplet 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 manipulation method for screening microalgae growth conditions according to any one of claims 1 to 8, characterized in that: The method is applied to a photoelectric microfluidic automatic control system for screening microalgae growth conditions. The control system includes 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 multiple light spots based on the tracking results of the droplet detection and tracking module, and guide the corresponding multiple culture medium droplets to move in parallel along the movement path to the corresponding algae droplets for fusion.

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