Microfluidic chip for microalgae growth stress detection and microscopic image analysis method

Abstract

The invention discloses a micro-fluidic chip for microalgae growth stress detection and a microscopic image analysis method, and belongs to the technical field of cell monitoring, the micro-fluidic chip comprises an inlet, the right side of the inlet is connected with a cell leading-in area, the right side of the cell leading-in area is connected with a cell arrangement area, the cell arrangement area comprises at least one micro-convex flow channel, and the micro-convex flow channel is connected with the micro-convex flow channel. The right port of the micro-convex flow channel is connected with a cell growth area, the cell growth area comprises a narrow flow channel and a culture flow channel arranged in the narrow flow channel, the cell growth area is connected with a discharged liquid collection area, the right end of the discharged liquid collection area is connected with an outlet, and a microscopic camera device is arranged above the culture flow channel. According to the micro-fluidic chip for detecting the growth stress of the microalgae and the microscopic image analysis method, a micro-fluidic environment is constructed on the micro-chip, fine regulation and control on the growth environment of the microalgae cells are realized, the form and behavior change of the microalgae cells can be observed in real time and at high resolution, and the micro-fluidic chip can be used for detecting the growth stress of the microalgae in combination with the microscopic image and the analysis method. The detection and the analysis on the microalgae cell growth extrusion stress are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cell monitoring, and in particular, to a microfluidic chip for detecting the growth stress of microalgae and a microscopic image analysis method. Background Art

[0002] As a type of single-celled photosynthetic organisms, microalgae have important values in the fields of ecosystem, energy, food, and biopharmaceutics. During the growth process of microalgae cells, the extrusion stress borne by the cells plays a key role in aspects such as their physiological functions, metabolic pathways, growth rates, and biomass accumulation. Microalgae cells do not exist in isolation in natural environments or artificial culture systems. They are subject to extrusion stresses generated by factors such as surrounding cells, the flow of the culture medium, and the boundaries of the culture container. These extrusion stresses can affect the morphology of microalgae cells, such as changing the shape and size of the cells; they can also have an impact on the physiological processes within the cells, such as photosynthesis efficiency, nutrient uptake, and metabolite synthesis. Appropriate extrusion stress may activate specific signal pathways within microalgae cells, prompting them to synthesize more oil, which is of great significance for the production of microalgae biodiesel; while excessive extrusion stress may lead to cell damage and inhibit cell growth and reproduction.

[0003] Traditional methods for detecting the growth extrusion stress of microalgae cells have many deficiencies. Conventional macroscopic detection means, such as inferring the stress borne by cells by measuring the overall physical parameters of the culture system, cannot be accurate to the single-cell level, making it difficult to obtain the true stress situation of cells and unable to simulate the diverse stress conditions faced by microalgae cells in complex environments. Microscopic detection techniques, such as atomic force microscopy, can perform mechanical measurements on single microalgae cells, but they have low throughput and complex operations, and cannot meet the needs of large-scale research and industrial applications. In addition, traditional methods are difficult to precisely control the growth environment of microalgae cells, making it difficult to study the response mechanisms of microalgae cells under different extrusion stress conditions. Summary of the Invention

[0004] The object of the present invention is to provide a microfluidic chip for detecting the growth stress of microalgae and a microscopic image analysis method, which can construct a precisely controllable microfluidic environment on a tiny chip, realize the fine regulation of the growth environment of microalgae cells, can observe the morphological and behavioral changes of microalgae cells in real time and with high resolution, and combine with microscopic images and analysis methods to achieve the precise detection and analysis of the growth extrusion stress of microalgae cells.

[0005] To achieve the above object, the present invention provides a microfluidic chip for detecting the growth stress of microalgae. The chip adopts a single-layer structure and is provided with different regions, including an inlet. The right side of the inlet is connected to the left port of the cell introduction region. The right side of the cell introduction region is connected to a cell arrangement region. The cell arrangement region includes at least one micro-convex flow channel. The right port of the micro-convex flow channel is connected to a cell growth region. The cell growth region includes a narrow flow channel. A culture flow channel is arranged in the narrow flow channel. A constriction is arranged at the right end of the culture flow channel. The cell growth region is connected to a liquid drainage and collection region. The right end of the liquid drainage and collection region is connected to an outlet. A microscopic imaging device is arranged above the culture flow channel.

[0006] Preferably, a micropump and an inlet micro-valve are arranged between the inlet and the cell introduction region. The micropump is driven by a micro motor.

[0007] Preferably, the micro-convex flow channel gradually narrows from the left port to the right port, and the right port of the micro-convex flow channel is connected to the narrow flow channel.

[0008] Preferably, an outlet micro-valve is arranged in the liquid drainage and collection region.

[0009] The present invention also provides a method for analyzing microscopic images for detecting the growth stress of microalgae, including the following steps:

[0010] S1. Image preprocessing;

[0011] S2. Boundary extraction;

[0012] S3. Deformation of the flow channel boundary and conversion of stress values.

[0013] Preferably, in S1, the image preprocessing includes the following steps:

[0014] S11. After obtaining the microscopic image, perform image grayscale processing. Convert the RGB three-channel color values into a single grayscale value by the weighted average method to highlight the key structural information in the image;

[0015] S12. Adopt the Gaussian filtering algorithm to perform weighted average calculation on each pixel point in the image and its neighboring pixels to remove Gaussian noise in the image;

[0016] S13. Perform image enhancement. Use the histogram equalization technology to adjust the grayscale histogram of the image to make the grayscale distribution of the image more uniform, enhance the contrast of the image, and highlight the detailed features of the narrow flow channel wall.

[0017] Preferably, in S2, the boundary extraction includes the following steps:

[0018] S21. Apply the Canny edge detection algorithm to calculate the gradient magnitude and direction of each pixel in the image. Through non-maximum suppression, retain the pixel with the largest gradient magnitude as an edge candidate point, and remove the blurred part of the edge to make the edge clearer.

[0019] S22. Use double-threshold detection to set two thresholds, high and low. Pixels above the high threshold are determined as edge points, pixels below the low threshold are excluded, and pixels between the high and low thresholds are judged as edge points based on their connectivity to the already determined edge points, and the boundary of the narrow channel wall is extracted.

[0020] Preferably, in S3, the conversion of the deformation and stress value of the channel boundary is specifically as follows: According to the principle of elasticity, for the narrow channel wall of an elastic thin plate structure, the relationship between its stress and deformation is calculated by the formula in the thin plate bending theory. The calculation formula is:

[0021] δ = k×Eh 2 / 12(1 - v 2 );

[0022] Where h is the thin wall thickness, E is the elastic modulus, v is the Poisson's ratio, k is the curvature change of the channel wall, and δ is the obtained stress.

[0023] Therefore, the present invention adopts the above-mentioned microalgae growth stress detection microfluidic chip and microscopic image analysis method, and has the following advantages:

[0024] (1) Real-time stress monitoring through image dynamic tracking: With the help of image dynamic tracking technology, synchronized with the stress application of the microfluidic chip, continuously observe microalgae cells. When the chip applies extrusion stress by regulating fluid pressure and flow rate, the microscopic imaging device captures the dynamic response of cells in real time, obtains growth data at different stress stages, breaks through the limitations of traditional static or intermittent detection, and provides key support for studying the correlation mechanism between stress and cell growth time.

[0025] (2) Image analysis to quantify the microfluidic extrusion stress: Use a dedicated image analysis algorithm to deeply analyze the images of microalgae cells under extrusion stress. Extract morphological parameters such as cell area and perimeter, and quantitatively calculate the extrusion stress in combination with a mechanical model, converting the difficult-to-quantify stress into data, which is more accurate and scientific than traditional empirical or macroscopic measurements, and provides a reliable data processing means for research.

[0026] The technical solution of the present invention will be further described in detail below through the accompanying drawings and embodiments. Brief Description of the Drawings

[0027] Figure 1 It is a schematic structural diagram of an embodiment of a microalgae growth stress detection microfluidic chip and microscopic image analysis method of the present invention.

[0028] Reference numerals

[0029] 1. Inlet; 2. Cell introduction area; 3. Cell arrangement area; 4. Cell growth area; 5. Microalgae cells; 6. Narrow flow channel; 7. Culture flow channel; 8. Microscopic imaging device; 9. Drainage collection area; 10. Outlet; 11. Micro-convex flow channel. Detailed implementation manners

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs. The "first", "second" and similar terms used in the present invention do not denote any order, quantity or importance, but are only used to distinguish different components. The terms such as "including" or "comprising" mean that the elements or objects appearing before this word cover the elements or objects listed after this word and their equivalents, without excluding other elements or objects. The terms such as "connected" or "coupled" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. The terms such as "upper", "lower", "left" and "right" are only used to represent relative positional relationships, and when the absolute position of the object being described changes, the relative positional relationship may also change accordingly.

[0032] Example 1

[0033] The present invention provides a microfluidic chip for detecting the growth stress of microalgae, which is tightly attached to a glass plane through lithography, etching and bonding techniques. The chip adopts a single-layer structure and is provided with different regions, such as a cell introduction area, a cell arrangement area, a cell growth area and a drainage collection area, etc. The introduction, arrangement, growth culture and waste liquid discharge and collection of microalgae cells are completed in sequence on the same chip, improving the experimental efficiency and data accuracy.

[0034] The specific structure is as Figure 1 shown, including an inlet 1, the right side of the inlet 1 is connected to the left port of the cell introduction area 2, a micropump and an inlet microvalve are arranged between the inlet 1 and the cell introduction area 2, and the micropump is driven by a micro motor. The inlet 1 is responsible for introducing the suspension of pre-cultured microalgae cells 5 into the subsequent regions, and the micropump and the inlet microvalve used in the introduction process are used to control the fluid on and off. The type of the micropump is preferably a peristaltic pump, a gear pump or a piezoelectric pump, which is driven by a micro motor.

[0035] On the right side of the cell introduction region 2 is connected to the cell arrangement region 3. The cell arrangement region 3 includes at least one micro-convex flow channel 11. The micro-convex flow channel 11 gradually narrows from the left port to the right port. The width and structure of the micro-convex flow channel 11 can be determined based on the size and characteristics of different types of microalgae cells 5 through numerical simulation and experimental verification. For example, for common Chlorella vulgaris, the width of the micro-convex flow channel 11 is designed to be between 5 - 10 μm to ensure that the microalgae cells 5 can pass through one by one in sequence. By adjusting the fluid velocity and pressure in the micro-convex flow channel 11 and using hydrodynamics, the microalgae cells are arranged one by one in the micro-convex flow channel 11, providing accurate single-cell samples for subsequent cell growth experiments. When the microalgae cell 5 suspension flows from the cell introduction region 2 into the cell arrangement region 3, local flow velocity changes and pressure differences are generated when the microalgae cell 5 suspension flows through the micro-convex flow channel 11, causing the microalgae cells 5 to be gradually separated and arranged to pass through the flow channel one by one as single cells.

[0036] The right port of the micro-convex flow channel 11 is connected to the cell growth region 4. The cell growth region 4 includes a narrow flow channel 6. The right port of the micro-convex flow channel 11 is connected to the narrow flow channel 6. A culture flow channel 7 is provided in the narrow flow channel 6. A constriction is provided at the right end of the culture flow channel 7 to prevent the microalgae cells 5 from flowing out of the culture flow channel 7. A microscopic imaging device 8 is provided above the culture flow channel 7. The cell growth region 4 is the core region for the growth and stress monitoring of microalgae cells 5. After passing through the micro-convex flow channel 11, the microalgae cells 5 flow into and are fixed in the culture flow channel 7 one by one. As the microalgae cells 5 grow in the culture flow channel 7, the volume of the microalgae cells 5 increases, generating pressure on the wall of the culture flow channel 7. Since the narrow flow channel 6 is adjacent to the culture flow channel 7 and has an elastic and deformable thin-walled structure, the pressure in the culture flow channel 7 causes the wall of the narrow flow channel 6 to deform. By observing and analyzing the deformation state of the wall of the narrow flow channel 6 through microscopic imaging and combining the principles of elasticity and the pre-established deformation-stress relationship model, the extrusion stress generated by the growth of the microalgae cells 5 can be calculated.

[0037] The end of the cell growth region 4 is connected to the drainage collection region 9. The drainage collection region 9 is provided with an outlet micro-valve. The right end of the drainage collection region 9 is connected to an outlet 10. The drainage collection region 9 is responsible for collecting waste such as carbon dioxide and lactic acid produced by the metabolism of microalgae cells 5 and excess culture medium. The outlet micro-valve can control the timing and flow rate of the waste liquid discharge to ensure that the waste liquid is discharged without disturbing the environment of the microalgae cell 5 growth region, maintaining the cleanliness and stability inside the chip.

[0038] The present invention also provides a method for analyzing microscopic images of microalgae growth stress, including the following steps:

[0039] S1. Image preprocessing, specifically including the following steps:

[0040] S11. After obtaining the microscopic image, perform image grayscale processing. Convert the RGB three-channel color values into a single grayscale value through the weighted average method to highlight the key structural information in the image and reduce the data volume for subsequent processing.

[0041] S12. Adopt the Gaussian filtering algorithm to perform weighted average calculation on each pixel point in the image and its neighboring pixels, remove the Gaussian noise in the image, make the image smoother, and provide a clearer basic image for subsequent analysis.

[0042] S13. Perform image enhancement. Use the histogram equalization technique to adjust the grayscale histogram of the image, make the grayscale distribution of the image more uniform, enhance the contrast of the image, and highlight the detailed features of the narrow channel wall.

[0043] S2. Boundary extraction, which specifically includes the following steps:

[0044] S21. Apply the Canny edge detection algorithm to calculate the gradient magnitude and direction of each pixel point in the image. Through non-maximum suppression, retain the pixel points with the largest gradient magnitude as edge candidate points, remove the blurred part of the edge, and make the edge clearer.

[0045] S22. Use double-threshold detection. Set two thresholds, high and low. Pixel points above the high threshold are determined as edge points, pixel points below the low threshold are excluded, and pixel points between the high and low thresholds are judged whether they are edge points according to their connectivity with the already determined edge points, and extract the boundary of the narrow channel wall.

[0046] S3. Deformation and stress value conversion of the channel boundary. According to the principle of elasticity mechanics, for the narrow channel wall with an elastic thin plate structure, the relationship between its stress and deformation is calculated through the relevant formulas in the thin plate bending theory. The calculation formula is:

[0047] δ = k × Eh 2 / 12(1 - v 2 );

[0048] Where h is the thin wall thickness, E is the elastic modulus, v is the Poisson's ratio, k is the curvature change of the channel wall, and δ is the obtained stress.

[0049] The microalgae growth stress detection microfluidic chip and microscopic image analysis method described in this embodiment are applicable to the following research fields:

[0050] (1) Research on cell physiological mechanisms

[0051] Analysis of stress response signal pathway: By precisely controlling the extrusion stress using a chip, study the changes in gene expression and protein activity of microalgae cells under different stress levels, clarify the signal transduction pathway by which microalgae cells sense and respond to extrusion stress, understand the stress physiological mechanism of cells, and provide key data for basic research in cell biology.

[0052] Response of organelle functions to stress: With the help of high-resolution microscopic imaging function, observe in real time the morphological and functional changes of organelles in microalgae cells under extrusion stress. For example, study the changes in photosynthesis efficiency of chloroplasts under stress and the adjustment of mitochondrial energy metabolism, and reveal the mechanism of action of organelles in the process of cells coping with extrusion stress.

[0053] (2) Innovation of microalgae biotechnology

[0054] Improvement of microalgae genetic transformation efficiency: During the microalgae genetic transformation process, apply specific extrusion stress through a chip to change the permeability of the microalgae cell wall and cell membrane, and promote the introduction of foreign genes. Study the effects of different stress conditions on genetic transformation efficiency, accelerate the genetic engineering transformation of microalgae, and cultivate new microalgae varieties with excellent traits.

[0055] Optimization of microalgae immobilization technology: Used to study the influence of extrusion stress on the interaction between microalgae and immobilization carriers. By regulating stress, enhance the attachment stability of microalgae on the carriers, improve the activity and service life of immobilized microalgae, and promote the application of microalgae immobilization technology in various fields.

[0056] (3) Monitoring and control of biological processes

[0057] Real-time monitoring of microalgae culture process: Combine real-time imaging and data analysis functions to conduct online monitoring of the large-scale microalgae culture process. By monitoring the growth state, morphological changes and extrusion stress suffered by microalgae cells, timely adjust the culture conditions to ensure the stability and high efficiency of the microalgae culture process.

[0058] Performance evaluation and improvement of bioreactors: Applied to the performance research of bioreactors, simulate the extrusion stress suffered by microalgae in the bioreactor under different operating conditions. By analyzing the response of microalgae cells, evaluate the hydrodynamic performance and mass transfer characteristics of the bioreactor, provide a basis for the optimized design of the bioreactor and the adjustment of operating parameters, and improve the operating efficiency of the bioreactor.

[0059] Therefore, by adopting the above-mentioned microalgae growth stress detection microfluidic chip and microscopic image analysis method, the present invention can construct a precisely controllable microfluidic environment on a tiny chip, realize the fine regulation of the growth environment of microalgae cells, can observe the morphological and behavioral changes of microalgae cells in real time and with high resolution, and combine microscopic images and analysis methods to realize the precise detection and analysis of the extrusion stress on microalgae cell growth.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements do not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A microfluidic chip for detecting the growth stress of microalgae, characterized in that: The chip adopts a single-layer structure and is provided with different regions, including an inlet. The right side of the inlet is connected to the left port of the cell introduction region. The right side of the cell introduction region is connected to a cell arrangement region. The cell arrangement region includes at least one micro-convex flow channel. The right port of the micro-convex flow channel is connected to a cell growth region. The cell growth region includes a narrow flow channel. A culture flow channel is arranged in the narrow flow channel. A constriction is arranged at the right end of the culture flow channel. The cell growth region is connected to a liquid discharge and collection region. The right end of the liquid discharge and collection region is connected to an outlet. A microscopic imaging device is arranged above the culture flow channel.

2. The microfluidic chip for detecting the growth stress of microalgae according to claim 1, characterized in that: A micropump and an inlet micro-valve are arranged between the inlet and the cell introduction region. The micropump is driven by a micro-motor.

3. The microfluidic chip for detecting the growth stress of microalgae according to claim 1, characterized in that: The micro-convex flow channel gradually narrows from the left port to the right port. The right port of the micro-convex flow channel is connected to the narrow flow channel.

4. A microfluidic chip for detecting the growth stress of microalgae according to claim 1, characterized in that: An outlet micro-valve is arranged in the liquid discharge and collection region.

5. A method for analyzing microscopic images of microalgae growth stress, characterized in that, It includes the following steps: S1. Image preprocessing; S2. Boundary extraction; S3. Transformation of the deformation and stress values of the flow channel boundary.

6. A method for analyzing microscopic images of microalgae growth stress detection according to claim 5, characterized in that: In S1, the image preprocessing includes the following steps: S11. After obtaining the microscopic image, perform image grayscale processing. Convert the RGB three-channel color values into a single grayscale value through the weighted average method to highlight the key structural information in the image; S12. Adopt the Gaussian filtering algorithm to perform weighted average calculation on each pixel point in the image and its neighboring pixels to remove Gaussian noise in the image; S13. Perform image enhancement. Use the histogram equalization technology to adjust the grayscale histogram of the image to make the grayscale distribution of the image more uniform, enhance the contrast of the image, and highlight the detailed features of the narrow flow channel wall.

7. A microscopic image analysis method for detecting the growth stress of microalgae according to claim 5, characterized in that: In S2, the boundary extraction includes the following steps: S21. Apply the Canny edge detection algorithm to calculate the gradient magnitude and direction of each pixel point in the image. Through non-maximum suppression, retain the pixel points with the largest gradient magnitude as edge candidate points, and remove the blurred part of the edge to make the edge clearer; S22. Use double-threshold detection. Set two thresholds, high and low. Pixel points higher than the high threshold are determined as edge points, pixel points lower than the low threshold are excluded, and pixel points between the high and low thresholds are judged whether they are edge points according to their connectivity with the already determined edge points to extract the boundary of the narrow flow channel wall.

8. A method for analyzing microscopic images of microalgae growth stress detection according to claim 5, characterized in that: In S3, the transformation of the deformation and stress values of the flow channel boundary is specifically: According to the principle of elasticity, for the narrow flow channel wall of the elastic thin plate structure, the relationship between its stress and deformation is calculated through the formula in the thin plate bending theory. The calculation formula is: δ = k × Eh 2 / 12(1 - v 2 ); Among them, h is the wall thickness, E is the elastic modulus, v is the Poisson's ratio, k is the curvature change of the flow channel wall, and δ is the obtained stress.

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