A micro-chamber microfluidic system for microbial growth image detection

[0062] Example 1

[0063] Such as Figure 1~4 As shown, this embodiment discloses a micro-chamber microfluidic chip for real-time cultivation and observation of microorganisms. The microfluidic chip includes a chip body and a PDMS film integrally molded therewith. The chip body includes four A microfluidic channel, the microfluidic channel is composed of a liquid inlet 1, a main channel 2 and a liquid outlet 3 that are connected in sequence; the middle part of the main channel 1 is the micro-chamber area of ​​the microfluidic chip, and the main channel The middle section of 1 is in the shape of a broken line, and the angle between each other is 90°. The main channel of the broken line extends outwards in the direction of the liquid flow at the corners to form an annular channel and a micro-chamber with a cofferdam structure. 4 A T-shaped corner 5 is provided at one end close to the main channel; a micro-pillar 6 is provided on the channel between the micro chamber 4 and the main channel 1; the cofferdam of the micro chamber 4 is semi-closed, incompletely closed or fully closed The openings are all arranged at one end of the microchamber away from the main channel, and at least three different types of microchambers 4 described above are provided on the chip body.

[0064] As a specific embodiment, the microchamber area has a total of 8 microchambers, among which 4-1 and 4-4 are microchambers with a semi-closed design, and their diameters can be divided into 250μm, 260μm and 150μm, and 160μm. Species; 4-2, 4-5 are incompletely enclosed microchambers with diameters of 260 µm and 160 µm, respectively; 4-3, 4-6 are completely enclosed microchambers with diameters of 250 µm and 150 µm. The microchamber with a diameter of 250µm~260µm is suitable for observation under a 20x objective lens, and 150µm~160µm is suitable for observation under a 40x objective lens.

[0065] As a specific embodiment, the size of the opening of the semi-closed microchamber is one third of the circumference of the microchamber.

[0066] As a specific embodiment, the size of the opening of the incompletely enclosed microchamber is slightly larger than the diameter of the bacteria, which is 8 μm.

[0067] As a specific implementation, the width of the main channel 1 is 100 μm, the diameter of the annular channel is 360 μm, and the distance from the adjacent main channel is 300 μm.

[0068] In a specific embodiment, the depth of the main channel is 35 µm and the depth of the micro chamber is 8 µm.

[0069] The microfluidic chip of this embodiment is provided with independent culturing microchambers, and the microchambers adopt three different designs according to different functions; among them, the semi-closed microchamber is used as the observation microchamber for the cultivation and observation of bacterial growth. Semi-closed design, when the culture medium passes through the circular channel, it can be fully mixed with the bacteria in the micro-chamber, which is suitable for the cultivation and observation of bacterial growth; the incompletely closed micro-chamber is used as a chemotaxis tracking micro-chamber and is suitable for the growth of bacteria Observation and chemotaxis tracking. When different media pass through the circular channel, the microchamber and the circular channel are not completely closed, so the bacteria in the microchamber can perceive the medium flowing through the circular channel, thereby generating chemotactic movement; The closed microchamber is used as a biofilm measurement microchamber. Because there is no opening between the annular channel and the microchamber, bacteria grow in the peripheral channel of the microchamber, and the entire microchamber plays a role of micropillar adhesion. In addition, the micro-column is a multi-column transmission structure, which functions as a valve with the T-shaped angle. By using the micro-column adhesion function, it can transfer the bacterial liquid and culture medium to the annular channel and the micro chamber. It plays a role of diversion and makes the liquid flow directional. It can flow in from one end of the annular channel and flow out from the other end. At the same time, the cofferdam of each micro-chamber also functions as a valve to ensure that bacteria and culture media can flow through the annular channel and Micro chamber. The micro-chamber microfluidic chip can culture bacteria in different three kinds of micro-chambers, and simultaneously observe different requirements.

[0070] The specific preparation parameters of the microchamber microfluidic chip are detailed in Figure 5 The preparation process is carried out in accordance with conventional techniques in the field.

[0071] Example 2

[0072] Such as Image 6 As shown, this embodiment discloses a micro-chamber micro-fluidic control system for microbial growth image detection, including a microscope, a constant-current injection pump, a syringe, a constant-temperature stage, the micro-chamber micro-fluidic chip described in Example 1, and a collector , CMOS video detection components, video recorders, mobile hard drives and displays; the constant temperature stage is placed on the microscope stage; the micro-chamber microfluidic chip is placed on the constant temperature stage to provide a constant culture for the chip The temperature and size are suitable for the microscope and the chip; the CMOS video detection component is fixed on the top of the microscope by connection, and the CMOS video detection component is used to measure the morphology of bacteria in each microchamber in the microchamber microfluidic chip. Change; the liquid inlet of the microchamber microfluidic chip is connected with a constant flow syringe pump, and a syringe is provided on the constant flow syringe pump; the liquid outlet of the microchamber microfluidic chip is connected with a collector; the video recorder is connected The display is connected to the COMS video detection component, the mobile hard disk and the display respectively.

[0073] As a specific implementation manner, the CMOS video detection component includes an arithmetic unit, which is based on the median filter principle and corresponding segmentation formulas and coefficients, and is used to obtain clear grayscale image information of microorganisms.

[0074] As a specific implementation, the arithmetic unit performs image filtering based on the mat2gray function and the imfilter function, and the image segmentation adopts the bwareaopen function and the bwareaclose function and the ring segmentation formula S=(R 2 -r 2 ).

[0075] Specifically, the imfilter function is used (to achieve a linear spatial filter function), the parameters are set to'corr' which means correlation and'replicate' means that the image size is expanded by copying the value of the outer boundary. The average filter window size is 30, and the minimum filter pixel threshold is 0.035. Before using the imfilter function, use the mat2gray function to normalize the image matrix, that is, divide all the data by the maximum value, and the maximum value of the matrix is ​​1, so that the matrix data is normalized between 0 and 1, which is convenient for later parameter setting .

[0076] For bwareaopen function (MATLAB) and bwareaclose function, the parameter threshold P is 0.3 and 5000, and the neighborhood is 8.

[0077] As a specific implementation, the CMOS video detection component is a digital camera; preferably a Nikon D7100 camera, which can realize a high-speed recording unit with a transmission pixel of 1080P and a frequency of 10 frames per second, which can clearly and accurately record high-speed moving microorganisms , To obtain more reliable image information, and the price is relatively low with the same performance industrial camera. Supporting image filtering and segmentation algorithms, mat2gray function and imfilter function can filter the image, remove image noise, smooth the uneven background, and bwareaopen function and bwareaclose function can segment and remove the image that is too small and large area of ​​non-microbial graphics Structure, so as to obtain accurate and true graphics data, the ring segmentation formula S=(R 2 -r 2 ) The biofilm area can be segmented.

[0078] As a specific embodiment, the microscope is a fluorescence microscope, and the fluorescence module is a CCD fluorescent lamp, which can emit blue, red, and green light.