A low-shear microfluidic chip, its fabrication method and application

Abstract

This invention discloses a low-shear microfluidic chip. The microchannel of the microfluidic chip has at least one microchamber, and each microchamber has a micropillar structure on each side; wherein the micropillar structure is inverted U-shaped; the microchamber is divided into an inlet area and a culture area; wherein the inlet area is square and has a cross-shaped baffle inside. This invention fabricates a low-shear microfluidic chip based on vortex flow for efficient bacterial capture. This chip employs a unique micropillar structure, achieving more efficient bacterial capture and minimizing the impact of shear force on bacterial growth within the microchamber.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture equipment manufacturing technology, specifically relating to a low-shear microfluidic chip, its preparation method, and its application. Background Technology

[0002] Antimicrobial drugs play an irreplaceable role in treating infectious diseases, preventing animal diseases, and ensuring public health safety. To facilitate research on antimicrobial drugs, researchers have designed various methods for antimicrobial susceptibility testing. Traditional antimicrobial susceptibility testing mainly includes whole-genome sequencing, mass spectrometry, PCR, broth dilution method, disk diffusion method, and E-test, all of which have been widely used. However, each method has its own shortcomings. For example, the E-test and disk diffusion method require visual observation of colony formation or measurement of the inhibition zone size with a ruler to determine the antimicrobial effect, inevitably leading to errors. Furthermore, these methods involve tedious manual operations and are very time-consuming, typically taking 24 to 48 hours. They are also labor-intensive and costly, representing a waste in both theoretical research and clinical application. In addition, when whole-genome sequencing approaches its detection limit, further increasing the sequencing depth may introduce low-frequency false-positive mutations. Simultaneously, with the increase in drug-resistant pathogens and the variation in resistance mechanisms, traditional antimicrobial susceptibility testing is gradually failing to meet the needs of rapid clinical diagnosis and treatment. Therefore, it has become more important to develop miniaturized, integrated, intelligent, and online monitoring biosensors.

[0003] In recent years, the use of microfluidic chips for bacterial culture and drug susceptibility testing has become an emerging research direction. Microfluidic technology has been widely applied in the field of biology, characterized by its small size, automation, high throughput, and precise fluid control. Polydimethylsiloxane (PDMS) is the most commonly used material in microfluidics, offering advantages such as ease of microfabrication, low cost, and good biocompatibility. Furthermore, PDMS possesses high elasticity and permeability, crucial for long-term cell culture within sealed microchannels. Compared to traditional culture dishes, PDMS chips provide a more controllable microenvironment, thus showing broad application prospects in bacterial culture. Experiments using PDMS chips can significantly shorten detection time and enable real-time microscopic detection of drug antibacterial effects. Moreover, PDMS chips have excellent sealing properties, preventing contamination after initial protection. The bacterial culture chamber connects bacteria to adjacent microchannels, continuously supplying nutrients and removing metabolites to achieve long-term dynamic bacterial culture within the chip. Long-term dynamic culture not only allows bacteria to adapt to dynamic survival conditions after entering the culture chamber, better simulating the intestinal environment, but also allows more drugs to come into contact with the bacteria, thus more realistically reflecting the actual antibacterial effect of the drugs. These advantages have driven the development of various microfluidic chips for bacterial testing to complete antimicrobial susceptibility testing (AST) and the study of drug resistance responses in drug-resistant strains. Zhang et al. used gradient microfluidic chips and MALDI-TOF mass spectrometry to investigate the filamentous changes of drug-resistant bacteria such as extended-spectrum β-lactamase *Escherichia coli* under the action of ampicillin and ceftriaxone sodium. Zhang et al. developed a novel microfluidic chip based on 3D printing technology to detect the resistance of *E. coli* to clinically representative antibiotics ampicillin, chloramphenicol, and kanamycin. This chip can rapidly perform drug susceptibility testing within 5 hours, further demonstrating the application prospects of microfluidic chips in bacterial AST. However, these studies did not involve long-term dynamic culture after bacterial perfusion, failing to reflect the survival status of bacteria after entering the culture chamber. Furthermore, they could not reflect the actual interaction between bacteria and drugs. Furthermore, during long-term dynamic culture, bacteria are subjected to certain fluid shear forces, leading to bacterial loss or damage. Therefore, constructing a low-shear-force bacterial culture environment is crucial, yet relevant research is scarce.

[0004] Therefore, how to provide a low-shear microfluidic chip, its fabrication method and application, and how to construct a low-shear bacterial culture environment are urgent problems to be solved. Summary of the Invention

[0005] In view of this, the present invention provides a low-shear microfluidic chip, its preparation method and application. By preparing a low-shear microfluidic chip based on vortex flow, bacteria can be efficiently captured. The chip adopts a unique micropillar structure, which achieves more efficient bacterial capture and minimizes the impact of shear force on bacterial growth in the microchamber.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A low-shear-force microfluidic chip, wherein the microchannel of the microfluidic chip is provided with at least one microchamber, and each side of the microchamber is provided with a micropillar structure;

[0008] The micropillar structure is inverted U-shaped.

[0009] This invention effectively reduces the dependence of fluid / cell manipulation on flow rate by employing a uniquely structured inertial microfluidic chip, optimizing the microfluidic device for evaluating the antibacterial effect of chlorogenic acid. This invention proposes a low-shear microfluidic chip based on vortex flow for efficient bacterial capture. The chip utilizes a unique micropillar structure to achieve more efficient bacterial capture and minimize the impact of shear force on bacterial growth within the microchamber. Simultaneously, by controlling the flow rate, transduced GFP *E. coli* TAc-IeGFP can be dynamically cultured for several hours, allowing for the study of the antibacterial effect of chlorogenic acid.

[0010] Preferably, the ratio of the width between the top of the micropillar structure and the top of the microchannel to the width of the microchannel is 1:10.

[0011] Preferably, the ratio of the width between the top of the micropillar structure and the top of the microchannel to the width of the microchannel is 20-100μm:200-1000μm.

[0012] Preferably, the ratio of the width of the micropillar structure to the width of the microchannel is 200μm:500μm.

[0013] Preferably, the ratio of the width between the top of the micropillar structure and the top of the microchannel to the width of the microchannel is 50μm:500μm.

[0014] Preferably, the microchamber is divided into an entrance area and a culture area;

[0015] The entrance to the entrance area is square and has a cross-shaped shield inside.

[0016] Preferably, the width of the cross-shaped baffle is smaller than the width of each side of the square.

[0017] Preferably, the culture area has a near-circular regular polygonal structure with a diameter of 300-800 μm.

[0018] Preferably, the microfluidic chip has two inlets and two outlets, wherein a filter column is provided in the pipe of the inlet.

[0019] Preferably, the width ratio of the micropillar structure to the microchannel is 1:10, and the ratio of the distance between the micropillar structures to the width of the microchannel is 1:2.

[0020] Preferably, the distance between the micropillar structures is 100-500 μm.

[0021] Preferably, the thickness of the microfluidic chip is 20-200 μm.

[0022] Preferably, it also includes a bacterial export structure.

[0023] The above-described method for fabricating a low-shear microfluidic chip specifically includes the following steps:

[0024] (1) Mold processing: After taking the negative photolithography coated silicon wafer, perform pre-baking, exposure, post-baking and development in sequence to obtain the mold for later use;

[0025] (2) Weigh out the polydimethylsiloxane prepolymer and the curing agent, mix them well, place them on the mold, and heat to cure;

[0026] (3) After the solidified mold is cut and punched, it is placed on a glass slide and baked.

[0027] The application of low-shear microfluidic chips in bacterial culture, as described above.

[0028] The application of the low-shear microfluidic chip in the antibacterial experiment, as described above.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides a low-shear microfluidic chip, its fabrication method, and its application. By fabricating a low-shear microfluidic chip based on vortex flow, bacteria can be efficiently captured. The chip adopts a unique micropillar structure, which achieves more efficient bacterial capture and minimizes the impact of shear force on bacterial growth in the microchamber. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 This is a structural diagram of a microfluidic chip according to the present invention;

[0033] Figure 2 This is an enlarged view of the structure of the micropillars and microcells of the present invention;

[0034] Figure 3 This is an enlarged view of the microchamber entrance region of the present invention;

[0035] Figure 4 Numerical simulation diagrams of microcells at different flow rates are shown in the application examples of this invention.

[0036] Figure 5 This is a schematic diagram simulating the diffusion effect of culture medium or drug at a flow rate of 3 μL / min, as an application example of the present invention.

[0037] Figure 6 Growth curve of Escherichia coli TAc-IeGFP in this invention;

[0038] Figure 7 The application examples of this invention are shown in the following figures after culturing in a microfluidic chip for 0h, 24h, and 72h. In the figure, A is the bright field image of Escherichia coli TAc-IeGFP, B is the fluorescence image under an inverted fluorescence microscope, and C is the fluorescence quantification analysis image.

[0039] Figure 8 The invention uses chlorogenic acid (concentration of 3 mg / ml) to inhibit the growth of Escherichia coli TAc-IeGFP at 2 h, 4 h, 6 h, and 6.5 h. In the figure, A is a bright field image, B is a fluorescence image under an inverted fluorescence microscope, and C is a fluorescence quantification analysis image.

[0040] Figure 9 The growth curves of Escherichia coli TAC-leGF exposed to different concentrations of chlorogenic acid are shown in the example of this invention.

[0041] Figure 10 An example of the application of this invention is an image of the inhibition zone of Escherichia coli TAc-IeGFP after culturing on agar plates with different concentrations of chlorogenic acid (1.25-10 mg / ml), kanamycin sulfate, and ampicillin for 24 h.

[0042] Figure 11 A structural diagram of a microfluidic chip according to Embodiment 2 of the present invention;

[0043] The structure includes: 1. Microchamber; 2. Microcolumn structure; 3. Inlet; 4. Outlet; 5. Filter column; 6. Inlet area; 7. Cross-shaped baffle; and 8. Bacterial extraction structure. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] A low-shear-force microfluidic chip structure, wherein the microchannel of the microfluidic chip is provided with at least one microchamber, and one micropillar structure is provided on each side of the microchamber;

[0046] The micropillar structure is inverted U-shaped, and the ratio of the width between the top of the micropillar structure and the top of the microchannel to the width of the microchannel is 1:10.

[0047] The microchamber is divided into an entrance area and a culture area; the entrance area has a square entrance and a cross-shaped baffle inside, the width of which is smaller than the width of each side of the square; the culture area has a near-circular regular polygonal structure.

[0048] The microfluidic chip has two inlets and two outlets, with a filter column installed in the pipe of the inlet.

[0049] Instruments and reagents

[0050] 2X2-2 type vacuum pump and DHG type electric heating constant temperature drying oven (Shanghai Jinghong Test Equipment Co., Ltd.); SW-CJ-2F type double-person double-sided clean bench (Suzhou Clean Equipment Co., Ltd.); JA2003 type electronic balance (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd.); KW-4 type spin coater (Research Department of Microelectronics Center, Chinese Academy of Sciences);

[0051] 75% ethanol solution, Hebei Kangji Pharmaceutical Equipment Co., Ltd.; polydimethylsiloxane (PDMS, RTV615), Momentive, Inc., USA; trichloro(1H,1H,2H,2H-perfluorooctyl)silane, Sigma-Aldrich Shanghai Trading Co., Ltd.; agar, BR grade, Beijing Solarbio Biotechnology Co., Ltd.; tryptone and yeast extract, BR grade, Beijing Aoboxing Biotechnology Co., Ltd.; chlorogenic acid (mass fraction >98%) purchased from Hefei Kemike Biochemical Technology Co., Ltd.

[0052] Example 1

[0053] like Figure 1 and 2 A low-shear-force microfluidic chip structure, the specific structure of which is as follows:

[0054] The chip is 25 μm thick and includes a microchannel with one microchamber and two micropillar structures. A cross-shaped shielding pillar is provided at the entrance of the microchamber. The microchamber is located between the two micropillar structures. The width of the microchannel is 500 μm. The micropillar structure is inverted U-shaped with a width of 200 μm. The distance between the micropillar structures is 390 μm. The distance between the micropillar structure and the microchamber is 150 μm. The width between the top of the micropillar structure and the top of the microchannel is 50 μm.

[0055] The microchamber is divided into an inlet zone and a culture zone. The culture zone is a nearly circular regular polygonal structure with a diameter of 300 μm. The microchamber is used for bacterial culture, while the microcolumn is used to create vortex flow under high-flow-rate perfusion conditions, ensuring that the bacterial solution enters the microchamber as much as possible; for example... Figure 3 The entrance area is a rectangle of 150μm*90μm, with a top and bottom opening width of 90μm. The inside of the rectangle is equipped with a cross-shaped baffle plate, and the length of each side of the cross-shaped baffle column is 30μm.

[0056] The microfluidic device has two inlets and two outlets, used for injecting the stock solution and discharging waste liquid, respectively. A filter column is installed in the inlet pipe to remove some impurities.

[0057] The specific steps for fabricating a microfluidic chip are as follows:

[0058] (1) Mold processing: After cleaning the silicon wafer, take 15mL of negative photoresist SU-82025 and place it in the center of the silicon wafer. Spin coat the silicon wafer with a spin coater. The spin-coated silicon wafer is then pre-baked, exposed, post-baked and developed. Finally, it is observed under an upright microscope. After checking that there are no errors, it is ready for use.

[0059] (2) Mold pretreatment: Fumigate the mold with TMSCl steam for 3 minutes to facilitate the separation of PDMS polymer from the mold;

[0060] (3) Preparation of PDMS: Weigh out polydimethylsiloxane prepolymer and curing agent in a certain mass ratio and mix them well;

[0061] (4) Degassing and curing: Pour the above-mentioned uniformly mixed PDMS mixture onto the mold, degas it under vacuum, and place it in an oven at a certain temperature for heating and curing;

[0062] (5) Cutting and drilling: Cut and drill the cured PDMS according to the designed chip structure, and clean it.

[0063] (6) Sealing of PDMS microfluidic chip: Place the prepared PDMS chip flat on the PDMS film on the glass slide and bake it in an oven to make it firmly bonded.

[0064] Example 2

[0065] like Figure 11 A low-shear microfluidic chip structure is provided, with 14 microchambers 1 and 15 micropillar structures 2 in the microchannel. The remaining parameters and structure are the same as in Example 1. It also includes a cross-shaped bacterial export structure 6 set below the microchambers 2, which is used to export bacterial samples for subsequent electron microscopy observation.

[0066] Application Example (Using the microfluidic chip structure of Example 1)

[0067] 1. Culture of Escherichia coli in microfluidic chips

[0068] 1) Activation and culture of bacterial strains

[0069] All bacterial experiments were conducted using GFP-transduced Escherichia coli TAc-IeGFP, which emits green fluorescence and can be easily observed under a fluorescence microscope.

[0070] Revival of cryopreserved bacterial strains: Escherichia coli TAc-IeGFP, which was frozen at -80℃, was taken out of the freezer and thawed at room temperature. The thawed bacteria were activated in prepared liquid LB medium and then evenly spread on solid LB medium. The culture plates were placed in a constant temperature incubator at 37℃ and cultured until single colonies appeared. Then, under aseptic conditions, the single colonies on the plates were inoculated into EP tubes (containing liquid LB medium) and cultured on a shaker at 37℃ (180 r / min) until the appropriate concentration was reached before sealing and storage. Before infusion, E. coli was cultured. Then, under aseptic conditions, 700 μL of the bacterial culture stored in the EP tube was transferred to 100 mL of LB liquid medium, shaken, and the absorbance at 600 nm was measured (multiple replicates were set). The growth curve of E. coli was then plotted, and the bacterial culture that entered the stationary phase after 12 h of culture was selected for the experiment. The bacterial culture after propagation should not be left for more than one week, because E. coli has entered the decline phase at this time, and its activity has decreased, making it unsuitable for the experiment.

[0071] 2) Culture of Escherichia coli in microfluidic chips

[0072] The microfluidic experimental platform was assembled in a sterile environment. 75% alcohol was drawn up with a sterile syringe and assembled with the chip. The chip was then disinfected with alcohol to remove impurities. The chip was then cleaned with ultrapure water to remove the alcohol. Finally, the chip was sterilized under ultraviolet light.

[0073] Perform the following operations under aseptic conditions: draw a certain amount of E. coli bacterial solution into a 10mL sterile syringe; then take another 2.5mL sterile syringe and draw a certain amount of E. coli bacterial solution, ensuring that the amount of E. coli drawn is balanced on the injection pump; connect the two syringes to the sterilized chip device; then place the syringes on the four-inlet injection pump to complete the installation; set the flow rate to inject the E. coli bacterial solution into the chip.

[0074] The specific perfusion procedure involves first perfusing at a high flow rate of 500 μL / min for 10 minutes, then reducing the flow rate to allow E. coli to stably enter the culture chamber. After perfusion, the syringe is replaced with a different syringe to perfuse the culture medium. LB medium is drawn into 2.5 mL and 10 mL syringes, respectively. The syringes are connected to the chip perfused with E. coli, and the flow rate is set to 3 μL / min. The chip is then wrapped and placed in a 37°C incubator for overnight culture of E. coli. Finally, the growth of E. coli is observed under a fluorescence microscope. Under the same irradiation interval (usually 15 min) and exposure time (usually 3 s), a series of fluorescence images of the same microchamber are taken to study bacterial colony growth. The images are analyzed using Image-Pro Plus 6.0 software (IPP, Media Cyternetics, Lilver Spring, MD). Assuming the total number of bacteria is proportional to the overall fluorescence intensity of the region, the fluorescence intensity value is obtained by subtracting the background value from the sum of all pixels inside the microchamber image. Fluorescence quantification analysis is performed using Origin 2021 (OriginLab Corporation, Northampton, MA). After culturing for a certain period of time, once the E. coli has fully grown and covered the entire chip, and stable fluorescence can be observed under a fluorescence microscope, the culture is considered successful, and the next step of the experiment can be carried out.

[0075] Different flow rates result in different Dean flow fields within the channel, leading to variations in solution diffusion and particle capture. This study selected two flow rates, 500 μL / min and 3 μL / min, for perfusion experiments. The high flow rate was used to generate vortex flow between the microcolumns to capture bacteria, while the low flow rate was used to perfuse culture medium and drugs. Figure 4Simulations of vortex flow distribution within the channel under different flow rates are presented to simulate molecular diffusion between the pipe and the microchamber. The distribution of vortex flow within the channel at flow rates of 500 μL / min (left) and 3 μL / min (right) is shown (black arrows represent bacterial movement trajectories within the capture chamber; white arrows represent bacterial movement trajectories within the small culture chamber). At the high flow rate of 500 μL / min, vortex flow is generated between the microcolumns. When bacterial suspension is injected into the microchamber, bacteria are gradually squeezed into the microchamber, thereby increasing the bacterial capture rate in a shorter time. However, at the low flow rate of 3 μL / min, no vortex flow occurs, and the flow velocity between the microcolumns and between the culture chambers is essentially zero. Thus, the bacteria in the microchamber are not affected by the liquid shear force, i.e., they are not physically damaged. Therefore, this study chose to perfuse culture medium or drugs at a low flow rate of 3 μL / min and simulate their diffusion within the microchamber. The simulation results are as follows: Figure 5 It was found that the liquid flow entered through the main channel within 15 seconds, gradually filled the main channel after 65 seconds, diffused to half its maximum volume in the microchamber at 255 seconds, and completely filled the culture chamber at 1000 seconds. This indicates that under low shear stress conditions, the culture medium and drugs can be adequately perfused, providing a theoretical basis and validation strategy for long-term monitoring of bacterial dynamic culture under low shear stress conditions. Furthermore, reducing the flow rate can prolong the drug's action time and slow down its action on *E. coli*, allowing for the observation of more detailed changes over a longer period. The distance between the micropillar structures cannot be increased indefinitely; at least the diffusion time should be less than the time required for bacterial division to occur in one generation. According to the diffusion formula for small molecules (e.g., amino acids, sucrose): t = L... 2 / D, where t is the diffusion time, L is the diffusion distance, and D is the diffusion constant (3-10×10⁻¹⁰). -6 cm 2 / s. Therefore, the time for material exchange between the main pipe and the microchamber is 200-800s, which is basically consistent with the results of the simulation.

[0076] Simultaneously, after preparing the monoclonal Escherichia coli TAc-IeGFP (E. coli TAc-IeGFP) bacterial suspension, the growth curve of fluorescent E. coli was plotted, as shown in the figure. Figure 6 The optical density of the bacterial suspension was recorded within 0-27 hours (four replicates). Figure 6It can be seen that the growth phase can be roughly divided into four stages: from the start of culture to 2 hours, the bacteria are in a latent phase with slow growth; from 2 hours to 12 hours, bacterial growth is in an exponential phase, with bacterial concentration increasing linearly for approximately 2 hours; from 12 hours to 24 hours, bacterial growth is in a plateau phase, with bacterial concentration remaining at a stable level; from 24 hours onwards, bacterial concentration shows a slight decreasing trend, and bacterial growth enters the decline phase. Within controllable differences, there are no significant differences in the trends of the four curves and the optical density of the bacterial solution. To reduce the error in fluorescence intensity caused by changes in bacterial density, this experiment used bacterial solutions in the 12-hour stable phase during perfusion, making the experimental results more reliable.

[0077] Recent studies have confirmed that using a flow rate of 60 μL / min for single-cell focusing is beneficial for maintaining the original morphology of cells. Based on computer simulation results, this study selected a flow rate of 500 μL / min for perfusing bacterial suspension with an OD600 of 0.5, and a flow rate of 3 μL / min for perfusing culture medium and drugs. Although the flow rate of 500 μL / min is relatively high, the overall duration is short, and no shear force was observed at the inlet to damage the bacteria. Both the bacterial suspension and drugs were also evenly distributed around the culture chamber. Furthermore, the bacterial suspension could be perfused within 10 minutes, and the perfusion time and effect were significantly better than those found in previous studies. Figure 7 Data from A and B at 0h showed that the bacterial density was relatively low after inoculation. Following perfusion, LB liquid medium was dynamically perfused at a low flow rate (3 μL / min) to ensure the replenishment of basic nutrients and the removal of metabolic waste without affecting bacterial growth conditions and location within the chip. After 72h of dynamic culture, the bacterial community filled the entire culture chamber and was arranged vertically, exhibiting a strong fluorescence signal. This indicates that the culture chip can meet the experimental needs of dynamic bacterial culture, drug screening, and high-throughput enrichment. We further performed fluorescence quantitative analysis on the reproduction process of *E. coli* in the chip. Figure 7 As shown in Figure C, *E. coli* grew well after long-term dynamic culture. The bacterial density was highest at the inlet of the culture chamber after 24 hours, while the bacterial density and activity were highest in the middle of the culture chamber after 72 hours of dynamic culture. These experimental results further demonstrate that the low-shear microfluidic chip constructed in this experiment can be used for long-term dynamic bacterial culture, laying the foundation for subsequent research on the antibacterial properties of chlorogenic acid.

[0078] 2. The antibacterial effect of chlorogenic acid on Escherichia coli cultured in chips.

[0079] Escherichia coli, which had been cultured and stably grown on the microchip for a period of time, and chlorogenic acid were prepared. The appropriate volumes of chlorogenic acid were drawn into 2.5 mL and 10 mL syringes, respectively, and connected to the microfluidic chip. To prevent light exposure from affecting the chlorogenic acid, a layer of aluminum foil was wrapped around the syringes. After wrapping the chip, it was placed in a 37°C incubator, and the flow rate of chlorogenic acid (concentration of 3 mg / mL) was set to 3 μL / min to conduct an antibacterial experiment on E. coli. The results were observed and photographed under a microscope at regular intervals.

[0080] This experiment used a microfluidic chip platform to evaluate the growth of fluorescently labeled *E. coli* by utilizing changes in fluorescence intensity, and investigated the antibacterial effect of chlorogenic acid at a concentration of 3 mg / ml on the cultured *E. coli*. The results are as follows: Figure 8 As shown, after approximately 2 hours of treatment with chlorogenic acid, the green fluorescence intensity of *E. coli* in the edge area of ​​the culture chamber decreased, indicating that its growth was inhibited. After 4 hours of treatment with chlorogenic acid, the green fluorescence intensity of *E. coli* in the culture chamber continued to decrease, indicating that chlorogenic acid continuously inhibited its growth. After 6 hours of treatment with chlorogenic acid, the fluorescence of *E. coli* in the culture chamber was very weak, indicating that its growth had been completely inhibited. After about 30 minutes, the green fluorescence in the culture chamber completely disappeared. The final results show that chlorogenic acid can completely inhibit the growth of *E. coli* TAc-IeGFP in about 6.5 hours. The gradual decrease in the fluorescence intensity of *E. coli* indicates that chlorogenic acid has a sustained inhibitory effect on its growth.

[0081] 3. Determination of the minimum inhibitory concentration of chlorogenic acid against Escherichia coli

[0082] The antibacterial effect of different concentrations of chlorogenic acid standard solutions on Escherichia coli TAc-IeGFP was detected by broth dilution method. The detection period in this experiment was 16 hours.

[0083] Specific steps: (1) Cultivate Escherichia coli TAc-IeGFP (pick a single colony and inoculate it into a 5ml test tube), place it in a 37℃, 180r / min incubator and shake for 4-6 hours. After cultivation, take 1ml of bacterial solution and add it into 3 prepared EP tubes (1.5ml specification) for later use.

[0084] (2) Prepare chlorogenic acid standard stock solution (4ml, 10mg / ml), sterilize by microporous membrane filtration, and then dilute with liquid LB medium to different concentrations (8.75, 7.5, 6.25, 5, 3.75, 2.5, 1.25mg / ml);

[0085] (3) Take 150 μl for a 96-well plate antibacterial experiment. Add 3 μl of bacterial suspension with OD600 = 0.5 to 0.6 to each well (at an inoculum rate of 2%). Perform 3 replicates for each gradient.

[0086] (4) Seal the 96-well plate and incubate it in a 37℃ incubator in the dark. At certain intervals, use a microplate reader to measure the absorbance value (OD600) at a wavelength of 600nm. Plot the growth curve of Escherichia coli TAc-IeGFP using the obtained data to determine the minimum inhibitory concentration of chlorogenic acid solution (when the OD600 value is 0.1, the concentration of the bacterial suspension is considered to be 1×10⁻⁶). 8 CFU·mL -1 ), the results are shown Figure 9 .

[0087] 4. The antibacterial effect of different concentrations of chlorogenic acid solutions on Escherichia coli

[0088] The inhibition zone experiment on agar plates was conducted using the paper disc diffusion method. Three sections were marked on the back of the agar plate using a marker and ruler: experimental group (1.25–10 mg / ml chlorogenic acid solution), positive control group (50 mg / ml kanamycin sulfate solution), and negative control group (50 mg / ml ampicillin solution). Filter paper discs were cut into small circles with a diameter of 0.7 mm using a punch. Sterilized filter paper discs were placed on the surface of LB agar plates coated with the experimental bacterial solution, with each disc positioned in one of the three marked sections. Then, 5 μl of the corresponding experimental or control solution was added to each disc. The agar plates were sealed with breathable sealing film and incubated upside down at 37°C for 24 hours. The inhibition zones were then observed. Results are shown in [Figure number missing]. Figure 10 .

[0089] As a control experiment, the traditional broth dilution method and paper disc diffusion method were used to detect the antibacterial effect of different concentrations of chlorogenic acid on *E. coli*. The broth dilution method can roughly determine the minimum inhibitory concentration (MIC) of chlorogenic acid. Different concentrations of chlorogenic acid solutions and bacterial cultures cultured to the stationary phase were sequentially added to 96-well plates, followed by static incubation at 37°C. The bacterial population was quantified by measuring the absorbance at 600 nm using a microplate reader at 2-hour intervals. Before use, the OD600 in the culture tubes was 0.5 (after multiple independent replicates, it was found that the absorbance at 600 nm did not change significantly at 2 hours, but the change was significant at 4 hours; therefore, the OD value after 4 hours of incubation was selected). The experimental results showed that chlorogenic acid can significantly inhibit the growth of *E. coli* TAc-IeGFP at a certain concentration, and the inhibitory effect increases with increasing chlorogenic acid concentration. The MIC of chlorogenic acid at this dilution gradient was approximately between 2.5 and 3.75 mg / ml. Figure 9 The results are consistent with those reported in the literature. However, compared to the microfluidic chip method, this method requires a cumbersome dilution step in a standard 96-well plate and is very time-consuming.

[0090] The antibacterial effect of chlorogenic acid standard solutions of different concentrations on *Escherichia coli* was studied using the paper disc diffusion method. The results showed that chlorogenic acid solutions with concentrations ranging from 1.25 to 10 mg / ml did not exhibit any antibacterial effect. Figure 10 Therefore, using microfluidic chips to study the antibacterial effect of chlorogenic acid offers higher sensitivity and shorter experimental time. Jain et al. also obtained similar experimental results, showing that the results differ under different drug susceptibility testing methods, further indicating the inaccuracy of the paper disc diffusion method when used for drug susceptibility testing. Differences exist between macroscopic and microscopic morphological observations; therefore, using microfluidic technology for drug susceptibility testing has unique advantages.

[0091] In summary, the low-shear microfluidic chip structure of this invention can generate vortex flow at high flow rates, enabling efficient capture and cultivation of *E. coli*. When the liquid flow is at a low velocity, its low shear force does not disturb the bacteria captured in the microchamber, making it suitable for long-term dynamic culture and drug administration observation. Cultivating *E. coli* using flowing liquid culture medium better simulates the in vivo growth environment of *E. coli*, making the experimental results more realistic. Observing the changes in the overall number and fluorescence intensity of *E. coli* at different time points under a fluorescence microscope using the microfluidic chip allows for a more intuitive and specific analysis of the antibacterial effect of the drug from a microscopic perspective. Furthermore, we also studied the antibacterial effect of chlorogenic acid on *E. coli* TAc-IeGFP using traditional antibacterial methods. Compared to traditional experiments, using microfluidic chips to study the antibacterial effect of drugs has many advantages. First, it improves the accuracy of the results and shortens the detection time, allowing drug sensitivity testing to be completed in approximately 6.5 hours. Furthermore, traditional antibacterial experiments are labor-intensive and material-intensive, resulting in resource waste. Therefore, using microfluidic chips for antibacterial experiments has excellent development prospects.

[0092] The various embodiments are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Similar or identical parts between the various embodiments can be referred to each other.

[0093] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A low-shear-force microfluidic chip, characterized in that, The microfluidic chip has at least one microchamber in its microchannel, and one micropillar structure is provided on each side of the microchamber; The micropillar structure is inverted U-shaped; The ratio of the width between the top of the micropillar structure and the top of the microchannel to the width of the microchannel is 1:10; The microchamber is divided into an entrance area and a culture area; the entrance area is square and has a cross-shaped baffle inside.

2. The low-shear microfluidic chip according to claim 1, characterized in that, The width of the cross-shaped baffle is smaller than the width of each side of the square.

3. A low-shear microfluidic chip according to claim 1, characterized in that, The culture area has a nearly circular regular polygonal structure.

4. A low-shear microfluidic chip according to claim 1, characterized in that, The microfluidic chip has two inlets and two outlets, wherein a filter column is provided in the pipe of the inlet.

5. A low-shear microfluidic chip according to claim 1, characterized in that, The ratio of the distance between the micropillar structures to the width of the microchannel is 1:

2.

6. The method for fabricating a low-shear microfluidic chip as described in any one of claims 1-5, characterized in that, Specifically, the following steps are included: (1) Mold processing: After taking the negative photolithography coated silicon wafer, perform pre-baking, exposure, post-baking and development in sequence to obtain the mold for later use; (2) Weigh out the polydimethylsiloxane prepolymer and the curing agent, mix them well, place them on the mold, and heat to cure; (3) After the solidified mold is cut and punched, it is placed on a glass slide and baked.

7. The application of the low-shear microfluidic chip as described in any one of claims 1-5 in bacterial culture, wherein the application is for non-therapeutic diagnostic purposes.

8. The application of the low shear force microfluidic chip as described in any one of claims 1-5 in an antibacterial experiment, wherein the application is for non-therapeutic diagnostic purposes.

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