A biofilm dynamic control system and method based on microfluidics and optogenetics

Abstract

The present invention belongs to the fields of biomedical engineering and synthetic biology, and specifically discloses a system and method for dynamically regulating biofilms based on microfluidics and optogenetics. The system and method comprise a microfluidic chip, a detection module, and an optogenetics control module. The detection module is electrically connected to the microfluidic chip via a multi-model detection interface, and the detection module includes a laser confocal microscope, a micro-CT three-dimensional reconstruction system, and a DESI-MSI mass spectrometer. The optogenetics control module includes Pseudomonas fluorescens, whose genome incorporates a red-light-responsive PhyB-PIF3 gene circuit. The present invention utilizes the aforementioned system and method for dynamically regulating biofilms based on microfluidics and optogenetics to precisely control the physicochemical properties of biofilms and the expression of quorum sensing (QS) signaling molecules. The system also enables real-time, in situ monitoring of biofilm thickness, porosity distribution, QS signaling molecules, and antimicrobial agent diffusion efficiency.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedical engineering and synthetic biology, and in particular to a system and method for dynamic regulation of biofilms based on microfluidics and optogenetics. Background Art

[0002] Biofilm culture models, as a type of probabilistic model, are often used to describe the environmental constraints under which changes in the physical and chemical properties of microorganisms occur. This requires determining the probability of such changes. Optogenetic control systems are cutting-edge biomedical tools that combine optical and genetic technologies. By genetically modifying specific cells or tissues to express light-sensitive proteins, they can precisely regulate cellular activity using light of specific wavelengths. Optogenetic control systems offer advantages such as high spatiotemporal resolution (millisecond-level, single-cell manipulation) and non-invasiveness (light can be transmitted through tissues), and are widely used in fields such as neuroscience, cell biology, and disease treatment.

[0003] Conventional biofilm culture models (such as static well-plate cultures) are unable to simulate real-world environmental parameters like dynamic salt gradients and shear stress, resulting in a single set of parameters. Furthermore, optogenetic control systems lack integration with microfluidic chips and are unable to simultaneously analyze porosity and diffusion rate in high-viscosity matrices, leading to low control precision and insufficient monitoring dimensionality. Furthermore, evaluation of antimicrobial penetration efficiency often relies on endpoint detection, which lacks spatial resolution. Summary of the Invention

[0004] The purpose of the present invention is to provide a biofilm dynamic regulation system and method based on microfluidics and optogenetics, which can be used to precisely regulate the physicochemical properties of biofilms and the expression of quorum sensing (QS) signal molecules, and to monitor biofilm thickness, porosity distribution, QS signal molecules, and antimicrobial agent diffusion efficiency in real time and in situ, breaking through the bottlenecks of the existing technology of single environmental parameters, low regulation accuracy, and insufficient monitoring dimensions.

[0005] To achieve the above objectives, the present invention provides a biofilm dynamic regulation system based on microfluidics and optogenetics, comprising a microfluidic chip, a detection module, and an optogenetic regulation module. The detection module is electrically connected to the microfluidic chip via a multi-model detection interface, and the detection module comprises a laser confocal microscope, a μCT three-dimensional reconstruction instrument, and a DESI-MSI mass spectrometer.

[0006] The microfluidic chip integrates a shear force control layer, a gradient generation layer, and a biofilm adhesion layer. The optogenetic regulation module is located in a Pseudomonas fluorescens cell system. The Pseudomonas fluorescens is located on the biofilm adhesion layer. The genome of the Pseudomonas fluorescens is integrated with a red light-responsive PhyB-PIF3 gene circuit.

[0007] Preferably, the microfluidic chip is a three-layer PDMS laminate structure, which is formed by casting PDMS using an SU-8 photoresist mold and then bonding by oxygen plasma to obtain a microchannel and a microfluidic network with a channel height of 50±2 μm;

[0008] The gradient generation layer generates a 0-5% linear NaCl gradient through a microfluidic network;

[0009] The shear force control layer is connected to a micro pump via a micro channel, and the micro pump controls the magnitude of the shear force by adjusting the flow rate;

[0010] The biofilm adhesion layer is also cast by mixing a high-viscosity matrix with PDMS.

[0011] Preferably, the gradient generating layer establishes a 0-5% linear NaCl gradient at a rate of 0.5% NaCl / min.

[0012] Preferably, the flow rate ranges from 0 to 500 μL / min, and the shear force ranges from 0.05 to 1.2 Pa.

[0013] Preferably, the high viscosity matrix is ​​1.5% sodium alginate, calculated by mass percentage.

[0014] Preferably, the genome of Pseudomonas fluorescens is integrated with a red light responsive PhyB-PIF3 gene circuit by electroporation, specifically:

[0015] T1. Pretreatment of bacteria: Pseudomonas fluorescens was inoculated into LB liquid medium, cultured at 30℃ with shaking until OD600=0.6, cooled in an ice bath for 15min, centrifuged at 4℃ for 5min, collected the bacteria, and washed three times with pre-cooled electroporation buffer with a sucrose concentration of ≥0.5M. Finally, the bacteria were resuspended to a concentration of 10 9 CFU / mL;

[0016] T2, DNA-bacteria mixture: The PhyB-PIF3 gene loop plasmid was linearized, and 80 μL of competent cells were mixed with 2 μg of the PhyB-PIF3 gene loop plasmid carrying the kanamycin resistance marker and incubated on ice for 10 min;

[0017] T3. Electroporation: Use a 2 mm electroporation cuvette with the following parameters: voltage 2.4-2.6 kV, capacitance 25 μF, resistance 200 Ω, and pulse time 5 ms. After electroporation, add 1 mL of preheated SOC recovery medium containing 0.2-0.5 M sucrose.

[0018] T4. Transformant screening: After 2 h of standing at 30°C for recovery, the cells were plated on LB solid medium containing 50 μg / mL kanamycin and verified by PCR.

[0019] Preferably, the laser confocal microscope is used to measure the thickness of the biofilm;

[0020] The μCT three-dimensional reconstruction instrument is used to analyze the porosity distribution;

[0021] The DESI-MSI mass spectrometry imager is used to detect the C4-HSL signal intensity with a spatial resolution of 50±5 μm.

[0022] The present invention also provides a method for dynamic regulation of biofilms based on microfluidics and optogenetics, the steps of which are as follows:

[0023] S1, dynamic salt gradient and shear stress conditions are applied through microfluidic chip;

[0024] S2, using blue light to activate lasI gene expression, or red light to inhibit lasI gene expression;

[0025] S3. Use μCT three-dimensional reconstruction instrument and DESI-MSI mass spectrometry imaging instrument to analyze the porosity of biofilm and the spatial distribution of C4-HSL signal molecules, and establish a porosity-bacteriocin diffusion rate model.

[0026] Preferably, in S2, the blue light activation condition is:

[0027] Wavelength 450±10nm, light intensity 10±0.5mW / cm 2 , activates lasI gene expression;

[0028] The red light suppression conditions are:

[0029] Wavelength 660±10nm, light intensity 5±0.3mW / cm 2 , inhibiting lasI gene expression.

[0030] Therefore, the present invention adopts the above-mentioned biofilm dynamic control system and method based on microfluidics and optogenetics, and the beneficial effects are as follows:

[0031] (1) The biofilm dynamic control system of the present invention integrates an environmental simulation module (salt gradient, shear force, high viscosity matrix), an optogenetic control module (PhyB-PIF3 system) and a multimodal detection module (μCT+DESI-MSI) through a three-layer PDMS microfluidic chip. Combined with the PhyB-PIF3 system, it realizes the spatiotemporal control of the quorum sensing gene lasI. It can synchronously and in situ monitor the biofilm thickness (accuracy ±5μm), porosity distribution (15-50μm) and antimicrobial agent diffusion efficiency (DESI-MSI imaging). It breaks through the bottleneck of the existing technology of single environmental parameters, low control accuracy and insufficient monitoring dimensions, significantly improves the environmental simulation authenticity and control accuracy of biofilm research, provides a quantitative basis for the optimization of antimicrobial agent delivery schemes, and has broad application prospects in the anti-fouling of industrial pipelines and the development of antimicrobial coatings for medical implants.

[0032] (2) The control effects of the biofilm dynamic control system of the present invention are as follows:

[0033] Dynamic environmental simulation module: Synchronous control of salt gradient (±0.1% accuracy), shear force (±0.01Pa) and matrix viscosity, biofilm thickness control range ±35%;

[0034] Optogenetics: Light-controlled gene expression in the optogenetics module: switching between red and blue light resulted in a 40% increase or decrease in lasI gene expression (p<0.01), and a 60% decrease in C4-HSL signal intensity.

[0035] Diffusion efficiency prediction of the multimodal detection module: A porosity-diffusion rate model was established (Pearson r=0.78).

[0036] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic structural diagram of an embodiment of a biofilm dynamic control system based on microfluidics and optogenetics of the present invention;

[0038] Figure 2 This is a schematic diagram of the structure of S2 of an embodiment of a method for dynamic regulation of biofilms based on microfluidics and optogenetics according to the present invention;

[0039] Figure 3This is the system verification result of an embodiment of the biofilm dynamic regulation method based on microfluidics and optogenetics of the present invention, wherein (a) is a bar graph of biomass changes under red and blue light irradiation, (b) is a comparison of biofilm thickness before and after red light treatment, (c) is a heat map of C4-HSL signal intensity displayed by DESI-MSI mass spectrometry imaging, and (d) is a scatter plot of the correlation between the porosity >30μm region and the bacteriocin diffusion rate, with Pearson r=0.78. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0041] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0042] Source of test materials:

[0043] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0044] Example 1

[0045] like Figure 1 A system for dynamic biofilm regulation based on microfluidics and optogenetics is shown. It includes a microfluidic chip, a detection module, and an optogenetic regulation module. The microfluidic chip is a three-layer polydimethylsiloxane (PDMS) laminate with a 50μm channel height. PDMS is cast using a SU-8 photoresist mold, and the three-layer structure is then bonded using oxygen plasma. The structure integrates a shear force control layer, a gradient generation layer, and a biofilm adhesion layer. The gradient generation layer generates a 0-5% linear NaCl gradient through a microfluidic network, maintaining a 0-5% linear NaCl gradient at a rate of 0.5% NaCl / min. The shear force control layer is connected to a micropump through microchannels. The micropump adjusts the flow rate from 0 to 500μL / min and controls the shear force between 0.05 and 1.2 Pa. The biofilm adhesion layer is also cast using a high-viscosity matrix mixed with PDMS. The high-viscosity matrix consists of 1.5% sodium alginate (w / v) to simulate the biofilm adhesion environment. The specific casting and oxygen plasma bonding procedures are as follows:

[0046] The PDMS casting process, based on the SU-8 mold, is as follows:

[0047] (1) Mold pretreatment: SU-8 2050 photoresist was used to photolithographically prepare a microchannel mold (channel height 50 ± 2 μm) on a silicon wafer. Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (FDTS) was vapor-deposited on the mold surface for 2 h to form an anti-sticking layer.

[0048] (2) PDMS mixing and degassing

[0049] PDMS (Sylgard 184) was mixed at a ratio of base glue to curing agent of 10:1 (w / w); after mechanical stirring for 5 minutes, vacuum degassing (-95 kPa) was performed until there were no bubbles (30 minutes).

[0050] (3) Casting and curing

[0051] Pour PDMS onto the SU-8 mold with a thickness controlled at 5 ± 0.2 mm.

[0052] Step curing procedure: pre-curing at 65℃ for 40 minutes, heating to 85℃ for main curing for 120 minutes. Step curing reduces channel deformation caused by thermal stress.

[0053] (4) Demolding and cutting: After cooling to room temperature, slowly peel off the PDMS from the edge

[0054] The three-layer structure (shear force control layer, gradient generation layer and biofilm adhesion layer) containing the microfluidic network was retained, and holes were punched to form a fluid interface (pore diameter 1.5 mm).

[0055] Oxygen plasma bonding process (three-layer PDMS integration), the steps are as follows:

[0056] (1) Surface activation treatment: The three PDMS structural layers (shear force control layer, gradient generation layer, biofilm adhesion layer) and the cover sheet were placed in the plasma chamber. The parameters were set as follows: power 50 W, oxygen flow rate 100 sccm, vacuum pressure 0.1 mbar, and treatment time 45 s.

[0057] (2) Precise alignment and bonding: Immediately contact and bond the activated surfaces (operation time <30s), and use a microscopic alignment system to ensure that the microchannel position error is ≤10μm and the 1.5% sodium alginate matrix (w / v) premixed in the biofilm adhesion layer does not overflow into the channel.

[0058] (3) Post-curing strengthening: After bonding, place on a hot plate and heat for 60 minutes at 80°C;

[0059] Bond strength test: withstand fluid pressure >300kPa (meets 0-500μL / min flow rate requirement).

[0060] The key process control points of pouring and oxygen plasma bonding are shown in Table 1.

[0061] Table 1 Key process control points

[0062] ;

[0063] The optogenetic control module is located in the Pseudomonas fluorescens cell system. Pseudomonas fluorescens is located on the biofilm adhesion layer. The genome of Pseudomonas fluorescens is integrated with the red light responsive PhyB-PIF3 gene circuit by electroporation. Figure 2 As shown, specifically:

[0064] T1. Pretreatment of bacteria: P.fluorescens was inoculated into LB liquid medium (containing 10 mM MgSO4), cultured at 30°C with shaking until OD600 = 0.6, cooled in an ice bath for 15 min, centrifuged at 5000 × g for 5 min at 4°C, and the bacteria were collected and washed three times with pre-cooled electroporation buffer (10% glycerol, 0.5 M sucrose, pH 7.0). Finally, the cells were resuspended to a concentration of 10 9 CFU / mL. The electroporation buffer must maintain osmotic pressure balance (sucrose concentration ≥ 0.5 M) to prevent lysis of Gram-negative bacteria.

[0065] T2. DNA-bacteria mixing: The PhyB-PIF3 gene loop plasmid is linearized. Using the restriction endonuclease EcoRI can increase the genome integration efficiency by 2.1 times. Take 80 μL of competent cells and 2 μg of the PhyB-PIF3 gene loop plasmid carrying the kanamycin resistance marker and incubate on ice for 10 minutes.

[0066] T3. Electroporation: Use a 2mm electroporation cuvette with the following parameters: voltage 2.5kV, capacitance 25μF, resistance 200Ω, and pulse time 5ms. After electroporation, add 1mL of preheated SOC recovery medium containing 0.3M sucrose. Adding 0.3M sucrose can increase the transformation efficiency to (3.2±0.4)×10 3 CFU / μg DNA.

[0067] T4. Transformant screening: After 2 h of standing at 30°C for recovery, the cells were plated on LB solid medium containing 50 μg / mL kanamycin, and single clones were picked for PCR verification.

[0068] Genomic integration was achieved by homologous recombination, with 1.5-kb homology arms flanking the plasmid (targeting the Pseudomonas fluorescens attB site). Transformation efficiency was verified by dot blotting and Southern blotting analysis using a digoxigenin-labeled lasI gene probe.

[0069] The detection module is electrically connected to the microfluidic chip via a multi-model detection interface and includes a laser confocal microscope, a μCT 3D reconstruction system, and a DESI-MSI mass spectrometer. The laser confocal microscope measures biofilm thickness with an accuracy of ±5 μm, the μCT 3D reconstruction system analyzes porosity distribution (15-50 μm), and the DESI-MSI mass spectrometer measures C4-HSL signal intensity with a spatial resolution of 50 μm.

[0070] Example 2

[0071] Using the system of Example 1, a method for dynamic regulation of biofilm based on microfluidics and optogenetics is provided, and the steps are as follows:

[0072] S1. Dynamic salt gradient and shear stress conditions were applied to the microfluidic chip. A linear NaCl gradient of 0-5% was established at a flow rate of 0.5% NaCl / min and a flow rate of 200 μL / min (corresponding to a shear stress of 0.8 Pa).

[0073] S2. Use 450nm blue light to activate lasI gene expression, or 660nm red light to inhibit lasI gene expression. Specific light control program: blue light irradiation twice a day (30 minutes each time), blue light activation condition is light intensity 10mW / cm 2 , activate lasI gene expression, red light continuous irradiation for 12h, red light inhibition condition is light intensity 5mW / cm 2 , inhibiting lasI gene expression, the principle is as follows Figure 2 Light intensity calibration is performed using a radiometer.

[0074] S3. A microCT 3D reconstruction system coupled with a DESI-MSI mass spectrometer was used to analyze biofilm porosity and the spatial distribution of C4-HSL signal molecules. System validation: The microCT 3D reconstruction system scanned samples with standard pore sizes (20-60 μm) with a measurement error of <3%. The DESI-MSI mass spectrometer was calibrated using a standard (C4-HSL, Sigma). A porosity-bacteriocin diffusion rate model was developed.

[0075] Experimental testing

[0076] Data analysis: ImageJ was used to quantify biofilm thickness, and t-tests were performed using SPSS 26.0 (with a significance threshold of p < 0.05). The system validation results are shown in the table below. Figure 3 shown.

[0077] Depend on Figure 3 As shown in (a), blue light (450±10nm) significantly increased the biofilm biomass to 135±4%, while red light (660±10nm) inhibited it to 75±3%. Figure 3As shown in (b), the biofilm thickness decreased from 50±2μm to 32±1.5μm (↓36%) under red light treatment, confirming the spatial regulation ability of light-controlled EPS synthesis. Figure 3 As shown in (c), the porosity measured by μCT (15-50 μm) is strongly positively correlated with the antimicrobial diffusion rate detected by DESI-MSI (Pearson r = 0.78), which is consistent with the model "diffusion rate = 0.023 × porosity + 0.41". Figure 3 As shown in (d), red light reduced the C4-HSL signal intensity by 60%, and DESI-MSI imaging (50μm resolution) revealed a signal distribution with edge enrichment and central attenuation. In summary, biofilm thickness can be controlled within a ±35% range. Light-controlled gene expression: Switching between red and blue light resulted in a 40% increase or decrease in lasI gene expression and a 60% decrease in C4-HSL signal intensity. Porosity-diffusion rate model (Pearson r = 0.78).

[0078] Therefore, the present invention adopts the above-mentioned microfluidics and optogenetics-based biofilm dynamic control system and method to accurately control the physical and chemical properties of biofilms and the expression of quorum sensing signal molecules, and monitor the diffusion efficiency of antibacterial agents in real time, breaking through the bottlenecks of the existing technology of single environmental parameters, low control accuracy, and insufficient monitoring dimensions.

[0079] 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 the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A biofilm dynamic control system based on microfluidics and optogenetics, characterized by: It includes a microfluidic chip, a detection module and an optogenetic regulation module. The detection module is electrically connected to the microfluidic chip through a multi-model detection interface. The detection module includes a laser confocal microscope, a μCT three-dimensional reconstruction instrument and a DESI-MSI mass spectrometer. The microfluidic chip integrates a shear force control layer, a gradient generation layer, and a biofilm adhesion layer. The optogenetic regulation module is located in a Pseudomonas fluorescens cell system. The Pseudomonas fluorescens is located on the biofilm adhesion layer. The genome of the Pseudomonas fluorescens is integrated with a red light-responsive PhyB-PIF3 gene circuit. The shear force control layer is connected to a micro pump via a micro channel, and the micro pump controls the magnitude of the shear force by adjusting the flow rate; The flow rate range is 0-500 μL / min, and the shear force range is 0.05-1.2 Pa; The gradient generation layer establishes a 0-5% linear NaCl gradient at a rate of 0.5% NaCl / min; The biofilm adhesion layer is also cast by mixing a high viscosity matrix with PDMS; The high viscosity matrix is ​​1.5% sodium alginate, calculated by mass percentage; The control method includes the following steps: S1, dynamic salt gradient and shear stress conditions are applied through microfluidic chip; S2, using blue light to activate lasI gene expression, or red light to inhibit lasI gene expression; S3. Use μCT three-dimensional reconstruction instrument and DESI-MSI mass spectrometry imaging instrument to analyze the porosity of biofilm and the spatial distribution of C4-HSL signal molecules, and establish a porosity-bacteriocin diffusion rate model.

2. The biofilm dynamic control system based on microfluidics and optogenetics according to claim 1, characterized in that: The microfluidic chip is a three-layer PDMS laminate structure, which is cast using an SU-8 photoresist mold and then bonded using oxygen plasma to obtain a microchannel with a channel height of 50±2μm and a microfluidic network; The gradient generation layer generates a 0-5% linear NaCl gradient through a microfluidic network.

3. The biofilm dynamic control system based on microfluidics and optogenetics according to claim 1, characterized in that: The genome of Pseudomonas fluorescens is integrated with a red light responsive PhyB-PIF3 gene circuit by electroporation, specifically: T1. Pretreatment of bacteria: Pseudomonas fluorescens was inoculated into LB liquid medium, cultured at 30℃ with shaking until OD600=0.6, cooled in an ice bath for 15min, centrifuged at 4℃ for 5min, collected the bacteria, and washed three times with pre-cooled electroporation buffer with a sucrose concentration of ≥0.5M. Finally, the bacteria were resuspended to a concentration of 10 9 CFU / mL; T2, DNA-bacteria mixture: The PhyB-PIF3 gene loop plasmid was linearized, and 80 μL of competent cells were mixed with 2 μg of the PhyB-PIF3 gene loop plasmid carrying the kanamycin resistance marker and incubated on ice for 10 min; T3. Electroporation: Use a 2 mm electroporation cuvette with the following parameters: voltage 2.4-2.6 kV, capacitance 25 μF, resistance 200 Ω, and pulse time 5 ms. After electroporation, add 1 mL of preheated SOC recovery medium containing 0.2-0.5 M sucrose. T4. Transformant screening: After 2 h of standing at 30°C for recovery, the cells were plated on LB solid medium containing 50 μg / mL kanamycin and verified by PCR.

4. The method of biofilm dynamic control system based on microfluidics and optogenetics according to claim 1, characterized in that: The laser confocal microscope is used to measure the thickness of the biofilm; The μCT three-dimensional reconstruction instrument is used to analyze the porosity distribution; The DESI-MSI mass spectrometry imager is used to detect the C4-HSL signal intensity with a spatial resolution of 50±5 μm.

5. The biofilm dynamic control system based on microfluidics and optogenetics according to claim 1, characterized in that: In S2, the blue light activation condition is: Wavelength 450±10nm, light intensity 10±0.5mW / cm 2 , activates lasI gene expression; The red light suppression conditions are: Wavelength 660±10nm, light intensity 5±0.3mW / cm 2 , inhibiting lasI gene expression.

Images