Microfluidic chip special for bacterial biofilm culture and bottom in-situ observation

Abstract

The invention discloses a micro-fluidic chip special for bacterial biofilm culture and bottom in-situ observation, and belongs to the technical field of micro-fluidic chips. The micro-fluidic chip is composed of a glass substrate and a micro-channel main body formed by pouring polydimethylsiloxane, and after the glass substrate and the micro-channel main body are bonded through UV glue, a sunken micro culture chamber used for fixing bacteria and promoting the formation of a biological membrane is formed in the micro-channel main body. The micro-fluidic chip is compatible with an inverted microscope and a high-content imaging analysis system (such as Operetta CLS), and can be directly used for bottom observation. A sunken micro culture cavity is integrated in the chip and is used for fixing bacteria; the height of the microchannel connected with the outlet is low, bacteria can be limited in the cavity, meanwhile, a nutrient solution can be permitted to be continuously poured, and a stable fluid environment is provided for biofilm formation. According to the structural design, long-time and in-situ real-time observation and research of bacterial biofilm growth are realized.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically relating to a microfluidic chip for bacterial biofilm culture and in-situ bottom observation. Background Technology

[0002] Microfluidic chips, with their precise microscale control, low sample volume, high throughput, and high degree of automation, are driving microbiology research to gradually shift from traditional large-scale culture to in-situ and real-time dynamic analysis at the single-cell level.

[0003] Despite the existence of various microfluidic chips for microbial culture on the market, existing products still face significant limitations when applied to bacterial biofilm research: First, limited application scenarios: current mainstream solutions mostly focus on observing single-layer bacteria, with a lack of dedicated designs for studying the formation and dynamics of multi-layer biofilms. Second, complex structures and high costs: existing chips generally have complex structures, relying on silicon-based photolithography microfabrication or complex multi-step PDMS replication processes, resulting in high manufacturing costs and long processing times, making it difficult to meet the needs of high-throughput or reproducible testing in laboratories. Third, design flaws in observation sites: a key limitation of most existing biofilm chips is that their observation sites are usually located on top of the chip, requiring an upright microscope. Although PDMS material itself has good light transmittance, compared to standard glass coverslips, it introduces additional optical interference and image quality loss as an optical medium; more importantly, this top-viewing method carries a high risk of the objective lens touching or even damaging the chip.

[0004] Therefore, this invention provides a novel microfluidic chip for biofilm culture that is simple to prepare and inexpensive, and can be observed in situ from the bottom, which has practical application significance. Summary of the Invention

[0005] The purpose of this invention is to propose a microfluidic chip specifically for bacterial biofilm culture and in-situ bottom observation. Its structure is reasonable, simple to manufacture, and inexpensive. It can be adapted to a standard inverted microscope stage for planar observation of biofilms, and can also be adapted to the high-content imaging analysis system Operetta CLS™ for 3D imaging observation of biofilms.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A microfluidic chip specifically designed for bacterial biofilm culture and in-situ bottom observation consists of a glass substrate and a microchannel body cast from polydimethylsiloxane. After the two are bonded together with UV adhesive, a recessed micro-culture chamber is formed inside the microchannel body to fix bacteria and promote biofilm formation. The two ends of the culture chamber are designed with an inlet and an outlet, respectively.

[0008] As a preferred embodiment of the present invention, the height of the culture chamber gradually decreases from the inlet to the outlet, ensuring that bacteria are trapped within the chamber and not easily carried away by the flow of the culture medium, while the culture medium can continuously flow through the chamber to provide nutrients for the bacteria. The heights of the culture chamber from the inlet to the outlet are 25~30μm, 15~20μm, and 0.1~0.5μm, respectively. The lower microchannel height connecting to the outlet can confine bacteria within the chamber while allowing continuous perfusion of the culture medium, providing a stable fluid environment for biofilm formation.

[0009] As a preferred embodiment of the present invention, there are two inlets arranged in a Y-shape, one for bacterial suspension and the other for culture medium; all liquids flow out through the outlet. The inlet and outlet are connected to the inside and outside of the microfluidic chip via stainless steel needles. A medical transparent tubing is connected to the outer end of the stainless steel needle, and the opening and closing of the tubing is controlled by a stainless steel paperclip on the medical transparent tubing.

[0010] As a preferred embodiment of the present invention, the glass substrate is made of quartz glass with a thickness of 0.17~0.35 mm.

[0011] Furthermore, this invention also proposes a method for fabricating and using a microfluidic chip specifically for bacterial biofilm culture and in-situ bottom observation, the steps of which are as follows:

[0012] (1) Mold preparation: Customize the mold for PDMS casting according to the cavity molding parameters. It should be noted that the mold material should not react with PDMS and that PDMS is easy to fall off after curing.

[0013] (2) PDMS casting: First, clean and dry the mold, mix Dow Corning 184 glue and coagulant evenly and degas it, then cast it into the chip mold. After drying and curing, peel off the PDMS layer with microchannel structure from the mold, and use a punch to punch holes at the liquid inlet and liquid outlet.

[0014] (3) Bonding PMDS layer to glass substrate: Use Dow Corning UV adhesive to bond PMDS layer to glass substrate. Place the bonded PMDS layer and glass substrate in a cleanroom and invert it so that the glass substrate is facing up. Use UV irradiation to fully cure the UV adhesive. Then soak it in PBS to remove residual UV adhesive at the bonding interface. Finally, apply a mixture of Dow Corning 184 adhesive and coagulant to the outer layer of the bonding area between PMDS and glass substrate and bake to cure.

[0015] (4) Installation and sterilization of microfluidic chip: Take the solidified microfluidic chip, insert one end of a stainless steel needle into its inlet and outlet, and connect the other end of the stainless steel needle to a medical transparent tube. The chip is then made. Before the microfluidic chip is put into operation, it needs to be sterilized. First, 75% alcohol is continuously introduced, and then the prepared and sterilized M9 culture medium is continuously introduced. The entire sterilization process is carried out in a clean room, and the clean room is in ultraviolet sterilization mode during the flow.

[0016] (5) Bacterial biofilm culture: When the microfluidic chip is working, close one inlet and continuously introduce mixed bacterial solution from the other inlet. After the bacteria are fixed in the culture chamber, stop introducing the solution and clamp the inlet tube with a stainless steel paperclip. Then open the other inlet and introduce the prepared and sterilized M9 culture medium. After continuously introducing the medium for a period of time, start calculating the culture time.

[0017] (6) Bottom in-situ observation: The microfluidic chip can directly perform high-resolution microscopic observation of the biofilm in the culture chamber from the bottom, and the chip structure perfectly fits the stage of the inverted fluorescence microscope; the microfluidic chip is also compatible with the observation stage of the high-content imaging analysis system Operetta CLS™, supporting the observation of biofilms with fluorescent labels and the generation of 3D structural diagrams.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) The height of the culture chamber inside the microfluidic chip is gradually reduced. The height of the culture chamber is lower than the height of the upper flow channel. At the same time, a channel with an even lower height is set on the lower side of the chamber to ensure that the bacteria are trapped in the chamber and are not easily carried away by the flow of the culture medium. Meanwhile, the culture medium can continuously flow through the chamber to provide nutrition for the bacteria.

[0020] (2) The glass substrate is made of very thin quartz glass, which is almost the same as the cover glass, so that the same effect can be achieved as observation from the cover glass. Unlike the observation from the traditional thick glass slide, better observation results can be obtained.

[0021] (3) The microfluidic chip can directly perform high-resolution microscopic observation of biofilms in the culture chamber from the bottom; this design does not require an additional cover slide on the PDMS layer, and the chip structure fits perfectly with the stage of the inverted fluorescence microscope, effectively avoiding the risk of the objective lens touching the chip when observing with an upright microscope, and significantly simplifying the chip fabrication and observation process; at the same time, the microfluidic chip is also compatible with the observation stage of the high-content imaging analysis system Operetta CLS™, supporting the observation of fluorescently labeled biofilms and the generation of 3D structural maps.

[0022] (4) The design of microfluidic chips takes into account both manufacturing simplicity and functionality. It is beneficial to prepare multi-layer molds through standard photolithography and perform PDMS replication molding. Moreover, due to its simple structure, it also reduces the PMDS thickness compared to other types of microfluidic chips.

[0023] (5) This invention simplifies the complex microfluidic chips on the market, changes the traditional observation method, removes the cover glass, makes the preparation of molds simpler and more convenient, and makes the preparation of microfluidic chips in the laboratory easier and more convenient. In addition, the microfluidic chip of this invention can also be compatible with the sampler and stage of the high-content imaging analysis system Operetta CLS™, making subsequent observation and quantitative experiments on biofilms more convenient. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the PDMS layer structure of the microfluidic chip of the present invention.

[0025] Figure 2 This is a cross-sectional view of the microfluidic chip of the present invention.

[0026] Figure 3 Images of biofilms cultured using the microfluidic chip of this invention, captured on the Operetta CLS™ high-content imaging analysis system; wherein, Figure 3 a and 3b represent the 2D biofilm structures of mixed cultures of E. coli WM3064 / pYYDT-AO-Psim-Gm-SPL-B0035-eGFP and P. aeruginosa PAO1-RFP under green and red fluorescence, respectively.

[0027] Figure 4 Images of biofilms cultured using the microfluidic chip of this invention, captured on the Operetta CLS™ high-content imaging analysis system; wherein, Figure 4 a and 4b show the 3D biofilm structures of a mixed culture of E. coli WM3064 / pYYDT-AO-Psim-Gm-SPL-B0035-eGFP and P. aeruginosa PAO1-RFP under green and red fluorescence.

[0028] Figure 1 , 2 The meanings of the reference numerals in the attached figures are as follows:

[0029] 1,2-Inlet, 3-Outlet, 4-Microchannel body, 5-Glass substrate, 6-Stainless steel needle, 7-Medical transparent tubing, 8-Cultivation chamber. Detailed Implementation

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

[0031] Example 1

[0032] Please see Figure 1 , 2 As shown, a microfluidic chip specifically designed for bacterial biofilm culture and in-situ bottom observation consists of a glass substrate 5 and a microchannel body 4 cast from polydimethylsiloxane (PDMS). After the two are bonded together with UV adhesive, a recessed micro-culture chamber 8 is formed inside the microchannel body 4 to immobilize bacteria and promote biofilm formation. The height of the culture chamber 8 gradually decreases from the inlet to the outlet, ensuring that the bacteria are trapped in the chamber and are not easily carried away by the flow of culture medium, while the culture medium can continuously flow through the chamber to provide nutrients for the bacteria.

[0033] In this embodiment, the heights of the culture chambers 8, which are distributed in a gradually decreasing manner, are 25~30μm, 15~20μm, and 0.1~0.5μm from the inlet to the outlet, respectively. The lower microchannel height connected to the outlet can restrict bacteria within the chamber while allowing continuous perfusion of the culture medium, thus providing a stable fluid environment for biofilm formation.

[0034] The culture chamber 8 has inlet ports 1 and 2 and outlet ports 3 at both ends. The two inlet ports are Y-shaped, one for bacterial culture and the other for culture medium; all liquids flow out through the outlet ports. Inlet ports 1 and 2 and outlet ports 3 are connected to the inside and outside of the microfluidic chip via stainless steel needles 6. A medical transparent tubing 7 is connected to the outer end of the stainless steel needle 6, and the opening and closing of the tubing is controlled by a stainless steel paperclip on the medical transparent tubing 7.

[0035] In this embodiment, the glass substrate 5 is made of quartz glass with a thickness of 1 to 2 times that of a standard cover glass, i.e., 0.17 to 0.35 mm.

[0036] Example 2

[0037] The fabrication and usage method of this microfluidic chip, specifically designed for bacterial biofilm culture and in-situ bottom observation, are as follows:

[0038] (1) Mold preparation: Customize the mold for PDMS casting according to the cavity molding parameters. Note that the mold material should not react with PDMS and that PDMS is easy to fall off after curing.

[0039] (2) PDMS casting: First, place the mold in ultrapure water for ultrasonic cleaning for 10-15 minutes, and then dry it with nitrogen or other laboratory safety gas. Mix Dow Corning 184 adhesive (Sylgard™ 184 silicone rubber) and coagulant at a volume ratio of 10:1, degas and then cast it into the chip mold. After drying and curing at 65°C for 2 hours, peel off the PDMS layer with microchannel structure from the mold and use a punch to make holes at the liquid inlet and outlet.

[0040] (3) Bonding of PMDS layer to glass substrate: Before bonding, immerse the PMDS layer and glass substrate in a 75% ethanol solution for cleaning, and then dry them with nitrogen. Use Dow Corning UV adhesive to bond the PMDS layer to the glass substrate. Place the bonded PMDS layer and glass substrate in a cleanroom, invert it so that the glass substrate faces upward, and irradiate with UV for 10-15 minutes to fully cure the UV adhesive. Then immerse it in PBS for 3-5 minutes to remove residual UV adhesive at the bonding interface. Finally, apply a mixture of Dow Corning 184 adhesive and curing agent at a volume ratio of 5:1 to the outer layer of the bonding area between PMDS and glass substrate, and bake at 65°C for 15 minutes to cure.

[0041] (4) Installation and sterilization of the microfluidic chip: Take the cured microfluidic chip and insert one end of a stainless steel needle into its inlet and outlet positions. Connect the other end of the stainless steel needle to a medical transparent tube. The chip is now complete. Before the microfluidic chip is put into operation, it needs to be sterilized. First, continuously flow through 75% alcohol for 30 minutes. Then, continuously flow through prepared and sterilized M9 culture medium for 30 minutes. The entire sterilization process is carried out in a cleanroom, and the cleanroom is in UV sterilization mode during the flow.

[0042] (5) Bacterial biofilm culture: When the microfluidic chip is working, close inlet 2 and continuously introduce a 1:1 mixture of E. coli WM3064 / pYYDT-AO-Psim-Gm-SPL-B0035-eGFP and P. aeruginosa PAO1-RFP bacterial solution through inlet 1 for 30 minutes. After the bacteria are fixed in the culture chamber, stop introducing the solution and close inlet 1 with a stainless steel paperclip. Then open inlet 2 and introduce the prepared and sterilized M9 medium for 30 minutes as the initial culture time.

[0043] (6) In-situ observation at the bottom: After 72 hours of incubation, the microfluidic chip was placed in the OperettaCLS™ high-content imaging analysis system for fluorescence imaging observation, such as... Figure 3 , 4 As shown.

[0044] Figure 3 a and 3b are 2D structures of the microfluidic chip obtained by single-layer scanning imaging on the Operetta CLS™ high-content imaging analysis system. They can clearly observe the distribution of single or multiple aggregated bacteria. The one with green fluorescence is E. coli WM3064 / pYYDT-AO-Psim-Gm-SPL-B0035-eGFP, and the one with red fluorescence is P. aeruginosa PAO1-RFP. The fluorescence can be used to determine the distribution and growth of the two bacteria within the microfluidic chip.

[0045] Figure 4 a and 4b are 3D structures of the microfluidic chip superimposed after multi-scanning on the Operetta CLS™ high-content imaging analysis system. The biofilms of E. coli WM3064 / pYYDT-AO-Psim-Gm-SPL-B0035-eGFP with green fluorescence and P. aeruginosa PAO1-RFP with red fluorescence can be clearly observed. The fluorescence can be used to determine the distribution and growth of the two bacterial biofilms within the microfluidic chip.

Claims

1. A microfluidic chip specifically designed for bacterial biofilm culture and in-situ bottom observation, characterized in that, It consists of a glass substrate (5) and a microchannel body (4) cast from polydimethylsiloxane (PDMS). After the two are bonded together with UV adhesive, a recessed micro-culture chamber (8) is formed in the microchannel body (4) to fix bacteria and promote biofilm formation. The two ends of the culture chamber (8) are designed with liquid inlet (1,2) and liquid outlet (3).

2. The microfluidic chip for bacterial biofilm culture and in-situ bottom observation as described in claim 1, characterized in that, The height of the culture chamber (8) gradually decreases from the inlet to the outlet, ensuring that the bacteria are trapped in the chamber and are not easily carried away by the flow of the culture medium, while the culture medium can continuously flow through the chamber to provide nutrients for the bacteria.

3. The microfluidic chip for bacterial biofilm culture and in-situ bottom observation as described in claim 2, characterized in that, The heights of the culture chambers (8) which are distributed in a gradually decreasing manner from the inlet to the outlet are 25~30μm, 15~20μm, and 0.1~0.5μm, respectively. The lower height of the microchannels connected to the outlet can restrict bacteria in the chamber while allowing continuous perfusion of culture medium, providing a stable fluid environment for biofilm formation.

4. The microfluidic chip for bacterial biofilm culture and in-situ bottom observation as described in claim 1, characterized in that, There are two inlets, arranged in a Y-shape. One inlet is for bacterial suspension only, and the other is for culture medium only. All liquids flow out through the outlet.

5. The microfluidic chip for bacterial biofilm culture and in-situ bottom observation as described in claim 4, characterized in that, The inlet (1,2) and outlet (3) are connected to the inside and outside of the microfluidic chip by stainless steel needles (6). A medical transparent tube (7) is connected to the outer end of the stainless steel needle (6). The opening and closing of the tube is controlled by a stainless steel paperclip on the medical transparent tube (7).

6. The microfluidic chip for bacterial biofilm culture and in-situ bottom observation as described in claim 1, characterized in that, The glass substrate (5) is made of quartz glass with a thickness of 0.17~0.35 mm.

7. A method for fabricating and using a microfluidic chip as described in any one of claims 1 to 6, specifically for bacterial biofilm culture and in-situ bottom observation, characterized in that, The steps are as follows: (1) Mold preparation: Customize the mold for PDMS casting according to the cavity molding parameters. It should be noted that the mold material should not react with PDMS and that PDMS is easy to fall off after curing. (2) PDMS casting: First, clean and dry the mold, mix Dow Corning 184 glue and coagulant evenly and degas it, then cast it into the chip mold. After drying and curing, peel off the PDMS layer with microchannel structure from the mold, and use a punch to punch holes at the liquid inlet and liquid outlet. (3) Bonding PMDS layer to glass substrate: Use Dow Corning UV adhesive to bond PMDS layer to glass substrate. Place the bonded PMDS layer and glass substrate in a cleanroom and invert it so that the glass substrate is facing up. Use UV irradiation to fully cure the UV adhesive. Then soak it in PBS to remove residual UV adhesive at the bonding interface. Finally, apply a mixture of Dow Corning 184 adhesive and coagulant to the outer layer of the bonding area between PMDS and glass substrate and bake to cure. (4) Installation and sterilization of microfluidic chip: Take the solidified microfluidic chip, insert one end of a stainless steel needle into its inlet and outlet, and connect the other end of the stainless steel needle to a medical transparent tube. The chip is then made. Before the microfluidic chip is put into operation, it needs to be sterilized. First, 75% alcohol is continuously introduced, and then the prepared and sterilized M9 culture medium is continuously introduced. The entire sterilization process is carried out in a clean room, and the clean room is in ultraviolet sterilization mode during the flow. (5) Bacterial biofilm culture: When the microfluidic chip is working, close one inlet and continuously introduce mixed bacterial solution from the other inlet. After the bacteria are fixed in the culture chamber, stop introducing the solution and clamp the inlet tube with a stainless steel paperclip. Then open the other inlet and introduce the prepared and sterilized M9 culture medium. After continuously introducing the medium for a period of time, start calculating the culture time. (6) Bottom in-situ observation: The microfluidic chip can directly perform high-resolution microscopic observation of the biofilm in the culture chamber from the bottom, and the chip structure perfectly fits the stage of the inverted fluorescence microscope; the microfluidic chip is also compatible with the observation stage of the high-content imaging analysis system Operetta CLS™, supporting the observation of biofilms with fluorescent labels and the generation of 3D structural diagrams.

Images