Nanofiber filled micro-fluidic chip as well as preparation method and application thereof

Abstract

The invention discloses a nanofiber filled micro-fluidic chip and a preparation method and application thereof, the preparation method comprises the following steps: 1) uniformly mixing a prepolymer A of PDMS and a curing agent B to obtain a solution C; (2) putting a prefabricated mold into a container, and pouring the solution C into the container; and 3) standing the container for a period of time, heating and curing, demolding, punching and bonding to obtain the PDMS micro-fluidic chip. And 4) sterilizing the PDMS micro-fluidic chip, inoculating the microchannel of the PDMS micro-fluidic chip with the bacterial liquid for fermentation, performing in-situ synthesis to fill the nanofiber, flushing the microchannel with a NaOH solution, and rinsing with deionized water until the pH value is neutral, thereby obtaining the nanofiber filled micro-fluidic chip. According to the invention, PDMS with air permeability is adopted to manufacture a microfluidic channel, in-situ filling in the microchannel is carried out through bacterial cellulose, a nanofiber network with high filling efficiency is formed, and separation of a target object is rapid and efficient.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip manufacturing technology, and in particular to a nanofiber-filled microfluidic chip, its preparation method, and its application. Background Technology

[0002] Microfluidic chips, characterized by their small size, compact structure, high integration, low sample and reagent consumption, and ease of operation, are widely used in environmental monitoring, chemical analysis, and biomedicine. Furthermore, the micron-scale channels of microfluidic chips are ideal for applications such as microparticle separation and purification. However, when traditional solid structures are used for capture and separation, the solid-liquid boundary effect limits the separation and enrichment efficiency. Literature reports the use of carbon nanotubes, silicon particles, and polydimethylsiloxane (PDMS) frameworks as filling materials within microfluidic channels. However, most currently available filled microfluidic chips suffer from limited fill rates and complex fabrication. Therefore, developing a microfluidic chip with high specific surface area, strong selectivity, and high capture efficiency is of great significance for separation and analysis applications. Summary of the Invention

[0003] In view of the above-mentioned defects or deficiencies in the prior art, it is desirable to provide a nanofiber-filled microfluidic chip, its preparation method and application, which uses breathable PDMS to make microfluidic channels, and uses bacterial cellulose to fill the microchannels in situ to form a nanofiber network with high filling efficiency, which can quickly and efficiently separate the target analytes.

[0004] The present invention provides a method for fabricating a nanofiber-filled microfluidic chip, comprising the following steps: 1) Mix PDMS prepolymer A and curing agent B evenly to obtain solution C; 2) Place the pre-made mold into the container, and then pour the solution C into the container; 3) After the container is left to stand for a period of time, it is heated and cured, and then demolded, punched, and bonded to obtain a PDMS microfluidic chip; 4) The PDMS microfluidic chip is sterilized, bacterial culture is inoculated into the microchannel of the PDMS microfluidic chip for fermentation, and nanofibers are synthesized in situ. Then, the microchannel is rinsed with NaOH solution and rinsed with deionized water until the pH value is neutral to obtain the nanofiber-filled microfluidic chip.

[0005] Furthermore, in step 1), the PDMS is Sylgard 184 or SE1700, and the mass ratio of prepolymer A to curing agent B is 2-16:1.

[0006] Furthermore, in step 2), the mold is formed by laser engraving of an acrylic sheet or glass sheet, with a laser engraving intensity of 5%-45%, a laser engraving speed of 10-200 mm / s, a thickness of 2.0-5.0 mm, and a laser engraving depth of 1.0-4.0 mm; the thickness of the solution C on the mold is 1.0-2.0 mm. Furthermore, in step 3), the PDMS microfluidic chip is formed by bonding an upper substrate and a lower substrate, and includes several parallel microchannels. One end of each microchannel is provided with an inlet, and the other end is provided with an outlet. Several fixing posts are equally spaced along the length of each microchannel. The radius of the inlet and outlet is 1.0-1.8 mm, the length of the microchannel is 5.0-30.0 mm, the width is 0.5-3.0 mm, and the height is 1.0-4.0 mm. The radius of the fixing posts is 0.2-0.3 mm, the number is 3-15, and the spacing is 1.0-2.0 mm.

[0007] Furthermore, in step 3), the container is left to stand at 20-25℃ for 2-12 hours; the heating curing temperature is 60-75℃, and the heating curing time is 4-6 hours.

[0008] Furthermore, in step 4), the microorganisms used for fermentation to prepare bacterial cellulose in the bacterial solution are selected from one of the genera *Acetobacter*, *Agrobacterium*, *Pseudomonas*, *Nitrogenobacter*, *Rhizobium*, or *Cladophora*, the fermentation temperature is 28-30℃, and the culture time is 1-7 days.

[0009] Furthermore, in step 4), the microchannel is rinsed with 0.1M NaOH solution at a flow rate of 0.5-2 mL / min for 10 min; the deionized water is rinsed at a flow rate of 0.5-2 mL / min.

[0010] Furthermore, in step 4), the prepared nanofibers are filled with microfluidic chips and doped with functional nanoparticles in situ or synthesized and then modified with corresponding functional groups in situ.

[0011] In addition, the present invention also provides a nanofiber-filled microfluidic chip prepared by the above-described method for preparing nanofiber-filled microfluidic chips.

[0012] In addition, the present invention also provides an application of the nanofiber-filled microfluidic chip as described above for the separation and enrichment of target substances in gas or liquid.

[0013] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention utilizes the characteristic of bacterial cellulose synthesis at the gas-liquid interface, and mediates the in-situ self-assembly of bacterial cellulose through polydimethylsiloxane (PDMS) breathable material to synthesize nanofiber-filled microfluidic chips in one step. The porosity of the filling material is adjustable and can be modified for multiple functions. The porosity can be controlled by changing the number of cultivation days; the functionality can be achieved through in-situ synthesis of fibers or chemical, physical, and biological modifications after fiber synthesis, making it suitable for the separation of different target substances. This nanofiber-filled microfluidic chip can be applied to the separation and enrichment of target substances in gases or liquids.

[0014] (2) The nanofiber-filled microfluidic chip prepared by the present invention has the advantages of being modifiable, having a high specific surface area, avoiding solid-liquid interface effects, and greatly improving the capture efficiency of target substances. It can be used for the separation of target substances in air, blood, and urine, and has extremely high application value in pollution detection, food safety, and disease diagnosis.

[0015] (3) The nanofiber-filled microfluidic chip prepared by the present invention is simple to manufacture, low in cost, green and environmentally friendly, and easy to mass-produce, and has extremely high promotion value.

[0016] (4) The nanofiber-filled microfluidic chip prepared by this invention can be used in conjunction with mass spectrometry for rapid analysis and detection. It is simple, fast and efficient, and suitable for point-of-care testing devices.

[0017] It should be understood that the description in the Summary of the Invention is not intended to limit the key or essential features of the embodiments of the present invention, nor is it intended to restrict the scope of the invention. Other features of the invention will become readily apparent from the following description. Attached Figure Description

[0018] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 A flowchart for fabricating nanofiber-filled microfluidic chips; Figure 2 This is a structural schematic diagram of one embodiment of the mold; Figure 3 Figure a shows a schematic diagram of a nanofiber-filled microfluidic chip structure, and Figure b shows a schematic diagram of local capture. Figure 4 Figure a shows a physical image of the bacterial cellulose-filled microfluidic chip in Example 1; Figure b shows a physical image of the TiO2-bacterial cellulose-filled microfluidic chip in Example 2. Figure 5 This is an electron microscope image of the TiO2-bacterial fiber composite material in Example 2; Figure 6Fluorescence capture image of cell vesicles in the TiO2-bacterial cellulose-filled chip in Example 3; Figure 7 This is a mass spectrometry analysis of cell vesicles separated from the TiO2-bacterial cellulose-filled chip in Example 4.

[0019] Labels in the figure: 1. Nanofiber-filled microfluidic chip; 11. Microchannel; 12. Inlet; 13. Outlet; 14. Fixed column. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0022] Example 1: Fabrication of a microfluidic chip filled with bacterial cellulose Please refer to Figures 1-3 The fabrication of microfluidic chips includes the following steps: 1) Design the microfluidic chip structure; The microfluidic chip consists of two layers, both made of polydimethylsiloxane (PDMS) substrate. The microfluidic chip includes three parallel microchannels 11. One end of each microchannel 11 has an inlet 12, and the other end has an outlet 13. Ten fixed posts 14 are evenly spaced along the length of each microchannel 11. The inlet and outlet have a radius of 1.8 mm. The microchannel has a length of 20.0 mm, a width of 2.5 mm, and a height of 2.0 mm. The fixed posts have a radius of 0.25 mm and a spacing of 1.5 mm.

[0023] 2) Precast molds; A 2.0mm thick acrylic sheet was selected, and the mold was laser-engraved according to the structure of the microchannel 11 using an engraving light intensity of 25% and an engraving speed of 45mm / s. 3) Fabrication of microfluidic chips; First, weigh out component A and component B of Sylgard 184 at a mass ratio of 10:1 and pour them into a beaker. Stir thoroughly with a glass rod for about 2 minutes to ensure that the mixture is uniform, and obtain solution C. Then, the vacuum pump was used to evacuate the solution and let it stand for 30 minutes to remove the air bubbles in solution C. Then, it was poured into a petri dish containing the mold and left to stand at room temperature for 4 hours. Then, the culture dish was placed in a 65℃ oven for 4 hours to heat and cure. After the culture dish was removed and cooled, it was demolded, punched, and bonded to obtain the PDMS microfluidic chip. The PDMS microfluidic chip was wrapped, sterilized, and dried at low temperature (65℃, 4h) for later use. Freshly prepared bacterial solution (inoculum: HS medium = 1:14) was added to the microchannels of the PDMS microfluidic chip. The chip was then incubated at 30℃ for 1-7 days to allow bacterial cellulose to synthesize within the microchannels. The microchannels were flushed with 0.1M NaOH solution at 1mL / min for 20min, followed by rinsing with deionized water at 1mL / min until the pH was neutral. The resulting microfluidic chip was filled with bacterial cellulose, as shown in the diagram. Figure 4 As shown in Figure a.

[0024] Example 2: Fabrication of TiO2-bacterial cellulose-filled microfluidic chip Please refer to Figures 1-3 The fabrication of microfluidic chips includes the following steps: 1) Design the microfluidic chip structure; Same as in Example 1; 2) Precast molds; Same as in Example 1; 3) Fabrication of microfluidic chips; Compared with Example 1, the difference in this example is that: freshly prepared bacterial solution (inoculum: HS medium = 1:14) and sterilized TiO2 nanoparticles are added into the microchannel of the PDMS microfluidic chip. The diameter of the TiO2 nanoparticles is 10-100 nm and the concentration is 0.1-2 mg / mL; TiO2-bacterial fiber composite material is synthesized in the microchannel. The final TiO2-bacterial cellulose-filled microfluidic chip is shown in the figure below. Figure 4 As shown in Figure b.

[0025] Example 3: Characterization and Test Result Analysis (1) Porosity test Based on microscopic images of bacterial cellulose taken by super-resolution field emission scanning electron microscopy (FE-SEM) from day 1 to day 5, ImageJ was used to calculate the daily porosity of bacterial cellulose, and the optimal number of days for bacterial cellulose growth was characterized.

[0026] (2) Scanning electron microscopy analysis TiO2-bacterial cellulose samples were characterized using a Verios 460L scanning electron microscope from FEI at a scanning voltage of 5 kV. The sample surface was sputter-coated with gold. Figure 5 As shown, a composite material uniformly doped with TiO2 and bacterial cellulose was synthesized in situ within a microchannel.

[0027] Example 4: Cell Vesicle Capture Experiment Take the cell culture medium supernatant and centrifuge it three times in gradients (300g, 10min), (2000g, 10min), and (10000g, 30min). After each centrifugation, take the supernatant and centrifuge it again. Then filter it through a 0.22μm filter membrane and store it at 4℃ for later use.

[0028] The treated cell supernatant is injected into the microchannel of the microfluidic chip at a rate of 100-300 μL / min, preferably 200 μL / min, with an injection volume of 10 mL, so that the cell vesicles in the cell supernatant are captured by titanium dioxide on the nanofibers filling the microchannel.

[0029] Staining was performed using DiI staining dye, and imaging was performed using a fluorescence microscope, such as... Figure 6 As shown, cell vesicles are captured within the microchannels. The enriched cell vesicles are then eluted using an eluent, which includes, but is not limited to, ammonia monohydrate (NH3·H2O), acetonitrile, methanol, or combinations thereof, at a flow rate of 100-300 μL / min, preferably 150 μL / min.

[0030] Example 5: Mass Spectrometry Detection and Analysis Experiment The cell vesicles prepared in Example 4 were injected into a Shimadzu LCMS-8045 liquid chromatography-mass spectrometry system to observe the composition of the cell vesicles. The results are as follows: Figure 7 The image shown is a mass spectrum of cell vesicles, demonstrating that the microfluidic chip of this application successfully detected small molecules of metabolites from cell vesicles in the cell supernatant.

[0031] Bacterial cellulose is a three-dimensional network of nanofibers secreted by bacteria. It has the characteristics of controllable self-assembly, chemical inertness, high porosity, high mechanical strength and hydrophilicity. It can completely solve the solid-liquid boundary effect in microchannels and is the preferred material for preparing filled microfluidic chips. Furthermore, this fiber can be functionalized through in-situ doping with nanoparticles and chemical reactions, making it suitable for the capture and separation of various target analytes. The nanofiber-filled microfluidic chip prepared by this invention can be multifunctionally modified, possessing advantages such as high specific surface area and high capture sites, making it suitable for the capture and separation of high-throughput particles. It can be applied to the separation and enrichment of pathogenic particles, cell vesicles, proteins, DNA, and other biological particles and macromolecules from air, urine, tears, blood, and cell supernatants, and has significant application value in fields such as biological particle separation and disease diagnosis.

[0032] In the description of this specification, the terms "one embodiment," "some embodiments," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0033] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for fabricating a nanofiber-filled microfluidic chip, characterized in that, It includes the following steps: 1) Mix PDMS prepolymer A and curing agent B evenly to obtain solution C; 2) Place the pre-made mold into the container, and then pour the solution C into the container; 3) After the container is left to stand for a period of time, it is heated and cured, and then demolded, punched, and bonded to obtain a PDMS microfluidic chip; 4) The PDMS microfluidic chip is sterilized, bacterial culture is inoculated into the microchannel of the PDMS microfluidic chip for fermentation, and nanofibers are synthesized in situ. Then, the microchannel is rinsed with NaOH solution and rinsed with deionized water until the pH value is neutral to obtain the nanofiber-filled microfluidic chip.

2. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 1), the PDMS is Sylgard 184 or SE1700, and the mass ratio of prepolymer A to curing agent B is 2-16:

1.

3. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 2), the mold is an acrylic sheet or glass sheet formed by laser engraving. The laser engraving intensity is 5%-45%, the laser engraving speed is 10-200mm / s, the thickness of the acrylic sheet or glass sheet is 2.0-5.0mm, and the laser engraving depth is 1.0-4.0mm; the thickness of the solution C on the mold is 1.0-2.0mm.

4. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 3), the PDMS microfluidic chip is formed by bonding an upper substrate and a lower substrate, and includes several parallel microchannels. One end of each microchannel is provided with an inlet and the other end with an outlet. Several fixing posts are evenly spaced along the length of each microchannel. The radius of the inlet and outlet is 1.0-1.8 mm, the length of the microchannel is 5.0-30.0 mm, the width is 0.5-3.0 mm, and the height is 1.0-4.0 mm. The radius of the fixing posts is 0.2-0.3 mm, the number is 3-15, and the spacing is 1.0-2.0 mm.

5. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 3), the container is left to stand at 20-25℃ for 2-12 hours; the heating curing temperature is 60-75℃ and the heating curing time is 4-6 hours.

6. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 4), the microorganisms used for fermentation to prepare bacterial cellulose in the bacterial solution are selected from one of the genera Acetic Acid Bacteria, Agrobacterium, Pseudomonas, Nitrogenobacteria, Rhizobium, or Cladophora. The fermentation temperature is 28-30℃ and the culture time is 1-7 days.

7. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 4), the microchannel is rinsed with 0.1M NaOH solution at a flow rate of 0.5-2 mL / min for 10 min; the deionized water is rinsed at a flow rate of 0.5-2 mL / min.

8. The method for preparing a nanofiber-filled microfluidic chip according to claim 1, characterized in that, In step 4), the prepared nanofibers are filled with microfluidic chips and doped with functional nanoparticles in situ or synthesized and then modified with corresponding functional groups in situ.

9. A nanofiber-filled microfluidic chip prepared by the method of any one of claims 1-8.

10. An application of the nanofiber-filled microfluidic chip as described in claim 8 for the separation and enrichment of target substances in gas or liquid.

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