Bionic stomach micro-fluidic chip based on liquid drop micro-fluidic control and mechanical force stimulation and application of bionic stomach micro-fluidic chip

Abstract

The invention provides a bionic stomach micro-fluidic chip based on liquid drop micro-fluidic control and mechanical force stimulation and application of the bionic stomach micro-fluidic chip. The bionic stomach micro-fluidic chip comprises a liquid drop generation module and a mechanical force stimulation module. The liquid drop generation module is used for dispersing artificial stomach digestive juice and a to-be-digested Newtonian fluid sample in an oil phase by utilizing a micro-fluidic chip structure to form monodispersed water-in-oil liquid drops to simulate a discrete chyme group; and the mechanical force stimulation module is provided with a micro-channel with a periodically variable cross section. When liquid drops flow through the flow channel, the liquid drops are extruded and deformed periodically, a complex flow field is generated in the liquid drops, and the mechanical kneading effect of stomach wall wriggling on chyme balls is simulated. By limiting biochemical reaction in micro-droplets and applying controllable physical shear force, collaborative high-fidelity simulation of chemical and mechanical effects in the gastric digestion process is realized, and the problems that a traditional static model is low in simulation degree and large dynamic equipment is low in flux and high in cost are solved. And a brand new high-throughput research platform is provided for the fields of food and medicine.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics and in vitro digestion simulation, and particularly to a biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation, and a method for simulating gastric digestion in vitro using the biomimetic gastric microfluidic chip. Background Technology

[0002] In vitro digestion models are key tools for studying the digestion, absorption, and transformation of food and drugs in the gastrointestinal tract. Traditional static models simply mix and incubate digestive fluids with samples in test tubes, completely ignoring the dynamic physical processes in the gastrointestinal tract (such as peristalsis and shearing), resulting in poor correlation between experimental results and real-world conditions.

[0003] To improve the physiological relevance of in vitro digestive models, dynamic in vitro models such as TNO gastrointestinal models (TIM) have been developed. These systems use computers to control the parameters of multiple reaction chambers, resulting in high simulation fidelity. However, they are typically large in size, expensive, complex to operate, and have extremely low throughput, making them unsuitable for large-scale screening.

[0004] In recent years, microfluidic technology has been introduced into this field due to its advantages such as miniaturization and precise fluid control. Some existing microfluidic gastric chips mainly simulate peristalsis by driving fluid to flow in contoured channels through pumps, but they often fail to adequately simulate the localized, high-intensity mechanical shear forces experienced by discrete chyme boluses in the stomach.

[0005] Droplet microfluidics can generate monodisperse droplets at the nanoliter to microliter level, with each droplet acting as an independent microreactor. It offers advantages such as high throughput, low consumption, and no cross-contamination, and has been widely applied in fields such as single-cell analysis and chemical synthesis. However, current technologies have not been effectively applied to simulating digestion processes that involve both complex chemical microenvironments and dynamic physical fields. In particular, there is a lack of technical solutions that can accurately replicate the periodic mechanical kneading action of gastric peristalsis on the chyme bolus using droplet systems. This technical challenge remains unresolved.

[0006] Therefore, there is an urgent need in this field for a novel in vitro digestion platform that can integrate microreactors and highly biomimetic mechanical stimulation, while also possessing the advantage of high throughput. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the present invention aims to provide a biomimetic gastric microfluidic chip that simulates the synergistic effect of chemical environment and physical mechanical force during gastric digestion. This biomimetic gastric microfluidic chip simulates the digestive environment of a chyme bolus in the stomach by enclosing the reaction system within a droplet and applying controllable physical compression, thus solving the problems of distortion in mechanical force simulation and low throughput in existing models.

[0008] This biomimetic gastric microfluidic chip, based on droplet microfluidics and mechanical stimulation, can be used in fields such as food science, nutrition, and pharmacy to study the digestive behavior of food or drugs in the stomach.

[0009] To achieve the above objectives, the present invention provides a biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation, comprising: The droplet generation module is used to form monodisperse water-in-oil droplets by flowing the aqueous phase of artificial gastric digestive fluid and the sample to be digested into the oil phase. A mechanical stimulation module is located downstream of the droplet generation module. It has a variable cross-section flow channel inside, which is used to apply periodic mechanical compression to the flowing water-in-oil droplets to simulate the shear force caused by gastric peristalsis.

[0010] Furthermore, the droplet generation module is configured as any one of a flow focusing structure, a T-junction structure, or a coaxial focusing structure.

[0011] Furthermore, the variable cross-section flow channel in the mechanical force stimulation module is a periodic contraction and expansion flow channel, and its cross-sectional shape is any one of sawtooth, sine wave, square wave or dumbbell shape.

[0012] Furthermore, the minimum width of the contraction section of the periodic contraction-expansion channel is 0.1 to 0.9 times the droplet diameter.

[0013] This invention also provides a method for simulating gastric digestion in vitro using a biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation as described in any of the foregoing technical solutions, comprising the following steps: S1, using a fluid drive device, artificial gastric digestive fluid, a Newtonian fluid sample to be digested, and an oil phase containing surfactant are injected into the biomimetic gastric microfluidic chip respectively; the artificial gastric digestive fluid, the Newtonian fluid sample to be digested, and the oil phase containing surfactant are generated in the droplet generation module to form monodisperse water-in-oil droplets containing gastric digestive fluid and sample. S2, the water-in-oil droplets flow into the mechanical stimulation module and are subjected to periodic compression as they flow through the variable cross-section channel, generating shear force and mixing inside; S3, collect the droplets flowing out of the mechanical stimulation module, perform demulsification and analyze the digestion products.

[0014] Further, in step S1, the fluid driving device is a precision injection pump, a pneumatic pump, or a piezoelectric pump, which is configured to adjust the flow rate of each phase fluid to control the diameter of the water-in-oil droplets between 300 μm and 1000 μm.

[0015] Furthermore, in step S1, the diameter of the water-in-oil droplets is controlled to be between 300 μm and 1000 μm by adjusting the flow rate ratio of the water phase to the oil phase.

[0016] Furthermore, in step S2, the flow rate of the water-in-oil droplets into the mechanical stimulation module is 0.01 mL / min to 0.6 mL / min.

[0017] Further, in step S1, the Newtonian fluid sample to be digested is any one of a protein solution, a carbohydrate solution, a fat emulsion, or a simulated nutrient solution; the artificial gastric digestive fluid contains hydrochloric acid and pepsin, and the hydrochloric acid concentration is 0.01 mol / L to 0.1 mol / L, and the pepsin activity is 1000 U / mL to 3000 U / mL; the oil phase is a surfactant-containing oil phase, which is any one of mineral oil, silicone oil, or fluorinated oil, and the surfactant is one of Span 80, PGPR, or PFPE-PEG block copolymer.

[0018] Furthermore, in step S3, the demulsification method is centrifugal demulsification, organic solvent demulsification, or heating demulsification. After demulsification, the composition and content of the digestion product are analyzed by gel electrophoresis, high performance liquid chromatography, mass spectrometry, laser particle size analyzer, or ultraviolet-visible spectrophotometry.

[0019] The beneficial effects of this invention are: 1. The biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation provided by this invention mainly includes two core modules: a droplet generation module and a mechanical stimulation module. The mechanical stimulation module is located downstream of the droplet generation module and consists of a microchannel designed with a periodically changing cross-section (variable cross-section flow channel, such as serrated or wavy). This application utilizes the passive deformation of droplets in the variable cross-section flow channel to simulate gastric peristaltic shearing, a clever method with low energy consumption and high simulation accuracy.

[0020] 2. The droplet generation module of the biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation provided by this invention can generate tens of thousands of microreactors (droplets) in parallel, achieving true high-throughput screening with efficiency far exceeding that of traditional methods.

[0021] 3. This invention integrates a complex digestion environment onto a centimeter-sized chip, resulting in extremely low sample and reagent consumption.

[0022] 4. The biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation proposed in this application has flexible and wide-ranging applications. By changing the components of the aqueous and oil phases, it can be applied to Newtonian fluids such as milk and soy milk, and has the potential to be extended to the research of non-Newtonian fluids and pharmaceutical preparations. This application can control the droplet size by adjusting the flow rate of the two phases, and control the mechanical force intensity by adjusting the droplet flow rate, thereby achieving precise and controllable digestion conditions. Attached Figure Description

[0023] Figure 1 The diagram shows the structural design and working principle of Example 1; where A is a structural diagram of the biomimetic gastric microfluidic chip, B is a flow rate diagram of the gastric fluid phase and the soy milk phase, and C is a partial enlarged view of the mechanical stimulation module.

[0024] Figure 2 This is a physical image of the biomimetic gastric microfluidic chip in Example 1.

[0025] Figure 3 This is an SDS-PAGE polyacrylamide gel electrophoresis image of soy milk samples digested at different times in Example 1.

[0026] Figure 4 The graph shows the content of 7S and 11S globulins and their subtypes in soy milk samples with different digestion times in Example 1.

[0027] Figure 5 The microstructure changes of soy milk samples digested for different times in Example 1 are shown. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0030] Please see Figures 1 to 5 As shown, this invention provides a biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation, comprising: The droplet generation module is used to form monodisperse water-in-oil droplets by flowing the aqueous phase of artificial gastric digestive fluid and the sample to be digested into the oil phase. The droplet generation module can be configured as one of a flow focusing structure, a T-shaped structure, or a coaxial focusing structure.

[0031] The mechanical stimulation module is located downstream of the droplet generation module. It has a variable cross-section flow channel inside, which is used to apply periodic mechanical compression to the flowing water-in-oil droplets to simulate the shear force caused by gastric peristalsis.

[0032] Among them, the variable cross-section flow channel in the mechanical force stimulation module is a periodic contraction and expansion flow channel, and its cross-sectional shape is any one of sawtooth, sine wave, square wave or dumbbell shape.

[0033] The minimum width of the contraction section of the periodically contracting and expanding flow channel is 0.1 to 0.9 times the droplet diameter.

[0034] The biomimetic gastric microfluidic chip of the present invention, based on droplet microfluidics and mechanical stimulation, mainly comprises two core modules: Droplet generation module: Using standard microfluidic structures (such as flow focusing, T-junction), gastric digestive fluid containing pepsin hydrochloric acid solution and the Newtonian fluid sample to be digested (aqueous phase) are dispersed in an oil phase containing biocompatible surfactants to form monodisperse water-in-oil droplets. Each droplet is a miniature gastric digestion reaction unit, simulating a chyme bolus in the stomach.

[0035] Mechanical stimulation module: Located downstream of the droplet generation module, it consists of a microchannel with a periodically varying cross-section (such as serrated or wavy). When the droplet flows through this area under drive, it undergoes periodic compression-relaxation deformation, thereby inducing circulating flow and shear force within it, simulating the mechanical kneading effect of gastric wall peristalsis on the chyme bolus with high fidelity.

[0036] This invention also provides a method for simulating gastric digestion in vitro using a biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation, comprising the following steps: S1, using a fluid drive device, artificial gastric digestive fluid, a Newtonian fluid sample to be digested, and an oil phase containing surfactant are injected into the biomimetic gastric microfluidic chip respectively; the artificial gastric digestive fluid, the Newtonian fluid sample to be digested, and the oil phase containing surfactant generate monodisperse water-in-oil droplets containing gastric digestive fluid and sample in the droplet generation module. The artificial gastric digestive fluid contains hydrochloric acid and pepsin, with the hydrochloric acid concentration ranging from 0.01 mol / L to 0.1 mol / L and the pepsin activity ranging from 1000 U / mL to 3000 U / mL. The pH value of the artificial gastric digestive fluid is maintained between 2.0 and 3.0.

[0037] The Newtonian fluid sample to be digested is any one of the following: protein solution, carbohydrate solution, fat emulsion, or simulated nutrient solution, such as soy milk or milk.

[0038] The concentration of the oil phase containing surfactant was configured to maintain the stability of droplets during transport in the flow channel while facilitating the demulsification and recovery of subsequent digestion products.

[0039] The oil phase contains surfactants and can be any one of mineral oil, silicone oil, or fluorinated oil.

[0040] The surfactant is generally one of Span 80, PGPR or PFPE-PEG block copolymer, and its addition concentration is 0.5% to 5% (by mass volume).

[0041] The fluid drive device is a precision injection pump, piezoelectric pump, or pneumatic pump, which is configured to adjust the flow rate of each phase fluid to control the oil package, with water droplets having a diameter between 300 μm and 1000 μm.

[0042] In some embodiments, in step S1, the diameter of the water-in-oil droplets is controlled to be between 300 μm and 1000 μm by adjusting the flow rate ratio of the aqueous phase to the oil phase.

[0043] S2, the water-in-oil droplets flow into the mechanical stimulation module and are periodically squeezed as they flow through the variable cross-section channel, generating shear force and mixing inside; The frequency and intensity of the mechanical stimulation of the droplets are controlled by adjusting the flow rate of the water-in-oil droplets into the mechanical stimulation module, with the flow rate ranging from 0.01 mL / min to 0.6 mL / min.

[0044] In step S2, the deformation and internal flow field of the droplets are observed in real time using microscopic imaging technology, or the progress of the digestion reaction is monitored using online spectroscopy technology.

[0045] S3 collects droplets flowing out of the mechanical stimulation module, performs demulsification, and analyzes the digestion products.

[0046] The demulsification methods include centrifugal demulsification, organic solvent demulsification, or heating demulsification. After demulsification, the composition and content of the digestion products are analyzed by gel electrophoresis, high performance liquid chromatography, mass spectrometry, laser particle size analyzer, or ultraviolet-visible spectrophotometry.

[0047] This method is used for high-throughput screening, which involves simultaneously generating droplets of gastric digestive fluids and / or samples with different compositions or concentrations, and performing digestion experiments in parallel.

[0048] The present invention will now be described with reference to specific embodiments.

[0049] Example 1 Please see Figures 1 to 5As shown, a PDMS microfluidic chip (a bionic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation) was fabricated using a CNC machine tool. In this embodiment, a physical image of the bionic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation is shown below. Figure 2 As shown, it includes an upstream flow focusing structure (droplet generation module) and a downstream mechanical stimulation module. The mechanical stimulation module is a periodic sawtooth-shaped flow channel up to 3 cm long, with a contraction width of 550 μm, an expansion width of 1000 μm (approximately 1.8 times the width of the contraction), and a period length of 500 μm.

[0050] The minimum width of the contraction is 0.1-0.9 times the droplet diameter.

[0051] Solution preparation: The oil phase is dimethyl silicone oil containing 2% (w / w) Span 80 surfactant.

[0052] One aqueous phase was a simulated artificial gastric juice (SGF, containing pepsin, pH=3.0), and the other aqueous phase was a soy milk solution with a protein concentration of 20 mg / mL.

[0053] Two precision syringe pumps were used to drive the oil and water phases respectively. By adjusting the different flow rates of the oil and water phases, monodisperse and stable oil-encapsulated soy milk / gastric juice mixture droplets were successfully generated at the flow focal point.

[0054] Simulating the stomach digestion of soy milk: System preparation: 1. Oil phase: Dimethyl silicone oil containing 2% (w / w) Span 80 surfactant.

[0055] 2. Aqueous phase 1 (artificial gastric digestive fluid): simulated gastric juice (SGF), containing pepsin (from porcine gastric mucosa), dissolved in 0.03 M NaCl solution, and the pH was adjusted to 3.0±0.1 with 1 M HCl.

[0056] 3. Aqueous phase 2 (soy milk sample): Prepare a commercially available soy milk solution with a protein concentration of 20 mg / mL and filter to remove large particles.

[0057] 4. Inject the three-phase solutions into the syringe pump and connect them to the inlet A (oil phase), inlet B1 (gastric fluid phase), and inlet B2 (soy milk phase) of the biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical stimulation, respectively.

[0058] Digestion process: 1. Set the flow rates: oil phase flow rate: 0.4 mL / h; aqueous phase 1 (artificial gastric juice) flow rate: 0.1 mL / h; aqueous phase 2 (soy milk) flow rate: 0.1 mL / h. Under these conditions, droplets with a diameter of approximately 600 μm are generated.

[0059] In this embodiment, the driving pump for aqueous phase 1 is controlled by a programmable control unit (a computer with custom control software installed) via an RS-232 or USB interface. The control software is written in LabVIEW or Python and can generate and send complex flow rate commands as shown in Table 1 below.

[0060] Table 1. Flow rate commands for water phase 1 at different stages in Example 1 2. After droplet generation, the droplets flow into the mechanical stimulation module inside a 37°C constant temperature chamber. The overall average flow rate of the droplets is set to 0.5 mL / h. The droplets flow through the serrated flow channel at a frequency of approximately 2 seconds per cycle, are periodically squeezed, and flow out from the outlet of the bionic gastric microfluidic chip in a complete and stable droplet shape.

[0061] 3. The digestion reaction lasts for 120 minutes.

[0062] Sample collection and analysis: 1. Collect the emulsion from the outlet of the bionic gastric microfluidic chip at time intervals (every 10 minutes).

[0063] 2. Centrifuge the emulsion (10,000 rpm, 2 min), and after demulsification, take the lower aqueous phase for analysis.

[0064] 3. SDS-PAGE polyacrylamide gel electrophoresis was used to analyze the degree of hydrolysis and band changes of soybean proteins (such as β-conglycinin and glycinin).

[0065] Figure 3 SDS-PAGE polyacrylamide gel electrophoresis images of soy milk samples digested at different times.

[0066] Figure 4 Figure showing the content of 7S and 11S globulins and their subtypes in soy milk samples at different digestion times.

[0067] The results showed that in the early stages of digestion (0-30 minutes), the intensity of the intact subunits (such as the 75α, 75β, and 52 kDa bands) of the high-molecular-weight major storage proteins -7S (β-conglycinin) and 11S (glycinin) rapidly decreased, indicating that they were highly sensitive to pepsin hydrolysis. As digestion time increased to 60-120 minutes, these intact subunit bands almost completely disappeared.

[0068] Figure 4 The content of 7S and 11S globulins and their subtypes in soybean milk decreases continuously with digestion time, indicating that the digestion process proceeds smoothly.

[0069] 4. LSCM confocal microscopy observation: Proteins and lipids in the sample are stained, and the morphology and size distribution of protein aggregates are observed under a confocal microscope. Results are as follows: Figure 5 As shown.

[0070] Confocal microscopy staining results showed that as the digestion time increased from 0 minutes to 120 minutes, the fluorescence intensity of Fast Green-labeled proteins and Nile Red-labeled lipids gradually decreased, the number of particles significantly reduced, and the morphology tended to be more diffuse. This indicates that under simulated gastric digestion conditions, proteins and lipids continuously undergo enzymatic hydrolysis and decomposition, the original complex structure gradually dissociates, and the overall system gradually transforms from an initial large aggregate state into smaller, more dispersed digestive products.

[0071] In summary, compared with digestion under static conditions, milk digested by the biomimetic gastric microfluidic chip (droplet + mechanical stimulation) of this invention has a significantly higher degree of protein hydrolysis, and the SDS-PAGE spectrum shows that the protein bands disappear faster, proving that mechanical stimulation significantly promotes the digestion of milk proteins by pepsin, and verifying the effectiveness and biomimetic superiority of this system.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical force stimulation, characterized in that, include: The droplet generation module is used to form monodisperse water-in-oil droplets by flowing the aqueous phase of artificial gastric digestive fluid and the sample to be digested into the oil phase. A mechanical stimulation module is located downstream of the droplet generation module. It has a variable cross-section flow channel inside, which is used to apply periodic mechanical compression to the flowing water-in-oil droplets to simulate the shear force caused by gastric peristalsis.

2. The droplet microfluidics and mechanical force stimulation based biomimetic stomach microfluidic chip according to claim 1, characterized in that: The droplet generation module is configured as any one of a flow focusing structure, a T-shaped structure, or a coaxial focusing structure.

3. The droplet microfluidics and mechanical force stimulation based biomimetic gastric microfluidic chip according to claim 1, characterized in that: The variable cross-section flow channel in the mechanical force stimulation module is a periodic contraction and expansion flow channel, and its cross-sectional shape is any one of sawtooth, sine wave, square wave or dumbbell shape.

4. The biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical force stimulation according to claim 3, characterized in that: The minimum width of the contraction section of the periodic contraction-expansion channel is 0.1 to 0.9 times the droplet diameter.

5. The use of a biomimetic gastric microfluidic chip based on droplet microfluidics and mechanical force stimulation, characterized in that, In vitro gastric digestion was simulated using the biomimetic gastric microfluidic chip according to any one of claims 1 to 4. Includes the following steps: S1, using a fluid drive device, artificial gastric digestive fluid, a sample of Newtonian fluid to be digested, and an oil phase containing surfactant are injected into the biomimetic gastric microfluidic chip respectively; Artificial gastric digestive fluid, a Newtonian fluid sample to be digested, and an oil phase containing surfactant are used in the droplet generation module to generate monodisperse water-in-oil droplets containing the gastric digestive fluid and the sample. S2, the water-in-oil droplets flow into the mechanical stimulation module and are subjected to periodic compression as they flow through the variable cross-section channel, generating shear force and mixing inside; S3, collect the droplets flowing out of the mechanical stimulation module, perform demulsification and analyze the digestion products.

6. The use of the droplet microfluidics and mechanical force stimulation based biomimetic gastric microfluidic chip according to claim 5, characterized in that: In step S1, the fluid driving device is a precision injection pump, a pneumatic pump, or a piezoelectric pump, which is configured to adjust the flow rate of each phase fluid to control the diameter of the water-in-oil droplets between 300 μm and 1000 μm.

7. The use of the droplet microfluidics and mechanical force stimulation based biomimetic gastric microfluidic chip according to claim 5, characterized in that: In step S1, the diameter of the water-in-oil droplets is controlled between 300 μm and 1000 μm by adjusting the flow rate ratio of the water phase to the oil phase. 8.The application of the biomimetic stomach microfluidic chip based on droplet microfluidics and mechanical force stimulation according to claim 5, wherein, In step S2, the flow rate of the water-in-oil droplets into the mechanical stimulation module is 0.01 mL / min to 0.6 mL / min. 9.The application of the biomimetic stomach microfluidic chip based on droplet microfluidics and mechanical force stimulation according to claim 5, characterized in that: In step S1, the Newtonian fluid sample to be digested is any one of a protein solution, a carbohydrate solution, a fat emulsion, or a simulated nutrient solution; the artificial gastric digestive fluid contains hydrochloric acid and pepsin, and the hydrochloric acid concentration is 0.01 mol / L to 0.1 mol / L, and the pepsin activity is 1000 U / mL to 3000 U / mL; the oil phase is a surfactant-containing oil phase, which is any one of mineral oil, silicone oil, or fluorinated oil, and the surfactant is one of Span 80, PGPR, or PFPE-PEG block copolymer.

10. The use of the droplet microfluidics and mechanical force stimulation based biomimetic gastric microfluidic chip according to claim 5, characterized in that: In step S3, the demulsification method is centrifugation, organic solvent demulsification, or heating demulsification. After demulsification, the composition and content of the digestion product are analyzed by gel electrophoresis, high performance liquid chromatography, mass spectrometry, laser particle size analyzer, or ultraviolet-visible spectrophotometry.

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