An in vitro dynamic intestinal digestion device in combination with a hydrogel and methods of use

Abstract

The application discloses an in-vitro dynamic intestinal digestion device combined with a hydrogel and a use method, and belongs to the technical field of in-vitro simulation digestion experimental devices.The device provided by the application completely simulates four intestinal segments of a duodenum, a jejunum, an ileum and a colon, is constructed according to real anatomical proportions, adopts a biocompatible hydrogel material, and has a bionic villus structure on an inner wall, so that a human intestinal physiological environment and absorption characteristics can be accurately reproduced; by regulating and controlling a hydrogel pore size, the device can reflect permeability differences of the intestinal segments and methods, can be used for tracing conversion and absorption behaviors of common metal forms in the environment in the whole intestinal digestion process, and can explore the harmfulness of the metal forms to a biological intestinal tract. The device integrates a 3D printing intestinal model and a functional module, while guaranteeing data reliability, greatly simplifies an operation process, improves experimental reproducibility, reduces artificial cost, and provides a more physiological real and efficient tool for intestinal migration and conversion research of metal forms.

Description

Technical Field

[0001] This invention belongs to the technical field of in vitro simulated digestion experimental devices, and particularly relates to an in vitro dynamic intestinal digestion device combined with hydrogel and its usage method. Background Technology

[0002] With the promotion of the 3Rs (Replacement, Reduction, and Improvement) principle and the pursuit of better control and reproducibility, in vitro models have gradually become the mainstream choice for research. The design of in vitro gastrointestinal models varies widely, mainly depending on the specific research or application objectives. To characterize the metabolism and bioavailability of heavy metals in the gastrointestinal tract, researchers have developed various in vitro simulation systems. Static models, with their advantages of speed, low labor intensity, and fewer bioethical and logistical constraints, are widely used to assess the bioavailability of orally administered metal forms. For example, this method has been used to study the oral vanadium form (V... IV and V V This includes studies on the transfer and digestion of metal-loaded microplastics, as well as the bioavailability of chromium adsorbed on microplastics (Science of the Total Environment. 2020. 703. 134805). Static models simulate the intestinal environment using constant variables to predict digestive behavior, including the ratio of food to enzymes, electrolytes, and pH at each stage of digestion. However, these models are too simplistic to simulate the fluid dynamics and regional dynamics of intestinal digestion, making them more suitable for specific applications such as screening, preliminary studies, and hypothesis development. Dynamic models, on the other hand, are conducted in rigid containers or glass sleeves with flexible inner walls, allowing for adjustments to pH and enzyme levels to promote digestion and absorption. For example, studies have reported using dynamic models to investigate the transfer and digestion of metal-loaded microplastics, finding significant differences compared to static models. Dynamic models have a unique advantage in simulating the bioavailability of heavy metals (Science of the Total Environment. 2011. 409. 604–611).

[0003] Due to significant differences in intestinal digestive conditions among different species and individuals (such as time, secretion, digestive enzymes, flora, and pH), coupled with ethical limitations, in vivo models are not suitable as a universal method for digestive research.

[0004] Chinese patent CN207619409U discloses an in vitro digestive system simulation device that uses a glass stirring jar to simulate the gastrointestinal tract. However, the model does not reflect the fine structure of the villi (such as intestinal villi) and their true geometry on the inner wall of the small intestine, nor the permeability differences in different parts of the small intestine, thus failing to realistically simulate the absorption function of the small intestine. Rigid containers (such as beakers and test tubes) cannot realize the true dynamics of the digestive tract, and there are no suitable pretreatment methods (including digestive and absorptive products) for analyzing the metal speciation to study the migration and transformation of metal speciation in the intestine (including the small and colon). Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an in vitro dynamic intestinal digestion simulation device and method based on polyacrylamide-sodium alginate double crosslinked network hydrogel (PAMSA). This device can be used to track the transformation and absorption behavior of common metal speciations in the environment throughout the intestinal digestion process and to explore their harmfulness to the intestinal tract. This invention combines an in vitro intestinal model with a multidimensional sampling-preprocessing module to build a dynamic intestinal simulation system suitable for the in vitro digestion of metal speciations. Combined with chromatography-ICPMS, it enables online real-time monitoring of the transformation and absorption of metal speciations in the intestine. By incorporating 3D printing to fabricate the intestine, sample loading interface, pH adjustment, temperature control, pressure control, absorption pipeline, sampling and preprocessing modules, it reproduces the actual anatomical structure, physical environment, and biochemical conditions of the biological intestine. Furthermore, a LabVIEW-based program is used to control the automated operation of digestion, absorption, sampling, and preprocessing. Intestinal extracts containing metals generated at different times and locations can be directly introduced into chromatography (including HPLC and capillary electrophoresis (CE))-ICPMS for speciation analysis after pretreatment. This effectively eliminates matrix interference and reduces operator intervention, providing a reliable tool for sensitive and accurate analysis of intestinal digestive behavior and its harm to the intestines after ingestion of metals.

[0006] An in vitro dynamic intestinal digestion device combined with hydrogel includes an electronic control unit, a hydrogel biomimetic intestine, a temperature control unit, a gas control unit, a reagent addition unit, an intestinal motility unit, and a pH control unit.

[0007] The electronic control unit includes a computer, an Arduino development board, a solenoid valve, an AC-DC switching power supply, a miniature peristaltic pump 15, and connecting wires. It uses LabVIEW and Arduino in combination to achieve semi-automatic computer control. The miniature peristaltic pump 15 is fixed on the two front door panels of the constant temperature chamber 16. One side is used for reagent injection, which is the sample injection miniature peristaltic pump. The other side is used for sampling, which is the sampling miniature peristaltic pump.

[0008] The hydrogel bionic intestine is prepared by molding. The inner wall of the hydrogel bionic intestine is provided with a raised bionic villous structure, and it is divided into four segments along the in vitro digestion process: duodenal segment 2, jejunal segment 7, ileal segment 10 and colonic segment 12.

[0009] The temperature control unit includes a constant temperature chamber 16, an electric heating constant temperature baking lamp 14, and a temperature sensor 11, which are used to regulate the digestive environment temperature of the device. The electric heating constant temperature baking lamp 14 is located on the two front door panels of the constant temperature chamber 16, and is fixed above or below the micro peristaltic pump 15. The temperature sensor 11 is fixed near the ileum segment 10.

[0010] The gas control unit includes an argon cylinder, a rotor flow meter, two solenoid valves and a silicone tube. One end of one solenoid valve is connected to the air inlet through the silicone tube, and the other end is connected to the rotor flow meter and the argon cylinder in sequence, for introducing argon into the hydrogel bionic intestine. The other solenoid valve is connected to the exhaust pipe of the exhaust port, for venting the air in the hydrogel bionic intestinal cavity.

[0011] The reagent addition unit includes a sample inlet micro-peristaltic pump, a silicone tube, and a storage bottle. The silicone tube connects the sample inlet micro-peristaltic pump and the reagent addition port 6, and is used to deliver gastric chyme, simulated intestinal fluid (SIF), microbial inoculum, and receiving fluid. Each segment of the intestine corresponds to a sampling port 5, which has four openings: top, bottom, left, and right. The horizontal openings of the sampling ports 5 in the duodenal segment 2, jejunal segment 7, and ileal segment 10 are connected sequentially to a primary filter 9, a micro-peristaltic pump 15, a secondary filter 18, and a quaternary filter 19 via silicone tubes. The sampling port 5 in the colon segment 12 is connected sequentially to a primary filter 9, a tertiary filter 13, a micro-peristaltic pump 15, and a quaternary filter 19 via silicone tubes. The two ports on the right side of the secondary filter 18 are used for reverse flushing. The other end of the horizontal opening of the sampling port 5 is connected to a one-way valve 8 and the reagent addition port 6 via silicone tubes, and then enters the next segment of the intestine. The sampling port 5 has vertical openings at the top and bottom, which are connected to the micro-peristaltic pump 15 via silicone tubes to collect the absorption fluid and digestive fluid.

[0012] The intestinal motility unit includes a fixed wheel 3, an eccentric wheel 4, a positioning shaft 17, a gear 20, and a stepper motor 21. The central shaft of the stepper motor 21 is connected to the 3D-printed eccentric wheel 4. The eccentric wheel 4 and the gear 20 are located on the front and back sides of the intestine, respectively. The eccentric wheel 4 and the gear 20 are coaxially connected through the positioning shaft 17. In the vertical plane on the front side of the intestine, the eccentric wheel 4 is located above the intestine, the fixed wheel 3 is located below the intestine, and the stepper motor 21 is located in the vertical plane on the back side of the intestine. Through gear transmission, a small number of stepper motors 21 control the rotation of a large number of eccentric wheels. The intestinal motility unit promotes the mixing and absorption of digestive products by squeezing the intestinal lumen to simulate intestinal peristalsis and segmented movement, and controls the food transport time to be consistent with the body.

[0013] The pH control unit includes a pH meter, a pH adjusting solution (acid / base), and a feeding port, and adds the pH adjusting solution via a micro peristaltic pump;

[0014] The pretreatment unit includes a primary filter 9, a secondary filter 18, a tertiary filter 13, and a quaternary filter 19; the primary filter 9 is a ceramic filter with a pore size of 300μm; the secondary filter 18 is a ceramic filter with a pore size of 5μm; the tertiary filter 13 is an absorbent sponge; and the quaternary filter 19 is an aqueous needle-type filter membrane with a pore size of 0.22μm.

[0015] The device is used as follows: The electric heating constant temperature baking lamp 14 and temperature sensor 11 are turned on to maintain the temperature of the entire constant temperature chamber 16 at 37±1℃. The mixture that has passed through the stomach digestion stage is input into the device through the sample inlet 1 to begin the small intestine digestion stage. Simulated intestinal fluid (SIF) is added using a sample injection micro-peristaltic pump, and receiving fluid is added through the reagent addition port 6. The mixture passes sequentially through the duodenal segment 2, jejunal segment 7, ileal segment 10, and colonic segment 12. Driven by the stepper motor 21, the eccentric wheel 4 and fixed wheel 3 promote the mixing and absorption of digested matter by squeezing the intestinal lumen to simulate intestinal peristalsis and segmental movement, and control food transport. The timing is consistent with in vivo. Samples digested from the duodenum (2), jejunum (7), and ileum (10) have high water content and, after passing through primary filter (9), can sequentially enter secondary filter (18) and quaternary filter (19). After digestion of the ileum, the micro-peristaltic pump is activated to introduce microbial nutrient culture medium into the colon (12) model. Since the colon digested sample has low water content, a tertiary filter (13) is placed after primary filter (9) to absorb the sample solution, which then enters quaternary filter (19). All absorbed samples, since they contain no food residue, can directly enter the quaternary filter for subsequent morphological analysis.

[0016] Further, the fabrication process of the hydrogel biomimetic intestine is as follows: First, sodium alginate (SA) and acrylamide (AAM) are added to deionized water at a mass ratio of (1:4)-(1:10), and the water content is fixed at 80wt%-86wt%. The mixture is stirred until completely dissolved to ensure that the system is uniform and free of particle precipitation. Then, ammonium persulfate and N,N'-ethylenebisacrylamide (MBAA) are added sequentially and stirred until dissolved to obtain a mixture. The mixture is then degassed under vacuum in an ice bath for 5-10 minutes. N,N,N',N'-tetramethylethylenediamine (TEMED) and calcium sulfate dihydrate are added and quickly mixed to form the hydrogel (PAMSA). The mixture is then immediately poured into a mold, irradiated with 365nm ultraviolet light for 10-20 minutes, and allowed to stand for 6-8 hours before demolding to obtain a soft hydrogel biomimetic intestine with a villous structure.

[0017] Further, the viscosity of the sodium alginate powder is 3 mPa·s-5 mPa·s; the ammonium persulfate is a photoinitiator, added at 0.0 to 0.05 times the mass of the acrylamide powder; the MBAA is a covalent crosslinking agent, added at 0.0006 to 0.0010 times the mass of the acrylamide powder; the N,N,N',N'-tetramethylethylenediamine is a crosslinking accelerator, added at 0.0025 to 0.0030 times the mass of the acrylamide powder; the calcium sulfate dihydrate is an ionic crosslinking agent, added at 0.132 to 0.1400 times the mass of the sodium alginate powder; the amount of N,N,N',N'-tetramethylethylenediamine added is 0.25% to 0.50% of the mass of the acrylamide powder; and the amount of calcium sulfate dihydrate added is 13.28% to 14.00% of the mass of the sodium alginate powder.

[0018] Furthermore, the hydrogel (PAMSA) is named PAMSA-n, where n represents the mass ratio of AAM to SA, and n = 4, 6, 8, 10. As the SA content decreases, the average pore size of the hydrogel will also change accordingly; the adjustable pore size of the hydrogel can be used to reflect the permeability differences of different intestinal segments.

[0019] A method of using the above-mentioned in vitro dynamic intestinal digestion device combined with hydrogel includes the following steps:

[0020] Step 1: Prepare the simulated digestive fluid needed for the sample digested in the dynamic simulated digestion system, and prepare the food sample to be digested.

[0021] Simulated salivary SSF: α-amylase (BR, 40−60 U·mg) -1 0.145 mg·mL -1 ), NaCl (0.117 mg·mL) -1 ), KCl (0.149 mg·mL) -1 ) and NaHCO3 (2.1 mg·mL -1 The pH was adjusted to 7.80 ± 0.44 using 0.5 M NaOH.

[0022] Simulated gastric juice SGF: pepsin (≥3000 U g) -1 1g·L -1 ), mucin (3g / L) -1 ), NaHCO3 (0.315 mg·mL) -1 ), NaCl (8.775 mg·mL) -1 The pH was adjusted to 1.63 ± 0.01 using 0.5 M HCl.

[0023] Simulated intestinal fluid SIF: Pancreatic enzyme (derived from porcine pancreas, P-1750, 4×USP specification, 0.9g / L) -1 ), bile salts (6 g·L) -1 ) and NaHCO3 (12.5 g·L -1 The pH was adjusted to 7.50 ± 0.01 using 0.5 M NaOH.

[0024] The simulated digestive fluid is preheated to 37°C before use and prepared fresh each time; the food sample to be digested is freeze-dried, ground, and then passed through a 20-mesh standard sieve.

[0025] Step 2: Simulate the oral digestive stage;

[0026] The food sample to be digested was mixed with Milli-Q water to prepare a suspension with a sample concentration of 0.1 g / mL. 1 mL of 37°C SSF was added and the mixture was stirred at 100-150 rpm for 1-2 minutes.

[0027] Step 3: Simulate the digestive process in the stomach;

[0028] The mixture after oral digestion was transferred to a gastric digestion container, and the secretion rate of SGF was set to 25 μL / min using a sample injection micro-peristaltic pump. Digestion was continued for 2-3 hours to obtain the mixture after gastric digestion.

[0029] Step 4: Preheat the device and purge it with argon gas for 5-10 minutes to remove air;

[0030] Turn on the electrothermal oven lamp 14 and temperature sensor 11 in the in vitro dynamic intestinal digestion device combined with hydrogel to maintain the entire constant temperature chamber 16 at 37±1℃; turn on the argon cylinder and solenoid valve to release gas at a rate of 0.5L·min. -1 -1L·min -1 The gas cylinder and solenoid valve are closed after 5-10 minutes of being introduced into the speed-introducing device.

[0031] Step 5: Simulate the digestive stages of the small intestine and colon;

[0032] Automatic reagent dispensing via a micro peristaltic pump: initially at a rate of 0.5 L / min. -1 -1.0L·min -1The receiving solution, in quantities of 10 mL to 50 mL, is introduced from reagent addition port 6 into the duodenal segment 2, jejunal segment 7, ileal segment 10, and colonic segment 12, respectively. Then, stepper motor 21 is activated, and the rotation speed of the eccentric wheel 4 in the duodenal segment 2, jejunal segment 7, and ileal segment 10 is set to 36 rpm to 40 rpm, while the rotation speed of the eccentric wheel 4 in the colonic segment is set to 8 rpm to 20 rpm. The mixture that has undergone gastric digestion is then input into the device through injection port 1 to begin small intestinal digestion. The SIF addition rate is set to 30-40 μL / min. -1 Digestion was carried out at 37±1℃ for 2-3 hours. During the digestion process, digestive fluid and absorptive fluid treated by the pretreatment system were collected separately. The digestion time was set according to the average in vivo transit time in the small intestine: 8-10 min for the duodenum, 70-110 min for the jejunum, and 40-50 min for the ileum.

[0033] After the ileum was digested, the micro-peristaltic pump was started, and microbial inoculum diluted with sterile PBS (20%, w / v) was added to the container at a ratio of 1:10 (v / v) to introduce the microbial nutrient medium into the colon model; the pH was adjusted to 5.6-5.9 with 0.5M HCl and maintained within this range, and fermented at 37±1℃ for 40-48 hours to obtain the sample;

[0034] Step 6: Store the sample in a -80°C freezer until analysis;

[0035] Step 7: Clean and dry the in vitro dynamic intestinal digestion device;

[0036] Step 8: Arsenic speciation analysis by HPLC-ICPMS:

[0037] The sample pretreated by the device was injected into the HPLC-ICPMS system using a 100μL flat-tip chromatographic syringe for arsenic speciation analysis.

[0038] The microbial nutrient culture medium is formulated as follows: 1 g / L arabinogalactan, 2 g / L apple pectin, 1 g / L xylan, 3 g / L potato starch, 0.4 g / L glucose, 3 g / L yeast extract, 1 g / L peptone, 0.5 g / L L-cysteine, and 4 g / L mucin.

[0039] In step 5, the receiving solution refers to phosphate buffer (1×PBS, pH 7.4), which is used to receive substances absorbed by the hydrogel biomimetic intestine. The composition and concentration of the phosphate buffer are: NaCl 137mM, KCl 2.7mM, Na2HPO4: 10mM, KH2PO4: 1.8mM.

[0040] In step 8, speciation analysis was performed using an Euron OIC-900 ion chromatograph equipped with a dual-plunger tandem pump. The chromatographic column was an anion exchange column: Hamilton PRP-X100, 250 mm × 4.1 mm, 10 μm; the guard column was also a Hamilton PRP-X100, 20 mm × 4.1 mm, 10 μm. The temperature was room temperature. The mobile phase consisted of 10 mM diammonium hydrogen phosphate aqueous solution and 10 mM ammonium nitrate aqueous solution, adjusted to pH 6.2 with 5% (v / v) dilute nitric acid. The flow rate was 1 mL / min, and the injection volume was 25 μL. The outlet is connected to the inlet of the concentric nebulizer of the inductively coupled plasma mass spectrometer (ICP-MS) via a 50cm long polyetheretherketone (PEEK) tube with an inner diameter of 0.125mm. An Agilent 8900 ICP-MS is used for detection in time-resolved mode. The instantaneous signal response of the analyte over time is captured and recorded to obtain the corresponding chromatographic peak. The peak area is calculated by combining the effective signal value and Gaussian fitting results to achieve quantitative analysis of the analyte. The carrier gas flow rate is 1L / min; the purity of the carrier gas and argon is 99.999%; the power of the Agilent 8900 ICP-MS is 1550W, and the integration time is 100ms.

[0041] The key point of this invention is:

[0042] (1) Most of the existing in vitro intestinal models use rigid materials or dialysis membranes to simulate the intestine, which cannot reproduce the villi and absorption mechanism of the real intestine very well. Constructing gel-based membranes and more comprehensive environmental conditions can more accurately simulate the differences in digestion and absorption in different parts of the intestine in vivo.

[0043] (2) Some metal forms in intestinal digestion products are easily transformed, and the matrix (concentration, solubility, acidity and alkalinity) varies greatly in different parts. Therefore, it is necessary to sample at different times and in different parts as needed, and design pretreatment units for samples with different matrix conditions to ensure the stability and accuracy of the metal forms.

[0044] (3) Existing in vitro dynamic models are difficult to apply on a large scale due to their complex operation and low reproducibility. By creating an automated control module based on 3D printing and LabVIEW programming environment, labor costs can be reduced and the reproducibility of analysis can be improved.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] (1) Segmented simulation of the entire intestine to reproduce physiological structure: The device fully simulates the four segments of the intestine: duodenum, jejunum, ileum and colon, and is constructed according to the actual anatomical proportions. It uses biocompatible hydrogel material and has a raised biomimetic villous structure on the inner wall, which can accurately reproduce the physiological environment and absorption characteristics of the human intestine. By adjusting the pore size of the hydrogel, the permeability differences of each intestinal segment can be reflected.

[0047] (2) Dynamic sampling design supports multi-time and space sampling: The device is equipped with an independent sampling port at the end of each intestinal segment, which can flexibly sample at different digestion times and different intestinal parts according to experimental needs. It is also equipped with a dedicated pretreatment unit, which can effectively maintain the stability of the metal morphology and the detection accuracy for different matrix samples.

[0048] (3) Semi-automatic control integration, simple and efficient operation: The system adopts an automated control system based on LabVIEW to achieve semi-automatic operation of the entire process of digestion, absorption, sampling and pretreatment. The system integrates 3D printed intestinal model and functional modules, which greatly simplifies the operation process, improves experimental reproducibility and reduces labor costs while ensuring data reliability.

[0049] (4) The operation is reliable and consistent with physiological data: Experimental verification shows that the digestive process simulated by this device has good consistency with the actual data in vivo, and it shows excellent colonization support for the colon microbiota, ensuring the biological relevance of the simulation process.

[0050] (5) Parameters are flexible and adjustable, and the applicability is wide: The motion parameters and temperature parameters of each intestinal segment can be adjusted in real time and independently through the LabVIEW control unit, so as to adapt to the in vitro digestion simulation and risk assessment of various metal / heavy metal pollutants in different types of samples such as food and environmental samples, and the scalability is strong.

[0051] (6) Overcoming the shortcomings of existing technologies: The overall design overcomes the problems that are common in existing simulation devices, such as incomplete intestinal segmentation, insufficient biocompatibility of materials, and inability of static batch processing to reflect dynamic processes, providing a more realistic and efficient tool for the study of intestinal migration and transformation in metal form. Attached Figure Description

[0052] Figure 1 This is a front view of an in vitro dynamic intestinal digestion device combined with hydrogel according to the present invention.

[0053] Figure 2 This is a rear view of an in vitro dynamic intestinal digestion device combined with hydrogel according to the present invention.

[0054] Figure 3 The image in the middle (ad) is a SEM image of the biomimetic intestinal hydrogel synthesized in Example 1.

[0055] Figure 4 Figure a shows the speciation and content of arsenic in the digestion solution detected by HPLC-ICPMS in Example 1, and figure b shows the speciation and content of arsenic in the receiving solution detected by HPLC-ICPMS in Example 1.

[0056] In the diagram: 1-Inlet, 2-Duodenum segment, 3-Fixing wheel, 4-Eccentric wheel, 5-Sampling port, 6-Reagent addition port, 7-Jejunum segment, 8-One-way valve, 9-Primary filter, 10-Ileum segment, 11-Temperature sensor, 12-Colon segment, 13-Tertiary filter, 14-Electrically heated constant temperature baking lamp, 15-Miniature peristaltic pump, 16-Constant temperature chamber, 17-Positioning shaft, 18-Secondary filter, 19-Fourth filter, 20-Gear, 21-Stepper motor. Detailed Implementation

[0057] The present invention will be described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0058] An in vitro dynamic intestinal digestion device combined with a hydrogel, such as Figure 1 and Figure 2 As shown, an in vitro dynamic intestinal digestion device combined with hydrogel includes an electronic control unit, a hydrogel biomimetic intestine, a temperature control unit, a gas control unit, a reagent addition unit, an intestinal motility unit, and a pH control unit.

[0059] The electronic control unit includes a computer, an Arduino development board, a solenoid valve, an AC-DC switching power supply, a miniature peristaltic pump 15, and connecting wires. It uses LabVIEW and Arduino in combination to achieve semi-automatic computer control. The miniature peristaltic pump 15 is fixed on the two front door panels of the constant temperature chamber 16. One side is used for reagent injection, which is the sample injection miniature peristaltic pump. The other side is used for sampling, which is the sampling miniature peristaltic pump.

[0060] The hydrogel bionic intestine is prepared by molding. The inner wall of the hydrogel bionic intestine has a raised bionic villous structure, and it is divided into four segments along the in vitro digestion process: duodenal segment 2, jejunal segment 7, ileal segment 10 and colonic segment 12. The villous structure increases the intestinal surface area, improves the colonization efficiency of intestinal flora, and enhances the laminar vortex transmission at the villous edge, thereby promoting the mixing of food and intestinal fluid and the absorption of nutrients.

[0061] The temperature control unit includes a constant temperature chamber 16, an electric heating constant temperature baking lamp 14, and a temperature sensor 11, which are used to regulate the digestive environment temperature of the device. The electric heating constant temperature baking lamp 14 is located on the two front door panels of the constant temperature chamber 16, and is fixed above or below the micro peristaltic pump 15. The temperature sensor 11 is fixed near the ileum segment 10.

[0062] The gas control unit includes an argon cylinder, a rotor flow meter, two solenoid valves and a silicone tube. One end of one solenoid valve is connected to the air inlet through the silicone tube, and the other end is connected to the rotor flow meter and the argon cylinder in sequence, for introducing argon into the hydrogel bionic intestine. The other solenoid valve is connected to the exhaust pipe of the exhaust port, for venting the air in the hydrogel bionic intestinal cavity.

[0063] The reagent addition unit includes a sample inlet micro-peristaltic pump, a silicone tube, and a storage bottle. The silicone tube connects the sample inlet micro-peristaltic pump and the reagent addition port 6, and is used to deliver gastric chyme, simulated intestinal fluid (SIF), microbial inoculum, and receiving fluid. Each segment of the intestine corresponds to a sampling port 5, which has four openings: top, bottom, left, and right. The horizontal openings of the sampling ports 5 in the duodenal segment 2, jejunal segment 7, and ileal segment 10 are connected sequentially to a primary filter 9, a micro-peristaltic pump 15, a secondary filter 18, and a quaternary filter 19 via silicone tubes. The sampling port 5 in the colon segment 12 is connected sequentially to a primary filter 9, a tertiary filter 13, a micro-peristaltic pump 15, and a quaternary filter 19 via silicone tubes. The two ports on the right side of the secondary filter 18 are used for reverse flushing. The other end of the horizontal opening of the sampling port 5 is connected to a one-way valve 8 and the reagent addition port 6 via silicone tubes, and then enters the next segment of the intestine. The sampling port 5 has vertical openings at the top and bottom, which are connected to the micro-peristaltic pump 15 via silicone tubes to collect the absorption fluid and digestive fluid.

[0064] The intestinal motility unit includes a fixed wheel 3, an eccentric wheel 4, a positioning shaft 17, a gear 20, and a stepper motor 21. The central shaft of the stepper motor 21 is connected to the 3D-printed eccentric wheel 4. The eccentric wheel 4 and the gear 20 are located on the front and back sides of the intestine, respectively. The eccentric wheel 4 and the gear 20 are coaxially connected through the positioning shaft 17. In the vertical plane on the front side of the intestine, the eccentric wheel 4 is located above the intestine, the fixed wheel 3 is located below the intestine, and the stepper motor 21 is located in the vertical plane on the back side of the intestine. Through gear transmission, a small number of stepper motors 21 control the rotation of a large number of eccentric wheels. The intestinal motility unit promotes the mixing and absorption of digestive products by squeezing the intestinal lumen to simulate intestinal peristalsis and segmented movement, and controls the food transport time to be consistent with the body.

[0065] The pH control unit includes a pH meter, a pH adjusting solution (acid / base), and a feeding port, and adds the pH adjusting solution via a micro peristaltic pump;

[0066] The pretreatment unit includes a primary filter 9, a secondary filter 18, a tertiary filter 13, and a quaternary filter 19; the primary filter 9 is a ceramic filter with a pore size of 300μm; the secondary filter 18 is a ceramic filter with a pore size of 5μm; the tertiary filter 13 is an absorbent sponge; and the quaternary filter 19 is an aqueous needle-type filter membrane with a pore size of 0.22μm.

[0067] The device is used as follows: The electric heating constant temperature baking lamp 14 and temperature sensor 11 are turned on to maintain the temperature of the entire constant temperature chamber 16 at 37±1℃. The mixture that has passed through the stomach digestion stage is input into the device through the sample inlet 1 to begin the small intestine digestion stage. Simulated intestinal fluid (SIF) is added using a sample injection micro-peristaltic pump, and receiving fluid is added through the reagent addition port 6. The mixture passes sequentially through the duodenal segment 2, jejunal segment 7, ileal segment 10, and colonic segment 12. Driven by the stepper motor 21, the eccentric wheel 4 and fixed wheel 3 promote the mixing and absorption of digested matter by squeezing the intestinal lumen to simulate intestinal peristalsis and segmental movement, and control the food transport time. Consistent with in vivo conditions; due to their high water content, the digested samples from the duodenum 2, jejunum 7, and ileum 10 can sequentially enter the secondary filter 18 and the quaternary filter 19 after passing through the primary filter 9; after the digestion of the ileum 10 is completed, the sample injection micro-peristaltic pump is activated to introduce the microbial nutrient culture medium into the colon 12 model. Due to their low water content, the colon digested samples need to be placed after the primary filter 9 in the tertiary filter 13, which is placed close to the ceramic plate of the primary filter 9 to absorb the sample solution, and finally enter the quaternary filter 19; all absorbed samples, since they do not contain food residue, can directly enter the quaternary filter 19 for subsequent morphological analysis.

[0068] Furthermore, the fabrication process of the hydrogel biomimetic intestine is as follows: First, sodium alginate powder (SA) and acrylamide powder (AAM) are added to deionized water in a designed ratio (mass ratio 1:4-1:10), fixing the water content at 80wt%-86wt%, and stirring until completely dissolved to ensure a uniform system without particle precipitation; then, ammonium persulfate and N,N'-ethylenebisacrylamide (MBAA) are added sequentially and stirred continuously until dissolved to obtain a mixture, avoiding uneven cross-linking caused by excessively high local concentrations; after the mixture is vacuum degassed in an ice bath for 5-10 minutes, N,N,N',N'-tetramethylethylenediamine (TEMED) and calcium sulfate dihydrate are added and quickly mixed to form the hydrogel (PAMSA); then, it is immediately poured into a mold, irradiated with 365nm ultraviolet light for 10-20 minutes, and allowed to stand for 6-8 hours before demolding to obtain a soft hydrogel biomimetic intestine with a villous structure.

[0069] Further, the viscosity of the sodium alginate powder is 3 mPa·s - 5 mPa·s; the ammonium persulfate is a photoinitiator, added at 0.0 to 0.05 times the mass of the acrylamide powder; the MBAA is a covalent crosslinking agent, added at 0.0006 to 0.0010 times the mass of the acrylamide powder; the N,N,N',N'-tetramethylethylenediamine is a crosslinking accelerator, added at 0.0025 to 0.0030 times the mass of the acrylamide powder; the calcium sulfate dihydrate is an ionic crosslinking agent, added at 0.132 to 0.1400 times the mass of the sodium alginate powder; the amount of N,N,N',N'-tetramethylethylenediamine added is 0.25% to 0.50% of the mass of the acrylamide powder; and the amount of calcium sulfate dihydrate added is 13.28% to 14.00% of the mass of the sodium alginate powder.

[0070] Furthermore, the hydrogel (PAMSA) is named PAMSA-n, where n represents the mass ratio of AAM to SA, and n = 4, 6, 8, 10. As the SA content decreases, the average pore size of the hydrogel will also change accordingly; the adjustable pore size of the hydrogel can be used to reflect the permeability differences of different intestinal segments.

[0071] Example 1

[0072] The fabrication process of the hydrogel biomimetic intestine is as follows: First, sodium alginate powder (SA) and acrylamide powder (AAM) are added to deionized water at a designed ratio (duodenal segment 1:4, jejunal segment 1:6, ileal segment 1:8, colonic segment 1:10), fixing the water content at 86wt%, and stirring until completely dissolved to ensure a homogeneous system without particle precipitation; then, ammonium persulfate and MBAA are added sequentially, and stirring is continued until dissolved to obtain a mixture; this avoids uneven cross-linking caused by excessively high local concentrations. After vacuum degassing the mixture for 8 minutes, N,N,N',N'-tetramethylethylenediamine (TEMED) and calcium sulfate dihydrate are added and quickly mixed to form the hydrogel (PAMSA); then, it is immediately poured into a mold, irradiated with 365nm ultraviolet light for 10 minutes, allowed to stand for 8 hours, and then demolded to obtain a soft hydrogel biomimetic intestine with a villous structure.

[0073] Furthermore, the ammonium persulfate is a photoinitiator, and its addition amount is 0.01 times the mass of the acrylamide powder; the MBAA is a covalent crosslinking agent, and its addition amount is 0.0006 times the mass of the acrylamide powder; the N,N,N',N'-tetramethylethylenediamine is a crosslinking promoter, and its addition amount is 0.0025 times the mass of the acrylamide powder; the calcium sulfate dihydrate is an ionic crosslinking agent, and its addition amount is 0.1328 times the mass of the alginate.

[0074] Furthermore, the hydrogel (PAMSA) is named PAMSA-n, where n represents the mass ratio of AAM to SA, and n = 4, 6, 8, 10. The adjustable pore size of the hydrogel reflects the permeability differences of different intestinal segments. Figure 3 The image in Figure 'ad' is a SEM image of the biomimetic intestinal hydrogel synthesized in Example 1. Figure 3 This shows that as the SA content decreases, the average pore size of the hydrogel decreases (20, 10, 5, 2 μm in sequence).

[0075] A method of using the above-mentioned in vitro dynamic intestinal digestion device combined with hydrogel includes the following steps:

[0076] Step 1: Prepare the simulated digestive solution needed for the sample digested in the dynamic simulated digestion system;

[0077] Simulated salivary SSF: α-amylase (BR, 40−60 U·mg) -1 0.145 mg·mL -1 ), NaCl (0.117 mg·mL) -1 ), KCl (0.149 mg·mL) -1 ) and NaHCO3 (2.1 mg·mL -1 The pH was adjusted to 7.80 ± 0.44 using 0.5 M NaOH.

[0078] Simulated gastric juice SGF: pepsin (≥3000 U g) -1 1g·L -1 ), mucin (3g / L) -1 ), NaHCO3 (0.315 mg·mL) -1 ), NaCl (8.775 mg·mL) -1 The pH was adjusted to 1.63 ± 0.01 using 0.5 M HCl.

[0079] Simulated intestinal fluid SIF: Pancreatic enzyme (derived from porcine pancreas, P-1750, 4×USP specification, 0.9g / L) -1 ), bile salts (6 g·L) -1 ) and NaHCO3 (12.5 g·L -1 The pH was adjusted to 7.50 ± 0.01 using 0.5 M NaOH.

[0080] The above-mentioned artificial digestive fluid should be preheated to 37°C before use and prepared fresh each time; the food sample to be digested should be freeze-dried, ground, and passed through a 20-mesh standard sieve.

[0081] Step 2: Simulate the oral digestive stage;

[0082] The food sample to be digested was mixed with Milli-Q water to prepare a suspension with a sample concentration of 0.1 g / mL. 1 mL of SSF at 37 °C was added and the mixture was vortexed for 1 minute.

[0083] Step 3: Simulate the digestive process in the stomach;

[0084] The mixture after oral digestion was transferred to a gastric digestion container, and the secretion rate of SGF was set to 25 μL / min using an injection pump, and digestion was continued for 2 hours.

[0085] Step 4: Preheat the apparatus and purge it with argon gas to remove air;

[0086] Turn on the electrothermal oven lamp 14 and temperature sensor 11 in the in vitro dynamic intestinal digestion device combined with hydrogel to maintain the entire constant temperature chamber 16 at 37±1℃; turn on the argon cylinder and solenoid valve to release gas at a rate of 1L·min. −1 The gas is introduced into the device at a certain speed, and after 10 minutes, the gas cylinder and solenoid valve are closed.

[0087] Step 5: Simulate the digestive stages of the small intestine and colon;

[0088] A controllable micro-peristaltic pump automatically adds reagents: initially at a rate of 1.0 L / min. -1 A flow rate of 20 mL of receiving solution (37°C) was introduced from reagent addition port 6 into duodenal segment 2, jejunum segment 7, ileum segment 10, and colon segment 12, respectively. The motor was then turned on, and the rotation speed of the eccentric wheel 4 in duodenal segment 2, jejunum segment 7, and ileum segment 10 was set to 36 rpm, while the rotation speed of the eccentric wheel 4 in colon segment 12 was set to 10 rpm. The mixture that had passed through the gastric digestion stage was then fed into the device to begin the small intestinal digestion stage, with the SIF addition rate set to 30 μL·min. -1 Digestion was carried out at 37°C for 2 hours. During digestion, digestive fluid and absorptive fluid, which had been pretreated by the system, were collected separately. Digestion times were set for the duodenum (10 minutes), jejunum (70 minutes), and ileum (40 minutes) based on the average transit time in the small intestine.

[0089] After small intestinal digestion, a micro-peristaltic pump was started, and microbial inoculum diluted with sterile PBS (20%, w / v) was added to the container at a ratio of 1:10 (v / v), introducing the microbial nutrient medium into the colon model. The pH was adjusted to 5.6-5.9 with 0.5M HCl and maintained within this range, and fermentation was carried out at 37±1℃ for 48 hours.

[0090] Step 6: Store the sample in a -80°C freezer until analysis;

[0091] Step 7: Clean and dry the in vitro dynamic intestinal digestion device;

[0092] Step 8: Arsenic speciation analysis by HPLC-ICPMS:

[0093] The sample pretreated by the device was injected into the HPLC-ICPMS system using a 100μL flat-tip chromatographic syringe for arsenic speciation analysis.

[0094] In step 8, speciation analysis was performed using an Euron OIC-900 ion chromatograph equipped with a dual-plunger tandem pump. The chromatographic column was an anion exchange column: Hamilton PRP-X100, 250 mm × 4.1 mm, 10 μm; the guard column was also a Hamilton PRP-X100, 20 mm × 4.1 mm, 10 μm. The temperature was room temperature. The mobile phase consisted of 10 mM diammonium hydrogen phosphate aqueous solution and 10 mM ammonium nitrate aqueous solution, adjusted to pH 6.2 with 5% (v / v) dilute nitric acid. The flow rate was 1 mL / min, and the injection volume was 25 μL. The outlet is connected to the inlet of the concentric nebulizer of the inductively coupled plasma mass spectrometer (ICP-MS) via a 50cm long polyetheretherketone (PEEK) tube with an inner diameter of 0.125mm. An Agilent 8900 ICP-MS is used for detection in time-resolved mode. The instantaneous signal response of the analyte over time is captured and recorded to obtain the corresponding chromatographic peak. The peak area is calculated by combining the effective signal value and Gaussian fitting results to achieve quantitative analysis of the analyte. The carrier gas flow rate is 1L / min; the purity of the carrier gas argon is 99.999%; the power of the Agilent 8900 ICP-MS is 1550W, and the integration time is 100ms.

[0095] Figure 4 Figure a shows the speciation and content of arsenic in the digestion solution detected by HPLC-ICPMS in Example 1, and figure b shows the speciation and content of arsenic in the receiving solution detected by HPLC-ICPMS in Example 1. Figure 4 The results show that by using the present invention to simulate the complete digestion process of rice in the duodenum to the colon, and combined with HPLC-ICPMS, the release, transformation and absorption of different forms of arsenic in rice by the intestine can be monitored online. The present invention provides a reliable tool for rapidly and accurately analyzing the digestive behavior of the small intestine and colon after ingesting foods containing different forms of arsenic, such as rice.

Claims

1. An in vitro dynamic intestinal digestion device combined with a hydrogel, characterized in that, It includes an electronic control unit, a hydrogel bionic intestine, a temperature control unit, a gas control unit, a reagent addition unit, an intestinal motility unit, and a pH control unit.

2. The in vitro dynamic intestinal digestion device combined with hydrogel according to claim 1, characterized in that, The electronic control unit includes a computer, an Arduino development board, a solenoid valve, an AC-DC switching power supply, a miniature peristaltic pump 15, and connecting wires. It uses LabVIEW and Arduino in combination to achieve semi-automatic computer control. The miniature peristaltic pump 15 is fixed on the two front door panels of the constant temperature chamber 16. One side is used for reagent injection, which is the sample injection miniature peristaltic pump. The other side is used for sampling, which is the sampling miniature peristaltic pump. The hydrogel bionic intestine is prepared by molding. The inner wall of the hydrogel bionic intestine is provided with a raised bionic villous structure, and it is divided into four segments along the in vitro digestion process: duodenal segment 2, jejunal segment 7, ileal segment 10 and colonic segment 12. The temperature control unit includes a constant temperature chamber 16, an electric heating constant temperature baking lamp 14, and a temperature sensor 11, which are used to regulate the digestive environment temperature of the device. The electric heating constant temperature baking lamp 14 is located on the two front door panels of the constant temperature chamber 16, and is fixed above or below the micro peristaltic pump 15. The temperature sensor 11 is fixed near the ileum segment 10. The gas control unit includes an argon cylinder, a rotor flow meter, two solenoid valves and a silicone tube. One end of one solenoid valve is connected to the air inlet through the silicone tube, and the other end is connected to the rotor flow meter and the argon cylinder in sequence, for introducing argon into the hydrogel bionic intestine. The other solenoid valve is connected to the exhaust pipe of the exhaust port, for venting the air in the hydrogel bionic intestinal cavity. The reagent addition unit includes a sample inlet micro-peristaltic pump, a silicone tube, and a storage bottle. The silicone tube connects the sample inlet micro-peristaltic pump and the reagent addition port 6, and is used to deliver gastric chyme, simulated intestinal fluid (SIF), microbial inoculum, and receiving fluid. Each segment of the intestine corresponds to a sampling port 5, which has four openings: top, bottom, left, and right. The horizontal openings of the sampling ports 5 in the duodenal segment 2, jejunal segment 7, and ileal segment 10 are connected sequentially to a primary filter 9, a micro-peristaltic pump 15, a secondary filter 18, and a quaternary filter 19 via silicone tubes. The sampling port 5 in the colon segment 12 is connected sequentially to a primary filter 9, a tertiary filter 13, a micro-peristaltic pump 15, and a quaternary filter 19 via silicone tubes. The two ports on the right side of the secondary filter 18 are used for reverse flushing. The other end of the horizontal opening of the sampling port 5 is connected to a one-way valve 8 and the reagent addition port 6 via silicone tubes, and then enters the next segment of the intestine. The sampling port 5 has vertical openings at the top and bottom, which are connected to the micro-peristaltic pump 15 via silicone tubes to collect the absorption fluid and digestive fluid. The intestinal motility unit includes a fixed wheel 3, an eccentric wheel 4, a positioning shaft 17, a gear 20, and a stepper motor 21. The central shaft of the stepper motor 21 is connected to the 3D-printed eccentric wheel 4. The eccentric wheel 4 and the gear 20 are located on the front and back sides of the intestine, respectively. The eccentric wheel 4 and the gear 20 are coaxially connected through the positioning shaft 17. In the vertical plane on the front side of the intestine, the eccentric wheel 4 is located above the intestine, the fixed wheel 3 is located below the intestine, and the stepper motor 21 is located in the vertical plane on the back side of the intestine. Through gear transmission, a small number of stepper motors 21 control the rotation of a large number of eccentric wheels. The intestinal motility unit promotes the mixing and absorption of digestive products by squeezing the intestinal lumen to simulate intestinal peristalsis and segmented movement, and controls the food transport time to be consistent with the body. The pH control unit includes a pH meter, a pH adjusting solution (acid / base), and a feeding port, and adds the pH adjusting solution via a micro peristaltic pump; The pretreatment unit includes a primary filter 9, a secondary filter 18, a tertiary filter 13, and a quaternary filter 19; the primary filter 9 is a ceramic filter with a pore size of 300μm; the secondary filter 18 is a ceramic filter with a pore size of 5μm; the tertiary filter 13 is an absorbent sponge; and the quaternary filter 19 is an aqueous needle-type filter membrane with a pore size of 0.22μm.

3. The in vitro dynamic intestinal digestion device combined with hydrogel according to claim 1, characterized in that, The device is used as follows: The electric heating constant temperature baking lamp 14 and temperature sensor 11 are turned on to maintain the temperature of the entire constant temperature chamber 16 at 37±1℃. The mixture that has passed through the stomach digestion stage is input into the device through the sample inlet 1 to begin the small intestine digestion stage. Simulated intestinal fluid (SIF) is added using a sample injection micro-peristaltic pump, and receiving fluid is added through the reagent addition port 6. The mixture passes sequentially through the duodenal segment 2, jejunal segment 7, ileal segment 10, and colonic segment 12. Driven by the stepper motor 21, the eccentric wheel 4 and fixed wheel 3 promote the mixing and absorption of digested matter by squeezing the intestinal lumen to simulate intestinal peristalsis and segmental movement, and control food transport. The timing is consistent with in vivo. Samples digested from the duodenum (2), jejunum (7), and ileum (10) have high water content and, after passing through primary filter (9), can sequentially enter secondary filter (18) and quaternary filter (19). After digestion of the ileum, the micro-peristaltic pump is activated to introduce microbial nutrient culture medium into the colon (12) model. Since the colon digested sample has low water content, a tertiary filter (13) is placed after primary filter (9) to absorb the sample solution, which then enters quaternary filter (19). All absorbed samples, since they contain no food residue, can directly enter the quaternary filter for subsequent morphological analysis.

4. The in vitro dynamic intestinal digestion device combined with hydrogel according to claim 1, characterized in that, The fabrication process of the hydrogel biomimetic intestine is as follows: First, sodium alginate (SA) and acrylamide (AAM) are added to deionized water at a mass ratio of (1:4)-(1:10), and the water content is fixed at 80wt%-86wt%. The mixture is stirred until completely dissolved to ensure that the system is uniform and free of particle precipitation. Then, ammonium persulfate and N,N'-ethylenebisacrylamide (MBAA) are added sequentially and stirred until dissolved to obtain a mixture. The mixture is then degassed under vacuum in an ice bath for 5-10 minutes. N,N,N',N'-tetramethylethylenediamine (TEMED) and calcium sulfate dihydrate are added and quickly mixed to form the hydrogel (PAMSA). The mixture is then immediately poured into a mold, irradiated with 365nm ultraviolet light for 10-20 minutes, and allowed to stand for 6-8 hours before demolding to obtain a soft hydrogel biomimetic intestine with a villous structure.

5. The in vitro dynamic intestinal digestion device combined with hydrogel according to claim 4, characterized in that, The viscosity of the sodium alginate powder is 3 mPa·s-5 mPa·s; the ammonium persulfate is a photoinitiator, added at 0.0 to 0.05 times the mass of the acrylamide powder; the MBAA is a covalent crosslinking agent, added at 0.0006 to 0.0010 times the mass of the acrylamide powder; the N,N,N',N'-tetramethylethylenediamine is a crosslinking accelerator, added at 0.0025 to 0.0030 times the mass of the acrylamide powder; the calcium sulfate dihydrate is an ionic crosslinking agent, added at 0.132 to 0.1400 times the mass of the sodium alginate powder; the amount of N,N,N',N'-tetramethylethylenediamine added is 0.25% to 0.50% of the mass of the acrylamide powder; and the amount of calcium sulfate dihydrate added is 13.28% to 14.00% of the mass of the sodium alginate powder.

6. The in vitro dynamic intestinal digestion device combined with hydrogel according to claim 4, characterized in that, The hydrogel (PAMSA) is named PAMSA-n, where n represents the mass ratio of AAM to SA, and n = 4, 6, 8, 10. As the SA content decreases, the average pore size of the hydrogel also changes. The adjustable pore size of the hydrogel is used to reflect the permeability differences of different intestinal segments.

7. A method of using the in vitro dynamic intestinal digestion device combined with hydrogel as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Prepare the simulated digestive fluid needed for the sample digested in the dynamic simulated digestion system, and prepare the food sample to be digested. Simulated salivary SSF: α-amylase (BR, 40−60 U·mg) -1 0.145 mg·mL -1 ), NaCl (0.117 mg·mL) -1 ), KCl (0.149 mg·mL) -1 ) and NaHCO3 (2.1 mg·mL -1 The pH was adjusted to 7.80 ± 0.44 using 0.5 M NaOH. Simulated gastric juice SGF: pepsin (≥3000 U g) -1 1g·L -1 ), mucin (3g / L) -1 ), NaHCO3 (0.315 mg·mL) -1 ), NaCl (8.775 mg·mL) -1 The pH was adjusted to 1.63 ± 0.01 using 0.5 M HCl. Simulated intestinal fluid SIF: Pancreatic enzyme (derived from porcine pancreas, P-1750, 4×USP specification, 0.9g / L) -1 ), bile salts (6 g·L) -1 ) and NaHCO3 (12.5 g·L -1 The pH was adjusted to 7.50 ± 0.01 using 0.5 M NaOH. The simulated digestive fluid is preheated to 37°C before use and prepared fresh each time; the food sample to be digested is freeze-dried, ground, and then passed through a 20-mesh standard sieve. Step 2: Simulate the oral digestive stage; The food sample to be digested was mixed with Milli-Q water to prepare a suspension with a sample concentration of 0.1 g / mL. 1 mL of 37°C SSF was added and the mixture was stirred at 100-150 rpm for 1-2 minutes. Step 3: Simulate the digestive process in the stomach; The mixture after oral digestion was transferred to a gastric digestion container, and the secretion rate of SGF was set to 25 μL / min using a sample injection micro-peristaltic pump. Digestion was continued for 2-3 hours to obtain the mixture after gastric digestion. Step 4: Preheat the device and purge it with argon gas for 5-10 minutes to remove air; Turn on the electrothermal oven lamp 14 and temperature sensor 11 in the in vitro dynamic intestinal digestion device combined with hydrogel to maintain the entire constant temperature chamber 16 at 37±1℃; turn on the argon cylinder and solenoid valve to release gas at a rate of 0.5-1 L / min. -1 The gas cylinder and solenoid valve are closed after 5-10 minutes of being introduced into the speed-introducing device. Step 5: Simulate the digestive stages of the small intestine and colon; Automatic reagent dispensing via a micro peristaltic pump: initially at a rate of 0.5 L / min. -1 -1.0L·min -1 The receiving solution, in quantities of 10 mL to 50 mL, is introduced from reagent addition port 6 into the duodenal segment 2, jejunal segment 7, ileal segment 10, and colonic segment 12, respectively. Then, stepper motor 21 is activated, and the rotation speed of the eccentric wheel 4 in the duodenal segment 2, jejunal segment 7, and ileal segment 10 is set to 36 rpm to 40 rpm, while the rotation speed of the eccentric wheel 4 in the colonic segment is set to 8 rpm to 20 rpm. The mixture that has undergone gastric digestion is then input into the device through injection port 1 to begin small intestinal digestion. The SIF addition rate is set to 30-40 μL / min. -1 Digestion was carried out at 37±1℃ for 2-3 hours. During the digestion process, digestive fluid and absorptive fluid treated by the pretreatment system were collected separately. The digestion time was set according to the average in vivo transit time in the small intestine: 8-10 min for the duodenum, 70-110 min for the jejunum, and 40-50 min for the ileum. After the ileum was digested, the micro-peristaltic pump was started, and microbial inoculum diluted with sterile PBS (20%, w / v) was added to the container at a ratio of 1:10 (v / v) to introduce the microbial nutrient medium into the colon model; the pH was adjusted to 5.6-5.9 with 0.5M HCl and maintained within this range, and fermented at 37±1℃ for 40-48 hours to obtain the sample; Step 6: Store the sample in a -80°C freezer until analysis; Step 7: Clean and dry the in vitro dynamic intestinal digestion device; Step 8: Arsenic speciation analysis by HPLC-ICPMS: The sample pretreated by the device was injected into the HPLC-ICPMS system using a 100μL flat-tip chromatographic syringe for arsenic speciation analysis.

8. The method of using the in vitro dynamic intestinal digestion device combined with hydrogel according to claim 7, characterized in that, The microbial nutrient culture medium is formulated as follows: 1 g / L arabinogalactan, 2 g / L apple pectin, 1 g / L xylan, 3 g / L potato starch, 0.4 g / L glucose, 3 g / L yeast extract, 1 g / L peptone, 0.5 g / L L-cysteine, and 4 g / L mucin.

9. The method of using the in vitro dynamic intestinal digestion device combined with hydrogel according to claim 7, characterized in that, In step 5, the receiving solution refers to phosphate buffer (1×PBS, pH 7.4), which is used to receive substances absorbed by the hydrogel biomimetic intestine. The composition and concentration of the phosphate buffer are: NaCl 137mM, KCl 2.7mM, Na2HPO4: 10mM, KH2PO4: 1.8mM.

10. The method of using the in vitro dynamic intestinal digestion device combined with hydrogel according to claim 7, characterized in that, In step 8, speciation analysis was performed using an Euron OIC-900 ion chromatograph equipped with a dual-plunger tandem pump. The chromatographic column was an anion exchange column: Hamilton PRP-X100, 250 mm × 4.1 mm, 10 μm; the guard column was also a Hamilton PRP-X100, 20 mm × 4.1 mm, 10 μm. The temperature was room temperature; the mobile phase consisted of 10 mM diammonium hydrogen phosphate aqueous solution and 10 mM ammonium nitrate aqueous solution, adjusted to pH 6.2 with 5% (v / v) dilute nitric acid; and the flow rate was 1 mL. The injection rate was 1 L / min, and the injection volume was 25 μL. The column outlet was connected to the inlet of the inductively coupled plasma mass spectrometer (ICP-MS) concentric nebulizer via a 50 cm long polyetheretherketone (PEEK) tube with an inner diameter of 0.125 mm. An Agilent 8900 ICP-MS was used for detection in time-resolved mode. The instantaneous signal response of the analyte over time was captured and recorded to obtain the corresponding chromatographic peak. The peak area was calculated by combining the effective signal value and Gaussian fitting results to achieve quantitative analysis of the analyte. The carrier gas flow rate was 1 L / min, and the purity of the carrier gas argon was 99.999%. The Agilent 8900ICP-MS has a power consumption of 1550W and an integration time of 100ms.

Images