WO2024007530A1 - 用于探索汞离子的肠器官芯片制备的优化方法 - Google Patents

用于探索汞离子的肠器官芯片制备的优化方法 Download PDF

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WO2024007530A1
WO2024007530A1 PCT/CN2022/137581 CN2022137581W WO2024007530A1 WO 2024007530 A1 WO2024007530 A1 WO 2024007530A1 CN 2022137581 W CN2022137581 W CN 2022137581W WO 2024007530 A1 WO2024007530 A1 WO 2024007530A1
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layer structure
intestinal organ
silver
organ chip
intestinal
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French (fr)
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王力
陈俊
韩军磊
苏伟光
李安庆
衣明东
许崇海
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齐鲁工业大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
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    • C12M23/00—Constructional details, e.g. recesses, hinges
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    • C—CHEMISTRY; METALLURGY
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept

Definitions

  • the invention belongs to the technical field of intestinal organ chips, and in particular relates to an optimized method for preparing intestinal organ chips for exploring mercury ions.
  • Hg 2+ Mercury ions
  • the absorption of Hg 2+ mainly occurs in the small intestine; therefore, a reasonable intestinal model is of great significance for exploring the transport and absorption mechanism of Hg 2+ into target cells, and can effectively provide a comprehensive method for treating the toxicological effects of Hg 2+ transport. information.
  • organ-on-a-chip can replicate the complex structure and physiological functions of human organs in microfluidic culture devices.
  • the intestinal organ chip can induce Caco-2 cells to form villus-like processes by applying mechanical stimulation to the epithelial cell monolayer, while producing four types of cells: absorptive cells, mucus-secreting cells, enteroendocrine cells and Paneth cells, partially completed Secretory and immune functions of the intestine; intestinal organ-on-a-chip has made great progress in drug screening, disease modeling, toxicology and personalized medicine.
  • intestinal organ-on-a-chip has made great progress in drug screening, disease modeling, toxicology and personalized medicine.
  • Ingber's team used oxygen-sensitive fluorescent probes to continuously monitor the microenvironment in the chip. Oxygen concentration gradient changes; in addition, other reactions including barrier integrity and inflammatory reactions (such as cytokine secretion) are also detected in situ through fluorescent probes, such as fluorophores, conjugated polymers, DNAzymes, and quantum dots. ;
  • fluorescent probes such as fluorophores, conjugated polymers, DNAzymes, and quantum dots.
  • problems with fluorophore bleaching, service life, and external environmental interference limit their application in intestinal organ chips, and current intestinal organ chips do not optimize in vitro biomimetic conditions, such as optimizing physiological movement and fluid flow.
  • the present invention proposes an optimized method for preparing an intestinal organ chip for exploring mercury ions.
  • the present invention optimizes and obtains a dual-channel, mechanically stretchable intestinal chip, a three-electrode sensor array and a silver- Silver chloride (Ag/AgCl) electrodes are integrated at different locations on the chip, allowing real-time, non-invasive monitoring of changes in transmembrane resistance and absorption of mercury ions.
  • the present invention proposes an optimized method for preparing intestinal organ chips for exploring mercury ions, adopting the following technical solutions:
  • An optimized method for the preparation of intestinal organ-on-a-chip for exploring mercury ions including:
  • an intestinal organ chip including a first layer structure and a second layer structure, channels are provided on both the first layer structure and the second layer structure; the channels are connected to a syringe pump through a silicone capillary tube;
  • intestinal organ chips includes:
  • the degassed base material and curing agent mixture are respectively poured on the first mold and the second mold preset with a plurality of channel patterns; heat-setting at a preset temperature for a second preset time; demoulding to obtain a channel pattern
  • Perforating holes in the first layer structure performing oxygen plasma cleaning on the perforated first layer structure, second layer structure and porous membrane, and the cleaning time is a third preset time length;
  • the porous membrane being located between the channels on the first layer structure and the channels on the second layer structure;
  • Each channel is connected to a silica gel capillary; the silica gel capillary is connected to a syringe pump.
  • the preset ratio of base material to curing agent is 10:1 by weight; the first preset time is 30 minutes; the second preset time is 4 hours; and the third preset time is 120 seconds.
  • the base material is polydimethylsiloxane base material.
  • the intestinal organ chip and the glass sheet with the three-electrode sensor are first cleaned by oxygen plasma.
  • the cleaning time of the intestinal organ chip and the glass slide equipped with the three-electrode sensor with oxygen plasma is 120 seconds.
  • semi-solid polydimethylsiloxane was used to seal the periphery of the intestinal organ chip; after a silver-silver chloride electrode was inserted into the through hole, a semi-solid polydimethylsiloxane was used to seal the intestinal organ chip. Solid polydimethylsiloxane seals through holes.
  • the silver-silver chloride electrode includes a first silver-silver chloride electrode and a second silver-silver chloride electrode; the first silver-silver chloride electrode is located in a through hole that communicates with the channel of the first layer structure. In the second silver-silver chloride electrode, the second silver-silver chloride electrode is located in a through hole that penetrates the channel of the second layer structure.
  • first silver-silver chloride electrode and the second silver-silver chloride electrode are connected to an electrochemical workstation.
  • the present invention also proposes an intestinal organ chip for exploring mercury ions, adopting the following technical solution:
  • An intestinal organ chip for exploring mercury ions is obtained using the optimized method for preparing an intestinal organ chip for exploring mercury ions described in the first aspect, including:
  • a porous membrane is located between the first layer structure and the second layer structure; the porous membrane separates the channels on the first layer structure and the channels on the second layer structure;
  • the glass piece equipped with the three-electrode sensor is fixed to the first layer structure, and the test end of the three-electrode sensor is located in the channel of the first layer structure;
  • the first silver-silver chloride electrode and the second silver-silver chloride electrode are respectively arranged on both sides of the intestinal organ chip through preset through holes.
  • the through holes on both sides are respectively connected with the channel on the first layer structure and the second layer structure.
  • the channels on the layer structure are connected.
  • channels are opened on both the first layer structure and the second layer structure, and the channels are connected to a syringe pump through a silicone capillary tube.
  • the flow rate and mechanical stretching are controlled by the syringe pump, which overcomes the current lack of optimized in vitro bionics of intestinal tubes on intestinal organ chips. shortcomings of conditions; a dual-channel, mechanically stretchable intestinal chip was optimized; at the same time, the three-electrode sensor array and silver-silver chloride electrodes were integrated at different locations on the chip, which can achieve real-time, non-invasive monitoring across Changes in membrane resistance and absorption of mercury ions.
  • Figure 1 is a partially enlarged structural schematic diagram of Embodiment 1 of the present invention.
  • FIG. 2 is a schematic structural diagram of Embodiment 1 of the present invention.
  • FIG. 3 is a schematic diagram of the actual effect of Embodiment 1 of the present invention.
  • Figure 4 is a schematic diagram of the simulation results of Embodiment 1 of the present invention.
  • Figure 5 is an analysis of the verification results of Embodiment 1 of the present invention.
  • Figure 6 is the deformation analysis of Embodiment 1 of the present invention.
  • Figure 7 is an analysis of hole diameter changes in Embodiment 1 of the present invention.
  • Figure 8 is the Young's modulus drift analysis of Example 1 of the present invention.
  • Figure 9 shows the steps of modifying AuNPs and detecting Hg 2+ in the three-electrode sensor of Embodiment 1 of the present invention.
  • Figure 10 is the peak current analysis of Embodiment 1 of the present invention.
  • Figure 11 is an analysis of the difference in oxidation peak current and the logarithmic concentration of Hg 2+ in Example 1 of the present invention.
  • Figure 12 is the Hg 2+ concentration analysis of Example 1 of the present invention.
  • Figure 13 is an analysis of Hg 2+ transport permeability in Example 1 of the present invention.
  • This embodiment provides an optimized method for preparing an intestinal organ chip for exploring mercury ions, including:
  • Producing an intestinal organ chip including a first layer structure and a second layer structure, both of which are provided with channels;
  • the purpose is that in the follow-up, when the first layer structure is fixed on the glass sheet, the test end of the three-electrode sensor can be located in the channel; the intestinal organ chip is fixed on the fixed three-electrode sensor. On the glass sheet of the array, the side of the first layer structure that is close to the channel without the outer wall is fixed to the glass sheet.
  • the test end of the three-electrode sensor is located in the channel of the first layer structure; the three-electrode sensor can be fixed on the glass sheet by adhesive.
  • the three-electrode sensor array may include 3 three-electrode sensors;
  • the intestinal organ chip when optimizing the intestinal organ chip, it can be mainly divided into three parts: manufacturing of the intestinal organ chip, electrochemical sensor integration and TEER sensor integration, specifically as follows:
  • the production of the intestinal organ chip includes: mixing a preset proportion of base material and curing agent, and degassing in a vacuum for a first preset time; pouring the degassed base material and curing agent mixture into preset multiple on the first mold and the second mold with channel patterns; heat-setting at a preset temperature for a second preset time; demoulding to obtain a first layer structure and a second layer structure with channels, when the first layer structure and the second layer
  • the first mold and the second mold can be set as one; when the first layer structure and the second layer structure are different, the first mold and the second mold are different; punch holes in the first layer structure;
  • the punched first layer structure, the second layer structure and the porous membrane are cleaned by oxygen plasma, and the cleaning time is a third preset time period; the cleaned first layer structure and the second layer structure are bonded, as described
  • the porous membrane is located between the channels on the first layer structure and the channels on the second layer structure; each channel is connected to a silica gel capillary;
  • the preset ratio of base material to curing agent may be a weight ratio of 10:1; the first preset time may be 30 minutes; the second preset time may be 4 hours; and the third preset time may be 120 seconds;
  • the base material may be a polydimethylsiloxane base material.
  • soft engraving technology can be used and flexible and transparent polydimethylsiloxane (PDMS; 184Silicone Elastomer, Dow Corning Co., Midland, MI, USA) can be used to make the intestinal organ chip; the intestinal organ chip consists of three parts: The top microchannel, the bottom microchannel and the porous membrane in the middle.
  • the first layer structure can be defined as the top layer
  • the second layer structure can be defined as the bottom layer.
  • the size of the microchannel can be set to 2.0mm wide ⁇ 0.25mm high; the thickness of the porous membrane It can be set to 20 ⁇ m and the pore size can be set to 5 ⁇ m.
  • the porous membrane separates the channels on the top layer and the bottom layer and plays a role in building a tissue interface.
  • a PDMS base material with a weight ratio of 10:1 (wt/wt) and a curing agent are mixed, degassed in a vacuum for 30 minutes, and then cast on a mold containing a microchannel pattern. After thermal curing at 70°C for 4 hours, The top and bottom layers with microchannels are demoulded. Use a hole punch to punch holes in the top layer and connect the silicone hoses to the channels of the organ chip.
  • the plasma cleaning machine can be CY-P2L-B, CY Scientific InstrumentCO., Ltd., China, to bond the top and bottom layers together, and use semi-solid PDMS to seal and solidify the peripheral gaps; finally, use stainless steel fine
  • the tube connects the silicone capillary to each channel, and the flow rate and mechanical stretching are controlled by a high-precision syringe pump, which can be LSP02-1B, Baoding Ditron Electronic Technology CO., Ltd, China.
  • Figure 1 shows a physical picture of the intestinal organ chip.
  • the silver-silver chloride electrode includes a first silver-silver chloride electrode and a second silver-silver chloride electrode; the first silver-silver chloride electrode is located with the channel of the first layer structure In the through hole, the second silver-silver chloride electrode is located in the through hole that passes through the channel of the second layer structure.
  • TEER transepithelial electrical resistance
  • R 1 is the actual measured value
  • R 0 is the base value resistance measured without cells
  • S is the surface area of the cell culture.
  • An inverted fluorescence microscope (Nikon Eclipse TI2) was used to record fluorescence images of living cells (green fluorescence) and dead cells (red fluorescence) at 495nm and 652nm respectively; live/dead cells were counted using ImageJ software (National Institutes of Health). Calculate cell viability.
  • this embodiment provides a dual-channel, mechanically stretchable intestinal chip.
  • the three-electrode electrochemical sensor array and Ag/AgCl electrode are integrated on the bottom channel surface, top surface and bottom surface of the chip porous membrane respectively; the sensor is used for real-time, non-invasive monitoring of changes in TEER and Hg 2+ absorption, as shown in Figure 1 to As shown in Figure 3.
  • Finite element analysis FEA, COMSOL MultiPhysical 5.5, trial version was used to simulate the bionic conditions of the internal physical parameters of the intestinal organ chip, and the appropriate perfusion flow and tensile strain were determined.
  • the physical parameters can be fluid shear stress, tensile stress and Strain, perfusion flow can be set to 160 ⁇ L/h; 0.02 dyne/cm2, and tensile strain can be set to 1%, as shown in Figure 4.
  • perfusion flow rate is 40-360 ⁇ L/h
  • shear stress range of the culture medium is 0.005dyne/cm 2 -0.05dyne/cm 2
  • flow field simulation, theoretical calculation and experiment were cross-validated, as shown in Figure 5.
  • the hole diameter increased slightly from 5.19 ⁇ 0.10 ⁇ m to 5.28 ⁇ 0.25 ⁇ m, as shown in Figure 7, and the Young’s modulus drifted from 2.54 ⁇ 0.06MPa to 2.54 ⁇ 0.05MPa, as shown in Figure 8. Therefore, long-term mechanical stretching within a certain strain range does not significantly change the physical properties of the pore size and Young's modulus of the porous membrane.
  • the upper and lower microchannels are separated by a porous membrane in the middle, and cells are seeded on its surface to form a biolayer interface.
  • the three-electrode sensor and Ag/AgCl electrode are integrated at the same time for in-situ detection of Hg 2+ and TEER;
  • Figure 3 is a photo of the intestinal organ chip with integrated sensor;
  • Figure 4 is a microchannel finite element analysis (flow rate, shear stress, tensile stress and strain);
  • Figure 5 shows the cross-validation of flow field simulation, theoretical calculation and experiment;
  • Figure 6 shows the quantification of the tensile strain of the porous membrane.
  • this embodiment integrates a three-electrode sensor array for detecting Hg 2+ in the intestinal bottom channel on the chip.
  • the three-electrode sensor mainly consists of four parts: glass substrate, 10nm thick Gr layer, 200nm thick Au layer and gold nanoparticles (AuNPs) layer.
  • AuNPs gold nanoparticles
  • the calibration curve of the three-electrode sensor for Hg 2+ determination was studied under optimal conditions.
  • the peak current of Hg 2+ appears at 0.02V, and the peak current continuously increases from 0.5 ⁇ A to 7.8 ⁇ A in the concentration range of 1nM-10 ⁇ M, as shown in Figure 10.
  • ⁇ i is the difference in oxidation peak current
  • lgC is the logarithmic concentration of Hg 2+ , as shown in Figure 11.
  • the sensitivity to Hg 2+ was calculated to be 1.78 ⁇ A/nM, with a detection limit of 0.1 nM.
  • this example detected Hg 2+ absorption by intestinal epithelial cells in Transwell and intestinal organ chips in situ within 180 minutes.
  • the adsorption concentrations of Hg 2+ by intestinal epithelial cells on the Transwell and the chip were 2.3 ⁇ M and 1.8 ⁇ M, respectively, and the absorption rates were 22% and 17.8%, respectively (initial Hg 2+ concentration in the top chamber/channel is 10 ⁇ M), as shown in Figure 12.
  • P app Hg 2+ transport permeability
  • the absorption rate of Hg 2+ was ⁇ 15.0%, which was lower than the absorption rate of Caco-2 cell experiment (17.8%). It should be taken into account that in vivo studies are characterized by the presence of luminal factors (bile salts, food components, etc.). This was not found in in vitro models and may affect Hg 2+ transport between intestinal epithelial cells. Therefore, this intestinal model can truly reflect the absorption of Hg 2+ by the human intestine.
  • luminal factors bile salts, food components, etc.
  • the Hg 2+ concentrations absorbed by intestinal epithelial cells in Transwell and chip were 2.3 ⁇ M and 1.8 ⁇ M respectively, and the absorption rates were 22% and 17.8% respectively.
  • Figure 13 it is a comparison of the Papp values of Hg 2+ absorbed by Transwell and intestinal organ chips.
  • This embodiment provides an intestinal organ chip for exploring mercury ions, which is obtained using the optimized method for preparing an intestinal organ chip for exploring mercury ions described in Example 1, including:
  • a porous membrane is located between the first layer structure and the second layer structure; the porous membrane separates the channels on the first layer structure and the channels on the second layer structure;
  • the glass piece equipped with the three-electrode sensor is fixed to the first layer structure, and the test end of the three-electrode sensor is located in the channel of the first layer structure;
  • the first silver-silver chloride electrode and the second silver-silver chloride electrode are respectively arranged on both sides of the intestinal organ chip through preset through holes.
  • the through holes on both sides are respectively connected with the channel on the first layer structure and the second layer structure.
  • the channels on the layer structure are connected.

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Abstract

本发明属于肠器官芯片技术领域，提出了一种用于探索汞离子的肠器官芯片制备的优化方法，本发明中在第一层结构和第二层结构上均开设通道，通道通过硅胶毛细管连接有注射泵，流量和机械拉伸由注射泵控制，克服了目前肠器官芯片上肠管没有优化体外仿生条件的缺点；优化得到了一种双通道、机械可伸展的肠道芯片；同时，三电极传感器阵列和银-氯化银电极分别集成在芯片的不同位置，可以实现实时、非侵入性监测跨膜电阻和吸收汞离子的变化。

Description

用于探索汞离子的肠器官芯片制备的优化方法
本发明要求于2022年7月4日提交中国专利局、申请号为202210778804.9、发明名称为“用于探索汞离子的肠器官芯片制备的优化方法”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于肠器官芯片技术领域，尤其涉及一种用于探索汞离子的肠器官芯片制备的优化方法。
背景技术
汞离子(Hg 2+)即使在超低浓度，比如在浓度为5μM的情况下也会在体内不可降解地积聚，这会损害心脏、肾脏、肠道和中枢神经系统。Hg 2+的吸收主要发生在小肠；因此，合理的肠道模型对探讨Hg 2+进入靶细胞的转运和吸收机制具有重要意义，可以有效地为治疗Hg 2+转运的毒理学效应提供全面的信息。
发明人发现，近年来，器官芯片可以在微流控培养装置中复制人体器官的复杂结构和生理功能。肠器官芯片可以通过对上皮细胞单层施加机械刺激来诱导Caco-2细胞形成绒毛状突起，同时会产生吸收细胞、粘液分泌细胞、肠道内分泌细胞和潘氏细胞四种类型的细胞，部分完成肠道的分泌和免疫功能；肠器官芯片在药物筛选、疾病建模、毒理学和个性化医学方面取得了很大进展。随着肠器官芯片中生物复杂性要求的不断提高，通过集成传感器实现微环境中物理化学参数的在线检测越来越受到人们的关注，例如，Ingber团队利用氧敏感荧光探针连续监测芯片内的氧浓度梯度变化；此外通过荧光探针，如荧光团、 共轭聚合物、脱氧核酶和量子点等探针对其他反应包括屏障完整性和炎症反应(如细胞因子分泌)也进行原位检测；但是，荧光团漂白、使用寿命和外部环境干扰的问题限制了它们在肠器官芯片中的应用，并且，目前的肠器官芯片没有优化体外仿生条件，比如优化生理运动和流体流动的问题。
发明内容
本发明为了解决上述问题，提出了一种用于探索汞离子的肠器官芯片制备的优化方法，本发明优化得到了一种双通道、机械可伸展的肠道芯片，三电极传感器阵列和银-氯化银(Ag/AgCl)电极分别集成在芯片的不同位置，可以实现实时、非侵入性监测跨膜电阻和吸收汞离子的变化。
为了实现上述目的，第一方面，本发明提出了一种用于探索汞离子的肠器官芯片制备的优化方法，采用如下技术方案：
一种用于探索汞离子的肠器官芯片制备的优化方法，包括：
制作包括第一层结构和第二层结构的肠器官芯片，所述第一层结构和所述第二层结构上均设置有通道；通道通过硅胶毛细管连接有注射泵；
将第一层结构中通道的一侧外壁去掉；将肠器官芯片固定在固定有三电极传感器阵列的玻璃片上，第一层结构靠近通道去掉外壁的一侧与玻璃片固定，三电极传感器的测试端位于第一层结构的通道中；
在所述肠器官芯片两侧，分别开设与通道平行且贯通的通孔，所述通孔内插入银-氯化银电极。
进一步的，肠器官芯片的制作包括：
将预设比例的基料与固化剂混合，在真空中脱气第一预设时长；
将脱气后的基料与固化剂混合料分别浇筑在预设有多个通道图案的第一模 具和第二模具上；在预设温度下热固第二预设时长；脱模得到具有通道的第一层结构和第二层结构；
在所述第一层结构上打孔；对打孔后的第一层结构、第二层结构和多孔膜进行氧等离子体清洗，清洗时长为第三预设时长；
将清洗后的第一层结构和第二层结构进行粘合，所述多孔膜位于所述第一层结构上的通道和所述第二层结构上的通道之间；
每个通道连通硅胶毛细管；硅胶毛细管连接有注射泵。
进一步的，基料与固化剂的预设比例为重量比是10：1；第一预设时间为30分钟；第二预设时长为4小时；第三预设时长为120秒。
进一步的，所述基料为聚二甲基硅氧烷基料。
进一步的，将肠器官芯片固定在固定有三电极传感器阵列的玻璃片上时，先将肠器官芯片和装有三电极传感器的玻璃片进行氧等离子体清洗。
进一步的，肠器官芯片和装有三电极传感器的玻璃片进行氧等离子体清洗的清洗时间为120秒。
进一步的，将肠器官芯片固定在装有三电极传感器的玻璃片上后，用半固态聚二甲基硅氧烷对肠器官芯片周围进行密封；通孔内插入银-氯化银电极后，用半固态聚二甲基硅氧烷对通孔处进行密封。
进一步的，银-氯化银电极包括第一银-氯化银电极和第二银-氯化银电极；所述第一银-氯化银电极位于和第一层结构的通道贯通的通孔内，所述第二银-氯化银电极位于和第二层结构的通道贯通的通孔内。
进一步的，所述第一银-氯化银电极和所述第二银-氯化银电极连接有电化学工作站。
为了实现上述目的，第二方面，本发明还提出了一种用于探索汞离子的肠器官芯片，采用如下技术方案：
一种用于探索汞离子的肠器官芯片，使用第一方面中所述的用于探索汞离子的肠器官芯片制备的优化方法得到，包括：
开设有通道的第一层结构和第二层结构；
多孔膜，位于第一层结构和第二层结构之间；多孔膜将第一层结构上的通道和第二层结构上的通道隔开；
装有三电极传感器的玻璃片，与第一层结构固定，三电极传感器的测试端位于第一层结构的通道内；
第一银-氯化银电极和第二银-氯化银电极，分别通过预设的通孔设置在肠器官芯片两侧，两侧的通孔分别与第一层结构上的通道和第二层结构上的通道贯通。
与现有技术相比，本发明的有益效果为：
本发明中，在第一层结构和第二层结构上均开设通道，通道通过硅胶毛细管连接有注射泵，流量和机械拉伸由注射泵控制，克服了目前肠器官芯片上肠管没有优化体外仿生条件的缺点；优化得到了一种双通道、机械可伸展的肠道芯片；同时，三电极传感器阵列和银-氯化银电极分别集成在芯片的不同位置，可以实现实时、非侵入性监测跨膜电阻和吸收汞离子的变化。
附图说明
构成本实施例的一部分的说明书附图用来提供对本实施例的进一步理解，本实施例的示意性实施例及其说明用于解释本实施例，并不构成对本实施例的不当限定。
图1为本发明实施例1的局部放大结构示意图；
图2为本发明实施例1的结构示意图；
图3为本发明实施例1的实物效果示意图；
图4为本发明实施例1的仿真结果示意图；
图5为本发明实施例1的验证结果分析；
图6为本发明实施例1的变形量分析；
图7为本发明实施例1的孔直径变化分析；
图8为本发明实施例1的杨氏模量漂移分析；
图9为本发明实施例1的三电极传感器修饰AuNPs和检测Hg 2+的步骤；
图10为本发明实施例1的峰电流分析；
图11为本发明实施例1的氧化峰电流之差和Hg 2+的对数浓度分析；
图12为本发明实施例1的Hg 2+浓度分析；
图13为本发明实施例1的Hg 2+的转运渗透率分析。
具体实施方式：
下面结合附图与实施例对本发明作进一步说明。
实施例1：
本实施例中提供了一种用于探索汞离子的肠器官芯片制备的优化方法，包括：
制作包括第一层结构和第二层结构的肠器官芯片，所述第一层结构和所述第二层结构上均设置有通道；
将第一层结构中通道的一侧外壁去掉，目的在于后续中，当第一层结构固定在玻璃片上时，三电极传感器的测试端可以位于通道内；将肠器官芯片固定 在固定有三电极传感器阵列的玻璃片上，第一层结构靠近通道去掉外壁的一侧与玻璃片固定，三电极传感器的测试端位于第一层结构的通道中；三电极传感器可以通过黏贴的方式固定在玻璃片上，三电极传感器阵列可以包括3个三电极传感器；
在所述肠器官芯片两侧，分别开设与通道平行且贯通的通孔，所述通孔内插入银-氯化银电极。
本实施例中，优化肠器官芯片时，主要可分为肠器官芯片的制造、电化学传感器集成和TEER传感器集成三部分，具体为：
肠器官芯片的制作包括：将预设比例的基料与固化剂混合，在真空中脱气第一预设时长；将脱气后的基料与固化剂混合料分别浇筑在预设有多个通道图案的第一模具和第二模具上；在预设温度下热固第二预设时长；脱模得到具有通道的第一层结构和第二层结构，当第一层结构和第二层结构相同时，第一模具和第二模具可以设置为一个，当第一层结构和第二层结构不同时，第一模具和第二模具不同；在所述第一层结构上打孔；对打孔后的第一层结构、第二层结构和多孔膜进行氧等离子体清洗，清洗时长为第三预设时长；将清洗后的第一层结构和第二层结构进行粘合，所述多孔膜位于所述第一层结构上的通道和所述第二层结构上的通道之间；每个通道连通硅胶毛细管；硅胶毛细管连接有注射泵。其中，基料与固化剂的预设比例可以为重量比是10：1；第一预设时间可以为30分钟；第二预设时长可以为4小时；第三预设时长可以为120秒；所述基料可以为聚二甲基硅氧烷基料。具体的，可以采用软刻技术并使用柔性透明的聚二甲基硅氧烷(PDMS；184Silicone Elastomer，Dow Corning Co.，Midland，MI，USA)制作肠器官芯片；肠器官芯片由三部分组成：顶层微通道、底层微通 道和中间的多孔膜，可以将第一层结构定义为顶层，第二层结构定义为底层，微通道的尺寸可以设置为宽2.0mm×高0.25mm；多孔膜的厚度可以设置为20μm，孔径可以设置为5μm。所述多孔膜将顶层和底层的通道隔开，起到构建组织界面的作用。首先，将重量比例为10：1(wt/wt)的PDMS基料与固化剂混合，在真空中脱气30min，然后浇注在含有微通道图案的模具上，在70℃下热固化4h后，脱模形成了具有微通道的顶层和底层，用打孔器在顶层打孔，将硅胶软管与器官芯片的通道连接；然后，将上下微通道和多孔膜放入氧等离子体清洗机中清洗120s，所述等离子体清洗机可以为CY-P2L-B，CY Scientific InstrumentCO.，Ltd，China，将顶层和底层们粘合在一起，并用半固态PDMS密封和固化周边缝隙；最后，用不锈钢细管将硅胶毛细管连接到每个通道，流量和机械拉伸由高精度注射泵控制，所述高精度注射泵可以为LSP02-1B，Baoding Ditron Electronic Technology CO.，Ltd，China。图1所示，展示了肠器官芯片的实物图片。
电化学传感器集成时，首先，可使用外科刀片沿着微通道边缘切割肠器官芯片的底层，保证底层黏贴到玻璃片后，三电极传感器的测试端可以位于通道内；将固定有三电极传感器阵列的玻璃片和肠器官芯片放入氧等离子体清洗机清洗120s后取出；然后，肠器官芯片的底层被牢固地粘合在玻璃片上，并用半固态PDMS密封在传感器周围；最后，放入75℃的烘干箱中固化2小时后完成。
TEER传感器集时，首先，可以使用打孔针在肠器官芯片的两侧制作出两个圆形通孔，通孔与上下通道相贯通并平行；然后，将两个直径为0.2mm的Ag/AgCl电极插入相应的通孔，用半固态PDMS密封；最后，两个Ag/AgCl电极位于多孔膜的两侧并连接到电化学工作站(PGSTAT302N，Herisau，Switzerland)，测量 多孔膜上下两侧的阻抗变化，可以理解的为，银-氯化银电极包括第一银-氯化银电极和第二银-氯化银电极；所述第一银-氯化银电极位于和第一层结构的通道贯通的通孔内，所述第二银-氯化银电极位于和第二层结构的通道贯通的通孔内。
本实施例中，对肠器官芯片内部参数进行了理论计算与仿真模拟，包括：
肠器官芯片中灌注流量和拉伸应变的标定，为了验证仿真结果的准确性与定量物理参数之间的关系，根据有限元分析(FEA)的结果，对肠器官芯片中的物理参数进行了实验验证；流量可以在10μL/h～400μL/h范围内用注射泵和2ml无菌注射器校准；用注射泵和1ml无菌注射器校准多孔膜的拉伸应变。
细胞培养，人结肠腺癌细胞系Caco-2(RuYao Biotechnology，Zhejiang，China)在DMEM(Gibco，Waltham，MA，USA)、10％胎牛血清(FBS；A3160801，Gibco，USA)和1％青霉素/链霉素(MA0110，Meilunbio，China)的T25培养瓶上培养；所有实验均采用第5～10代之间的Caco-2细胞；对细胞进行了支原体污染的常规检测，结果为阴性；细胞培养可以采用常规或现有技术实现，在此不再详述。
上皮屏障功能的检测，通过共聚焦免疫荧光显微镜对紧密连接蛋白ZO-1的染色和通过测量跨上皮电阻(TEER)来评估人类肠上皮细胞单层的完整性。用Autolab电化学工作站(PGSTAT302N，Herisau，Switzerland)与Ag/AgCl电极线耦合，测量了培养在肠器官芯片和Transwell中的Caco-2单层的TEER。TEER值使用以下公式计算：
TEER＝(R 1-R 0)·S
其中，R 1为实际测量值；R 0是在没有细胞的情况下测量的基值电阻；S是细胞培养的表面积。
暴露在不同浓度汞离子中细胞活性检测，为研究不同浓度Hg 2+对Caco-2细胞活性的影响，采用活/死细胞染色试剂盒(MA0361，Dalian Meilun Biotechnology Co.，Ltd，China)进行图像分析；1mMHg 2+在无血清培养基中稀释为0.5μM、1μM、10μM、30μM、50μM、100μM和200μM，每个浓度分别作用5h、12h和24h。200μL活/死细胞染色试剂盒孵育3min后，用胰蛋白酶/EDTA(0.25％，25200-056，USA)处理Caco-2细胞。用倒置荧光显微镜(Nikon Eclipse TI2)分别在495nm和652nm处记录活细胞(绿色荧光)和死亡细胞(红色荧光)的荧光图像；使用ImageJ软件(National Institutes of Health)对活/死细胞进行计数，计算细胞存活率。
为了建立体外肠道微环境，克服目前芯片上肠管没有优化体外仿生条件(如生理运动和流体流动)的缺点，本实施例提供了一种双通道、机械可伸展的肠道芯片。三电极电化学传感器阵列和Ag/AgCl电极分别集成在芯片多孔膜的底沟道表面、顶面和底面；传感器用于实时、非侵入性监测TEER和吸收Hg 2+的变化，如图1到图3所示。通过有限元分析(FEA，COMSOL MultiPhysical 5.5，试用版)对肠器官芯片内部物理参数的仿生条件进行了模拟，确定了合适的灌注流量和拉伸应变，物理参数可以为流体剪应力、拉应力和应变，灌注流量可以设置为160μL/h；0.02dyne/cm2，拉伸应变可以设置为1％，如图4所示。当灌注流量为40～360μL/h时，培养液的剪应力范围为0.005dyne/cm 2-0.05dyne/cm 2，并流场模拟、理论计算和实验进行了交叉验证，如图5所示。通过有限元分析和实验验证，证明了当吸气压力从0kPa增加到20kPa时，多孔膜和细胞团的变形量都可以从0％线性增加到5.5％，如图6所示，从而使上皮细胞单层产生有节奏的机械变形(类似于人体肠道的蠕动运动)。为了确定多孔膜在长期拉伸下是否 会发生疲劳破坏，本实施例中测量了1.4×10 5循环拉伸(10days)下多孔膜的孔径和杨氏模量的变化。孔直径由5.19±0.10μm略微增大至5.28±0.25μm，如图7所示，杨氏模量由2.54±0.06MPa漂移至2.54±0.05MPa，如图8所示。因此，在一定应变范围内的长期机械拉伸对多孔膜的孔径和杨氏模量的物理性能没有显著改变。
如图1和图2所示，上下微通道由中间多孔膜隔开，细胞接种于其表面，形成生物层界面。三电极传感器和Ag/AgCl电极同时集成，用于原位检测Hg 2+和TEER；图3为集成传感器的肠器官芯片照片；图4为微通道有限元分析(流速、剪应力、拉应力和应变)；图5为流场模拟、理论计算和实验的交叉验证；图6为多孔膜拉伸应变的量化。可以得到，通过机械模拟和实验验证，证明随着吸入压力从0增加到20kPa，多孔膜和细胞团的变形从0％线性增加到5.5％(n＝3)；如图7所示，在1％拉伸应变下10天内多孔膜的孔径变化。RSD＝1.81％(n＝3)；如图8所示，多孔膜在1％拉伸应变下10天内杨氏模量的变化。RSD＝0.07％(n＝3)。
肠器官芯片对吸收汞离子的原位和实时检测，为了实现小肠上皮细胞对Hg 2+吸收的实时检测，本实施例在芯片上肠底通道中集成了一个检测Hg 2+的三电极传感器阵列；三电极传感器主要由四部分组成：玻璃衬底、10nm厚的Gr层、200nm厚的Au层和金纳米颗粒(AuNPs)层。如图9所示，展示了检测系统的照片以及三电极传感器修饰AuNPs和检测Hg 2+的步骤。检测基于Hg 2+的氧化还原反应原理。
在最佳条件下，研究了三电极传感器测定Hg 2+的校准曲线。Hg 2+的峰电流出现在0.02V，在1nM-10μM浓度范围内，峰电流从0.5μA持续增加到7.8μA，如图10所示，。汞离子的拟合曲线为Δi＝0.16976+1.78083lgC(R2＝0.983)。其 中Δi为氧化峰电流之差，lgC为Hg 2+的对数浓度，如图11所示，。计算出对Hg 2+的灵敏度为1.78μA/nM，检出限为0.1nM。
接下来，本实施例在180min内原位检测了Transwell和肠器官芯片中肠上皮细胞对Hg 2+的吸收。在180min的检测过程中，Transwell和芯片上的肠上皮细胞对Hg 2+的吸附浓度分别为2.3μM和1.8μM，吸收率分别为22％和17.8％(顶室/通道的初始Hg 2+浓度为10μM)，如图12所示，。此外，通过计算两种培养方法对Hg 2+的转运渗透率(P app)，得出Transwell的P app值是肠器官芯片的4倍，如图13所示，。这是因为单层细胞在机械刺激下形成更高的紧密连接。
Hg 2+的吸收率<15.0％，低于Caco-2细胞实验的吸收率(17.8％)。应该考虑到，活体研究的特点是存在管腔因素(胆盐、食物成分等)。这在体外模型中没有发现，可能会影响Hg 2+在肠上皮细胞间的转运。因此，该肠道模型能真实反映人体肠道对Hg 2+的吸收情况。
如图9到图10所示，为上皮细胞对汞离子的吸收；其中，如图9所示，为检测装置实物图和三电极传感器检测Hg 2+示意图；如图10所示，为三电极传感器在1nM至10μM不同Hg 2+浓度下的DPV响应；如图11所示，为ΔI(峰值电流与背景电流之差)与Hg 2+浓度的拟合结果；ΔI＝0.16976+1.78083lgC(R 2＝0.983)；如图12所示，为Transwell和肠器官芯片培养180分钟的Hg 2+吸收变化曲线。在180分钟的检测过程中，Transwell和芯片中肠上皮细胞吸收的Hg 2+浓度分别为2.3μM和1.8μM，吸收率分别为22％和17.8％，加入的初始浓度为10μM，n＝3；如图13所示，为Transwell和肠器官芯片吸收Hg 2+的Papp值比较。Transwell的Papp值是肠器官芯片的4倍(n＝3)。
实施例2：
本实施例提供了一种用于探索汞离子的肠器官芯片，使用实施例1中所述的用于探索汞离子的肠器官芯片制备的优化方法得到，包括：
开设有通道的第一层结构和第二层结构；
多孔膜，位于第一层结构和第二层结构之间；多孔膜将第一层结构上的通道和第二层结构上的通道隔开；
装有三电极传感器的玻璃片，与第一层结构固定，三电极传感器的测试端位于第一层结构的通道内；
第一银-氯化银电极和第二银-氯化银电极，分别通过预设的通孔设置在肠器官芯片两侧，两侧的通孔分别与第一层结构上的通道和第二层结构上的通道贯通。
以上所述仅为本实施例的优选实施例而已，并不用于限制本实施例，对于本领域的技术人员来说，本实施例可以有各种更改和变化。凡在本实施例的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本实施例的保护范围之内。

Claims (10)

  1. 一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，包括：
    制作包括第一层结构和第二层结构的肠器官芯片，所述第一层结构和所述第二层结构上均设置有通道；通道通过硅胶毛细管连接有注射泵；
    将第一层结构中通道的一侧外壁去掉；将肠器官芯片固定在固定有三电极传感器阵列的玻璃片上，第一层结构靠近通道去掉外壁的一侧与玻璃片固定，三电极传感器的测试端位于第一层结构的通道中；
    在所述肠器官芯片两侧，分别开设与通道平行且贯通的通孔，所述通孔内插入银-氯化银电极。
  2. 如权利要求1所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，肠器官芯片的制作包括：
    将预设比例的基料与固化剂混合，在真空中脱气第一预设时长；
    将脱气后的基料与固化剂混合料分别浇筑在预设有多个通道图案的第一模具和第二模具上；在预设温度下热固第二预设时长；脱模得到具有通道的第一层结构和第二层结构；
    在所述第一层结构上打孔；对打孔后的第一层结构、第二层结构和多孔膜进行氧等离子体清洗，清洗时长为第三预设时长；
    将清洗后的第一层结构和第二层结构进行粘合，所述多孔膜位于所述第一层结构上的通道和所述第二层结构上的通道之间；
    每个通道连通硅胶毛细管；硅胶毛细管连接有注射泵。
  3. 如权利要求2所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，基料与固化剂的预设比例为重量比是10：1；第一预设时间为30分钟；第二预设时长为4小时；第三预设时长为120秒。
  4. 如权利要求2所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，所述基料为聚二甲基硅氧烷基料。
  5. 如权利要求1所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，将肠器官芯片固定在固定有三电极传感器阵列的玻璃片上时，先将肠器官芯片和装有三电极传感器的玻璃片进行氧等离子体清洗。
  6. 如权利要求5所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，肠器官芯片和装有三电极传感器的玻璃片进行氧等离子体清洗的清洗时间为120秒。
  7. 如权利要求1所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，将肠器官芯片固定在装有三电极传感器的玻璃片上后，用半固态聚二甲基硅氧烷对肠器官芯片周围进行密封；通孔内插入银-氯化银电极后，用半固态聚二甲基硅氧烷对通孔处进行密封。
  8. 如权利要求1所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，银-氯化银电极包括第一银-氯化银电极和第二银-氯化银电极；所述第一银-氯化银电极位于和第一层结构的通道贯通的通孔内，所述第二银-氯化银电极位于和第二层结构的通道贯通的通孔内。
  9. 如权利要求8所述的一种用于探索汞离子的肠器官芯片制备的优化方法，其特征在于，所述第一银-氯化银电极和所述第二银-氯化银电极连接有电化学工作站。
  10. 一种用于探索汞离子的肠器官芯片，其特征在于，使用权利要求1-9任一项所述的用于探索汞离子的肠器官芯片制备的优化方法得到，包括：
    开设有通道的第一层结构和第二层结构；
    多孔膜，位于第一层结构和第二层结构之间；多孔膜将第一层结构上的通道和第二层结构上的通道隔开；
    装有三电极传感器的玻璃片，与第一层结构固定，三电极传感器的测试端位于第一层结构的通道内；
    第一银-氯化银电极和第二银-氯化银电极，分别通过预设的通孔设置在肠器官芯片两侧，两侧的通孔分别与第一层结构上的通道和第二层结构上的通道贯通。
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