Intestine-on-a-chip integrated with biosensor, preparation method and application thereof

Abstract

The application discloses an intestinal organ chip integrated with a biosensor, a preparation method and application, and comprises a substrate layer, wherein a cell culture area and a CEA detection area are arranged on the substrate layer; the cell culture area comprises a top microchannel and a bottom microchannel, and an intermediate porous membrane for constructing a tissue interface is arranged between the top microchannel and the bottom microchannel; the CEA detection area comprises at least two chambers, and a biosensor is integrated in each chamber; the biosensor is subjected to functionalization treatment, so that CEA secreted in the cell culture area can be attached to the surface of the biosensor; and the bottom microchannel is in communication with a microchannel of the CEA detection area. The application can dynamically detect CEA secretion in the differentiation process of Caco-2 cells, and on the basis of detecting the CEA secretion level and the relationship between CEA and the differentiation of Caco-2 cells, the difference and applicability of Caco-2 cells in the construction of GOC are further explored.

Description

Technical Field

[0001] This invention relates to the field of intestinal organ-on-a-chip technology, and in particular to an intestinal organ-on-a-chip integrated with a biosensor, its fabrication method, and its applications. Background Technology

[0002] The statements in this section are merely background information related to the present invention and do not necessarily constitute prior art.

[0003] Organ-on-a-chip (GOC) has shown great potential in studying drug action, microbes, and mechanisms of human disease. The dynamic culture conditions in GOCs enhance the intestinal endothelial barrier, helping to maintain microbiome homeostasis and promoting metabolite transport under homeostatic conditions. In vitro GOC models show promise in overcoming many limitations of traditional cell culture techniques. For example, mechanically stimulated GOCs can promote the development of human intestinal-like villi and the formation of a functional mucus layer.

[0004] Human colorectal cells (Caco-2) are commonly used (>66%) on various in vitro intestinal platforms because their structure and function are similar to differentiated intestinal epithelial cells. Studies have shown that Caco-2 can form villous-like basal proliferative crypts in GOCs. Compared to statically cultured Caco-2, this mechanically active and microengineered intestinal epithelium more closely resembles in vivo intestinal physiology in terms of CYP3A4 activity, glucose reuptake, and mucus secretion. However, the carriers and transporters secreted on the cell membrane differ significantly from those expressed in in vivo intestinal epithelium. P-glycoprotein (P-gp), an efflux transporter on the apical side, is overexpressed in Caco-2 cells. This reduces cellular drug uptake, leading to multidrug resistance. Similarly, low cholesterol secretion is observed in Caco-2 cells, which increases cell membrane fluidity. This may lead to the overexpression and extracellular migration of carcinoembryonic antigen (CEA), a glycosylphosphatidylinositol-anchored cell surface glycoprotein. Overexpression of CEA protects human colon cancer cells from apoptosis induced by drug therapy, ultraviolet radiation, and fusion growth. Furthermore, CEA-associated cell adhesion molecule 6 (CEA-CAM6) promotes cancer cell differentiation and invasion, leading to the unlimited proliferation of Caco-2 cells in the absence of contact inhibition. Therefore, it is necessary to investigate the differences and applicability of CEA secretion by Caco-2 cells at different growth and differentiation stages in GOCs. However, existing GOCs cannot dynamically detect CEA secretion during Caco-2 cell differentiation. Summary of the Invention

[0005] To address the aforementioned issues, this invention proposes an intestinal organ-on-a-chip (GOC) integrated with a biosensor, its preparation method, and its application. This OCC can dynamically detect CEA secretion during Caco-2 cell differentiation. Furthermore, based on the detection of CEA secretion levels and the relationship between CEA and Caco-2 cell differentiation, the applicability of Caco-2 cells in constructing GOCs is further explored.

[0006] In some implementations, the following technical solutions are adopted:

[0007] An intestinal organ-on-a-chip integrated with a biosensor includes: a base layer, wherein a cell culture region and a CEA detection region are respectively provided on the base layer;

[0008] The cell culture area includes a top microchannel and a bottom microchannel, and an intermediate porous membrane for constructing a tissue interface is provided between the top microchannel and the bottom microchannel.

[0009] The CEA detection area includes at least two chambers, and biosensors are integrated into each chamber. The biosensors are functionalized so that CEA secreted in the cell culture area can adhere to the surface of the biosensors. The microchannels of the CEA detection area pass through the two chambers in sequence. The bottom microchannel is connected to the microchannels of the CEA detection area.

[0010] Vacuum chambers are provided on both sides of the cell culture area. Applying negative pressure in the vacuum chambers can achieve mechanical stretching of the porous membrane in the middle.

[0011] Gold nanoparticles are deposited on the surface of the working electrode of the biosensor. The gold nanoparticles are modified with MUA / MPA and EDC / NHS, respectively, and then conjugated with carcinoembryonic antigen (CEA) antibody.

[0012] Caco-2 cell culture medium is injected into the top microchannel of the cell culture zone, and CEA secreted by Caco-2 cells seeps into the culture medium in the bottom microchannel through the middle porous membrane; the culture medium in the bottom microchannel is then sent into the CEA detection zone microchannel at set time intervals.

[0013] When the culture medium flows through the microchannels in the CEA detection zone, CEA can adhere to the surface of the biosensor; R is continuously measured at set time intervals. ct The changes in CEA values ​​and CEA concentrations were studied to determine the relationship between CEA and Caco-2 cell differentiation.

[0014] In the above process, the intermediate porous membrane is mechanically stretched by an injection pump to simulate intestinal movement.

[0015] In other embodiments, the following technical solutions are adopted:

[0016] A method for fabricating an intestinal organ-on-a-chip integrated with a biosensor, comprising:

[0017] Top and bottom microchannels of the cell culture area in the intestinal organ-on-a-chip (GOC) were prepared, and an intermediate porous membrane for constructing the tissue interface was set between the top and bottom microchannels.

[0018] A microchannel for the CEA detection region in the intestinal organ-on-a-chip (GOC) was prepared; the CEA detection region microchannel includes at least two chambers;

[0019] Each chamber integrates a biosensor, which is then functionalized.

[0020] Connect the bottom microchannel to the CEA detection area microchannel; connect the top microchannel, bottom microchannel, and CEA detection area microchannel to the microfluidic device respectively.

[0021] As a further step, the biosensor is functionalized, and the specific process is as follows:

[0022] A set amount of HAuCl4 was injected into the detection area, and AuNPs were electrodeposited on the working electrode by chronoamperometry at a constant potential. After a set time, the solution was drained and washed with anhydrous ethanol.

[0023] The MUA / MPA was incubated for a set time to allow carboxyl groups to be introduced onto the sensor surface; the solution was washed with anhydrous ethanol; the mixing ratio of MUA and MPA was 7:3.

[0024] The EDC / NHS was incubated for a set time to activate the carboxyl groups and promote the binding of amino groups to the carboxyl groups on the sensor; the solution was washed with anhydrous ethanol; the mixing ratio of EDC and NHS was 1:1.

[0025] The carcinoembryonic antigen (CEA) antibody was incubated for a set time, and the solution was washed with anhydrous ethanol.

[0026] As a further step, the top and bottom microchannels of the cell culture area in the intestinal organ-on-a-chip (GOC) were prepared, and the specific process is as follows:

[0027] Mix the base elastomer and curing agent of PDMS in a set ratio;

[0028] After degassing for a set time, PDMS is poured into a mold with a microchannel pattern and cured at 60℃-80℃ to form the top and bottom layers of the microchannels. A hole is punched in the top layer. The top layer, the middle porous membrane and the bottom layer are then bonded together by oxygen plasma to form the top microchannel and the bottom microchannel separated by the middle porous membrane. At the same time, a vacuum cavity is formed in the middle of the top microchannel and the bottom microchannel.

[0029] As a further embodiment, the fabrication process of the biosensor is as follows:

[0030] The glass substrate was cleaned using an ultrasonic cleaner, and the surface was dried with nitrogen gas.

[0031] Clean the glass surface using an oxygen plasma cleaner, cover the glass substrate with a patterned shadow mask and place it in a vacuum evaporator;

[0032] Using Cr as an adhesion promoter for the gold film, Au of a set thickness is deposited on the glass using vacuum evaporation; after peeling the mask off the wafer, the desired pattern is obtained.

[0033] In other embodiments, the following technical solutions are adopted:

[0034] Application of an intestinal organ-on-a-chip integrated with a biosensor in the dynamic detection of CEA secretion during Caco-2 cell differentiation.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] (1) This invention integrates a biosensor into the intestinal organ chip GOC and performs functional processing on the biosensor so that the intestinal organ chip GOC can dynamically detect CEA secretion during the differentiation process of Caco-2 cells. At the same time, based on the detection of CEA secretion level and the relationship between CEA and Caco-2 cell differentiation, the differences and applicability of Caco-2 cells in the construction of GOC are further explored.

[0037] (2) The process of functionalizing the biosensor in this invention can effectively improve the stability and sensitivity of the sensor in detecting CEA.

[0038] Other features and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the fabrication process of the intestinal organ chip integrated with a biosensor in an embodiment of the present invention; wherein, A-1 represents the cell detection area and A-2 represents the CEA detection area;

[0040] Figure 2This is a schematic diagram of fluid flow and micromechanical stretching in the GOC in an embodiment of the present invention; wherein, B-1 shows SEM images of the intermediate porous membrane (left, scale bar 5 μm) before and after stretching (0% vs 10%), and microscopic images of Caco-2 cells cultured on the membrane surface (right, scale bar 20 μm) before and after stretching (0% vs 10%); B-2 shows the external contours of the porous membrane and Caco-2 cells before and after stretching (0% vs 10%).

[0041] Figure 3 This is a schematic diagram of the composition of the electrochemical biosensor in the GOC in the embodiments of the present invention (scale bar 1mm);

[0042] Figure 4 This is a physical image of the intestinal organ chip integrated with a biosensor according to an embodiment of the present invention;

[0043] Figure 5 This is a schematic diagram of the biosensor functionalization process in an embodiment of the present invention;

[0044] Figures 6(a)-(b) are SEM images (scale bar, 1 μm) of the bare Au electrode surface and the AuNP modified on the electrode surface, respectively, in the embodiments of the present invention.

[0045] Figure 7 This is a schematic diagram of the FTIR spectra of MUA-MPA and EDC-NHS in an embodiment of the present invention;

[0046] Figure 8 This is the XPS spectrum of S-2p in an embodiment of the present invention;

[0047] Figure 9 This is a schematic diagram of the C-1S binding energy of MUA-MPA modified on the gold electrode in an embodiment of the present invention;

[0048] Figure 10 This is the XPS spectrum of the N-1s core level of the MUA-MPA activated by the Au electrode modified with EDC-NHS in this embodiment of the invention.

[0049] Figure 11 A-1 shows the proposed GOC and its monitoring and culture components; A-2 shows a micrograph of cells cultured on a porous membrane;

[0050] Figure 12(a) shows the R values ​​of CEA secreted by Caco-2 cells on different culture days in this embodiment of the invention. ct Schematic diagram of value changes;

[0051] Figure 12(b) shows the change in CEA concentration corresponding to the measurement results in Figure 12(a). Detailed Implementation

[0052] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0054] Terminology Explanation:

[0055] R ct , is the charge transfer resistance, or electrochemical polarization resistance;

[0056] GOC, Intestinal Organ Microarray;

[0057] Caco-2 cells, human colorectal cells;

[0058] CEA, carcinoembryonic antigen;

[0059] HAuCl4, tetrachloroauric acid;

[0060] AuNPs, gold nanoparticles;

[0061] MPA, 3-mercaptopropionic acid;

[0062] MUA, 11-mercaptoalkyl acid;

[0063] EDC, 1-Ethyl-(3-dimethylaminopropyl)carbodiimide;

[0064] NHS, N-hydroxysuccinimide;

[0065] BSA, bovine serum albumin.

[0066] Example 1

[0067] In one or more embodiments, an intestinal organ-on-a-chip integrated with a biosensor is disclosed, combining... Figure 1 and Figure 4 Specifically, it includes: a basal layer, on which a cell culture area and a CEA detection area are respectively set;

[0068] in, Figure 1A-1 in the diagram shows the structure of the cell detection zone; the cell culture zone includes a top microchannel and a bottom microchannel, with an intermediate porous membrane between the top and bottom microchannels for constructing a tissue interface;

[0069] In this embodiment, the middle porous membrane separates the top microchannel and the bottom microchannel, and the porous membrane divides the upper and lower parts, thus forming a tissue interface; the top microchannel is perfused with Caco-2 cell culture medium, and the bottom microchannel is perfused with culture medium; Caco-2 cells can secrete CEA during growth and development, and CEA can permeate into the culture medium of the bottom microchannel through the middle porous membrane; as a specific implementation, the dimensions of the top microchannel and the bottom microchannel are 2.0 mm wide × 0.25 mm high; the dimensions of the middle porous membrane are 20 μm thick and 5 μm pore size.

[0070] Figure 1 Figure A-2 shows the structure of the CEA detection area, which includes two circular chambers, each of which integrates a biosensor. The CEA detection area microchannel passes through the two chambers in sequence. The bottom microchannel is connected to the CEA detection area microchannel, and the culture medium in the bottom microchannel can enter the CEA detection area microchannel and contact the biosensor through the two chambers.

[0071] In this embodiment, vacuum chambers are provided on both sides of the cell culture area. Using a high-precision injection pump (LSP02-1B, Baoding Ditron Electronic Technology CO., Ltd.) to apply negative pressure in the vacuum chamber can achieve mechanical stretching of the intermediate porous membrane, thereby simulating intestinal movement and promoting the secretion of CEA by Caco-2 cells.

[0072] Figure 2 A schematic diagram of fluid flow and micromechanical stretching in GOC is given, wherein, Figure 2 The left side of B-1 shows SEM images of the porous membrane (scale bar 5 μm) before and after stretching (0% vs 10%), and the right side shows microscopic images of Caco-2 cells cultured on the membrane surface before and after stretching (0% vs 10%) (scale bar 20 μm). Figure 2 B-2 in the diagram shows the external contours of the porous membrane and Caco-2 cells before and after stretching (0% vs 10%). This demonstrates that the intestinal organ-on-a-chip of this embodiment can achieve the application of mechanical stimulation.

[0073] In this embodiment, combined with Figure 3The sensor for long-term monitoring of CEA secretion employs a three-electrode configuration: an Au reference electrode (RE), an Au counter electrode (CE), and an Au working electrode (WE). Gold nanoparticles are deposited on the surface of the biosensor electrodes, and the gold nanoparticles are modified with MUA / MPA and EDC / NHS, respectively, before being conjugated with carcinoembryonic antigen (CEA) antibodies.

[0074] In this embodiment, the top microchannel, bottom microchannel, and CEA detection area microchannel are each connected to a microfluidic device via thin tubes. The flow rate of the cell culture medium is controlled by the microfluidic device.

[0075] In this embodiment, the intestinal organ chip integrated with the biosensor is used by injecting Caco-2 cell culture medium into the top microchannel of the cell culture area, and CEA secreted by Caco-2 cells seeps into the culture medium in the bottom microchannel through the middle porous membrane; the culture medium in the bottom microchannel is sent into the CEA detection area microchannel at set time intervals.

[0076] When the culture medium flows through the microchannels in the CEA detection zone, CEA can adhere to the surface of the biosensor; R is continuously measured at set time intervals. ct The changes in CEA values ​​and CEA concentrations were studied to determine the relationship between CEA and Caco-2 cell differentiation.

[0077] In the above process, the intermediate porous membrane is mechanically stretched by an injection pump to simulate intestinal movement.

[0078] The principle of CEA detection by biosensors is as follows:

[0079] After functionalizing the sensor, it is necessary to prepare CEA solutions of different concentrations (0.01 ng / ml-100 ng / ml) to calibrate the sensor, that is, to obtain the impedance values ​​under different CEA concentrations and fit a linear equation between CEA concentration and Rct value.

[0080] In subsequent experiments, it is only necessary to detect the Rct value and substitute it into the linear equation to obtain the concentration of CEA.

[0081] The intestinal organ-on-a-chip integrated with the biosensor in this embodiment can fully simulate intestinal motility and dynamically detect CEA secretion during the differentiation process of Caco-2 cells through the integrated biosensor.

[0082] Example 2

[0083] In one or more embodiments, a method for fabricating an intestinal organ-on-a-chip integrated with a biosensor is disclosed, combining... Figure 1 and Figure 5 Specifically, it includes the following process:

[0084] (1) Prepare the top and bottom microchannels of the cell culture area in the intestinal organ-on-a-chip (GOC), and set an intermediate porous membrane between the top and bottom microchannels to construct the tissue interface.

[0085] In this embodiment, GOC is made of polydimethylsiloxane (PDMS, 184SiliconeElastomer, Dow Corning Co., USA).

[0086] Combination Figure 1 First, the base elastomer and curing agent of PDMS were mixed at a weight ratio of 10:1 (wt / wt). After degassing for 30 minutes, the PDMS was poured into a mold with a microchannel pattern. After curing at 70°C for 4 hours, a top and bottom layer with microchannels were formed. Holes were punched in the top layer to provide cell culture medium. The top, porous membrane, and bottom layer were then bonded together by oxygen plasma for 120 seconds (CY-P2L-B, CY Scientific Instrument CO., Ltd, China). Finally, the microchannels in the chip were connected to silicone capillaries using stainless steel capillaries. Flow rate and mechanical stretching were controlled using a high-precision injection pump (LSP02-1B, Baoding Ditron Electronic Technology CO., Ltd.).

[0087] (2) Preparation of CEA detection area microchannel in intestinal organ-on-a-chip (GOC); the CEA detection area microchannel includes two chambers.

[0088] (3) Prepare biosensors by integrating biosensors into the two chambers of the CEA detection area; and perform functionalization on the biosensors.

[0089] In this embodiment, the sensor used for long-term monitoring of CEA secretion adopts a three-electrode configuration: Au reference electrode (RE), Au counter electrode (CE), and Au working electrode (WE).

[0090] Of course, a two-electrode configuration can also be selected as needed, consisting of a working electrode and a counter electrode.

[0091] The sensor was fabricated using a shadow mask process and vacuum vapor deposition. The glass substrate was cleaned for 30 minutes using an ultrasonic cleaner (CR-010S, China) and then dried with nitrogen. An oxygen plasma cleaner (CY-P2L-B, CY Scientific Instrument CO., Ltd, China) was used to clean the glass surface for 120 seconds. A patterned shadow mask was then applied to the glass substrate and placed in a vacuum evaporator (Angstrom Engineering, Canada). A 10 nm thick Cr layer (acting as an adhesion promoter for the gold film) and a 100 nm thick Au layer were deposited on the glass using vacuum evaporation. After the mask was peeled off from the wafer, the desired pattern was achieved without any wet processing.

[0092] The biosensor and CEA detection area were cleaned with oxygen plasma for 120 seconds and then bonded together. Finally, the prepared GOC was connected to the microfluidic device via a silicone capillary.

[0093] Functionalization of biosensors, such as Figure 5 As shown, the specific process includes the following:

[0094] ① Inject 2 mM HAuCl4 into the detection area, and electrodeposit AuNPs (gold nanoparticles) onto the working electrode for 160 seconds at a constant potential of -0.2 V using a chronoamperometry method. The solution is then drained and washed with anhydrous ethanol.

[0095] ② Infuse MUA / MPA (7:3 v / v; 5 mM Sigma prepared in anhydrous ethanol) for 12 hours. Carboxyl groups are introduced onto the sensor surface. The solution is washed with anhydrous ethanol.

[0096] ③ EDC / NHS (EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide) (volume ratio 1:1; v / v; in 50 mM PBS buffer solution at pH 4.5, Sigma) perfusion incubation for 1 h to activate carboxyl groups and promote the binding of amino groups to carboxyl groups on the sensor. The washing procedure is the same as the above operation.

[0097] ④ Infuse with carcinoembryonic antigen (CEA) antibody (10 μg / mL) and incubate for 1 hour. Repeat the washing steps.

[0098] ⑤ Blocking was performed using bovine serum albumin (BSA).

[0099] In this embodiment, the surface of the biosensor was characterized using scanning electron microscopy (SEM, Hitachi SU8100), Fourier transform infrared spectroscopy (FTIR, BRUKERALPHA, Germany), and X-ray photoelectron spectroscopy (XPS, Escalab 250Xi, Thermo Fisher, USA). Furthermore, each modification step was characterized using an electrochemical workstation (PGSTAT302N, Switzerland).

[0100] Specifically, the surface morphology of the electrode was analyzed by scanning electron microscopy (SEM) before and after modification with gold nanoparticles (AuNPs). Figures 6(a)-(b) show SEM images (scale bar, 1 μm) of the bare Au electrode surface and AuNPs modified on the electrode surface, respectively. The figures show that the diameter of the AuNPs is concentrated between 20 nm and 70 nm.

[0101] In Figure 6(a), the bare electrode has a flat and uniform surface, while in Figure 6(b), the surface of the modified electrode is densely deposited with spherical AuNPs with a diameter of 20 nm-70 nm; AuNPs help to expand the electrochemical response and provide a basis for fixing MUA-MPA through Au-S bonds.

[0102] Fourier transform infrared spectroscopy (FTIR) is used to confirm the presence of functional groups on the electrodes. Figure 7 The FTIR spectra of MUA-MPA and EDC-NHS are shown. Stretching vibrations appear at 2914 cm⁻¹. -1 2848cm -1 and 1695cm -1 Corresponding to -CH2 and C=O. EDC-NHS (1638cm) -1 and 1556cm -1 The peaks at these locations correspond to the amide I band (C=O stretching vibration) and the amide III band (CN stretching vibration), respectively.

[0103] Figure 8 The XPS spectrum of S-2p is shown, with the first binding energy at 161.5 eV representing the gold-sulfur bond (Au-S). The second binding energy at 162.3 eV is due to the non-covalent adsorption of free thiol groups onto the Au / AuNPs electrode.

[0104] Figure 9 The C-1S binding energy of MUA-MPA modified on the gold electrode is shown. The binding energy at 284.2 eV is attributed to the -CH2 groups in MUA and MPA, while the peak at 287.7 eV represents the characteristic carboxyl group after binding.

[0105] Figure 10XPS spectra of the N-1s core level of MUA-MPA activated by an Au electrode modified with EDC-NHS are shown. A nitrogen binding energy of 399.8 eV at the activated surface was found in the N-1s spectrum, indicating the presence of an O-acylisourea intermediate formed by the reaction of EDC with the carboxylic acid moieties of MUA and MPA.

[0106] X-ray photoelectron spectroscopy (XPS) and Figures 8-10 Further confirmation indicates that after modifying the Au / AuNPs electrode surface with MUA-MPA, FTIR values ​​at 2914, 2848, and 1695 cm⁻¹ corresponding to -CH₂ and C=O are improved. -1 Stretching vibrations occurred at that location. This result is consistent with... Figure 7 161.2 eV and in the XPS spectrum Figure 9 The peak value at 284.2 eV showed a consistent change. Therefore, the formation of an alkylthiol self-assembled film (SAM) on the electrode was confirmed. After EDC-NHS modification, the peak value at 1638 cm⁻¹ was [missing value]. -1 and 1556cm -1 The peaks at these locations represent the amide I band (C=O stretching vibration) and the amide III band (CN stretching vibration), respectively; furthermore, Figure 10 XPS analysis showed a nitrogen 1s (N-1s) peak (399.8 eV), indicating the presence of an O-acylisourea intermediate formed by the reaction of EDC with the carboxylic acid moieties of MUA and MPA.

[0107] The cell culture process in this embodiment is as follows:

[0108] Human colon adenocarcinoma cell line Caco-2 (e.g., Meilunbio, Zhejiang, China) was cultured in DMEM (Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS; A3160801, Gibco, USA) and 1% penicillin / streptomycin (MA0110, Meilunbio, China). Caco-2 cells between passage 5 and passage 10 were used for all experiments. Routine mycoplasma contamination testing of the cells was performed, and the results were negative.

[0109] To dynamically measure CEA secretion from GOCs, GOCs and their monitoring and culture components were constructed, such as... Figure 11 As shown in A-1. To dynamically perfuse culture medium in the GOC, control valves and microfluidic channels were programmed to allow solution to enter at predetermined times. Caco-2 cells (1.5 × 10⁻⁶) were then introduced. 5 pcs / cm 2 A micrograph of cells cultured on the porous membrane after injection into the top layer of the GOC porous membrane is shown below. Figure 11 As shown in A-2 (scale bar, 50 μm).

[0110] R was measured continuously for 10 days every 24 hours.ct The changes in values ​​are shown in Figure 12(a), n=3; *P<0.05, **P<0.01;

[0111] After 1-5 days of cultivation, R ct The value increased from 17.75±1.52kΩ to 24.44±1.57kΩ (ΔR). ct =6.69±1.54kΩ), and the difference was not statistically significant (P=0.06>0.05).

[0112] Within 6-10 days of cultivation, R ct The value increased from 30.35±1.65kΩ to 39.30±1.42kΩ (ΔR) ct =8.95±1.53kΩ), the difference was significant (P<0.05).

[0113] Accordingly, the CEA concentration trend curve was transformed by the regression equation, as shown in Figure 12(b), n=3; *P<0.05, **P<0.01.

[0114] When a fused epithelial monolayer has not formed (≤5 days), CEA secretion is less than 3.6 × 10⁻⁶. -3 ±1.2×10 -3 The concentration of CEA increased from 0.03±0.01 ng / ml to 0.22±0.06 ng / ml after 6-10 days of culture (after the formation of a complete cell barrier), an increase of 7.3 times (day 10 compared to day 6). This indicates that CEA secretion is related to the growth and development of Caco-2 cells.

[0115] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. An intestinal organ-on-a-chip integrated with a biosensor, characterized in that, Includes: a basal layer, on which a cell culture area and a CEA detection area are respectively provided; The cell culture area includes a top microchannel and a bottom microchannel, with an intermediate porous membrane for constructing a tissue interface provided between the top and bottom microchannels; vacuum chambers are provided on both sides of the cell culture area, and applying negative pressure in the vacuum chambers can achieve mechanical stretching of the intermediate porous membrane; The CEA detection area includes at least two chambers, and biosensors are integrated into each chamber. The biosensors are functionalized so that CEA secreted in the cell culture area can adhere to the surface of the biosensors. Microchannels in the CEA detection area pass through the two chambers in sequence. The bottom microchannel is connected to the microchannels in the CEA detection area. The specific process for functionalizing biosensors is as follows: A set amount of HAuCl4 was injected into the detection area, and AuNPs were electrodeposited on the working electrode by chronoamperometry at a constant potential. After a set time, the solution was drained and washed with anhydrous ethanol. The MUA / MPA was incubated for a set time to allow carboxyl groups to be introduced onto the sensor surface; the solution was washed with anhydrous ethanol; the mixing ratio of MUA and MPA was 7:

3. The EDC / NHS was incubated for a set time to activate the carboxyl groups and promote the binding of amino groups to the carboxyl groups on the sensor; the solution was washed with anhydrous ethanol; the mixing ratio of EDC and NHS was 1:

1. The carcinoembryonic antigen (CEA) antibody was incubated for a set time, and the solution was washed with anhydrous ethanol.

2. The intestinal organ-on-a-chip integrated with a biosensor as described in claim 1, characterized in that, Gold nanoparticles are deposited on the surface of the working electrode of the biosensor. The gold nanoparticles are modified with MUA / MPA and EDC / NHS, respectively, and then conjugated with carcinoembryonic antigen (CEA) antibody.

3. The intestinal organ-on-a-chip integrated with a biosensor as described in claim 1, characterized in that, The top microchannel, bottom microchannel, and CEA detection area microchannel are each connected to the microfluidic device via thin tubes.

4. The intestinal organ-on-a-chip integrated with a biosensor as described in claim 1, characterized in that, Caco-2 cell culture medium is injected into the top microchannel of the cell culture zone, and CEA secreted by Caco-2 cells seeps into the culture medium in the bottom microchannel through the middle porous membrane; the culture medium in the bottom microchannel is then sent into the CEA detection zone microchannel at set time intervals. When the culture medium flows through the microchannels in the CEA detection zone, CEA can adhere to the surface of the biosensor; R is continuously measured at set time intervals. ct The changes in CEA values ​​and CEA concentrations were studied to determine the relationship between CEA and Caco-2 cell differentiation. In the above process, the intermediate porous membrane is mechanically stretched by an injection pump to simulate intestinal movement.

5. A method for fabricating an intestinal organ-on-a-chip integrated with a biosensor, characterized in that, include: The top and bottom microchannels of the cell culture area in the intestinal organ-on-a-chip (GOC) are prepared, and an intermediate porous membrane for constructing the tissue interface is set between the top and bottom microchannels; vacuum cavities are provided on both sides of the cell culture area, and applying negative pressure in the vacuum cavities can achieve mechanical stretching of the intermediate porous membrane; A microchannel for the CEA detection region in the intestinal organ-on-a-chip (GOC) was prepared; the CEA detection region microchannel includes at least two chambers; Each chamber integrates a biosensor, which is then functionalized. Connect the bottom microchannel to the CEA detection area microchannel; connect the top microchannel, bottom microchannel, and CEA detection area microchannel to the microfluidic device respectively; perform functionalization processing on the biosensor, the specific process of which is as follows: A set amount of HAuCl4 was injected into the detection area, and AuNPs were electrodeposited on the working electrode by chronoamperometry at a constant potential. After a set time, the solution was drained and washed with anhydrous ethanol. The MUA / MPA incubation time is set to allow carboxyl groups to be introduced onto the surface of the sensor; The solution was washed with anhydrous ethanol; The mixing ratio of MUA and MPA is 7:3; The EDC / NHS was incubated for a set time to activate the carboxyl groups and promote the binding of amino groups to the carboxyl groups on the sensor; the solution was washed with anhydrous ethanol; the mixing ratio of EDC and NHS was 1:

1. The carcinoembryonic antigen (CEA) antibody was incubated for a set time, and the solution was washed with anhydrous ethanol.

6. The method for fabricating an intestinal organ-on-a-chip integrated with a biosensor as described in claim 5, characterized in that, The specific process for preparing the top and bottom microchannels of the cell culture region in the intestinal organ-on-a-chip (GOC) is as follows: Mix the base elastomer and curing agent of PDMS in a set ratio; After degassing for a set time, PDMS is poured into a mold with a microchannel pattern and cured at 60℃-80℃ to form the top and bottom layers of the microchannels. A hole is punched in the top layer. The top layer, the middle porous membrane and the bottom layer are then bonded together by oxygen plasma to form the top microchannel and the bottom microchannel separated by the middle porous membrane. At the same time, a vacuum cavity is formed in the middle of the top microchannel and the bottom microchannel.

7. The method for fabricating an intestinal organ-on-a-chip integrated with a biosensor as described in claim 5, characterized in that, The fabrication process of the biosensor is as follows: The glass substrate was cleaned using an ultrasonic cleaner, and the surface was dried with nitrogen gas. Clean the glass surface using an oxygen plasma cleaner, cover the glass substrate with a patterned shadow mask and place it in a vacuum evaporator; Using Cr as an adhesion promoter for the gold film, Au of a set thickness is deposited on the glass using vacuum evaporation; after peeling the mask off the wafer, the desired pattern is obtained.

8. The application of an intestinal organ-on-a-chip integrated with a biosensor according to any one of claims 1-4 in the dynamic detection of CEA secretion during Caco-2 cell differentiation.

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