A multi-parameter microenvironment combined analysis gastrointestinal capsule and detection method thereof

Abstract

The application discloses a gastrointestinal capsule for multi-parameter microenvironment combined analysis and a detection method thereof, and belongs to the technical field of biosensing. The gastrointestinal capsule is used for microenvironment multi-parameter detection of serotonin, ORP and pH value in the gastrointestinal tract, can comprehensively reflect the multidimensional microenvironment physiological state of neurotransmitters, oxidative stress and acid-base environment and the like in the gastrointestinal tract, and through aptamer functional modification of an electrochemical sensor, the influence of biological pollution, electrode surface passivation and signal drift on the work of the electrochemical sensor in the gastrointestinal environment is improved, so that the detection stability and specificity in the complex gastrointestinal environment are enhanced. Through modification of a filter layer, a water-soluble protective layer and an enteric response layer from the inside to the outside of a sampling hole, particle impurities are filtered, the exposure of a sensing area is controlled, the pollution of the sensing area in the complex environment is reduced, and the directional sampling function in the intestinal tract is realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensing, specifically relating to a gastrointestinal capsule for multi-parameter microenvironment joint analysis and its detection method. Background Technology

[0002] Disruptions in the homeostasis of the gastrointestinal tract microenvironment can lead to symptoms such as diarrhea, abdominal pain, and gastrointestinal bleeding. Current biochemical diagnostic methods for the gastrointestinal tract mainly include endoscopy and stool examination. Endoscopy is invasive, has poor patient compliance, and cannot be repeated within a short period; stool examination cannot achieve in-situ real-time monitoring, physiological indicators are lagging, and it is difficult to reflect the dynamic changes in the microenvironment of different segments of the gastrointestinal tract. To overcome these limitations, ingestible sensor capsules have become a research hotspot in recent years. As background technology, the following related patent schemes have been disclosed in China:

[0003] A detection device for the physiological environment of the gastrointestinal tract, disclosed in patent publication number CN118121195A, is mounted on an endoscope and includes a housing installed on the endoscope body. The housing is characterized by having a sampling mechanism for aspirating and placing gastric fluid samples and an image acquisition component for magnifying the image of the gastric fluid sample from the sampling mechanism and acquiring the magnified image. This device solves the problem of morphological analysis of microbiota in multiple gastric fluid samples from the gastrointestinal tract through the sampling mechanism mounted on the endoscope. However, microscopic imaging only provides qualitative information and lacks quantitative judgment of key physiological and biochemical indicators, making it difficult to accurately assess the course of gastrointestinal diseases.

[0004] Patent publication number CN113616200A discloses a wireless capsule sensing device and method for detecting gastrointestinal pH, which solves the problem of difficulty in detecting gastrointestinal pH under clinical non-anesthesia conditions. However, the pH detection method disclosed in this technology can only reflect the most basic physiological parameters. The detection of a single indicator cannot intuitively and comprehensively reflect the complex physiological state of the microenvironment in the gastrointestinal tract. For example, key pathological information such as oxidative stress level and neurotransmitter disorder cannot be obtained, and it cannot accurately determine the physiological state of the microenvironment in the gastrointestinal tract.

[0005] Therefore, existing swallowable capsule sensors generally only target single or a few physical parameters (such as pH or temperature), making it difficult to comprehensively reflect the complex physiological and pathological microenvironment in the gastrointestinal tract shaped by microbial metabolism, inflammatory responses, oxidative stress, and neuroendocrine factors. Furthermore, while commercially available capsule endoscopes (such as the PillCam series) can acquire images of the digestive tract, and multifunctional capsules (such as SmartPill) can simultaneously detect pH, temperature, and pressure, they all focus on physical parameters or imaging information, lacking the ability to jointly analyze molecular-level biomarkers. This invention is therefore proposed. Summary of the Invention

[0006] To address the limitations of existing endoscopic and in vitro analysis methods, such as high invasiveness and inability to achieve in-situ monitoring, as well as the insufficient multi-parameter integrated detection capabilities in current gastrointestinal detection technologies, this invention provides a gastrointestinal capsule and its detection method for multi-parameter microenvironment joint analysis. This capsule is an ingestible electrochemical sensor that enables non-invasive, in-situ joint analysis and sensing of multiple parameters. The capsule integrates serotonin, ORP, and pH detection sensing electrodes, as well as signal acquisition, processing, and wireless communication units. A multi-parameter in-situ detection method for serotonin, ORP, and pH values ​​in the in vivo gastrointestinal tract has been developed, comprehensively reflecting the multi-dimensional microenvironmental physiological state of neurotransmitters, oxidative stress, and acid-base environment within the gastrointestinal tract. By functionalizing the electrochemical sensor with aptamers, its detection specificity in the gastrointestinal environment is improved. The sample inlet is equipped with a filter membrane, a water-soluble protective layer, and an enteric-coated response layer to reduce contamination from impurities in complex environments and to achieve directional sampling within the intestine.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A gastrointestinal capsule for multi-parameter microenvironment joint analysis includes a capsule shell, which includes a first shell and a second shell connected to each other. The first shell and the second shell are connected by a light-curing adhesive to form a sealed cavity. One end of the capsule shell is provided with a sampling hole, and the capsule shell is provided with a first, second, third, fourth and fifth circular discs in sequence from the sampling hole end to the other end.

[0009] An electrochemical interface unit is provided on the first disc for electrical connection with an electrochemical sensor. The electrochemical sensor includes a common counter electrode CE, a common reference electrode RE, and three independent working electrodes WE1, WE2, and WE3 disposed on a PET plastic substrate. The WE1 electrode, the common counter electrode, and the common reference electrode constitute a serotonin sensor for serotonin detection; the WE2 electrode, the common counter electrode, and the common reference electrode constitute an ORP sensor for ORP detection; and the WE3 electrode, the common counter electrode, and the common reference electrode constitute a pH sensor for pH detection.

[0010] The surface of the first working electrode WE1 is modified with a recognition layer capable of specifically recognizing serotonin to improve selectivity in complex intestinal environments. The recognition layer includes aptamers, antibodies, and molecularly imprinted materials.

[0011] The second working electrode WE2 is used to detect the redox state in the gastrointestinal environment. Its surface is provided with a redox-responsive functional layer, which includes noble metal nanomaterials to improve the electron exchange capacity of the electrode interface and improve the potential response stability and response speed.

[0012] The third working electrode WE3 is used for detecting the acid-base state in the gastrointestinal environment, and its surface is modified with an ion-electron conversion layer and a hydrogen ion response layer. The ion-electron conversion layer is used to improve the charge transport capability of the electrode interface and reduce potential drift; the hydrogen ion response layer is used to sense changes in the hydrogen ion concentration in the environment to achieve pH parameter detection.

[0013] The second disc is equipped with an analog front-end unit, which includes a multi-channel operational amplifier, an analog switch, and peripheral resistor-capacitor components. The different operational amplification channels of the multi-channel operational amplifier and the peripheral resistor-capacitor components constitute a constant potential control circuit, a transimpedance amplifier circuit (TIA), and a low-pass filter circuit. The analog switch is connected to the main control chip, and the main control chip controls the analog switch to be turned on or off according to a preset detection timing sequence.

[0014] The third wafer is equipped with a main control unit (MCU), which includes a main control chip for data acquisition. The main control chip integrates an analog-to-digital converter (ADC) and a digital-to-analog converter (DAC). The main control chip outputs preset step potential and pulse potential signals through its internal DAC. The output of the DAC is connected to the input of the constant potential control circuit in the multi-channel operational amplifier. The ADC performs analog-to-digital conversion on the analog signals of the three working electrodes. The main control chip further performs detection timing control, data processing, and result calculation.

[0015] The analog front-end unit is electrically connected to the electrochemical interface unit and the main control MCU unit, respectively.

[0016] The fourth disc is equipped with a power supply unit, which includes a low dropout linear regulator (LDO). A battery is disposed between the fourth and fifth discs. The LDO is electrically connected to the battery, and its regulated output terminal is connected to the power input terminals of the analog front-end unit, the main control unit, and the wireless communication unit, respectively, to provide a stable operating voltage to each functional unit.

[0017] The fifth disc is equipped with a wireless communication unit, which includes a BLE wireless communication module. The BLE module connects to the main control chip via a UART communication interface and receives serotonin, ORP, and pH detection data processed by the main control chip. It then wirelessly transmits this data to an external terminal or mobile app, enabling wireless transmission, real-time display, and monitoring of multi-parameter electrochemical signals in the gastrointestinal tract. All electrical connections are achieved through flexible conductive lines.

[0018] Furthermore, the sampling port is sequentially covered from the inside out with a filter layer, a water-soluble protective layer, and an enteric-coating response layer. The capsule shell is made of photocurable resin. The multi-channel operational amplifier is an LTC2065; the analog switch is an ADG842; the main control chip is an STM32L151; the wafer is made of FPC; and the battery is a button cell. The enteric-coating response layer is designed to dissolve or undergo structural changes under specific gastrointestinal environmental conditions, exposing the internal sensing area and thus enabling selective sampling of the target gastrointestinal region.

[0019] The water-soluble protective layer is used to control the exposure process of the sensing area;

[0020] The filter layer is used to block large molecular pollutants in intestinal fluid from entering the sensing area.

[0021] Furthermore, the capsule shell is made of BMF HTL resin by 3D printing. After printing, the shell is ultrasonically cleaned in anhydrous ethanol for 20-40 minutes. The diameter of the capsule shell is 9-11 mm and the length is 15-20 mm.

[0022] Further, the sampling well diameter is 2.5~3.5 mm, the filter layer is a polyvinylidene fluoride microporous membrane with a diameter of 5 mm and a pore size of 50 μm; the water-soluble protective layer is obtained by uniformly coating the outside of the polyvinylidene fluoride microporous membrane with 8~12 μL of 10 mg / mL PVA aqueous solution, drying at room temperature for at least 30 min, and repeating 2~4 times; the enteric response layer is obtained by dissolving Eudragit L100 in anhydrous ethanol to a mass fraction of 8~12%, adding triethyl citrate at a mass of 8~12% of Eudragit L100, stirring for 20~40 min to form a uniform coating solution; taking 8~12 μL of the coating solution and coating the outside of the water-soluble membrane, drying at room temperature for at least 30 min, and repeating 2~4 times.

[0023] Further, silver paste is screen-printed on a PET plastic substrate to form wires and a reference electrode area. The reference electrode area is chlorinated with FeCl3 aqueous solution to form a silver / silver chloride reference electrode. Then, carbon paste is screen-printed to form the first, second, and third carbon working electrodes and the carbon counter electrode. The working electrode has a diameter of 1 mm, the counter electrode has a diameter of 1.4 mm, and the reference electrode has a diameter of 0.5 mm. The reference electrode needs to be modified by the following process: polyvinyl butyral and sodium chloride are dissolved in methanol to obtain a reference solution. The concentration of polyvinyl butyral is 79~80 mg / mL, and the concentration of sodium chloride is 50 mg / mL. Then, 0.5 µL of the reference solution is drop-coated onto the reference electrode and dried at room temperature to obtain the final product.

[0024] Furthermore, the fabrication process of the WE1 working electrode is as follows:

[0025] Gold nanoparticle electrodes were electrodeposited on a first carbon working electrode using cyclic voltammetry in a mixed solution of 1 mM HAuCl4·4H2O and 100 mM KCl. The parameters were set to -0.35 V to -0.6 V, scan rate 0.1 V / s, and 40 cycles. After drying, gold nanoparticle electrodes were obtained. Subsequently, serotonin aptamer was dissolved in 1× Tris-EDTA buffer to prepare a 10 µM concentration, aliquoted, and stored at -20 °C. The gold nanoparticle electrodes were electrochemically cleaned using cyclic voltammetry in 0.5 M H2SO4 solution at a scan rate of 0.1 V / s. -1 The voltage range was -1 V to 1.6 V, and the cycles were repeated 15–25 times. The electrode was then rinsed with deionized water to obtain a clean working electrode. 5 µL of a 10 µM aptamer solution was mixed with 5 µL of a 1 mM TCEP solution and reacted at room temperature in the dark for 1 h. The solution was then diluted to 2.5 µM with 1× PBS buffer. The aptamer solution was then heated at 95 °C for 5 min using a PCR instrument, allowed to return to room temperature, and then 2.5 µL of the 2.5 µM aptamer solution was added dropwise to the clean working electrode and incubated at 4 °C in the dark for 12 h. Unbound aptamers were washed away with 1× PBS buffer, and the electrode was then incubated with 0.5 mM mercaptohexanol solution for 30 h. min is used to block the unbound electroactive surface on the gold electrode. Finally, the aptamer-modified electrode is thoroughly rinsed with 1×PBS buffer to obtain the serotonin aptamer sequence as follows: 5'-Thiol-C6-Linker-CGACTGGTAGGCAGATAGGGGAAGCTGATTCGATGCGTGGGTCG-MB-3'.

[0026] Furthermore, the preparation process of the WE2 working electrode is as follows: A mixed solution containing 1 mM H2PtCl6 and 100 mM H2SO4 is used as the electrodeposition system. Platinum nanoparticles are deposited on the second carbon working electrode by cyclic voltammetry. The electrodeposition potential range is -0.35 V to 0.6 V, the scan rate is 0.05 V / s, and the cycle is 15 to 25 times. After the deposition is completed, the electrode is dried to obtain the final product.

[0027] Furthermore, the fabrication process of the WE3 working electrode is as follows:

[0028] (1) Preparation of PEDOT:PSS layer: 10.6 µL EDOT, 206 mg NaPSS and 36.8 mg K4Fe(CN)6 were dissolved in 10 mL deionized water to obtain electrodeposition solution. Electrodeposition was performed on the third carbon working electrode using a multi-potential step method. The specific process was as follows: first step voltage and time: 0.465 V and 0.75 s; second step voltage and time: 0.865 V and 0.25 s; number of cycles: 840. The electrode was dried at 55~65 ℃ for 1~3 h.

[0029] (2) Preparation of polyaniline hydrogen ion response layer: In a mixed solution of 0.1 M aniline and 1 M HCl, cyclic voltammetry was performed for 40 cycles in a potential range of -0.2 V to 1 V relative to a commercial Ag / AgCl reference electrode at a scan rate of 0.1 V / s. The solution was then dried at 55~65 ℃ for 1~3 h to obtain the final product.

[0030] A method for detecting gastrointestinal capsules using the above-mentioned multi-parameter microenvironment joint analysis, wherein serotonin is detected by differential pulse voltammetry (DPV), and ORP and pH are detected by open circuit potential-time method (OCPT).

[0031] When the capsule is immersed in gastric or intestinal fluid for serotonin DPV detection, the constant potential control circuit receives the DPV set potential signal output by the digital-to-analog converter (DAC) inside the main control chip MCU. At the same time, the constant potential control circuit receives the potential signal fed back by the common reference electrode RE, and adjusts the driving potential of the common counter electrode CE according to the relationship between the set potential and the feedback potential, thereby controlling the potential difference between the serotonin working electrode WE1 and the reference electrode RE to change according to the preset DPV waveform.

[0032] The weak current signal generated by the serotonin working electrode WE1 is input to the transimpedance amplifier circuit TIA. The transimpedance amplifier circuit TIA converts the current signal generated by the working electrode into a corresponding voltage signal. The converted voltage signal is filtered by a low-pass filter circuit and signal conditioned before being input to the ADC inside the main control chip in the third wafer for analog-to-digital conversion to obtain the serotonin current signal.

[0033] When performing ORP and pH detection, the working electrodes WE2 and WE3 form an open-circuit potential detection loop with the common reference electrode RE. The open-circuit potential signals generated by the working electrodes WE2 and WE3 relative to the reference electrode RE are input to a multi-channel operational amplifier, filtered by a low-pass filter circuit, and then input to the ADC inside the main control chip to obtain the ORP potential signal and pH potential signal.

[0034] After processing the three-channel detection data, the main control chip sends the data to the BLE wireless communication module via UART, and then further wirelessly to an external terminal. The collected data is substituted into the standard curve equation of the corresponding parameters to obtain the serotonin concentration, ORP value, or pH value.

[0035] Compared with the prior art, the present invention has the following significant advantages:

[0036] An integrated sensing capsule based on electrochemical sensors enables molecular-level investigation of various conditions in the gastrointestinal tract, including neurotransmitters, oxidative stress, and acid-base balance, allowing for in-situ sensing of the gastrointestinal microenvironment. Compared to existing technologies, this invention integrates serotonin, ORP, and pH detection functions into a single device, equipped with corresponding signal acquisition, processing, and wireless communication units to provide multi-parameter microenvironment detection information. The electrochemical sensor, functionalized with materials such as aptamers and noble metal nanoparticles, enhances sensing selectivity in the complex intestinal environment, enabling precise analysis of multidimensional parameters of the gastrointestinal tract in vivo. A multi-layer enteric-coated filter membrane at the sampling port improves filtration of the complex biological matrix of the gastrointestinal tract and controls the exposure of the sensing area. Detection results from the supernatant of isolated mouse intestinal contents show that the capsule can detect serotonin, redox potential, and pH parameters from actual source samples, providing experimental evidence for its application in gastrointestinal microenvironment detection. These technical features can be modified by altering the modifying elements, such as glucose oxidase or antibodies, to meet the in-situ screening needs for intestinal metabolic and inflammatory markers. Attached Figure Description

[0037] To more clearly illustrate the embodiments or technical solutions of the present invention, the embodiments and related drawings will be briefly described below. It should be understood that the drawings described below are only some embodiments of the present invention, and those skilled in the art can obtain other drawings without creative effort.

[0038] Figure 1 Here are the overall schematic diagram and exploded view of the electrochemical sensing capsule;

[0039] Figure 2 It is an integral component of the electrochemical sensing capsule;

[0040] Figure 3This is a system block diagram of an electrochemical sensing capsule;

[0041] Figure 4 A schematic diagram illustrating the construction of a responsive sampling window for the gastrointestinal region;

[0042] Figure 5 This shows the arrangement of the electrochemical sensors;

[0043] Figure 6 The signal response of the capsule sensor in simulated gastric fluid (pH2) and simulated intestinal fluid (pH7);

[0044] Figure 7 DPV response, linear fitting standard curve and selectivity of serotonin sensor (n=3).

[0045] Figure 8 The OCPT response and linear standard curve of the ORP sensor (n=3);

[0046] Figure 9 The OCPT response and linear standard curve of the pH sensor (n=3).

[0047] Figure 10 The colon morphology, length, and histology of normal mice and IBD mice were compared (n=6).

[0048] Figure 11 The results of capsule sensor analysis of 5-HT, ORP and pH in intestinal fluid of normal mice and IBD mice (n=6). Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0050] (1) Overall capsule structure design and preparation

[0051] like Figures 1 to 3 As shown, this embodiment provides a gastrointestinal capsule for multi-parameter microenvironment joint analysis, the sensing capsule comprising:

[0052] The capsule shell includes a first shell and a second shell that are connected to each other. The first shell and the second shell are connected by a light-curing adhesive to form a sealed cavity. One end of the capsule shell is provided with a sampling hole. The capsule shell contains a first, second, third, fourth and fifth circular discs arranged sequentially from the sampling hole end to the other end.

[0053] An electrochemical interface unit is provided on the first disc for electrical connection with an electrochemical sensor. The electrochemical sensor includes a common counter electrode CE, a common reference electrode RE, and three independent working electrodes WE1, WE2, and WE3, all disposed on a flexible transparent PET plastic substrate. The WE1 electrode, the common counter electrode, and the common reference electrode constitute a serotonin sensor for serotonin detection; the WE2 electrode, the common counter electrode, and the common reference electrode constitute an ORP sensor for ORP detection; and the WE3 electrode, the common counter electrode, and the common reference electrode constitute a pH sensor for pH detection.

[0054] The second disc is equipped with an analog front-end unit, which includes a multi-channel operational amplifier, an analog switch, and peripheral resistive and capacitive components. Different operational amplification channels of the multi-channel operational amplifier and the peripheral resistive and capacitive components constitute a constant potential control circuit, a transimpedance amplifier (TIA) circuit, and a low-pass filter circuit. The low-pass filter circuit is used to filter out high-frequency noise in the analog signal, improve signal stability, and output the voltage signal to the ADC inside the main control chip for acquisition. The analog switch is connected to the main control chip, which controls the analog switch to be turned on or off according to a preset detection timing sequence, thereby achieving switching or isolation of the working mode between the DPV detection path and the OCPT detection path.

[0055] The third wafer is equipped with a main control unit (MCU), which includes a main control chip for data acquisition. The main control chip integrates an analog-to-digital converter (ADC) and a digital-to-analog converter (DAC). The main control chip outputs preset step potential and pulse potential signals through its internal DAC. The output of the DAC is connected to the input of the constant potential control circuit in the multi-channel operational amplifier. The ADC performs analog-to-digital conversion on the analog signals of the three working electrodes. The main control chip further performs detection timing control, data processing, and result calculation.

[0056] The analog front-end unit is electrically connected to the electrochemical interface unit and the main control MCU unit, respectively.

[0057] The fourth disc is equipped with a power supply unit, which includes a low dropout linear regulator (LDO). A battery is disposed between the fourth and fifth discs. The LDO is electrically connected to the battery, and its regulated output terminal is connected to the power input terminals of the analog front-end unit, the main control unit, and the wireless communication unit, respectively, to provide a stable operating voltage to each functional unit.

[0058] The fifth disc is equipped with a wireless communication unit, which includes a BLE wireless communication module. The BLE wireless communication module is connected to the main control chip through a UART communication interface. It is used to receive serotonin, ORP and pH detection data processed by the main control chip and wirelessly transmit the detection data to an external terminal or mobile APP to realize wireless transmission, real-time display and monitoring of multi-parameter electrochemical signals of the gastrointestinal tract.

[0059] The capsule shell serves to provide encapsulation space for internal electronic components and protect the sensing components during gastrointestinal movement. First, the capsule shell was designed and fabricated using a photocurable resin material (BMF HTLResin, Boston Micro Fabrication) via 3D printing. After printing, the shell was ultrasonically cleaned in anhydrous ethanol at a frequency of 45 kHz, a power of 200 W, and a time of 30 min to remove residual resin material.

[0060] In this embodiment, the prepared capsule is preferably 9.9 mm in diameter and 18 mm in length.

[0061] The multi-channel operational amplifier is model LTC2065; the analog switch is model ADG842; the main control chip is model STM32L151; the wafer material is FPC; and the battery is a button cell battery.

[0062] During serotonin DPV detection, the constant potential control circuit receives the DPV set potential signal output from the DAC inside the main control chip MCU. The DPV set potential signal includes a stepped scan potential and a pulse potential superimposed on the stepped scan potential. Simultaneously, the constant potential control circuit receives the potential signal fed back from the reference electrode RE, and adjusts the driving potential of the counter electrode CE according to the relationship between the set potential and the feedback potential, thereby controlling the potential difference between the serotonin working electrode WE1 and the reference electrode RE to change according to the preset DPV waveform. The weak current signal generated by the serotonin working electrode WE1 is input to a transimpedance amplifier (TIA) circuit composed of an LTC2065 multi-channel operational amplifier and external RC components. The TIA converts the current signal generated by the working electrode into a corresponding voltage signal. The converted voltage signal is filtered by a low-pass filter circuit and then input to the ADC inside the main control chip in the third wafer for analog-to-digital conversion.

[0063] When detecting ORP and pH, the working electrodes WE2 and WE3 form open-circuit potential detection loops with the common reference electrode RE. The open-circuit potential signals generated by WE2 and WE3 relative to RE are input to the LTC2065, filtered by a low-pass filter circuit, and then input to the internal ADC of the main control chip to obtain the ORP potential signal and pH value.

[0064] In this embodiment, the entire signal transmission process can be simply represented as follows: For serotonin DPV detection, the DAC inside the main control chip outputs the DPV set potential, which is controlled by the analog front-end constant potential control circuit to form a feedback loop composed of CE and RE. The current generated by WE1 is amplified by transimpedance amplification (TIA) and filtered before being input into the ADC inside the main control chip. For ORP and pH detection, WE2 and WE3 respectively generate open-circuit potential signals relative to the shared RE, which are then filtered before being input into the ADC inside the main control chip. After processing the three detection data, the main control chip sends them to the BLE wireless communication module via UART and further wirelessly to an external terminal.

[0065] (2) Construction of a gastrointestinal region response sampling window

[0066] like Figure 4 As shown, a gastrointestinal responsive protective membrane structure is set at the sampling port position of the capsule shell, with the sampling port diameter preferably being 3 mm. First, a polyvinylidene fluoride (PVDF) microporous membrane (preferably 5 mm in diameter and 50 μm in pore size) is placed over the sample inlet to block some larger particles and suspended impurities. Subsequently, a polyvinyl alcohol (PVA) water-soluble protective layer is coated on the outside of the PVDF membrane to regulate the exposure process of the sensing area. Finally, an enteric-coating responsive layer composed of Eudragit L100 is coated on the outside of the PVA layer.

[0067] In this embodiment, 10 μL of a 10 mg / mL PVA aqueous solution was uniformly coated onto the outer side of a PVDF filter membrane and dried at room temperature, repeated three times. Subsequently, Eudragit L100 was used as the enteric polymer material, and triethyl citrate was used as a plasticizer to prepare an enteric-responsive coating. Specifically, 10 wt% Eudragit L100 was dissolved in 100 mL of anhydrous ethanol, and 10 wt% triethyl citrate was added. The mixture was magnetically stirred for 30 min to plasticize and form a uniform coating solution. 10 μL of this coating solution was coated onto the outer side of the PVA water-soluble protective layer of the sampling window and dried at room temperature for 30 min to form an enteric protective layer, repeated three times. The enteric protective layer remains stable in the acidic environment of the stomach and dissolves in the neutral to slightly alkaline environment of the intestine, thereby achieving selective sampling of the gastrointestinal region.

[0068] (3) Sensor construction

[0069] The sensor was fabricated using screen printing technology and includes a wire connection layer, a working electrode, a counter electrode, and a reference electrode. The wire connection layer is used for electrical signal transmission. The working and counter electrodes are made of carbon-based conductive materials, and the reference electrode is made of silver / silver chloride. Two screen printing stencils were first customized, and printing was performed in two steps. First, silver paste was printed to form the wires and reference electrode, followed by carbon paste to form the working and counter electrodes. 1 μL of a 10 mM FeCl3 aqueous solution was added dropwise to the reference electrode area for chlorination for 1 min, followed immediately by rinsing with deionized water and drying at 60°C for 30 min to obtain the silver / silver chloride reference electrode.

[0070] like Figure 5 As shown, the conductive wires are made of silver paste, the working and counter electrodes are made of carbon, and the reference electrode is made of silver / silver chloride. The diameter of the circular working electrode is 1 mm, the diameter of the circular counter electrode is 1.4 mm, and the diameter of the circular reference electrode is 0.5 mm.

[0071] Reference electrode modification: 79.1 mg of polyvinyl butyral (PVB) and 50 mg of sodium chloride (NaCl) were dissolved in 1 mL of methanol to form a reference solution. Then, 0.5 µL of the reference solution was drop-coated onto the reference electrode and dried at room temperature for 1 h.

[0072] Preparation of serotonin working electrode WE1

[0073] First, a gold nanoparticle-modified layer is formed on the surface of a carbon-based working electrode via electrochemical deposition to provide binding sites suitable for immobilizing thiol-modified biorecognition molecules and to improve the electron transport capability of the electrode surface. A target molecule recognition layer, including a serotonin aptamer, is disposed on the surface of the first working electrode.

[0074] In this embodiment, the serotonin aptamer used includes a thiol-modified end, which is used to bind to the gold nanoparticle-modified layer through gold-sulfur bonding.

[0075] The serotonin aptamer sequence is as follows:

[0076] 5'-Thiol-C6-Linker-CGACTGGTAGGCAGATAGGGGAAGCTGATTCGATGCGTGGGTCG-MB-3' (purchased from Sangon Biotech (Shanghai) Co., Ltd.)

[0077] Among them, 5'-Thiol-C6 Linker refers to the 5'-terminal C6 thiol linker (a hexa-thiol linker), with the structure: HS-(CH2)6-

[0078] Applications: The thiol group (-SH) can be coupled to gold surfaces, maleimide, and disulfide crosslinking groups, and is commonly used in DNA fixation, biosensors, DNA chips, and gold nanoparticle modification; the C6 long linker reduces steric hindrance; MB = Methylene Blue.

[0079] The specific fixing process is as follows:

[0080] Electrodeposition was performed using cyclic voltammetry (CV) in a mixed solution of 1 mM HAuCl4·4H2O and 100 mM KCl, with parameters set to -0.35 V to -0.6 V, a scan rate of 0.1 V / s, and 40 cycles. The electrode was then dried at 60 °C for 30 min. Subsequently, the aptamer was dissolved in 1× Tris-EDTA buffer to prepare a 10 µM concentration, aliquoted, and stored at -20 °C. The gold nanoparticle electrode was electrochemically cleaned using cyclic voltammetry in 0.5 M H2SO4 solution at a scan rate of 0.1 V / s. -1 The voltage range was -1 V to 1.6 V, and the cycle was repeated 20 times, followed by rinsing with deionized water. 5 µL of a 10 µM aptamer solution was mixed with 5 µL of a 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution and reacted at room temperature in the dark for 1 h to reduce the terminal disulfide bond (-SS-) of the aptamer, generating an active thiol group (-SH). The mixture was then diluted to 2.5 µM with 1× PBS buffer (pH 7.4). Subsequently, the aptamer was heated at 95 °C for 5 min using a PCR instrument and gradually brought to room temperature. The aptamer (2.5 µM, 2.5 µL) was then added dropwise to the working electrode and incubated at 4 °C in the dark for 12 h to immobilize it on a clean working electrode. Unbound aptamer was washed away with 1× PBS buffer. Subsequently, the electrode was incubated with 0.5 mM mercaptohexanol (MCH) solution to block the unbound electroactive surface on the gold electrode for 30 min. Finally, the aptamer-modified electrode was thoroughly rinsed with 1×PBS buffer to obtain the serotonin aptamer electrode WE1 (5-HT). The serotonin-specific recognition working electrode was thus obtained.

[0081] Preparation of working electrode for oxidation-reduction potential (ORP) detection

[0082] To address the need for detecting the redox state of the gastrointestinal tract, the second working electrode in the electrode array was functionalized to construct an ORP sensing probe.

[0083] First, a platinum nanostructure modification layer is constructed on the surface of a carbon-based working electrode to improve the electron transport capability of the electrode surface.

[0084] Specifically, the working electrode is placed in an electrodeposition solution containing a platinum precursor, and a platinum nanoparticle modified layer is formed by electrochemical deposition.

[0085] In this embodiment, a mixed solution containing 1 mM H2PtCl6 and 100 mM H2SO4 was used as the electrodeposition system. Platinum nanoparticles were deposited by cyclic voltammetry. The electrodeposition potential range was -0.35 V to 0.6 V, the scan rate was 0.05 V / s, and the cycle was 20 times.

[0086] After deposition, the electrode was dried to obtain the platinum nanoparticle-modified ORP detection working electrode WE2.

[0087] Preparation of pH detection working electrode

[0088] To address the need for detecting changes in the gastrointestinal acid-base environment, the third working electrode in the electrode array was functionalized to construct a pH sensor. The pH-sensing working electrode includes an ion-electron conversion layer and a hydrogen ion response layer.

[0089] First, a PEDOT:PSS layer is constructed on the surface of a carbon-based working electrode as an ion-electron conversion interface to improve the charge transport capacity of the electrode interface and reduce potential drift during the detection process.

[0090] The PEDOT:PSS layer was electrodeposited using the step-multiple potential (STEP) method. The electrodeposition solution was 10 mL of deionized water containing 10.6 µL of EDOT, 206 mg of NaPSS, and 36.8 mg of K₄Fe(CN)₆. The parameters were as follows: Step 1 voltage and time: 0.465 V and 0.75 s; Step 2 voltage and time: 0.865 V and 0.25 s; Number of cycles: 840. The electrode was then dried at 60 °C for 2 h.

[0091] Subsequently, a polyaniline (PANI) hydrogen ion responsive layer was further constructed on the surface of the PEDOT:PSS conversion layer.

[0092] PANI was electrodeposited onto the working electrode in a mixed solution of 0.1 M aniline and 1 M HCl by cyclic voltammetry (CV) at a scan rate of 0.1 V / s for 40 cycles in a potential range of -0.2 V to 1 V (relative to a commercial Ag / AgCl reference electrode). The electrode was then dried at 60 °C for 2 h to obtain the PEDOT:PSS / PANI composite pH sensing working electrode.

[0093] Capsule performance testing:

[0094] Preparation method of simulated gastric juice: Prepare a mixed solution containing 5.76 mM KCl, 1.44 mM KH2 PO4, 115.4 mM NaCl, 8 mM Na2HPO4 and 0.33 mM MgCl2 with deionized water, and then adjust the pH of the solution to 2 with 1M hydrochloric acid solution.

[0095] Preparation method of simulated intestinal fluid: Prepare a mixed solution containing 5.76 mM KCl, 1.44 mM KH2 PO4, 115.4 mM NaCl, 8 mM Na2HPO4 and 0.33 mM MgCl2 with deionized water, and then adjust the pH of the solution to 7 with 1M hydrochloric acid solution.

[0096] (1) Performance verification of enteric-coated response layer

[0097] The capsules were immersed in simulated gastric fluid at pH 2 and simulated intestinal fluid at pH 7, respectively, and changes in electrode signals were recorded. Figure 6 As shown, when the capsule was immersed in simulated gastric fluid at pH 2, the signal remained stable and showed no significant changes within 80 minutes, indicating that the coating remained intact and there were no signal fluctuations. When the capsule was immersed in simulated intestinal fluid at pH 7, the signal began to change after about 10 minutes, indicating that the coating began to dissolve, and the signal tended to stabilize after about 40 minutes, indicating that the coating was about to dissolve completely.

[0098] (2) Performance verification of serotonin detection

[0099] First, using simulated intestinal fluid as the detection medium, the detection solution was prepared according to the actual simulated intestinal fluid formula. A 10 mM stock solution of serotonin (5-hydroxytryptamine, 5-HT) was prepared and serially diluted using simulated intestinal fluid to prepare serotonin standard solutions with final concentrations of 10 nM, 100 nM, 500 nM, 1 μM, 10 μM, and 100 μM, respectively. Simulated intestinal fluid without serotonin served as a blank control, i.e., the serotonin concentration was 0.

[0100] During detection, 100 μL of the same volume of serotonin solutions of different concentrations were dropped onto a PET plastic substrate, ensuring the solution fully covered the electrode surface. Differential pulse voltammetry (DPV) was used to record the electrochemical response before and after serotonin aptamer recognition, within a voltage range of 0 to -0.5 V. The characteristic reduction peak current generated by the methylene blue (MB) probe molecule labeling was read at approximately -0.30 V, and this peak current was used as the response signal for changes in serotonin concentration. All concentrations were detected under the same conditions.

[0101] Test results as follows Figure 7As shown in the left image, in blank simulated intestinal fluid and serotonin solutions of different concentrations from 10 nM to 100 μM, the sensing electrode exhibited a significant MB characteristic reduction peak around -0.30 V. As the serotonin concentration increased from 10 nM to 100 μM, the position of the characteristic peak remained relatively stable, while the absolute value of the reduction peak current gradually decreased, i.e., the peak current gradually changed from a relatively negative current value towards zero. This result indicates that after serotonin specifically binds to the aptamer recognition layer on the electrode surface, it causes changes in the aptamer conformation and the electron transfer efficiency between the MB label and the electrode surface, thereby reducing the electrochemical peak current of MB, exhibiting a typical signal-off response.

[0102] Further, using the common logarithm of serotonin concentration (Log) 10 (5-HT) Linear fitting was performed with (μM) as the x-axis and the corresponding DPV characteristic peak current as the y-axis. A blank simulated intestinal fluid with a concentration of 0 was used to obtain the blank response and was not included in the linear fitting on the logarithmic scale. The results are as follows: Figure 7 As shown in the middle, within the serotonin concentration range of 10 nM to 100 μM, the peak current exhibits a good linear correlation with the logarithm of the serotonin concentration. The linear fitting equation is: Y = 19.15X - 208.36, where X is the logarithm of the serotonin concentration. 10 (5-HT) Y represents the DPV peak current of the serotonin aptamer electrode, and the correlation coefficient R is... 2 = 0.953.

[0103] According to the limit of detection (LOD) formula, The calculated LOD is 4.68 nM.

[0104] To further evaluate the selectivity of the constructed serotonin-sensing working electrode, 1 μM dopamine (DA), 1 μM melatonin (MT), 1 μM norepinephrine (NE), 100 μM ascorbic acid (AA), 100 μM uric acid (UA), and 1 μM serotonin were detected under the same detection conditions within a voltage range of 0 to -0.5 V. Using the initial response S0 in a blank simulated intestinal fluid as a baseline, the relative signal change ΔS / S0 caused by each analyte was calculated to evaluate the effect of different substances on the serotonin-sensing working electrode.

[0105] The results are as follows Figure 7As shown on the right, the relative signal changes caused by dopamine (DA), melatonin (MT), norepinephrine (NP), ascorbic acid (AA), and uric acid (UA) were all relatively small, with the largest relative change rate being approximately 3.6%. However, when 1 μM serotonin was added, the sensor produced a significant electrochemical response, with a relative signal change of approximately 42%, about 11.7 times the maximum interference response. The results indicate that even when the test concentrations of ascorbic acid and uric acid reach 100 μM, significantly higher than the test concentration of serotonin, the interference responses they produce are still significantly lower than the serotonin response. This demonstrates that the constructed serotonin aptamer sensing electrode has good specific recognition ability and anti-interference performance for serotonin, and can be used to detect changes in serotonin concentration in the gastrointestinal environment.

[0106] (3) ORP performance verification

[0107] The fabricated ORP sensor exhibits a stable potential response to different redox states and can be used for monitoring the redox state of the gastrointestinal microenvironment. First, the sensor was calibrated using commercially available ORP standard solutions with known redox potentials (86 mV, 220 mV, 256 mV, and 430 mV, respectively). During detection, 100 μL of the standard solution with different ORP values ​​was added dropwise to a PET plastic substrate, ensuring the solution fully covered the electrode surface. The electrode potential was then recorded using open circuit potential-time (OCPT) mode. Each ORP standard solution was tested under the same conditions, and the measurements were repeated three times in parallel.

[0108] like Figure 8 As shown in the left image, when the sensing electrode is sequentially contacted with standard solutions of different ORP values, its open-circuit potential exhibits a significant stepwise change and reaches a relatively stable state after a certain response time. The stable potentials corresponding to different ORP standard solutions show a clear stepwise distinction, and the measured potentials are basically consistent with the nominal ORP values ​​of the standard solutions, indicating that the prepared ORP sensing electrode can produce a stable response to different redox states.

[0109] A calibration curve was further established with the potential value of the ORP standard solution on the x-axis and the potential value detected by the sensing working electrode on the y-axis. For example... Figure 8 As shown in the right figure, there is a good linear relationship between the measured potential and the standard ORP value, and the linear fitting equation is: Y = 0.98X-0.92.

[0110] Where X is the redox potential value (mV) of the ORP standard solution, Y is the measured potential value (mV), and the correlation coefficient R is... 2 = 0.999. The fitting slope is 0.98, which is close to the ideal value of 1, indicating that the measurement results of the sensing working electrode have high consistency and accuracy with the standard ORP value.

[0111] In this embodiment, the lowest ORP value verified by the standard solution is 86 mV. Since the lowest calibration value of the ORP standard solution used in the experiment is 86 mV, 86 mV represents the lower limit of the verified measurement range in this embodiment, rather than the theoretical lowest detection limit of the sensing working electrode.

[0112] (4) pH performance verification

[0113] like Figure 9 As shown, the potential response of the prepared pH sensor under different acidic and alkaline environments was tested. During the test, 100 μL of standard phosphate buffer solution (PBS) with pH values ​​of 4, 5, 6, 7, and 8 were sequentially added to a PET plastic substrate to ensure that the test solution fully covered the electrode surface. The potential response of the electrode was continuously recorded using the OCPT method. When the potential signal under the pH conditions tended to stabilize, the corresponding stable potential value was recorded. Each pH standard buffer solution was tested under the same test conditions, and the measurements were repeated three times in parallel.

[0114] like Figure 9 As shown in the left image, when detecting solutions with different pH values ​​(pH 4-8), the detection potential of the pH sensor exhibits a stepwise decreasing trend, and a stable and clearly distinguishable potential gradient can be formed under different pH conditions. This result indicates that the prepared pH sensor can produce a rapid and stable potential response to pH changes in the detection environment.

[0115] A calibration curve was then constructed with the pH value of the standard buffer solution on the x-axis and the steady-state potential value measured by the pH sensor on the y-axis. The results are as follows: Figure 9 As shown on the right. Within the pH range of 4–8, the sensor response potential exhibits a strong linear negative correlation with pH value, and its linear fitting equation is: Y = -65.79X + 464.19.

[0116] Where X is the pH value of the solution to be tested, Y is the steady-state potential value (mV) measured by the pH sensor, and the correlation coefficient R0 2 =0.994. The obtained potential response sensitivity is -65.79 mV / pH, indicating that for every 1 unit increase in solution pH, the electrode response potential decreases by an average of approximately 65.79 mV, exhibiting good near-Nernst potential response characteristics.

[0117] Therefore, within the pH range of 4 to 8 verified in this embodiment, the prepared pH sensor can produce a stable and linear potential response to different acidity and alkalinity in the environment, and can be used to monitor pH changes in the gastrointestinal microenvironment.

[0118] (5) Detection process and results of isolated mouse intestinal contents

[0119] To further verify the detection capability of the constructed multi-parameter electrochemical capsule detection device on actual intestinal samples, an inflammatory bowel disease (IBD) mouse model was established by inducing dextran sulfate sodium (DSS), and the serotonin, ORP, and pH values ​​in the intestinal contents of normal mice and IBD model mice were detected and compared.

[0120] C57BL / 6 mice were selected as experimental animals and randomly divided into a normal control group and an IBD model group. The normal control group was given normal drinking water daily; the IBD model group was given drinking water containing 2.5 wt% DSS for free access, and both groups were fed continuously for 10 days. During the model establishment period, the mice's condition was observed daily, and changes in weight, fecal characteristics, and the presence of symptoms such as bloody stools and diarrhea were recorded. After continuous DSS feeding, the IBD model group showed varying degrees of weight loss, loose stools, and bloody stools, indicating that the IBD model was successfully established.

[0121] After completing the animal experiment on day 10, the mice were euthanized, and intestinal contents were subsequently collected from both the normal control group and the IBD model group. The anatomical results are as follows: Figure 10 As shown, the colon length of IBD mice was significantly shorter than that of the normal control group. Furthermore, H&E staining results indicated that the colonic mucosa structure of the normal group was relatively intact, with regular and orderly arranged crypt morphology; while the crypt structure of the colonic tissue of IBD mice was disordered, exhibiting localized crypt damage and inflammatory cell infiltration, among other inflammatory histological features. These results demonstrate the successful establishment of the mouse IBD model.

[0122] Next, the intestinal contents of the collected colon segment were placed in a centrifuge tube and centrifuged at 10,000 rpm for 10 min to remove fecal residue, cells and other larger particles. The supernatant was then collected as the intestinal sample to be tested.

[0123] During testing, 50 μL of the supernatant of intestinal contents from mice in the normal control group and IBD model group were added dropwise onto the PET plastic substrate of the capsule device, ensuring the sample fully covered the five sensing electrodes. The serotonin DPV peak current signal was substituted into the standard curve equation Y = 19.15X - 208.36 to obtain the serotonin concentration. The detected potential value (mV) was substituted into the standard curve equation Y = 0.98X - 0.92 to obtain the redox potential value (mV). The detected potential value (mV) was substituted into the standard curve equation Y = -65.79X + 464.19 to obtain the pH value. For detailed results, see [link to results]. Figure 10 .

[0124] like Figure 11 As shown, the detection results of serotonin, ORP and pH in the intestinal contents of mice in the normal control group and the IBD model group were compared respectively.

[0125] from Figure 11 (Left) It can be seen that the serotonin level in the IBD model group (11.5 μM) was significantly higher than that in the normal control group (4.07 μM) (p<0.001).

[0126] from Figure 11 (In the middle) it can be seen that the ORP of intestinal contents in the IBD model group (166.74 mV) was significantly different from that in the normal group (69.18 mV), indicating that the redox state of the intestine changed after DSS-induced inflammation, and the ORP changed in the positive direction with a significant difference (p<0.01).

[0127] from Figure 11 (Right) It can be seen that the pH of the intestinal contents in the IBD model group (7.21) showed a decreasing trend compared with the normal group (7.71), which further indicates that the local chemical microenvironment of the intestine changes under inflammatory conditions and the mean pH of the IBD model group is reduced. However, there was no significant difference under the experimental conditions (p>0.05).

[0128] The above results indicate that the constructed multi-parameter electrochemical capsule detection device can jointly detect serotonin, ORP and pH in actual intestinal samples, and can distinguish the differences in the intestinal microenvironment between normal and IBD inflammatory states.

Claims

1. A gastrointestinal capsule for multi-parameter microenvironment joint analysis, characterized in that, The capsule shell includes a first shell and a second shell that are connected to each other. The first shell and the second shell are connected by a light-curing adhesive to form a sealed cavity. One end of the capsule shell is provided with a sampling hole. The capsule shell contains a first, second, third, fourth and fifth circular discs arranged sequentially from the sampling hole end to the other end. An electrochemical interface unit is provided on the first disc for electrical connection with an electrochemical sensor. The electrochemical sensor includes a common counter electrode CE, a common reference electrode RE, and three independent working electrodes WE1, WE2, and WE3 disposed on a PET plastic substrate. The WE1 electrode, the common counter electrode, and the common reference electrode constitute a serotonin sensor for serotonin detection; the WE2 electrode, the common counter electrode, and the common reference electrode constitute an ORP sensor for ORP detection; and the WE3 electrode, the common counter electrode, and the common reference electrode constitute a pH sensor for pH detection. The second disc is equipped with an analog front-end unit, which includes a multi-channel operational amplifier, an analog switch, and peripheral resistor-capacitor components. The different operational amplification channels of the multi-channel operational amplifier and the peripheral resistor-capacitor components constitute a constant potential control circuit, a transimpedance amplifier circuit (TIA), and a low-pass filter circuit. The analog switch is connected to the main control chip, and the main control chip controls the analog switch to be turned on or off according to a preset detection timing sequence. The third wafer is equipped with a main control unit (MCU). The MCU includes a main control chip for data acquisition. The main control chip integrates an analog-to-digital converter (ADC) and a digital-to-analog converter (DAC). The output of the DAC is connected to the input of the constant potential control circuit in the multi-channel operational amplifier. The ADC performs analog-to-digital conversion on the analog signals of the three working electrodes. The main control chip further performs detection timing control, data processing, and result calculation. The analog front-end unit is electrically connected to the electrochemical interface unit and the main control MCU unit, respectively. The fourth disc is equipped with a power supply unit, which includes a low dropout linear regulator (LDO). A battery is disposed between the fourth and fifth discs. The LDO is electrically connected to the battery, and its regulated output terminal is connected to the power input terminals of the analog front-end unit, the main control unit, and the wireless communication unit, respectively, to provide a stable operating voltage to each functional unit. The fifth disc is equipped with a wireless communication unit, which includes a BLE wireless communication module. The BLE wireless communication module is connected to the main control chip through a UART communication interface. It is used to receive serotonin, ORP and pH detection data processed by the main control chip and wirelessly transmit the detection data to an external terminal or mobile APP to realize wireless transmission, real-time display and monitoring of multi-parameter electrochemical signals of the gastrointestinal tract.

2. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 1, characterized in that, The sampling port is covered from the inside out with a filter layer, a water-soluble protective layer, and an enteric-coated response layer. The capsule shell is made of light-cured resin. The multi-channel operational amplifier is an LTC2065; the analog switch is an ADG842; the main control chip is an STM32L151; the disc is made of FPC; and the battery is a button cell.

3. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 2, characterized in that, The capsule shell is made of BMF HTL resin by 3D printing. After printing, the shell is ultrasonically cleaned in anhydrous ethanol for 20-40 minutes. The diameter of the capsule shell is 9-11 mm and the length is 15-20 mm.

4. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 2, characterized in that, The sampling well diameter is 2.5~3.5 mm, and the filter layer is a polyvinylidene fluoride microporous membrane with a diameter of 5 mm and a pore size of 50 μm. The water-soluble protective layer is obtained by uniformly coating 8-12 μL of 10 mg / mL PVA aqueous solution onto the outer side of the polyvinylidene fluoride microporous membrane and drying it at room temperature for at least 30 min, repeating 2-4 times. The enteric-coated response layer is obtained through the following process: Eudragit L100 is dissolved in anhydrous ethanol to a mass fraction of 8-12%, and triethyl citrate is added at a mass fraction of 8-12% of Eudragit L100. The mixture is stirred for 20-40 min to form a uniform coating solution. 8-12 μL of the coating solution is then coated onto the outer side of the water-soluble membrane and dried at room temperature for at least 30 min. This process is repeated 2-4 times.

5. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 1, characterized in that, Silver paste is screen-printed onto a PET plastic substrate to form wires and a reference electrode region. The reference electrode region is chlorinated with FeCl3 aqueous solution to form a silver / silver chloride reference electrode. Then, carbon paste is screen-printed to form the first, second, and third carbon working electrodes and the carbon counter electrode. The working electrode has a diameter of 1 mm, the counter electrode has a diameter of 1.4 mm, and the reference electrode has a diameter of 0.5 mm. The reference electrode needs to be modified by the following process: polyvinyl butyral and sodium chloride are dissolved in methanol to obtain a reference solution. The concentration of polyvinyl butyral is 79~80 mg / mL, and the concentration of sodium chloride is 50 mg / mL. Then, 0.5 µL of the reference solution is drop-coated onto the reference electrode and dried at room temperature to obtain the final product.

6. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 5, characterized in that, The preparation process of the WE1 working electrode is as follows: Gold nanoparticle electrodes were electrodeposited on a first carbon working electrode using cyclic voltammetry in a mixed solution of 1 mM HAuCl4·4H2O and 100 mM KCl. The parameters were set to -0.35 V to -0.6 V, scan rate 0.1 V / s, and 40 cycles. After drying, gold nanoparticle electrodes were obtained. Subsequently, serotonin aptamer was dissolved in 1× Tris-EDTA buffer to prepare a 10 µM concentration, aliquoted, and stored at -20 °C. The gold nanoparticle electrodes were electrochemically cleaned using cyclic voltammetry in 0.5 M H2SO4 solution at a scan rate of 0.1 V / s. -1 The voltage range was -1 V to 1.6 V, and the cycles were repeated 15–25 times. The electrode was then rinsed with deionized water to obtain a clean working electrode. 5 µL of a 10 µM aptamer solution was mixed with 5 µL of a 1 mM TCEP solution and reacted at room temperature in the dark for 1 h. The solution was then diluted to 2.5 µM with 1× PBS buffer. The aptamer solution was then heated at 95 °C for 5 min using a PCR instrument, allowed to return to room temperature, and then 2.5 µL of the 2.5 µM aptamer solution was added dropwise to the clean working electrode and incubated at 4 °C in the dark for 12 h. Unbound aptamers were washed away with 1× PBS buffer, and the electrode was then incubated with 0.5 mM mercaptohexanol solution for 30 h. min is used to block the unbound electroactive surface on the gold electrode. Finally, the aptamer-modified electrode is thoroughly rinsed with 1×PBS buffer to obtain the serotonin aptamer sequence as follows: 5'-Thiol-C6-Linker-CGACTGGTAGGCAGATAGGGGAAGCTGATTCGATGCGTGGGTCG-MB-3'.

7. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 5, characterized in that, The preparation process of the WE2 working electrode is as follows: A mixed solution containing 1 mM H2PtCl6 and 100 mM H2SO4 is used as the electrodeposition system. Platinum nanoparticles are deposited on the second carbon working electrode by cyclic voltammetry. The electrodeposition potential range is -0.35 V to 0.6 V, the scan rate is 0.05 V / s, and the cycle is 15 to 25 times. After the deposition is completed, the electrode is dried to obtain the final electrode.

8. The gastrointestinal capsule for multi-parameter microenvironment joint analysis according to claim 5, characterized in that, The preparation process of the WE3 working electrode is as follows: (1) Preparation of PEDOT:PSS layer: 10.6 µL EDOT, 206 mg NaPSS and 36.8 mg K4Fe(CN)6 were dissolved in 10 mL of deionized water to obtain electrodeposition solution. Electrodeposition was performed on the third carbon working electrode using a multi-potential step method. The specific process was as follows: first step voltage and time: 0.465 V and 0.75 s; second step voltage and time: 0.865 V and 0.25 s; number of cycles:

840. The electrode was dried at 55~65 ℃ for 1~3 h. (2) Preparation of polyaniline hydrogen ion response layer: In a mixed solution of 0.1 M aniline and 1 M HCl, cyclic voltammetry was performed for 40 cycles in a potential range of -0.2 V to 1 V relative to a commercial Ag / AgCl reference electrode at a scan rate of 0.1 V / s, and then dried at 55~65℃ for 1~3 h to obtain the product.

9. A method for detecting gastrointestinal capsules using multi-parameter microenvironment joint analysis as described in any one of claims 1 to 8, characterized in that, Serum was detected using differential pulse voltammetry (DPV), while ORP and pH were detected using open circuit potential-time (OCPT). When the capsule is immersed in gastric or intestinal fluid for serotonin DPV detection, the constant potential control circuit receives the DPV set potential signal output by the digital-to-analog converter (DAC) inside the main control chip MCU. At the same time, the constant potential control circuit receives the potential signal fed back by the common reference electrode RE, and adjusts the driving potential of the common counter electrode CE according to the relationship between the set potential and the feedback potential, thereby controlling the potential difference between the serotonin working electrode WE1 and the reference electrode RE to change according to the preset DPV waveform. The weak current signal generated by the serotonin working electrode WE1 is input to the transimpedance amplifier circuit TIA. The transimpedance amplifier circuit TIA converts the current signal generated by the working electrode into a corresponding voltage signal. The converted voltage signal is filtered by a low-pass filter circuit and signal conditioned before being input to the ADC inside the main control chip in the third wafer for analog-to-digital conversion to obtain the serotonin current signal. When performing ORP and pH detection, the working electrodes WE2 and WE3 form an open-circuit potential detection loop with the common reference electrode RE. The open-circuit potential signals generated by the working electrodes WE2 and WE3 relative to the reference electrode RE are input to a multi-channel operational amplifier, filtered by a low-pass filter circuit, and then input to the ADC inside the main control chip to obtain the ORP potential signal and pH potential signal. After processing the three-channel detection data, the main control chip sends the data to the BLE wireless communication module via UART, and then further wirelessly to an external terminal. The collected data is substituted into the standard curve equation of the corresponding parameters to obtain the serotonin concentration, ORP value, or pH value.

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