Integrated kidney organ chip system and its application in online monitoring of pesticide nephrotoxicity

Abstract

This invention belongs to the field of biomaterials technology, specifically relating to an integrated kidney organ-on-a-chip system and its application in online monitoring of pesticide nephrotoxicity. The integrated kidney organ-on-a-chip system includes a sample introduction device, a renal tubular microfluidic chip, a Raman detection platform, and a Raman spectrometer. The labeled antibody probe in the Raman detection platform includes a SERS substrate, and recognition antibodies and signal molecules bound to the SERS substrate. The SERS substrate is a gallium-doped zinc oxide superlattice nanocube material. This invention uses a gallium-doped zinc oxide superlattice nanocube material as the SERS substrate, integrating semiconductor surface-enhanced Raman detection technology into a kidney organ-on-a-chip, providing an online monitoring system for pesticide nephrotoxicity with advantages such as fast analysis speed, high molecular selectivity, and high detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to an integrated kidney organ-on-a-chip system and its application in online monitoring of pesticide nephrotoxicity. Background Technology

[0002] With the widespread use of pesticides in modern agricultural production, their accumulation in the environment and food chain, and their potential threats to human health, have attracted considerable attention. In particular, some pesticide metabolites may have chronic nephrotoxicity, leading to kidney damage and even irreversible structural damage. Therefore, establishing a highly sensitive, multi-indicator in vitro nephrotoxicity evaluation system is of great significance for pesticide safety research and guiding the development of green pesticides.

[0003] Currently, pesticide nephrotoxicity evaluation mainly relies on animal experimental models and two-dimensional (2D) cell culture models. However, both of these traditional methods have significant limitations. While animal experiments can reflect overall physiological responses, the results often deviate from human responses due to differences in metabolic pathways, gene expression, and physiological structures among different species. Furthermore, these methods are time-consuming, costly, and subject to ethical controversies. Although traditional 2D cell culture models are simple to operate and low in cost, their cell growth environment differs fundamentally from the in vivo microenvironment, making it difficult to accurately reflect the physiological functions and pathological changes of complex organs such as the kidneys.

[0004] To overcome the aforementioned shortcomings, organ-on-a-chip (OoC) technology, which has emerged in recent years, provides a new approach for in vitro physiological modeling and toxicological detection. OoC utilizes microfluidics to co-culture multiple cell types in a controlled three-dimensional microenvironment, simulating the structure, function, and fluid flow environment of human organs. This technology has significant advantages in maintaining cell viability and functional differentiation, enabling intercellular signal exchange, dynamic transfer of nutrients and metabolites, and thus more realistically reproducing key physiological processes such as filtration, reabsorption, and excretion in the kidneys. Compared to animal models, OoC offers advantages such as humanization, reproducibility, rapid response, and lack of ethical restrictions. Compared to traditional two-dimensional cell culture, OoC can better reflect the toxic reactions and damage mechanisms of glomerular and tubular cells exposed to drugs or pesticides. Especially with multi-channel designs, different chip units can detect multiple samples or different concentration gradients in parallel, significantly improving throughput and data reliability.

[0005] In the acquisition of toxicity detection signals, surface-enhanced Raman scattering (SERS) technology has attracted widespread attention due to its high sensitivity and molecular specificity. SERS can perform fingerprint-like detection of target molecules without damaging the sample, offering advantages such as speed, label-free operation, and high throughput. Traditional SERS substrates mainly employ noble metal nanomaterials (such as gold, silver, and copper), whose surface plasmon resonance effect can significantly enhance the Raman signal. However, noble metal SERS substrates have several drawbacks: their preparation cost is high, and noble metal nanomaterials may cause biocompatibility issues in organ-on-a-chip microenvironments, even exhibiting toxicity to cells, limiting their application in long-term physiological monitoring; more importantly, their stability is poor, easily agglomerating or being encapsulated by proteins in biological systems, leading to signal attenuation and low detection sensitivity. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide an integrated kidney organ-on-a-chip system and its application in online monitoring of pesticide nephrotoxicity. The integrated kidney organ-on-a-chip system provided by this invention uses gallium-doped zinc oxide superlattice nanocube material as the SERS substrate for its Raman detection platform, resulting in high detection sensitivity.

[0007] This invention provides an integrated kidney organ-on-a-chip system, including a sample introduction device, a renal tubular microfluidic chip, a Raman detection platform, and a Raman spectrometer; The flow path outlet of the renal tubular microfluidic chip is connected to the flow path inlet of the Raman detection platform; The Raman detection platform includes a bonded substrate and a cover plate. The substrate has a groove, and a silicon wafer with a capture antibody is placed at the bottom of the groove. The cover plate has a through-hole opposite to the groove for Raman spectroscopy detection. The groove is filled with a suspension containing labeled antibody probes; The labeled antibody probe includes a SERS substrate, and a recognition antibody and a signaling molecule bound to the SERS substrate; the SERS substrate is a gallium-doped zinc oxide superlattice nanocube material. The method for preparing the gallium-doped zinc oxide superlattice nanocube material includes the following steps: An alkaline alcohol solution was added dropwise to a precursor solution containing gallium and zinc to carry out an alcoholysis-condensation reaction. The resulting solid was then dried and annealed sequentially to obtain a gallium-doped zinc oxide superlattice nanocube material. The molar ratio of gallium to zinc in the precursor solution containing gallium and zinc was 1:1 to 10.

[0008] Preferably, the capture antibody includes NGAL antibody, OPN antibody, IL-6 antibody and LDH antibody.

[0009] Preferably, the recognition antibody includes NGAL antibody, OPN antibody, IL-6 antibody and LDH antibody; the signaling molecule includes CuPc, 4-MBA, TCNQ and PB.

[0010] Preferably, the method for preparing the Raman detection platform includes the following steps: By binding the capture antibody onto the silicon wafer, a silicon wafer with the capture antibody is obtained; By binding recognition antibodies and signaling molecules onto a SERS substrate, labeled antibody probes are obtained. A silicon wafer with the capture antibody is placed at the bottom of a groove in a substrate. A suspension containing the labeled antibody probe is injected into the inlet of the Raman detection platform flow path until the groove is filled, thus obtaining the Raman detection platform.

[0011] Preferably, the process of binding the capture antibody onto the silicon wafer includes the following steps: After the silicon wafer is first immersed in a piranha solution, it is then second immersed in a 3-aminopropyltriethoxysilanol solution and third immersed in a succinic anhydride solution to obtain a modified silicon wafer. The modified silicon wafer was mixed with a solution containing capture antibodies and incubated to obtain a silicon wafer bound with capture antibodies.

[0012] Preferably, the binding of the recognition antibody and signaling molecule to the SERS substrate includes the following steps: The SERS substrate was silanized to obtain a modified SERS substrate; the recognition antibody and signal molecule were then bound to obtain the binding product. The modified SERS substrate and the suspension containing the binding product were mixed and incubated to obtain a labeled antibody probe.

[0013] Preferably, the renal tubular microfluidic chip comprises an upper and lower two-layer structure divided by a porous PET membrane. The upper layer contains renal tubular channels with a length of 24 mm, a width of 1 mm, and a height of 1 mm. The lower layer contains vascular channels with a length of 24 mm, a width of 1 mm, and a height of 0.2 mm.

[0014] Preferably, the upper layer contains renal tubular channels seeded with renal tubular epithelial cells, and the lower layer contains vascular channels seeded with human umbilical vein endothelial cells; culture medium is continuously perfused into the renal tubular channels and vascular channels at a flow rate of 2 μL / min.

[0015] This invention also provides the application of the integrated kidney organ-on-a-chip system described above in online monitoring of pesticide nephrotoxicity.

[0016] Preferably, the method of application includes the following steps: The pesticide to be tested is introduced into the renal tubule microfluidic chip, and the resulting biomarkers enter the Raman detection platform, where the Raman spectrometer is used to collect the signals on the silicon wafer.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an integrated kidney organ-on-a-chip system, including a sample introduction device, a renal tubular microfluidic chip, a Raman detection platform, and a Raman spectrometer.

[0018] This invention uses gallium-doped zinc oxide superlattice nanocube material (GZO SL) as the substrate material for surface-enhanced Raman spectroscopy and integrates surface-enhanced Raman spectroscopy detection technology into a kidney organ-on-a-chip system. This enables the preparation of a new Raman detection and evaluation system for nephrotoxicity biomarkers with multiple indicators (capable of simultaneously detecting four toxicity biomarkers), high sensitivity, and online quantitative analysis. This system can not only assess the nephrotoxicity of pesticides but also guide the development of green pesticides.

[0019] This invention verifies that the bilayer renal tubular microfluidic chip can simulate the physiological activities of human renal tubules. Furthermore, based on the renal tubular microfluidic chip, this invention integrates a SERS detection platform, using gallium-doped zinc oxide superlattice nanocube material as the SERS substrate. This provides a highly sensitive, selective, and stable bioanalytical system for analyzing multiple trace biomarkers in the toxicological process of pesticide-induced nephrotoxicity. Using CuPc, 4-MBA, TCNQ, and PB as response signal molecules and the GZO SL material internal standard peak as the reference signal, high sensitivity, high selectivity, and rapid quantitative analysis of multiple indicators are achieved, with a detection limit as low as 0.1 ng / mL. This renal organ-on-a-chip system not only accurately simulates the physiological activities of human renal tubules but also detects multiple toxicity biomarkers, providing guidance for pesticide toxicity assessment and the development of new green pesticides. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Electron micrograph of GZO SL prepared in Example 1; Figure 2 EDS spectrum of GZO SL prepared in Example 1; Figure 3 This is a design diagram of the renal tubular microfluidic chip and the sandwich-type immunoRaman detection platform in Example 3; Figure 4 This is a physical image of the renal tubular microfluidic chip and the sandwich-type immunoRaman detection platform used in Example 3; Figure 5 This is a side view of the renal tubular microfluidic chip in Example 3; Figure 6 This is a physical diagram of the kidney organ-on-a-chip system integrating the SERS platform in Example 3; Figure 7 This is a flow diagram illustrating the kidney organ-on-a-chip system integrating the SERS platform in Example 3; Figure 8 This is a fluorescence image of cell proliferation in the renal tubule microfluidic chip in Example 3; Figure 9 This is a bright-field image of cells seeded in the renal tubular microfluidic chip in Example 3; Figure 10 This is a diagram verifying the glucose and albumin reabsorption function of the renal tubular microfluidic chip in Example 3; Figure 11 This is a schematic diagram of the SERS detection platform in Example 4; Figure 12 The Raman signal molecular energy level diagram used in the SERS detection platform for toxic biomarkers in Example 4 is shown. Figure 13 This is a graph showing the selective test results of the SERS detection platform for toxic biomarkers in Example 4; Figure 14 This is a graph showing the linear test results of the SERS detection platform for toxic biomarkers in Example 4; Figure 15 This is a graph showing the response time test results of the SERS detection platform for toxic biomarkers in Example 4; Figure 16 This is a graph showing the stability test results of the labeled antibody probe in Example 4; Figure 17 This is a graph showing the test results of the SERS detection platform for nephrotoxicity biomarkers of three commercial pesticides in Example 5; Figure 18 This is an image of the live / dead staining test of cells within the renal tubular microfluidic chip in Example 5. Detailed Implementation

[0022] This invention provides an integrated kidney organ-on-a-chip system, including a sample introduction device, a renal tubular microfluidic chip, a Raman detection platform, and a Raman spectrometer; The flow path outlet of the renal tubular microfluidic chip is connected to the flow path inlet of the Raman detection platform; The Raman detection platform includes a bonded substrate and a cover plate. The substrate has a groove, and a silicon wafer with a capture antibody is placed at the bottom of the groove. The cover plate has a through-hole opposite to the groove for Raman spectroscopy detection. The groove is filled with a suspension containing labeled antibody probes; The labeled antibody probe includes a SERS substrate, and a recognition antibody and a signaling molecule bound to the SERS substrate; the SERS substrate is a gallium-doped zinc oxide superlattice nanocube material. The method for preparing the gallium-doped zinc oxide superlattice nanocube material includes the following steps: An alkaline alcohol solution was added dropwise to a precursor solution containing gallium and zinc to carry out an alcoholysis-condensation reaction. The resulting solid was then dried and annealed sequentially to obtain a gallium-doped zinc oxide superlattice nanocube material. The molar ratio of gallium to zinc in the precursor solution containing gallium and zinc was 1:1 to 10.

[0023] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0024] In this invention, the solutes of the gallium and zinc-containing precursor solution are preferably zinc nitrate and gallium nitrate, and the solvent is preferably ethylene glycol; the zinc nitrate is preferably zinc nitrate hexahydrate (Zn(NO3)2·6H2O), and the gallium nitrate is preferably gallium nitrate hydrate (Ga(NO3)3·xH2O); the concentrations of the zinc nitrate and gallium nitrate are independently preferably 0.4~0.6 mol / L, specifically 0.5 mol / L.

[0025] In this invention, the solute in the alkaline alcohol solution is preferably sodium hydroxide, and the solvent is preferably ethylene glycol; the concentration of the alkaline alcohol solution is preferably 0.8~1.2 mol / L, specifically 1 mol / L. The volume ratio of the gallium and zinc-containing precursor solution to the alkaline alcohol solution is preferably 1:1.

[0026] In this invention, the dropwise addition is preferably carried out under stirring; the temperature of the alcoholysis-condensation reaction is preferably 130~150℃, specifically 140℃, and the time is preferably 3~5h, specifically 4h; the alcoholysis-condensation reaction is preferably carried out under reflux heating.

[0027] In this invention, the drying temperature is preferably 80°C, and the drying time is preferably 24 hours; the annealing temperature is preferably 500~700°C, specifically 600°C, and the annealing time is preferably 8~12 hours, specifically 10 hours. During the annealing process, the precursor decomposes, removes residual organic matter, and rearranges and crystallizes to finally form gallium-doped zinc oxide crystals.

[0028] This invention produces gallium-doped zinc oxide superlattice nanocube materials by doping semiconductors (zinc oxide), which have excellent chemical stability and mechanical strength, making them suitable for long-term use in microfluidic chips. The SERS substrate signal enhancement of this invention mainly originates from the charge transfer mechanism (CT), which can achieve selective enhancement under specific molecular energy level matching, thereby improving the detection sensitivity of specific pesticide metabolites (neutrophil gelatinase-associated lipid carrier protein NGAL, osteopontin OPN, interleukin-6 IL-6, and lactate dehydrogenase LDH).

[0029] In this invention, the capture antibody preferably includes NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody. The recognition antibody preferably includes NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody. The signaling molecule preferably includes CuPc (copper phthalocyanine), 4-MBA (4-mercaptobenzoic acid), TCNQ (7,7,8,8-tetracyanoquinoxaline dimethyl ether), and PB (Prussian blue). When an antigen is present, the capture antibody captures the antigen, and the recognition antibody binds to the antigen, forming a sandwich structure, in which the signaling molecule is easily detected by Raman spectroscopy.

[0030] In this invention, the depth of the groove is preferably 1 mm, and the area is preferably 25 mm². 2 The diameter of the through-hole is preferably 2 mm, and the through-hole is a Raman detection point.

[0031] In this invention, the method for preparing the Raman detection platform includes the following steps: By binding the capture antibody onto the silicon wafer, a silicon wafer with the capture antibody is obtained; Labeled antibody probes are obtained by binding labeled antibodies and signal molecules onto a SERS substrate. A silicon wafer with the capture antibody is placed at the bottom of a groove in a substrate. A suspension containing the labeled antibody probe is injected into the inlet of the Raman detection platform flow path until the groove is filled, thus obtaining the Raman detection platform.

[0032] In this invention, the step of binding the capture antibody onto the silicon wafer preferably includes the following steps: After the silicon wafer is first immersed in a piranha solution, it is then second immersed in a 3-aminopropyltriethoxysilanol solution and third immersed in a succinic anhydride solution to obtain a modified silicon wafer. The modified silicon wafer was mixed with a solution containing capture antibodies and incubated to obtain a silicon wafer bound with capture antibodies.

[0033] In this invention, the silicon wafer is preferably 5mm × 5mm × 0.5mm in size, and is preferably rinsed with deionized water before use. The piranha solution comprises 30wt% hydrogen peroxide aqueous solution and 98wt% sulfuric acid, with a volume ratio of 1:3. The first soaking time is preferably 30 minutes. This first soaking increases the number of silanol groups (Si-OH) on the surface of the silicon wafer. After the first soaking, the silicon wafer is preferably rinsed sequentially with deionized water and then with ethanol.

[0034] In this invention, the solvent for the 3-aminopropyltriethoxysilane solution is preferably anhydrous ethanol, and the volume ratio of 3-aminopropyltriethoxysilane (APTES) to anhydrous ethanol is preferably 1:10. The second immersion is preferably carried out at room temperature for a duration of 2 hours; after the second immersion, an aminated silicon wafer is formed. Preferably, the second immersion also includes rinsing the silicon wafer three times with anhydrous ethanol.

[0035] In this invention, the succinic anhydride solution is preferably a saturated succinic anhydride solution; the third soaking is preferably carried out at room temperature for 3 hours; after the third soaking, the succinic anhydride will undergo a ring-opening acylation reaction with the -NH2 on the surface to generate an amide bond, while leaving a free carboxyl group at the other end of the molecule, and the surface amino group is converted into a carboxyl group to form a carboxyl functionalized silicon wafer.

[0036] In this invention, the concentrations of NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody in the solution containing the capture antibody are preferably all 10 mmol / L; the incubation temperature is preferably 4°C, and the incubation time is preferably 12-18 h, specifically overnight. The incubation process preferably includes sequential washing with PBS solution and incubation in PBS solution containing 1% BSA; the washing with PBS solution is preferably performed three times to remove unbound antibodies; the incubation time is preferably 1 h, and incubation in PBS solution containing 1% BSA can block non-specific binding sites.

[0037] In this invention, the binding of the recognition antibody and signaling molecule to the SERS substrate preferably includes the following steps: The SERS substrate was silanized to obtain a modified SERS substrate; the recognition antibody and signal molecule were then bound to obtain the binding product. The modified SERS substrate and the suspension containing the binding product were mixed and incubated to obtain a labeled antibody probe.

[0038] In this invention, the silanization treatment preferably includes: treating the SERS substrate with oxygen plasma, then immersing it in a 3-aminopropyltriethoxysilane alcohol solution and removing it for curing to obtain a modified SERS substrate. Preferably, before the oxygen plasma treatment, the SERS substrate is further immersed in ethanol and deionized water for 10 minutes each, followed by drying under nitrogen. The oxygen plasma treatment power is preferably 100 W, and the time is preferably 10 minutes. The volume fraction of 3-aminopropyltriethoxysilane alcohol in the 3-aminopropyltriethoxysilane alcohol solution is preferably 2%, and the solvent is preferably ethanol; the immersion is preferably carried out at room temperature for 2 hours. The curing temperature is preferably 110°C, and the time is preferably 1 hour. The silanization treatment of this invention can introduce surface amino groups.

[0039] In this invention, the binding of the recognition antibody and the signal molecule preferably includes: activating the signal molecule with its carboxyl group and then mixing it with the recognition antibody for incubation; the molar ratio of the signal molecule to the recognition antibody is preferably 100:1; the incubation temperature is 4°C, and the incubation time is preferably overnight; the incubation is preferably carried out in PBS solution; and the incubation process preferably further includes: ultrafiltration of the resulting reaction solution, wherein the molecular weight cutoff of the ultrafiltration is preferably 3 kDa. The binding products include CuPc-NGAL antibody, 4-MBA-OPN antibody, TCNQ-IL-6 antibody, and PB-LDH antibody (each recognition antibody corresponds to a signal molecule).

[0040] In this invention, the molar ratio of the modified SERS substrate to each binding product is preferably 1:100-200. The concentration of each binding product in the suspension containing the binding product is preferably 100 mmol / L, and the volume ratio of the modified SERS substrate to the suspension is preferably 1 μmol:1 mL. The incubation temperature for mixing the modified SERS substrate and the suspension containing the binding product is preferably 37°C, and the incubation time is preferably 2 hours.

[0041] The present invention places a silicon wafer with a capture antibody at the bottom of a groove in a substrate, and injects a suspension containing the labeled antibody probe into the inlet of the Raman detection platform flow path until the groove is filled, thereby obtaining a Raman detection platform.

[0042] In this invention, the flow rate of the suspension containing the labeled antibody probe is preferably 2 μL / min, and the suspension containing the labeled antibody probe is preferably a suspension of four mixed labeled antibody probes, with the concentration of each labeled antibody probe preferably being 10 mmol / L.

[0043] In this invention, the sample introduction device preferably includes a syringe and a flow pump.

[0044] In this invention, the renal tubular microfluidic chip comprises a porous PET (polyethylene terephthalate) membrane and PDMS (polydimethylsiloxane) clamps disposed on the upper and lower sides of the porous PET membrane; the pore size of the porous PET membrane is preferably 2 μm, and the thickness is preferably 22 μm. The renal tubular microfluidic chip comprises an upper and lower two-layer structure divided by the porous PET membrane. The upper layer contains renal tubular channels with a length of 24 mm, a width of 1 mm, and a height of 1 mm; the lower layer contains vascular channels with a length of 24 mm, a width of 1 mm, and a height of 0.2 mm. Renal tubular epithelial cells (HK-2) are seeded in the upper renal tubular channels, and human umbilical vein endothelial cells (HUVECs) are seeded in the lower vascular channels. Culture medium is perfused into the renal tubular channels and vascular channels by a flow pump to maintain cell viability. The flow rate of the culture medium is preferably 2 μL / min, and the culture medium is preferably DMEM medium. The Raman detection platform can detect and quantitatively analyze secretions in the urine and blood channels.

[0045] This invention also provides the application of the integrated kidney organ-on-a-chip system described above in online monitoring of pesticide nephrotoxicity.

[0046] In this invention, the method of application includes the following steps: The pesticide to be tested is introduced into the renal tubule microfluidic chip, and the resulting biomarkers enter the Raman detection platform, where the Raman spectrometer is used to collect the signals on the silicon wafer.

[0047] In this invention, the pesticide preferably includes one or more of diazinon, trichlorfon, and lambda-cyhalothrin. The pesticide to be tested is used in solution form, preferably with a culture medium as the solvent, preferably DMEM medium; the concentration of diazinon is preferably 0.9 g / L, the concentration of trichlorfon is preferably 0.6 g / L, and the concentration of lambda-cyhalothrin is preferably 0.01 g / L.

[0048] In this invention, the flow rate of the pesticide to be tested into the renal tubule microfluidic chip is preferably 2 μL / min, and the time is preferably 30 min.

[0049] In this invention, the preferred conditions for acquiring signals on the silicon wafer using a Raman spectrometer include: using a 633nm laser as the excitation source, a laser power of 0.5mW, and an exposure time of 10s.

[0050] This invention introduces a toxic biomarker generated after pesticides are infused into a renal tubular microfluidic chip and then fed into a sandwich-type immunoRaman detection platform for pesticide nephrotoxicity detection.

[0051] This invention provides a kidney organ-on-a-chip system integrating a highly sensitive online monitoring platform for multiple physiological indicators. Firstly, regarding analysis speed, the renal tubular chip can simulate the real microenvironment of renal tubular epithelial cells under microfluidic conditions, achieving integrated continuous analysis of pesticide exposure, cellular response, and signal acquisition, reducing complex pretreatment steps such as sampling, lysis, and staining in traditional toxicity testing. Simultaneously, Raman detection is rapid, in-situ, and label-free, allowing direct real-time or near-real-time reading of target molecules or cell damage-related biomarkers, thus significantly shortening the detection cycle. Secondly, regarding molecular selectivity, semiconductor surface-enhanced Raman spectroscopy differs from metal substrates that rely solely on electromagnetic enhancement. Its enhancement mechanism includes a significant charge transfer enhancement effect. The band structure of the semiconductor material can match the molecular orbitals of the analyte, thereby selectively enhancing specific molecules or vibrational modes. This "energy level matching-selective charge transfer" mechanism helps improve the recognition ability of pesticide molecules, nephrotoxicity-related metabolites, and damage biomarkers, while reducing interference from complex biological matrix backgrounds. Furthermore, regarding detection sensitivity, semiconductor surface-enhanced Raman spectroscopy can significantly amplify the characteristic vibrational signals of target molecules, enabling the effective identification of even low-abundance toxicity-related molecules. Simultaneously, the chip's microscale structure allows for the local enrichment of analytes, improving the contact efficiency between target molecules and the enhancement substrate and reducing sample consumption, thereby further enhancing the detection response intensity and signal-to-noise ratio. Compared to traditional biochemical analysis methods, this invention's detection system also possesses the advantages of being non-destructive, providing high information content, and enabling dynamic monitoring. It can more realistically reflect the early damage process and molecular changes of pesticides on renal tubules, which is of great significance for achieving rapid screening, mechanism research, and efficient evaluation of pesticide nephrotoxicity.

[0052] To further illustrate the present invention, the integrated kidney organ-on-a-chip system with integrated high sensitivity and multi-index online monitoring platform provided by the present invention and its application in online monitoring of pesticide nephrotoxicity are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1: Preparation of SERS substrate material (GZO SL) 1. Preparation of precursor solution: Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and gallium nitrate hydrate (Ga(NO3)3·xH2O) were dissolved in ethylene glycol to prepare precursor solutions with a concentration of 0.5 M and a volume of 25 mL.

[0054] 2. Preparation of GZO SL: Under continuous stirring, a 1 M sodium hydroxide ethylene glycol solution (25 mL) was added dropwise to the precursor solution. The reaction mixture was refluxed at 140 °C for 4 h, and the resulting solid was dried at 80 °C for 24 h. Finally, the sample was annealed at 600 °C for 10 h to obtain GZO SL (gallium-doped zinc oxide superlattice nanocube material).

[0055] Figure 1 The image shows an electron microscope (TEM) image of the GZO SL prepared in Example 1. The image, obtained using a transmission electron microscope, reveals the morphology and size of the material. The sample consists of regular cubic nanoparticles with a particle size distribution ranging from 10 to 14 nm. The particles are uniformly distributed without significant agglomeration, indicating good crystallinity and dispersibility. The dense, smooth surface and clear boundary edges demonstrate that the annealing process effectively controlled the grain growth direction and size.

[0056] Figure 2 The EDS spectrum of GZO SL prepared in Example 1 is shown. The elemental composition and distribution of the material can be seen from the energy-dispersive X-ray spectroscopy analysis. Semi-quantitative elemental analysis using an energy-dispersive X-ray spectroscopy (EDX, X-Max 150T) showed that Ga, Zn, and O were uniformly distributed in the sample, and the Ga content was close to the theoretical molar ratio, indicating good doping efficiency.

[0057] Example 2: Preparation of a sandwich-style immunoRaman detection platform 1. Fixation of captured antibodies: First, a 5 mm × 5 mm × 0.5 mm silicon wafer was rinsed with deionized water. Then, it was immersed in a piranha solution (30 wt% hydrogen peroxide aqueous solution: 98 wt% sulfuric acid = 1:3, volume ratio) for 30 min to increase the number of silanol groups on the wafer surface. Next, the wafer was rinsed sequentially with deionized water and ethanol, then immersed in a mixture of 3-aminopropyltriethoxysilane (APTES) and anhydrous ethanol (APTES: anhydrous ethanol = 1:10, volume ratio) and incubated at room temperature for 2 h with gentle stirring. Afterward, it was rinsed three times with ethanol and incubated at room temperature for 3 h with gentle stirring. Finally, the wafer was stored in ethanol, resulting in the modified silicon wafer.

[0058] Subsequently, the modified silicon wafers were incubated overnight at 4°C in a solution containing NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody (all at a concentration of 10 mM). After incubation, the wafers were washed three times with PBS to remove unbound antibodies. To block non-specific binding sites, the silicon wafers immobilized with the capture antibodies were incubated with PBS solution containing 1% BSA at room temperature for 1 h, then washed three times with PBS solution and stored in PBS solution at 4°C.

[0059] 2. Preparation of labeled antibody probes: The GZO SL substrate material prepared in Example 1 was immersed in ethanol and deionized water for 10 min each, then dried under nitrogen and treated with oxygen plasma (100 W for 10 min). After plasma treatment, the substrate material was immersed in a 2% (v / v) solution of (3-aminopropyl)triethoxysilane (APTES) in ethanol and silanized at room temperature for 2 h. The substrate was thoroughly rinsed with ethanol and cured at 110 °C for 1 h.

[0060] Four Raman reporter molecules, namely signal molecules CuPc (copper phthalocyanine), 4-MBA (4-mercaptobenzoic acid), TCNQ (7,7,8,8-tetracyanoquinoxaline dimethyl ether), and PB (Prussian blue), were activated at room temperature for 30 min by soaking them in PBS buffer containing 5 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 5 mM NHS (N-hydroxysuccinimide). The activated CuPc, 4-MBA, TCNQ, and PB were then incubated overnight at 4 °C in PBS solutions (10 μM each) containing NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody (each antibody corresponds to a signal molecule), at a molar ratio of 100:1 (reporter molecule: antibody). The binding products were purified by ultrafiltration (molecular weight cutoff of 3 kDa) and resuspended in PBS solution at a concentration of 100 mM.

[0061] Each 1 μmol of silanized GZO SL substrate was incubated with 1 mL of suspensions of NGAL, OPN, IL-6, and OPN binding products at 37 °C for 2 h. The suspensions of the four labeled antibody probes were then mixed to obtain a suspension containing mixed labeled antibody probes at a concentration of 10 mM.

[0062] 3. Construction of the sandwich-style immunoassay Raman spectroscopy (SERS) platform: A 1mm deep, 25mm area hole is cut in the middle of a PDMS substrate with a flow path inlet and outlet. 2 The substrate and cover plate are bonded together. The silicon wafer with the captured antibody fixed in step 1 is placed in the groove. A hole with a diameter of 2 mm is punched in the middle of the cover plate as a fixed Raman detection point. After the substrate and cover plate are bonded together, the suspension containing the mixed labeled antibody probe is perfused at a flow rate of 2 μL / min until the entire Raman detection area is filled.

[0063] Example 3: Construction and functional characterization of an integrated kidney organ-on-a-chip system The integrated kidney organ-on-a-chip system includes a syringe, a flow pump, a renal tubular microfluidic chip, a sandwich-type immunoRaman detection platform prepared in Example 2, a Raman spectrometer, and a waste collection bottle.

[0064] The renal tubular microfluidic chip consists of a porous polyethylene terephthalate (PET) membrane and two polydimethylsiloxane (PDMS) clamps. The porous PET membrane has a pore size of 2 μm and a thickness of 22 μm. The microfluidic chip is divided into upper and lower layers: the upper layer contains renal tubular channels, and the lower layer contains vascular channels (used for cell culture). Both the renal tubular and vascular channels are 24 mm long and 1 mm wide, with a height of 1 mm for the renal tubular channels and 0.2 mm for the vascular channels. Renal tubular epithelial cells (HK-2) are seeded in the upper renal tubular channels, and human umbilical vein endothelial cells (HUVECs) are seeded in the lower vascular channels. The upper and lower channels are equipped with inlets and outlets, respectively. DMEM culture medium is pumped into the channels to maintain cell viability at a flow rate of 2 μL / min.

[0065] Figure 3 This is a design diagram of the renal tubular microfluidic chip and sandwich-type immunoRaman detection platform in Example 3; it was drawn using drawing software, and the size of the design can be seen.

[0066] Figure 4 The image shows the renal tubular microfluidic chip and sandwich-type immunoRaman detection platform in Example 3. It was taken with a camera and can be seen that the overall size is small and portable.

[0067] Figure 5 This is a side view of the renal tubular microfluidic chip in Example 3; taken by a camera, it can be seen that the chip has an upper PDMS clamp 1, a middle PET membrane 2, and a lower PDMS clamp 3.

[0068] Figure 6 This is a physical diagram of the kidney organ-on-a-chip system integrating the SERS platform in Example 3; it was taken by a camera and includes a syringe 4 (only one is shown), a flow pump 5, a renal tubular microfluidic chip, a Raman detection platform, a Raman spectrometer 6, and a waste collection bottle 7, wherein the renal tubular microfluidic chip and the Raman detection platform are obscured by the Raman spectrometer 6.

[0069] Figure 7This is a flow path demonstration diagram of the kidney organ-on-a-chip system integrating the SERS platform in Example 3; drawn using graphics software, the flow paths can be seen. In the renal tubular microfluidic chip 8, the upper channel of the PET membrane (only the flow path is shown, the PET membrane is not drawn) is the renal tubular channel, and the lower channel is the vascular channel; urine is injected into the renal tubular channel and blood is injected into the vascular channel using the first syringe 42 and the second syringe 41, respectively. After flowing out from each channel, they flow into the Raman detection platform 9 for detection, and are then collected by the waste liquid collection bottle 7.

[0070] 1. Cell culture: Human renal tubular epithelial cells (HK-2) and human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin solution. First, the microarray was sterilized with UV light for 30 min, then loaded with extracellular matrix protein type IV collagen, incubated for 1 h, washed three times with PBS, and then DMEM was perfused into the channels. Next, HK-2 cells were extracted from T25 cell culture flasks using trypsin and cultured at approximately 5 × 10⁻⁶ cells / mL. 5 Injected into the upper channel at a density of 10 cells / mL. After 24 h, at a rate of approximately 1×10⁻⁶ cells / mL... 6 HUVEC cells were injected into the lower channel at a density of 1 cell / mL. Finally, the chip was incubated in a 37°C, 5% CO2 incubator.

[0071] 2. Monitoring of cell proliferation and the process of cell monolayer formation: Immunofluorescence staining was performed on cells on days 1, 3, 5, and 7 after cell seeding.

[0072] First, cells on the chip were fixed with 4% paraformaldehyde for 15 min, then washed with PBS. To prevent nonspecific binding, cells were incubated at 37°C in PBS solution containing 5% normal goat serum for 30 min, and the blocking solution was removed. Primary antibodies against tight junction protein ZO-1 (1:500 volume dilution in commercial immunofluorescence antibody dilution buffer), the HK-2 cell marker protein AQP1 (1:200 volume dilution in antibody dilution buffer), and the HUVEC cell marker protein von Willebrand factor vWF (1:500 volume dilution in antibody dilution buffer) were added to the cell channels and incubated overnight at 4°C. The next day, cells were washed with PBS, and then Alexa Fluor 594, Alexa Fluor 488, and Cy5 fluorescently labeled secondary antibodies were added, respectively, and incubated at 37°C in the dark for 1 h for staining. Cells were then washed again with PBS. Cell nuclei were stained with Hoechst 33342 solution at room temperature for 10 min. Finally, the cells were imaged and analyzed using confocal laser scanning microscopy (CLSM). Green fluorescence represented ZO-1 protein, red fluorescence represented AQP1 protein and vWF factor, and blue fluorescence represented the cell nucleus.

[0073] 3. Glucose and albumin reabsorption analysis: Reabsorption experiments were conducted on days 1, 2, 3, 4, 5, 6, and 7.

[0074] To investigate the efficiency of glucose reabsorption in a renal tubular microfluidic chip, a 17.5 mM glucose solution was continuously perfused into the renal tubular channels at a rate of 0.5 μL / min. Effluent from the proximal tubular channels was collected after 2 h until the glucose concentration in the perfusion fluid stabilized. Glucose concentration was measured at 505 nm using a glucose detection kit and a spectrophotometer. The glucose reabsorption rate (Rg) was calculated using the following formula: Rg = ((Cp-Ce) / Cp) × 100%; Where Cp is the initial glucose concentration in the perfusion fluid, and Ce is the glucose concentration in the effluent from the proximal tubule channel.

[0075] The experimental method for detecting albumin reabsorption is the same as that for glucose.

[0076] Figure 8The image shows the fluorescence of cell proliferation in the renal tubular microfluidic chip in Example 3. It was obtained by confocal fluorescence microscopy. The blue fluorescence represents the cell nucleus, the green fluorescence represents the tight junction protein ZO-1, and the red fluorescence represents the specific expression proteins of the two cell types (human renal tubular epithelial cells HK-2 expressing aquaporin AQP1 and human umbilical vein endothelial cells HUVEC expressing von Willebrand factor vWF), which proves the types of cells seeded in the chip and that the cells are growing well.

[0077] Figure 9 This is a bright-field image of cells seeded in the renal tubular microfluidic chip in Example 3; it was obtained by confocal fluorescence microscopy, indicating that the cells in the chip are growing well.

[0078] Figure 10 This is a verification diagram of the glucose and albumin reabsorption function of the renal tubular microfluidic chip in Example 3; the diagram is drawn from experimental data obtained from the glucose and albumin detection kit, demonstrating that the chip can reproduce the reabsorption function of the renal tubules.

[0079] Example 4: SERS detection procedure for four toxicity markers 1. Raman signal acquisition, data recording and processing The Raman detection platform (silicon wafer) was fixed on a Raman microscopy imaging platform. A 633 nm laser was used as the excitation source, with a laser power set to 0.5 mW and an exposure time of 10 s. The characteristic peak position of the target Raman probe was recorded, specifically at 437 cm⁻¹ on the GZO SL laser. -1 1099 cm of 4-MBA -1 1529cm of CuPc -1 PB's 2155 cm -1 2228 cm of TCNQ -1 .

[0080] The acquired spectral data underwent background subtraction and normalization using built-in software. A standard curve was constructed using the average intensity of each peak as a quantification index, comparing Raman intensity with biomarkers. The 437 cm⁻¹ peak of GZO SL was used as the reference. -1 As an internal standard peak, the signal hardly changes with concentration. The other four signal molecules correspond to four biomarkers. The concentration of the corresponding biomarker is determined by the ratio of the characteristic peak height of the signal molecule to the peak height of the internal standard peak, i.e., I / I0.05. 437 This enables quantitative analysis.

[0081] 2. Validation of detection sensitivity and specificity Four toxicity biomarkers were detected in a concentration range of 0 to 350 ng / mL. Standards of the four proteins were prepared with PBS buffer at concentrations of 50, 100, 150, 200, 250, 300, and 350 ng / mL, respectively, and the results were obtained using a sandwich immunoRaman assay platform.

[0082] 500 μL of mixed standard solutions of NGAL, OPN, IL-6, and LDH at different concentrations were passed into the Raman detection platform at a flow rate of 2 μL / min and incubated together at 37 °C for 30 min. Raman spectroscopy was performed under 633 nm laser irradiation, and the signal of the SERS probe on the silicon wafer was acquired. A 50x eyepiece, a laser power of 0.5 mW, and an exposure time of 10 s were used in the experiment.

[0083] The results showed that the characteristic peak could still be detected stably below 1 ng / mL, and the limit of detection (LOD) was approximately 0.1 ng / mL.

[0084] To verify specificity, non-target biomarkers (Kim-1, Urea, etc., see...) were used respectively. Figure 13 The system was tested with labeled antibody probes, and no target peak response was observed, indicating that the system has good specific recognition ability and low cross-reactivity.

[0085] Figure 11 This is a schematic diagram of the SERS detection platform in Example 4. Drawn using drawing software, it illustrates the detection principle of the SERS platform and demonstrates the structure and composition of the sandwich-type immunoRaman platform. The binding order of the recognition antibody (labeled antibody), signal molecule, and substrate can be altered. Renal tubular microfluidic chip: Type IV collagen 11 is loaded into the upper and lower channels on both sides of the PET membrane 2. HK-2 cells 10 are seeded in the upper renal tubular channel, and HUVEC cells 12 are seeded in the lower vascular channel. Raman detection area: Capture antibodies (including NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody) are bound to an amino-modified silicon wafer, which can specifically capture antigens after blocking. The amino-modified SERS substrate (GZO SL) is bound to labeled antibodies (including NGAL antibody, OPN antibody, IL-6 antibody, and LDH antibody) and signal molecules (including CuPc, 4-MBA, TCNQ, and PB). After the labeled antibody binds to the captured antigen, a sandwich structure is formed. Simultaneous quantitative analysis of four toxicity markers can be achieved through signal molecule detection.

[0086] Figure 12 The energy level diagram of the Raman signal molecules selected for the SERS detection platform for toxic biomarkers in Example 4 is shown. It was drawn by plotting software and illustrates the energy matching of the four Raman signal molecules.

[0087] Figure 13 This is a graph showing the selective test results of the SERS detection platform for toxic biomarkers in Example 4. The graph is drawn from the selective test experimental data, which illustrates the high selectivity of the detection platform and its ability to exclude interference from other metal ions, proteins, and other molecules.

[0088] Figure 14 The graph shows the linearity test results of the SERS detection platform for toxic biomarkers in Example 4. The graph, plotted from the linearity test data, demonstrates that the linear range of the detection platform is 0.1~350 ng / mL, providing a computational basis for quantitative analysis. The linear equations are as follows: IL-6: y=0.0088x+0.61, LDH: y=0.0133x+0.68, NGAL: y=0.0160x+0.86, OPN: y=0.0138x+0.92.

[0089] 3. Response time verification of Raman detection platform After the sandwich-type immunoRaman detection platform is constructed, protein standard samples are added, and then detection is performed every minute until the signal reaches its maximum value and becomes basically stable. This time is the response time of the platform.

[0090] The response time of 50 samples was tested, and the normal distribution plot showed that the response time was basically within 10 minutes. Figure 15 The graph shows the response time test results of the SERS detection platform for toxic biomarkers in Example 4 (i-OPN, ii-IL-6, iii-LDH, iv-NGAL). The graph, plotted from the response time experimental data, demonstrates that the signal response time of the detection platform is within 10 minutes, indicating that the platform has the advantage of rapid detection.

[0091] 4. Stability verification of the Raman detection platform To verify the stability of the entire sandwich-type immunoRaman detection platform, after successful setup, samples were added, and four protein standards, each at a concentration of 20 ng / mL, were used for detection. Detection was performed every 15 days, and after 180 days, the I / I ratio was calculated based on the Raman spectra obtained from each test. 437 The value is used to compare the results of the last 12 tests with the results of the test on day 0.

[0092] Figure 16 The stability test of the labeled antibody probe in Example 4 is shown in the stability test data. The stability test data shows that the labeled antibody probe still retains more than 95% of the signal after 180 days, which demonstrates the stability of the platform.

[0093] Example 5: Detection of nephrotoxicity after pesticide application The pesticide, prepared at a working concentration, is injected into the upper and lower channels of a renal tubular microfluidic chip. The degree of nephrotoxicity is assessed by the release of toxic biomarkers. The concentrations of these biomarkers are calculated based on Raman spectroscopy data. Simultaneously, the cells within the channels are stained for live and dead cell detection, and the cell mortality rate is calculated. This data is then cross-referenced with the toxic biomarker concentrations to demonstrate the accuracy of the organ-on-a-chip system in assessing pesticide nephrotoxicity.

[0094] 1. Preparation of culture medium containing pesticides Approximately 100L of water is needed to prepare pesticide spraying solution for one acre of land. Based on the instructions for the purchased commercial pesticides, the application rates for each pesticide (diaziphos 160mL / acre, trichlorfon 200g / acre, lambda-cyhalothrin 5mL / acre) are calculated, and the working concentrations of each pesticide are standardized to g / L. Using a culture medium as a solvent, the corresponding pesticide concentrations are prepared as follows: diazinon 0.9g / L, trichlorfon 0.6g / L, and lambda-cyhalothrin 0.01g / L.

[0095] 2. Application of pesticides and detection of nephrotoxicity Culture medium containing the working concentration of pesticide was injected into the upper and lower channels of the renal tubular microfluidic chip for 30 min (the total perfusion volume for both channels was 60 μL). The medium was then replaced with normal culture medium (DMEM). Raman spectroscopy was performed at 24 h, 48 h, and 72 h after pesticide application, and the concentrations of these biomarkers were calculated based on the Raman spectroscopy data. Live and dead cell staining was also performed on the cells within the channels at 24 h, 48 h, and 72 h after pesticide application to calculate the cell mortality rate.

[0096] Figure 17The results of the SERS detection platform in Example 5 on the nephrotoxicity biomarkers of three commercial pesticides are shown in the figure. The data, plotted from the platform's Raman spectroscopy, demonstrate that the platform can quantitatively detect four toxicity biomarkers and perform toxicity assessments. After 24 hours of application, the concentrations of all four biomarkers increased significantly. By 72 hours, the concentrations of each biomarker had risen to abnormal levels. Specifically, after 72 hours of diazinon application, NGAL increased to 121 ng / mL; OPN increased to 89 ng / mL; IL-6 increased to 106 ng / mL; and LDH increased to 98 ng / mL. After 72 hours of trichlorfon application, NGAL increased to 176 ng / mL; OPN increased to 128 ng / mL; IL-6 increased to 148 ng / mL; and LDH increased to 165 ng / mL. After 72 hours of application of lambda-cyhalothrin, NGAL increased to 83 ng / mL; OPN increased to 55 ng / mL; IL-6 increased to 66 ng / mL; and LDH increased to 48 ng / mL. These findings indicate the presence of toxic effects. In a cross-sectional comparison of the three pesticides, trichlorfon showed the strongest toxicity, followed by diazinon, while lambda-cyhalothrin showed the weakest toxicity.

[0097] Figure 18 Images from Example 5 show the cell viability and mortality tests performed on the renal tubular microfluidic chip. These images, captured using confocal fluorescence microscopy, visually illustrate cell viability and corroborate Raman spectroscopy data, demonstrating the pesticide toxicity. The mortality rate was calculated by dividing the number of dead cells by the total number of cells based on the cell viability and mortality staining images. Specifically, after 72 hours of diazinon application, the HK-2 cell mortality rate reached 97%, and the HUVEC cell mortality rate reached 95%; after 72 hours of trichlorfon application, the HK-2 cell mortality rate reached 98%, and the HUVEC cell mortality rate reached 97%; after 72 hours of lambda-cyhalothrin application, the HK-2 cell mortality rate reached 60%, and the HUVEC cell mortality rate reached 82%. The mortality rate data also indicates that all three pesticides are toxic, with trichlorfon being the most toxic, followed by diazinon, and lambda-cyhalothrin being the least toxic, corroborating the Raman spectroscopy data.

[0098] This invention combines the biomimetic culture advantages of microfluidic chips with the high-sensitivity detection characteristics of semiconductor SERS to construct a highly sensitive, multi-indicator pesticide nephrotoxicity detection system (kidney organ-on-a-chip system). The system can simulate the physiological structure and function of the kidney in an in vitro microenvironment, enabling rapid assessment of pesticide nephrotoxicity through online detection of toxic biomarkers. Compared with traditional methods, this invention's system has advantages such as high sensitivity, high throughput, good stability, and strong scalability, providing a new technical approach for pesticide safety evaluation.

[0099] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An integrated kidney organ-on-a-chip system, characterized in that, Includes a sample introduction device, a renal tubular microfluidic chip, a Raman detection platform, and a Raman spectrometer; The flow path outlet of the renal tubular microfluidic chip is connected to the flow path inlet of the Raman detection platform; The Raman detection platform includes a bonded substrate and a cover plate. The substrate has a groove, and a silicon wafer with a capture antibody is placed at the bottom of the groove. The cover plate has a through-hole opposite to the groove for Raman spectroscopy detection. The groove is filled with a suspension containing labeled antibody probes; The labeled antibody probe includes a SERS substrate, and a recognition antibody and a signaling molecule bound to the SERS substrate; The SERS substrate is a gallium-doped zinc oxide superlattice nanocube material; The method for preparing the gallium-doped zinc oxide superlattice nanocube material includes the following steps: An alkaline alcohol solution was added dropwise to a precursor solution containing gallium and zinc to carry out an alcoholysis-condensation reaction. The resulting solid was then dried and annealed to obtain gallium-doped zinc oxide superlattice nanocube material. The molar ratio of gallium to zinc in the gallium- and zinc-containing precursor solution is 1:

1.

2. The system according to claim 1, characterized in that, The capture antibodies include NGAL antibody, OPN antibody, IL-6 antibody and LDH antibody.

3. The system according to claim 1 or 2, characterized in that, The recognition antibodies include NGAL antibody, OPN antibody, IL-6 antibody and LDH antibody; the signaling molecules include CuPc, 4-MBA, TCNQ and PB.

4. The system according to claim 1, characterized in that, The method for preparing the Raman detection platform includes the following steps: By binding the capture antibody onto the silicon wafer, a silicon wafer with the capture antibody is obtained. By binding recognition antibodies and signaling molecules onto a SERS substrate, labeled antibody probes are obtained. A silicon wafer with the capture antibody is placed at the bottom of a groove in a substrate. A suspension containing the labeled antibody probe is injected into the inlet of the Raman detection platform flow path until the groove is filled, thus obtaining the Raman detection platform.

5. The system according to claim 4, characterized in that, The process of binding the capture antibody onto the silicon wafer includes the following steps: After the silicon wafer is first immersed in a piranha solution, it is then second immersed in a 3-aminopropyltriethoxysilanol solution and third immersed in a succinic anhydride solution to obtain a modified silicon wafer. The modified silicon wafer was mixed with a solution containing capture antibodies and incubated to obtain a silicon wafer bound with capture antibodies.

6. The system according to claim 4, characterized in that, The process of binding recognition antibodies and signaling molecules onto a SERS substrate includes the following steps: The SERS substrate was silanized to obtain a modified SERS substrate; the recognition antibody and signal molecule were then bound to obtain the binding product. The modified SERS substrate and the suspension containing the binding product were mixed and incubated to obtain a labeled antibody probe.

7. The system according to claim 1, characterized in that, The renal tubular microfluidic chip comprises an upper and lower two-layer structure divided by a porous PET membrane. The upper layer contains renal tubular channels with a length of 24 mm, a width of 1 mm, and a height of 1 mm. The lower layer contains vascular channels with a length of 24 mm, a width of 1 mm, and a height of 0.2 mm.

8. The system according to claim 7, characterized in that, The upper layer contains renal tubular channels seeded with renal tubular epithelial cells, and the lower layer contains vascular channels seeded with human umbilical vein endothelial cells; culture medium is continuously perfused into the renal tubular channels and vascular channels at a flow rate of 2 μL / min.

9. The application of the integrated kidney organ-on-a-chip system according to any one of claims 1 to 8 in online monitoring of pesticide nephrotoxicity.

10. The application according to claim 9, characterized in that, The method of application includes the following steps: The pesticide to be tested is introduced into the renal tubule microfluidic chip, and the resulting biomarkers enter the Raman detection platform, where the Raman spectrometer is used to collect the signals on the silicon wafer.

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