Integrated electrochemical detection device for stomach cancer postoperative MRD auxiliary detection as well as preparation method and application of integrated electrochemical detection device
By constructing an integrated electrochemical detection device, utilizing functionalized nanofiber sensing interfaces and lyophilized reagent layers, the problems of rapid, accurate, and low-cost MRD detection after gastric cancer surgery were solved, achieving efficient capture and sensitive detection of exosomes in minimal residual lesions.
Patent Information
- Application Number
- CN202511939062.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies are insufficient for the rapid, accurate, and low-cost detection of minimal residual disease (MRD) after gastric cancer surgery. Traditional methods are cumbersome, time-consuming, lack sufficient sensitivity, or rely on expensive and large instruments, making it difficult to meet clinical needs.
An integrated electrochemical detection device was developed, which uses a screen-printed electrode (SPE) as a substrate to construct a functionalized nanofiber sensing interface layer and a lyophilized reagent layer, and integrates an exosome antibody and a signal reporter system to achieve efficient capture and detection of the gastric cancer-specific target Claudin18.2.
It achieves sensitive detection of exosomes in extremely low abundance and tiny residual lesions, with the detection limit reduced to thousands of particles per microliter. The detection process is simplified to two steps: "sample addition - reading value", making it suitable for use in primary care and at home. The detection results are accurate and reliable.
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Figure CN121762652A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, specifically to an integrated electrochemical detection device, its preparation method, and its application for auxiliary MRD detection after gastric cancer surgery. Background Technology
[0002] Minimal residual disease (MRD) refers to trace amounts of tumor cells or related components remaining in the body after cancer treatment, and is the root cause of disease recurrence and metastasis. For solid tumors (such as gastric cancer), traditional imaging methods can only detect residual cells at the millimeter-level molecular level, making it impossible to provide timely warnings of recurrence risk.
[0003] In the field of molecular detection, besides circulating tumor DNA (ctDNA), tumor-derived exosomes, as nanoscale vesicles carrying abundant tumor-specific membrane proteins, have become a promising new target for MRD liquid biopsy. However, current exosome MRD detection technologies face significant challenges: First, traditional exosome separation methods based on ultracentrifugation are cumbersome, time-consuming, and have low yields, making it difficult to meet the needs of rapid clinical detection; second, the gold standard methods used for subsequent analysis, such as Western blotting or enzyme-linked immunosorbent assay (ELISA), suffer from insufficient sensitivity, complex operation, and inability to accurately quantify; third, while emerging single-particle analysis technologies (such as nanoflow cytometry) have high sensitivity, they rely on expensive large-scale instruments and professional operators, resulting in high costs and hindering their widespread clinical application. Summary of the Invention
[0004] The purpose of this invention is to address the above-mentioned technical problems by providing an integrated electrochemical detection device for postoperative MRD auxiliary detection of gastric cancer and its preparation method.
[0005] This invention provides an integrated electrochemical detection device for auxiliary detection of MRD after gastric cancer surgery. The sensing structure of the device uses a screen-printed electrode (SPE) as a substrate, on which a functionalized nanofiber sensing interface layer is constructed. A lyophilized reagent layer is integrated on the functionalized nanofiber sensing interface layer. The functionalized nanofiber sensing interface layer includes a polyacrylic acid (PAA) electrospun nanofiber membrane located on the SPE working electrode, and an exosome-mediated anti-human CD63 capture antibody immobilized on the nanofiber membrane. The lyophilized reagent layer contains detection antibodies targeting specific gastric cancer targets, lyophilization protectants, and a signal reporting system; The specific target for gastric cancer is Claudin 18.2; The detection antibody is an anti-Claudin18.2 antibody; The freeze-drying protectant is trehalose, bovine serum albumin (BSA), and polyethylene glycol (PEG). The signal reporting system is a streptavidin-horseradish peroxidase conjugate.
[0006] This invention also provides a method for preparing an integrated electrochemical detection device for postoperative MRD auxiliary detection of gastric cancer, specifically including the following steps: Construction of the functionalized nanofiber sensing interface layer: A 3 mm diameter SPE (Sensitive Polymer Electrode) was used as the working electrode. Its non-working area, including the counter electrode, reference electrode, and conductive leads, was covered using a precision mask, leaving only the working electrode exposed. An 8% (w / v) PAA aqueous solution was electrospun in situ on the working electrode surface of the SPE for 20 min under conditions of 18 kV high voltage, 0.8 mL / h feed rate, and 15 cm receiving distance to form a uniform nanofiber membrane. This yielded SPE@PAA. Subsequently, under mask protection, 20 μL of an activation mixture of 0.1 M EDC and 0.05 M NHS was added dropwise, and the reaction was carried out at room temperature. Next, 20 μL of mouse anti-human CD63 capture antibody solution was immobilized in 0.01 M, pH 7.4 PBS and incubated at 4 ℃ for 2 h. Finally, the interface was blocked with PBS buffer containing 2% BSA at 37 ℃ for 45 min, completing the biofunctionalization of the sensing interface and yielding SPE@PAA / CD63. Construction of the lyophilized reagent layer: The detection reagent mixture contained 2 μg / mL streptavidin-HRP, biotinylated anti-Claudin18.2 antibody, and a lyophilization protectant consisting of 5% trehalose, 1% BSA, and 0.1% PEG-8000. 1.0 μL of this mixture was spotted onto SPE@PAA / CD63 in a low-temperature, low-humidity environment. The sample was then pre-frozen at -80 ℃ for 1 h, followed by primary drying at -40 ℃ under a vacuum of less than 10 Pa for 12 h to form the lyophilized reagent layer, yielding SPE@PAA / CD63 / C18.2. Finally, the sample was vacuum-sealed in a dry nitrogen environment with humidity less than 10% RH using a high-barrier aluminum foil bag, and the finished product was stored at 4 ℃ in the dark.
[0007] Furthermore, the concentration of the mouse anti-human CD63 capture antibody solution in step (1) is 20 μg / mL.
[0008] Furthermore, the room temperature reaction time in step (1) is 30 min.
[0009] Furthermore, the concentration of the biotinylated anti-Claudin18.2 antibody in step (2) is 5 μg / mL.
[0010] The present invention also provides the use of an integrated electrochemical detection device for auxiliary detection of MRD after gastric cancer surgery in the preparation of a kit for auxiliary detection of MRD after gastric cancer surgery.
[0011] The advantages of this invention are: This invention is based on a three-dimensional nanofiber sensing interface, which significantly improves the amount of captured antibody immobilized due to its huge specific surface area, and achieves efficient capture and enrichment of target exosomes, thereby reducing the detection limit to thousands of particles per microliter, and realizing sensitive detection of exosomes in extremely low abundance and tiny residual lesions.
[0012] This invention, through the integrated design of pre-loaded lyophilized reagents, condenses the detection process into two steps: "sample addition - value reading," eliminating the need for large laboratory instruments and making it suitable for grassroots and home settings, thus making postoperative MRD-assisted detection for gastric cancer convenient and accessible to all.
[0013] The invention's sophisticated mask protection process, combined with specific sandwich immune recognition, effectively eliminates interference from complex samples, ensuring the accuracy and reliability of the test results. Attached Figure Description
[0014] Figure 1 This is a top view schematic diagram of an integrated electrochemical detection device for postoperative MRD auxiliary detection of gastric cancer according to the present invention.
[0015] Figure 2 This is the interface characterization of the device of the present invention.
[0016] Figure 3a This is a graph showing the test results of the exosome concentration using the device of the present invention; Figure 3b This is a graph showing the linear data analysis of the concentration logarithm of the device of the present invention.
[0017] Figure 4 This is a characterization diagram of the detection specificity of the device of the present invention; Figure 5 This is a clinical sample test analysis diagram of the device of the present invention. Detailed Implementation
[0018] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.
[0019] Example 1: Preparation of an integrated device for assisting in the detection of postoperative MRD in gastric cancer patients Construction of the functionalized nanofiber sensing interface layer: A 3 mm diameter SPE (Synthetic Element Electrode) was used as the working electrode. Its non-working area, including the counter electrode, reference electrode, and conductive leads, was covered using a precision mask, leaving only the working electrode exposed. An 8% (w / v) PAA aqueous solution was electrospun in situ on the working electrode surface of the SPE for 20 min under conditions of 18 kV high voltage, 0.8 mL / h feed rate, and 15 cm receiving distance to form a uniform nanofiber membrane, thus preparing SPE@PAA. Subsequently, under mask protection, 20 μL of an activation mixture consisting of 0.1 M EDC and 0.05 M NHS was added dropwise, and the reaction was carried out at room temperature for 30 min. Next, 20 μL of a 20 μg / mL mouse anti-human CD63 capture antibody solution was fixed in 0.01 M, pH 7.4 PBS and incubated at 4 ℃ for 2 h. Finally, the membrane was blocked with PBS buffer containing 2% BSA at 37 ℃ for 45 h. min, complete the biofunctionalization of the sensing interface, and obtain SPE@PAA / CD63; Construction of the lyophilized reagent layer: The detection reagent mixture contained 2 μg / mL streptavidin-HRP, 5 μg / mL biotinylated anti-Claudin18.2 antibody, and a lyophilization protectant consisting of 5% trehalose, 1% BSA, and 0.1% PEG-8000. 1.0 μL of this mixture was spotted onto SPE@PAA / CD63 in a low-temperature, low-humidity environment. The sample was then pre-frozen at -80 ℃ for 1 h, followed by primary drying at -40 ℃ under a vacuum of less than 10 Pa for 12 h to form the lyophilized reagent layer, yielding SPE@PAA / CD63 / C18.2. Finally, the sample was vacuum-sealed in a dry nitrogen environment with humidity less than 10% RH using a high-barrier aluminum foil bag, and the finished product was stored at 4 ℃ in the dark.
[0020] This embodiment presents an integrated electrochemical detection device for auxiliary MRD detection after gastric cancer surgery, the top view of which is shown below. Figure 1 As shown, the sensing structure of the device is based on the working electrode 1 of the SPE, on which a functionalized nanofiber sensing interface layer 2 is constructed, and a lyophilized reagent layer 3 is integrated on the interface layer.
[0021] Comparative Example 1: Preparation of an integrated device for assisting in the detection of postoperative MRD in gastric cancer without nanofiber membranes The difference between this comparative example and Example 1 is that a nanofiber membrane was not prepared by electrospinning on the SPE working electrode; instead, the captured antibody was directly immobilized on the bare SPE working electrode.
[0022] Comparative Example 2: Preparation of an integrated device for auxiliary detection of postoperative MRD in gastric cancer without pre-prepared lyophilized detection reagents The difference between this comparative example and Example 1 is that, in step 2, the test reagent mixture was not spotted onto the SPE@CD63 surface for lyophilization. When using the device prepared in this comparative example, the test reagent mixture must be manually added dropwise sequentially, followed by incubation and washing.
[0023] Comparative Example 3: Preparation of an integrated device for maskless auxiliary detection of postoperative MRD in gastric cancer The difference between this comparative example and Example 1 is that no mask was used to protect the counter electrode and the reference electrode throughout the entire preparation process (electrospinning, activation, spotting of the reagent mixture, etc.).
[0024] Comparative Example 4: Preparation of an integrated device for auxiliary detection of postoperative MRD in gastric cancer without lyophilization protectant. The difference between this comparative example and Example 1 is that the trehalose / BSA / PEG-8000 lyophilization protectant system was not used when preparing the test reagent mixture in step 2.
[0025] Experimental Example 1: Interface Characterization of the Integrated Device for Assisting Detection of Postoperative MRD in Gastric Cancer Prepared in Example 1 The stepwise construction process of the sensing interface was systematically characterized using electrochemical impedance spectroscopy. Specifically, using PBS buffer (pH 7.4) containing 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] and 0.1 M KCl as the electrolyte, the SPE, SPE@PAA, and SPE@PAA / CD63 prepared in Example 1 were sequentially tested at open-circuit potential with a perturbation amplitude of 5 mV within a frequency range of 0.1 Hz to 100 kHz.
[0026] The results are as follows Figure 2 As shown, by analyzing the Nyquist plot and fitting the data using the equivalent circuit model, it was observed that the electron transfer resistance of SPE, SPE@PAA and SPE@PAA / CD63 showed a significant increasing trend at each level. This indicates that the non-conductive nanofiber membrane and biomolecular layer were successfully and densely fixed on the electrode surface, forming a complete functional sensing interface, which provides key evidence for the reliability and effectiveness of the device prepared in Example 1.
[0027] Experimental Example 2: Characterization of the detection sensitivity of the integrated device for assisting in the detection of postoperative MRD in gastric cancer prepared in Example 1. To evaluate the detection sensitivity of the devices prepared in Example 1, Comparative Example 1, and Comparative Example 4, samples containing different concentrations of gastric cancer exosomes (concentration gradient of particles / μL) were added to the two sets of sensors respectively. After incubation at 37 °C for 30 minutes, TMB substrate solution was added, and the steady-state current value was immediately detected at a potential of -0.1 V using chronoamperometry. A calibration curve was plotted with the logarithm of the target concentration as the abscissa and the corresponding current signal value as the ordinate, and the linear range and detection limit were calculated accordingly.
[0028] The results are as follows Figure 3a , Figure 3b As shown, the device of Example 1 exhibits significantly better detection performance than Comparative Example 1, while the device prepared in Comparative Example 4, due to the lack of a protective agent, results in the inactivation of enzymes and antibodies during lyophilization storage, leading to almost no signal during testing. Example 1, based on a nanofiber interface, has a calibration curve at 2.0 × 10⁻⁶. 3 Up to 5.0×10 6 The particles / μL concentration exhibits a good linear relationship, with R0 2 =0.956, and the calculated detection limit is 1.5×10. 3 The detection limit of the device prepared in Comparative Example 1 was significantly higher than that of the device prepared in Example 1. In summary, the device prepared in Example 1, by constructing a three-dimensional nanofiber sensing interface, greatly improved antibody immobilization capacity and exosome capture efficiency, exhibiting excellent detection sensitivity.
[0029] Experimental Example 3: Specific characterization of the integrated device for assisting in the detection of postoperative MRD in gastric cancer prepared in Example 1. To evaluate the specificity of the devices prepared in Example 1, Comparative Example 2, and Comparative Example 3, cross-reactivity tests were performed using high concentrations of non-target exosomes and common blood interfering substances. The sensing devices prepared in the same batch were divided into five groups for detecting: (1) Target analyte: concentration of 1×10⁻⁶. 6 (2) Non-target exosomes A: concentration of 1×10⁻⁶ particles / μL (Claudin18.2 positive) 7 (3) Non-target exosomes B: concentration of 1×10⁻⁶ particles / μL; 7 Hepatocellular carcinoma-derived exosomes (Claudin 18.2 negative) with particles / μL; (4) high-concentration human serum albumin solution, with a concentration of 1 mg / mL; (5) high-concentration human immunoglobulin G solution, with a concentration of 1 mg / mL. All test samples were uniformly 50 μL in volume, and their steady-state current signals were recorded using the chronoamperometry method. The specificity of the device was evaluated by comparing the significant differences in the signals of each group.
[0030] The results are as follows Figure 4 As shown, the device prepared in Example 1 produced a strong current response to the target analyte, with a signal value reaching 253 nA, significantly higher than all other control groups. However, in tests with non-target exosomes (healthy and liver cancer sources) at concentrations 10 times higher, as well as high concentrations of human serum albumin and immunoglobulin G, the generated current signals were all below 40 nA. While the device prepared in Comparative Example 2 could distinguish between target and non-target samples, its signal value standard deviation was significantly increased. This indicates that the cumbersome manual pipetting, incubation, and washing steps introduced significant human error and operational inconsistencies, leading to decreased precision and repeatability of the detection results. Furthermore, the device in Comparative Example 3 produced abnormally elevated background signals in all samples (including non-target exosomes and interfering proteins), easily leading to false positives. This demonstrates that mask protection is crucial for maintaining the functional purity of the reference / counter electrode and ensuring accurate electrochemical readings. Therefore, the device prepared in Example 1 exhibits good specificity, effectively eliminating interference from other cell-derived exosomes and common blood proteins, ensuring the accuracy of auxiliary detection of postoperative MRD in complex clinical samples.
[0031] Experimental Example 4: Clinical sample testing and characterization of the integrated device for assisting in the detection of postoperative MRD in gastric cancer prepared in Example 1. To evaluate the clinical pre-detection capability of the device prepared in Example 1, we collected postoperative plasma samples from pathologically confirmed gastric cancer patients (n=5) and healthy volunteers (n=5). The device prepared in Example 1 was compared in parallel with the existing gold standard (flow cytometry for exosome epitope analysis). First, each plasma sample was divided into two parts. One part was tested using the device prepared in Example 1, and the generated current signal value was recorded. The other part was used to extract total exosomes by ultracentrifugation, and fluorescently labeled with anti-CD63 and anti-Claudin18.2 antibodies. Finally, the proportion of Claudin18.2 positive exosomes was counted by flow cytometry. The entire experiment was conducted under double-blind conditions. The detection results obtained by the two methods were statistically analyzed. For direct comparison, the current value measured by the device prepared in Example 1 was converted to exosome concentration using its own calibration curve. Simultaneously, the fluorescence count from flow cytometry was converted to absolute concentration using built-in fluorescent microsphere standards.
[0032] The results are as follows Figure 5As shown, the concentration of gastric cancer exosomes measured by the device prepared in Example 1 was correlated with the concentration measured by flow cytometry. All data points (including samples from gastric cancer patients and healthy volunteers) were closely distributed on both sides of the fitted line, exhibiting an excellent linear relationship. Linear regression analysis yielded the fitted equation y = 1.04x - 0.02, where y represents the concentration measured by flow cytometry and x represents the concentration measured by the device in Example 1. R² = 0.996, indicating that the detection results of the device prepared in Example 1 are consistent with the gold standard method and can accurately quantify the content of target exosomes in clinical samples.
[0033] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. An integrated electrochemical detection device for postoperative MRD assisted detection of gastric cancer, characterized in that, The device sensing structure takes the working electrode (1) of a screen-printed electrode as a substrate, a functionalized nanofiber sensing interface layer (2) is constructed on the working electrode, and a lyophilized reagent layer (3) is integrated on the functionalized nanofiber sensing interface layer (2): The functionalized nanofiber sensing interface layer (2) comprises a polyacrylic acid electrospun nanofiber membrane on the working electrode (1) of the screen-printed electrode, and an exosome anti-human CD63 capture antibody fixed on the nanofiber membrane; The lyophilized reagent layer (3) comprises a detection antibody for a gastric cancer specific target, a lyophilized protective agent, and a signal reporting system.
2. The integrated electrochemical detection device of claim 1, wherein, The gastric cancer specific target is Claudin18.2, and the detection antibody is an anti-Claudin18.2 antibody.
3. The integrated electrochemical detection device of claim 1, wherein the at least one electrode is a working electrode. The lyophilized protective agent is trehalose, bovine serum albumin, and polyethylene glycol.
4. The integrated electrochemical detection device of claim 1, wherein the at least one electrode is a working electrode. The signal reporting system is a streptavidin-horseradish peroxidase conjugate.
5. A method for preparing the integrated electrochemical detection device for MRD assisted detection of gastric cancer post-operation according to any one of claims 1-4, characterized in that, The preparation method comprises the following steps: Construction of the functionalized nanofiber sensing interface layer: on the surface of the working electrode of the screen-printed electrode protected by a mask for the non-working area, a polyacrylic acid nanofiber membrane is formed by electrospinning; then carboxyl activation, fixation of the anti-human CD63 capture antibody, and bovine serum albumin blocking are sequentially performed; Construction of the lyophilized reagent layer: a detection reagent mixture solution comprising a streptavidin-horseradish peroxidase conjugate, a biotinylated anti-Claudin18.2 antibody, and a lyophilized protective agent composed of trehalose, bovine serum albumin, and PEG-8000 is prepared, then the detection reagent mixture solution is spotted on the functionalized nanofiber sensing interface layer, and lyophilization is performed to form a lyophilized reagent layer, and finally vacuum sealing packaging is performed in a dry nitrogen environment.
6. The production method according to claim 5, wherein The concentration of the mouse anti-human CD63 capture antibody solution in step (1) is 20 μg / mL.
7. The production method according to claim 5, wherein The carboxyl activation in step (1) is performed at room temperature for 30 min.
8. The production method according to claim 5, wherein The concentration of the biotinylated anti-Claudin18.2 antibody in step (2) is 5 μg / mL.
9. Use of the integrated electrochemical detection device according to any one of claims 1-4 in the preparation of a kit for assisting in the detection of postoperative molecular residual lesions of gastric cancer.