Universal electrochemical impedance sensor based on two-dimensional transition metal boride linked appropriate ligand, preparation method of universal electrochemical impedance sensor and application of universal electrochemical impedance sensor in cancer detection

Abstract

The invention discloses a universal electrochemical impedance sensor based on a two-dimensional transition metal boride linked appropriate ligand, a preparation method of the universal electrochemical impedance sensor and application of the universal electrochemical impedance sensor in cancer detection, and belongs to the technical field of crossing of biomedical detection and electrochemical sensors. An instant throwing type screen printing electrode is used as a transducer for signal conversion, and a two-dimensional MBene nanometer material is modified on the surface of the instant throwing type screen printing electrode to serve as a signal sensitization layer; a thiolated aptamer subjected to reduction treatment is used as a modular recognition element and is fixed on the surface of a matrix through a gold-sulfur bond or a physical anchoring effect, non-specific interference is eliminated in combination with a small molecule sealing agent, and the universal electrochemical impedance sensor is obtained. According to the invention, the excellent conductivity of MBene and the high-specificity recognition capability of the aptamer are combined, and the change of the interface impedance of the sensor is monitored through the electrochemical impedance spectroscopy, so that the rapid, high-sensitivity and label-free detection of tumor cells can be realized, and reliable technical support is provided for early screening and clinical instant detection of cancers.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedical detection and electrochemical sensors, specifically relating to a general-purpose electrochemical impedance sensor based on a two-dimensional transition metal boride aptamer, its preparation method, and its application in cancer detection. Background Technology

[0002] Malignant tumors, as a prevalent and deadly disease worldwide, have become a major threat to human life and health. Studies have shown that early detection, early diagnosis, and early treatment of cancer are decisive factors in improving patients' five-year survival rates and prognoses. Therefore, developing efficient and accurate early cancer detection technologies has significant clinical value and social implications for improving cancer prevention and control and safeguarding national health.

[0003] However, current clinical cancer detection technologies still have many limitations, making it difficult to meet the needs of very early screening and accurate clinical diagnosis. While imaging techniques (such as CT, MRI, and ultrasound) are non-invasive, their ability to identify early microlesions smaller than 1 cm in diameter is limited, and they are easily affected by tissue anatomy, making it difficult to capture trace lesions early. Often, a definitive diagnosis is only made when the tumor has progressed to an advanced stage, missing the optimal treatment window. Biochemical and immunological detection techniques (such as enzyme-linked immunosorbent assays and chemiluminescent immunoassays) rely on specific antibodies for target recognition, but they require expensive fluorescent, enzyme, or radioactive markers, increasing detection costs and making the process cumbersome and time-consuming. Furthermore, due to limitations in antibody specificity and labeling efficiency, the detection limit is usually at the ng / mL level, making it difficult to meet the need for capturing trace tumor markers at the pg / mL or even fg / mL level in very early-stage cancer. Tissue biopsy, as a cancer detection method... While the "gold standard" for diagnosis can clearly identify the pathological type and differentiation degree of a tumor, it is an invasive examination that is prone to complications such as bleeding and infection, resulting in poor patient compliance. Furthermore, the sampling range is limited, posing a risk of sampling bias. It also cannot achieve dynamic monitoring of tumor progression and treatment effectiveness, making it difficult to meet the clinical needs of individualized treatment. Cytological testing methods (such as cervical liquid-based cytology and sputum exfoliative cytology) are relatively simple to operate, but they are highly dependent on the professional experience of the operators, and the test results are highly subjective and have poor repeatability. In addition, the testing cycle is long, the cost per test is high, and the sensitivity and specificity of the tests are limited, making them unsuitable for widespread application in primary healthcare institutions and large-scale population screening.

[0004] In summary, existing cancer detection technologies generally suffer from technical bottlenecks such as high cost, low detection sensitivity, complex operation procedures, poor universality, and difficulty in achieving early capture of trace targets. Therefore, developing a low-cost, high-sensitivity, easy-to-operate, and universally applicable detection technology that can meet the needs of very early cancer screening and dynamic monitoring has become a critical issue that urgently needs to be addressed in the field of biomedical detection. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a general electrochemical impedance sensor based on a two-dimensional transition metal boride aptamer, its preparation method and its application in cancer detection, so as to solve the technical problems of high cost, low sensitivity, complex operation and poor universality of existing cancer detection methods.

[0006] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials, comprising: Electrode units serve as the basis for electrical signal conversion; The nano-sensitizing unit is composed of two-dimensional layered MBene nanosheets and is modified on the surface of the electrode unit. The specific recognition unit is composed of aptamers targeting cancer-related molecules. The aptamers have thiol functional groups at their ends and are fixed to the surface of the MBene nanosheet-modified electrode unit through gold-sulfur bonds or physical adsorption for the specific capture of tumor cells. A sealing unit is disposed on the outer layer of the specific recognition unit to seal the unoccupied active sites on the surface of the electrode unit, thereby reducing the interference of non-specific adsorption on the detection signal.

[0007] Preferably, the electrode unit uses screen-printed gold electrodes.

[0008] Preferably, the sealing unit is a 6-mercapto-1-hexanol solution or a bovine serum albumin solution.

[0009] More preferably, the concentration of the 6-mercapto-1-hexanol solution is 10~1000 μM, and the concentration of the bovine serum albumin solution is 0.1~10 mg / mL.

[0010] In a second aspect, the present invention discloses a method for preparing a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials. MBene powder dispersion is drop-coated onto the surface of an electrode unit and dried to form a film, resulting in a modified electrode. An aptamer solution activated by a reducing agent is drop-added onto the surface of the modified electrode, incubated, dried to form a film, and then sealed to obtain a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials.

[0011] Preferably, the concentration of the MBene powder dispersion is 0.5~2.0 mg / mL.

[0012] Preferably, the preparation method of MBene powder dispersion is as follows: the transition metal boron precursor powder is immersed in inorganic acid at a mass-to-volume ratio of 1:(20~50), selectively etched, centrifuged, washed until the pH value is stable between 6.5 and 7.5, and then freeze-dried under vacuum to obtain two-dimensional MBene powder; the two-dimensional MBene powder is ultrasonically dispersed in ultrapure water or anhydrous ethanol to obtain MBene powder dispersion.

[0013] More preferably, the aptamer solution is changed according to different cancer types.

[0014] More preferably, if the target is endometrial cancer, liver cancer, breast cancer, or lung cancer, an EpCAM aptamer solution is used; if the target is gastrointestinal malignancy or lung cancer, a CEA aptamer solution is used; and if the target is prostate cancer, a PSA aptamer solution is used.

[0015] A third aspect of the present invention discloses a general electrochemical impedance spectroscopy platform based on two-dimensional MBene nanomaterials, comprising the aforementioned general electrochemical impedance sensor based on two-dimensional MBene nanomaterials, a detection solution, and an electrochemical impedance detection unit. The electrochemical impedance detection unit is connected to the electrode unit in a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials. By acquiring electrochemical signals, it measures and calculates the impedance change at the sensor interface and outputs the detection impedance result.

[0016] Preferably, the detection solution is a phosphate buffer solution containing K3[Fe(CN)6] / K4[Fe(CN)6].

[0017] In a fourth aspect, the present invention discloses a method for preparing a general electrochemical impedance spectroscopy platform based on two-dimensional MBene nanomaterials. The method involves connecting an electrode unit of a general electrochemical impedance sensor based on two-dimensional MBene nanomaterials with an electrochemical impedance detection unit, and adding a detection solution to the surface of the general electrochemical impedance sensor based on two-dimensional MBene nanomaterials to obtain the general electrochemical impedance spectroscopy platform based on two-dimensional MBene nanomaterials.

[0018] The fifth aspect of the present invention discloses the application of a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials or a general-purpose electrochemical impedance sensing platform based on two-dimensional MBene nanomaterials in cancer detection.

[0019] The sixth aspect of the present invention discloses a cancer detection method based on a universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials. The sample suspension to be tested is centrifuged and the supernatant is discarded. The suspension is then resuspended in phosphate buffer and incubated on the surface of the universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials. Then, the electrode unit is connected to the electrochemical impedance detection unit, and a phosphate buffer solution containing 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] is added. Impedance measurement is performed, and the detection of tumor cells is achieved based on the impedance change amplitude.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials, which organically combines the signal-enhancing properties of MBene with the specific trapping ability of aptamers. 1) It uses a disposable screen-printed electrode as the transducer for signal conversion; 2) By modifying the surface of the electrode unit with a two-dimensional transition metal boride (MBene) having metal-like conductivity and a large specific surface area as a signal-enhancing layer, MBene material has excellent conductivity, a large specific surface area, and abundant surface chemical active sites, which can significantly enhance the electron transport efficiency of the electrode interface and increase the loading of aptamers, thereby effectively amplifying the impedance response signal; 3) Utilizing the reduction treatment... The aptamer is used as a modular recognition element. The aptamer recognition interface gives the sensor a high selectivity for tumor cells that express the corresponding markers, reducing non-specific interference. Compared with traditional antibodies, it has the advantages of small molecular weight, high stability, easy modification, strong specificity and low cost. When in use, only the specific aptamer needs to be replaced to achieve highly sensitive detection of various cancer markers such as endometrial cancer, lung cancer, breast cancer, colorectal cancer and prostate cancer; 4) It is fixed on the matrix surface by gold-sulfur bond or physical anchoring effect, and combined with small molecule blocking agent to eliminate non-specific interference, to obtain a general electrochemical impedance sensor based on two-dimensional MBene nanomaterials. This universal electrochemical impedance sensor requires no fluorescent or enzyme labeling during the entire detection process, featuring simple operation, short detection time, and good repeatability. Combined with disposable screen-printed electrode technology, it offers advantages such as low cost, label-free operation, rapid response, high sensitivity, wide detection range, strong specificity, and ease of integration. By combining the excellent conductivity of MBene with the high specificity of aptamers, and monitoring changes in sensor interface impedance through electrochemical impedance spectroscopy, it enables rapid, highly sensitive, and label-free detection of tumor cells, providing reliable technical support for pan-cancer early screening and clinical point-of-care testing. Attached Figure Description

[0021] Figure 1This is a schematic diagram of the sensing interface of the universal electrochemical impedance sensor based on a two-dimensional transition metal boride co-aptamer of the present invention; wherein, 1-electrode unit, 2-nanometer sensitization unit, 3-specific recognition unit, 4-blocking unit, 5-target cell; Figure 2 This is a scanning electron microscope image of the highly porous two-dimensional MBene powder with large interlayer spacing of the present invention. Figure 3 The Nyquist comparison diagrams of the electrochemical impedance response during the stepwise modification and final target cell detection process of the universal electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers of the present invention are shown. Among them, A is the Nyquist comparison diagram of the electrochemical impedance response during the stepwise construction and final detection process of the universal electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers of the present invention, and B is an enlarged view of the boxed part in A. Figure 4 The graph shows the sensitivity detection results of the universal electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers of the present invention. Figure 5 This is a graph showing the specific detection results of the universal electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers of the present invention. Figure 6 The graph shows the repeatability test results of the universal electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers of the present invention. Figure 7 The graph shows the stability test results of the universal electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers of the present invention. Figure 8 This is a diagram showing the clinical sample detection results of the universal electrochemical impedance sensor based on a two-dimensional transition metal boride co-aptamer of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0023] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0024] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0025] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0026] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0027] In this article, the term "electrochemical impedance detection unit" refers to the detection module of an electrochemical workstation. This unit acquires electrochemical signals, measures the impedance changes at the sensor interface, calculates and outputs the impedance results. The term "electrochemical workstation" refers to an integrated instrument for electrochemical testing, capable of controlling electrode potential / current and simultaneously measuring changes in current / potential over time or frequency. It is a core device for electrochemical research and sensor testing.

[0028] This invention provides a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials, such as... Figure 1 As shown, it includes: Electrode unit 1, serving as the substrate for electrical signal conversion, is preferably a screen-printed gold electrode (SPGE) with an integrated three-electrode system, which features structural stability, ease of mass production, and single-use capability. The nano-sensitizing unit 2, composed of two-dimensional layered MBene nanosheets, is modified on the surface of electrode unit 1 and prepared by etching. It serves as an electrochemical signal sensitizing carrier. Its two-dimensional layered structure provides a high-speed channel for electron transfer and acts as a loading matrix for biorecognition molecules. The specific recognition unit 3 is composed of an aptamer targeting cancer-related molecules. The aptamer has a thiol functional group at its end and is fixed to the surface of the electrode unit 1 modified by the nano-sensitization unit 2 through gold-sulfur bonds or physical adsorption, for the specific capture of tumor markers or tumor cells. The sealing unit 4 is located on the outer layer of the specific recognition unit 3. It uses small molecule thiol compounds or inert proteins to seal the unoccupied active sites on the electrode surface, so as to reduce the interference of non-specific adsorption on the detection signal.

[0029] This invention also provides a method for fabricating a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials, the steps of which are as follows: 1. Synthesis and Regulation of General-Purpose Two-Dimensional MBene Nanomaterials Transition boron precursor powder was immersed in 1-4 M inorganic acid at a mass-to-volume ratio of 1:(20-50) and etched by continuous stirring at 20-30°C for 72-120 hours. The product was then centrifuged at 8000-10000 rpm and repeatedly washed with high-purity deionized water until the pH stabilized between 6.5 and 7.5. The product was then freeze-dried under vacuum to obtain highly porous two-dimensional MBene powder with large interlayer spacing.

[0030] 2. Construction of a general electrochemical impedance sensor based on two-dimensional transition metal boride co-aptamers 1) Clean the SPGE electrode and then blow it dry with nitrogen to ensure that there are no organic contaminants on the electrode surface.

[0031] 2) Nanomatrix modification: The two-dimensional MBene powder obtained in step 1 is dispersed in a solvent to prepare a dispersion of 0.5~2.0 mg / mL, which is then drop-coated onto the surface of the SPGE electrode and dried at 30~40℃ to form a film.

[0032] 3) The aptamer solution activated by the reducing agent is dropped onto the electrode surface modified in step 2), incubated, and dried to form a film; wherein, the reducing agent is preferably tris(2-carboxyethyl)phosphine (TCEP); the aptamer solution is changed according to different cancer types: for example, for the detection of endometrial cancer, liver cancer, breast cancer, or lung cancer, the aptamer solution used includes, but is not limited to, EpCAM aptamer solution; for the detection of gastrointestinal malignancies such as colorectal cancer and lung cancer, the aptamer solution used includes, but is not limited to, CEA aptamer solution; for the detection of prostate cancer, the aptamer solution used includes, but is not limited to, PSA aptamer solution.

[0033] 4) The electrode was sealed for 20 minutes to 1 hour with 10~1000 μM 6-mercapto-1-hexanol (MCH) solution or 0.1~10 mg / mL bovine serum albumin (BSA) solution to obtain a general electrochemical impedance sensor based on two-dimensional MBene nanomaterials.

[0034] This invention provides a general electrochemical impedance sensing platform based on two-dimensional transition metal boride co-aptamers, comprising: A general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials is used for highly sensitive detection of cancer biomarkers; A phosphate buffer solution of K3[Fe(CN)6] / K4[Fe(CN)6] is used as the detection solution to provide an ion conduction pathway and stabilize the solution pH, thereby reducing the impact of environmental changes on the response. The electrochemical impedance detection unit is connected to electrode unit 1 in a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials. By acquiring electrochemical signals, it measures and calculates the impedance change at the sensor interface and outputs the detection impedance result.

[0035] This invention also provides a method for cancer detection using a universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials, comprising the following steps: 1) Prepare a suspension of the sample to be tested (such as a cell line suspension or a clinical cytology sample). 2) Centrifuge the sample suspension obtained in step 1) and discard the supernatant. Resuspend the sample in phosphate buffered saline (PBS) and incubate it on the surface of a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials. Incubate at 37°C for 30 to 90 minutes. Gently wash the surface with PBS. Connect the SPGE electrode to the electrochemical impedance detection unit. Add a phosphate buffer solution containing 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] to the surface of the SPGE electrode of the general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials. Perform impedance measurement in a Faraday cage. The detection frequency is 0.1 Hz to 100 kHz and the open-circuit voltage is 5 mV. Measure the impedance change at the electrode interface by electrochemical impedance spectroscopy and detect tumor cells based on the impedance change amplitude.

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0037] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0038] I. Fabrication of a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials 1. Synthesis of two-dimensional layered MBene nanomaterials Weigh 1.0 g of Cr2AlB2 powder (purchased from Foshan Xinxi Technology Co., Ltd.; purity ≥99%), add 30 mL of 2 M HCl, and stir continuously at room temperature (25℃) for 96 hours. During the reaction, the suspension color gradually changes from grayish-white to dark black. Centrifuge at 8000 rpm and wash repeatedly with high-purity deionized water until the pH value stabilizes between 6.5 and 7.5. Collect the solid and freeze-dry it in a vacuum freeze dryer to obtain highly porous two-dimensional MBene powder with large interlayer spacing.

[0039] Scanning electron microscopy images of two-dimensional MBene powder are shown below. Figure 2 As shown, it exhibits a distinctly rough porous surface with an open layered structure. This morphological change is attributed to the selective exfoliation of the intermediate aluminum layer. This process weakens the interlayer bonding and promotes the exfoliation of the precursor material into two-dimensional nanosheets, indicating the successful synthesis of two-dimensional layered MBene nanomaterials.

[0040] 2. Construction of a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials 1) The SPGE electrode (purchased from Botan Technology (Weihai) Co., Ltd.) was washed with ethanol and ultrapure water and dried with nitrogen to ensure that there were no organic contaminants on the electrode surface.

[0041] 2) Nanomatrix modification: The MBene powder obtained in step 1 was ultrasonically dispersed in ultrapure water to prepare a 1 mg / mL dispersion, which was then drop-coated onto the surface of the SPGE electrode and dried at 37°C to form a film.

[0042] 3) Take 1.0 μM thiolized aptamer solution activated by TCEP (purchased from Sangon Biotech (Shanghai) Co., Ltd.), drop it onto the electrode surface after step 2), incubate at 4°C in the dark for 12 hours, and dry to form a film.

[0043] 4) Based on step 3), add 100 μL of 1 mM mercaptohexanol (MCH) and incubate at 37°C for 1 hour to block non-specific binding sites. After blocking, rinse the surface with phosphate buffer solution to remove unbound substances and obtain a general electrochemical impedance sensor based on two-dimensional MBene nanomaterials.

[0044] II. Cancer Detection Using a General-Purpose Electrochemical Impedance Sensor Based on Two-Dimensional MBene Nanomaterials 1. Detection system setup and signal processing: 1) After resuscitating the endometrial cancer cell line Ishikawa (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.), the cells were inoculated into DMEM high-glucose complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. The cells were incubated at 37°C in a 5% CO2 incubator, with the medium being replaced regularly. When the cell confluence reached 80%–90%, the cells were gently rinsed twice with sterile PBS buffer (pH=7.4) to remove residual medium. Then, 2 mL of 0.25% trypsin-EDTA digestion solution was added to the culture flask, and the flask was incubated at 37°C for 2 min. Observation was performed under an inverted microscope. When the cell morphology changed from spindle-shaped to round and the intercellular connections loosened, 3 mL of DMEM complete medium containing 10% FBS was immediately added to terminate the digestion. The bottom of the culture flask was gently agitated with a sterile pipette to ensure complete cell detachment and even dispersion, avoiding air bubbles. The cell suspension was then transferred to sterile centrifuge tubes and centrifuged at 800 rpm at room temperature for 5 minutes. After 1 minute, discard the supernatant; add 1 mL of sterile PBS buffer to the centrifuge tube, resuspend the cells by pipetting, dilute to a certain ratio, and count using a hemocytometer. Serially dilute the cell stock solution to different concentration gradients (1 × 10⁻⁶) using sterile PBS buffer. 5 1×10 4 1×10 3 1×10 2 Ishikawa endometrial cancer cell line suspensions of different concentrations (10 cells / mL) were obtained and stored in a 4°C refrigerator for later use.

[0045] 2) To clarify the impedance response changes during the stepwise construction of a general-purpose electrochemical impedance sensor, and to verify the effectiveness of aptamer loading and MCH blocking, electrochemical detection and recording were performed throughout the stepwise modification process, including the following steps: Bare SPGE (unmodified bare screen-printed gold electrode): Take SPGE electrodes without any modification, without MBene powder modification, aptamer loading and any incubation treatment, and use them directly for impedance measurement after routine cleaning and nitrogen drying. MBene powder-modified SPGE electrode (MBene): The bare SPGE electrode after testing was modified with MBene powder only on its surface. No aptamer loading, MCH blocking and cell incubation were performed. After modification, it was washed twice with sterile PBS buffer and dried with nitrogen gas for impedance measurement. MBene / aptamer modified SPGE electrode (MBene / Apt): The above-mentioned SPGE electrode modified with MBene powder was loaded with 1.0 μM of TCEP-activated thiolized aptamer solution on its surface, incubated at 4°C in the dark for 12 hours, and dried to form a film (aptamer loading conditions are the same as in step one of the functionalization process). After incubation, unbound free aptamers were removed by rinsing with PBS. MCH blocking and cell incubation were not performed. The treated electrode was then used for impedance measurement. MCH-blocked MBene / aptamer-modified SPGE electrode (MBene / Apt / MCH): The SPGE electrode modified with MBene powder and TCEP-activated thiolated aptamer was incubated with 100 μL of 1 mM MCH solution at 37°C for 1 hour to block the non-specific binding sites of the aptamer on the electrode surface. After incubation, the unbound MCH was removed by PBS washing. Cell incubation was not performed. The treated electrode was then used for impedance measurement. MBene / aptamer-modified SPGE electrode (MBene / Apt / MCH / Cell) with MCH-blocked cells after target cell capture: Take the Ishikawa endometrial cancer cell line suspensions of different concentrations obtained in step 1), gently pipette to ensure uniform cell dispersion; slowly drop the cell suspension onto the surface of the universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials prepared in step 1, ensuring that the suspension completely covers the sensing interface, and incubate in a 37°C constant temperature incubator for 1 h to allow Ishikawa cells (i.e., target cells 5) to specifically bind to the aptamers on the surface of the universal electrochemical impedance sensor; after incubation, gently rinse the surface of the universal electrochemical impedance sensor three times with sterile PBS buffer (pH=7.4), dry with nitrogen, and then use it for impedance measurement.

[0046] After each modification step and after target cell capture, the electrodes were connected to the SPGE electrode and the electrochemical impedance detection unit to check the stability of the circuit connection and ensure that there was no poor contact.

[0047] 3) Place the universal electrochemical impedance sensor system obtained step by step in step 2) after each modification step and target cell capture in a Faraday cage to avoid external electromagnetic interference. Add a phosphate buffer solution (PBS, pH=7.4) containing 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] (concentration ratio 1:1) before impedance measurement. Measurement parameters are set as follows: frequency range 0.1 Hz~100 kHz, open circuit voltage 5 mV, AC signal amplitude 5 mV, and measurement temperature maintained at room temperature (25±1℃). Three batches of electrodes were independently fabricated, and three parallel measurements were performed, with the average value taken to reduce experimental error. After measurement, the electrochemical impedance spectra (Nyquist curves) for each modification step were recorded, and the impedance data were fitted and analyzed using the software accompanying the electrochemical workstation. The charge transfer resistance (Rct) value was extracted as the core detection signal.

[0048] Electrochemical impedance spectroscopy as follows Figure 3 As shown, the Nyquist curve radius of the MBene control group is significantly smaller than that of the BareSPGE group. This phenomenon confirms that the two-dimensional MBene material has excellent conductivity, providing a channel for electron transfer and effectively reducing the charge transfer resistance at the electrode interface. Simultaneously, the MBene material possesses a large specific surface area and good biocompatibility, providing a stable matrix for efficient aptamer loading, which is beneficial for connecting more specific aptamers at the sensing interface and improving the capture capability of the general-purpose electrochemical impedance sensor. With aptamer loading (MBene / Apt), MCH blocking (MBene / Apt / MCH), and target cell capture (MBene / Apt / Cell), the radius of the Nyquist curve gradually increases, and the corresponding charge transfer resistance (Rct) value also gradually increases. The regular changes in the impedance signal not only clearly present the gradual construction process of the general-purpose electrochemical impedance sensor but also directly prove that the general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials has successfully completed interface functionalization modification and can specifically capture target cells, providing reliable experimental evidence for subsequent precise cancer detection.

[0049] It should be noted that, for different cancer needs, by changing the aptamer (such as for CEA detection of gastrointestinal malignancies such as colorectal cancer, and for PSA detection of prostate cancer), a corresponding universal electrochemical impedance sensor can be prepared, and by testing and recording, sensitive detection of different tumor targets can be achieved.

[0050] III. Performance Evaluation of a General-Purpose Electrochemical Impedance Sensor Based on Two-Dimensional MBene Nanomaterials 1. Sensitivity Detection Experiment Ishikawa endometrial cancer cell line in logarithmic growth phase was harvested, washed twice with PBS buffer, digested with trypsin, and centrifuged after stopping digestion with complete culture medium. The cell pellet was resuspended in PBS. After counting using a hemocytometer, the cells were serially diluted to 10⁻⁶. 5 10 4 1×10 3 1×10 2 Five concentration gradients were established, with 10 cells / mL as the base. The prepared universal electrochemical impedance spectroscopy (EIS) sensor based on two-dimensional MBene nanomaterials was incubated with cell suspensions of each concentration gradient at 37°C for 60 minutes. After incubation, the surface of the EIS sensor was gently rinsed with PBS to remove unbound cells. The EIS signal was immediately detected on an electrochemical workstation, and the ΔRct value (Rct (after cell capture) - Rct (after MCH blocking)) was recorded for each concentration. A scatter plot was plotted with the logarithm of cell concentration (cells / mL) on the x-axis and the corresponding ΔRct on the y-axis. The standard curve equation and correlation coefficient R were obtained through linear fitting. 2 To verify the linear range and goodness of fit.

[0051] Test results as follows Figure 4 As shown, ΔRct and Lg (Ishikawa cell concentration) exhibit a good linear relationship, with a linear correlation coefficient R. 2 =0.99, indicating that in the range of 10~10 5 Within the cell / mL concentration range, the ΔRct signal is highly linearly correlated with the logarithm of the cell concentration. This indicates that the general-purpose electrochemical impedance sensor has high sensitivity, and the detection limit is calculated to be 7 cells / mL based on the 3σ principle.

[0052] 2. Specificity detection experiment Experimental groups were formed as follows: Blank control group: PBS buffer only, containing no cells; HEK293T negative cell group: EpCAM-negative HEK293T cells, concentration adjusted to 10-1 5 cell / mL; Positive cell group (Ishikawa): EpCAM-positive Ishikawa cells, concentration adjusted to 102 5 cell / mL; Mixed cell group: Ishikawa cells (10 5 cell / mL) and HEK293T cells (10 5 (cell / mL) are mixed at a volume ratio of 1:1.

[0053] Each group had three parallel samples. A general-purpose electrochemical impedance spectroscopy (EIS) sensor based on two-dimensional MBene nanomaterials was incubated with each group's samples at 37°C for 60 minutes. After rinsing, the EIS signal was detected, and the ΔRct value for each group was recorded. One-way ANOVA was used to compare the differences in ΔRct between the two groups, and **** P <0.0001 indicates a highly significant difference, and "ns" indicates no significant difference. P >0.05).

[0054] Test results as follows Figure 5 As shown, the ΔRct of the EpCAM-positive Ishikawa cell group was significantly higher than that of the blank control group and the HEK293T-negative cell group. P <0.0001); There was no significant difference between the mixed cell group (Ishikawa+HEK293T) and the Ishikawa-only group (ns, P >0.05), indicating that this general-purpose electrochemical impedance sensor can specifically identify EpCAM-positive cells without interference from negative cells.

[0055] 3. Repeatability test Three representative concentrations were selected: low, medium, and high: 10, 10... 3 10 5 Cells / mL of Ishikawa cell suspension were prepared independently. Three batches of identical electrodes were prepared, and each electrode was tested three times. Incubation and detection were performed using three batches of general-purpose electrochemical impedance sensors based on two-dimensional MBene nanomaterials. Electrode modification, incubation time, and detection conditions were kept completely consistent to minimize systematic errors. The mean and standard deviation (SD) of the three parallel detections at each concentration were calculated. (Formula follows) Calculate the relative standard deviation and verify repeatability.

[0056] Test results as follows Figure 6 As shown, at 10, 10 3 10 5 At three concentrations per cell / mL, the relative standard deviations (RSDs) for parallel detections were 4.99%, 2.02%, and 4.98%, respectively. All RSDs were less than 5%, indicating that the detection results of this general-purpose electrochemical impedance sensor have good repeatability.

[0057] 4. Stability testing experiment Eighteen general-purpose electrochemical impedance sensors based on two-dimensional MBene nanomaterials, prepared under the same conditions, were stored in a 4°C freezer for drying. Three sensors were removed on days 0, 3, 5, 7, 9, and 14 after storage. The general-purpose electrochemical impedance sensors were then compared with 10...4 Ishikawa cell suspension at 1 / mL was incubated at 37°C for 60 minutes. The ΔRct value was measured and recorded at each time point. Using the ΔRct on day 0 as a baseline, the signal retention rate at subsequent time points was calculated. The long-term stability of the sensing interface was assessed by comparing the ΔRct fluctuation amplitude at each time point.

[0058] Test results as follows Figure 7 As shown, after 14 days of storage at 4°C, the modified electrode did not show a significant decrease in the ΔRct signal of cells at the same concentration. P The signal fluctuation amplitude is >0.05, indicating a small amplitude. This result demonstrates the good stability of the general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials, which can maintain effective detection performance for 14 days.

[0059] 5. Clinical sample validation experiment Endometrial cytology samples were collected from patients with postoperative pathologically confirmed malignant endometrial lesions and patients with benign endometrial lesions, with no fewer than 20 cases in each group. After sampling, the specimens were placed in a suitable cell preservation solution, centrifuged before testing, the supernatant was discarded, and the bottom cell pellet was resuspended in PBS. All samples were numbered, and a blinded testing method was used to avoid subjective bias. A general-purpose electrochemical impedance spectroscopy (EIS) sensor based on two-dimensional MBene nanomaterials was incubated with each clinical sample at 37°C for 60 minutes. After rinsing, the EIS signal was measured, and the ΔRct value was recorded. Box plots were used to display the ΔRct distribution of the two groups of samples, and the differences between groups were compared using an independent samples t-test. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC), sensitivity, and specificity were calculated to evaluate the clinical diagnostic value of the general-purpose electrochemical impedance spectroscopy sensor.

[0060] Test results as follows Figure 8 As shown, the ΔRct in the malignant endometrial lesion group was significantly higher than that in the benign control group ( P <0.0001). In clinical sample testing, there was a significant difference in ΔRct between the malignant and benign groups. This result indicates that a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials can effectively distinguish malignant cells in clinical samples and has potential clinical diagnostic value.

[0061] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials, characterized in that, include: Electrode unit (1) serves as the base for electrical signal conversion; The nano-sensitizing unit (2) is composed of two-dimensional layered MBene nanosheets and is modified on the surface of the electrode unit (1); The specific recognition unit (3) is composed of an aptamer targeting cancer-related molecules. The aptamer has a thiol functional group at its end and is fixed to the surface of the electrode unit (1) modified with MBene nanosheets by gold-sulfur bonds or physical adsorption, for the specific capture of tumor cells. The sealing unit (4) is disposed on the outer layer of the specific recognition unit (3) to seal the unoccupied active sites on the surface of the electrode unit (1) in order to reduce the interference of non-specific adsorption on the detection signal.

2. The universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials according to claim 1, characterized in that, The electrode unit (1) uses screen-printed gold electrodes.

3. The universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials according to claim 1, characterized in that, The sealing unit (4) is made of 6-mercapto-1-hexanol solution or bovine serum albumin solution.

4. The method for preparing a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials as described in any one of claims 1 to 3, characterized in that, MBene powder dispersion was drop-coated onto the surface of electrode unit (1), dried to form a film, and the modified electrode was obtained. The aptamer solution activated by the reducing agent was drop-added onto the surface of the modified electrode, incubated and dried to form a film, and then sealed to obtain a general electrochemical impedance sensor based on two-dimensional MBene nanomaterials.

5. The method for fabricating a general-purpose electrochemical impedance sensor based on two-dimensional MBene nanomaterials according to claim 4, characterized in that, The aptamer solution is changed according to different cancer types.

6. A general-purpose electrochemical impedance sensing platform based on two-dimensional MBene nanomaterials, characterized in that, Includes the general-purpose electrochemical impedance sensor, electrochemical impedance detection unit, and detection solution based on two-dimensional MBene nanomaterials as described in any one of claims 1 to 3; The electrochemical impedance detection unit is connected to the electrode unit (1) in the general electrochemical impedance sensor based on two-dimensional MBene nanomaterials. By acquiring electrochemical signals, it measures and calculates the impedance change at the sensor interface and outputs the detection impedance result.

7. The universal electrochemical impedance sensing platform based on two-dimensional MBene nanomaterials according to claim 6, characterized in that, The detection solution is a phosphate buffer solution containing K3[Fe(CN)6] / K4[Fe(CN)6].

8. The method for preparing the general-purpose electrochemical impedance sensing platform based on two-dimensional MBene nanomaterials as described in claim 6 or 7, characterized in that, Connect the electrode unit (1) of the general electrochemical impedance sensor based on two-dimensional MBene nanomaterials to the electrochemical impedance detection unit, and drop a detection solution onto the surface of the general electrochemical impedance sensor based on two-dimensional MBene nanomaterials to obtain a general electrochemical impedance sensing platform based on two-dimensional MBene nanomaterials.

9. The application of the universal electrochemical impedance sensor based on two-dimensional MBene nanomaterials as described in any one of claims 1 to 3, or the universal electrochemical impedance sensing platform based on two-dimensional MBene nanomaterials as described in claim 6 or 7, in cancer detection.

10. A cancer detection method based on a general-purpose electrochemical impedance sensor using two-dimensional MBene nanomaterials, characterized in that, Centrifuge the sample suspension to be tested, discard the supernatant, resuspend in phosphate buffer, and incubate on the surface of the general electrochemical impedance sensor based on two-dimensional MBene nanomaterials as described in any one of claims 1 to 3. Connect the electrode unit (1) to the electrochemical impedance detection unit, and then add a phosphate buffer solution containing 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] to perform impedance measurement. The detection of tumor cells is achieved based on the impedance change amplitude.

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