Biosensor based on MXene (at) MnCoZDH (at) Au NPs and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and relates to a biosensor, in particular to a biosensor based on MXene (at) MnCoZDH (at) Au NPs and a preparation method of the biosensor. The biosensor comprises a capture probe and a labeling probe, magnetic beads wrapped by nucleic acid aptamer functionalized dopamine serve as the capture probe, circulating tumor cells are screened and enriched, meanwhile, based on the dual catalytic effect of a composite nanomaterial MXene (at) MnCoZDH and gold nanoparticles, MXene (at) MnCoZDH (at) Au NPs serves as the labeling probe, and the visual aptamer sensor is constructed. The biosensor has good peroxidase simulation activity, the nucleic acid aptamer is used for functionalizing the multifunctional composite nano material, the detection performance of the sensor is greatly improved, and circulating tumor cells can be efficiently detected.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a biosensor, in particular to a biosensor based on MXenes@MnCoZDH@Au NPs and a preparation method thereof. Background Art

[0002] Early screening for tumors is crucial for diagnosis, treatment, and prognosis. Circulating tumor cells (CTCs) originate from primary or metastatic tumors and contain a wealth of tumor-related information. CTCs can enter the bloodstream through the vascular or lymphatic systems and contribute to the formation of distant metastases. Previous studies have shown that CTCs can be used to predict disease progression and survival in patients with early-stage cancer, predict treatment response and risk of disease recurrence, better understand mechanisms of treatment resistance, and identify new therapeutic targets. Therefore, CTCs are crucial for assisting in the early screening and diagnosis of tumors, predicting treatment response, and identifying new therapeutic targets.

[0003] Conventional methods for detecting circulating tumor cells mainly include flow cytometry, immunomicroarrays, and polymerase chain reaction (PCR). These methods are expensive, time-consuming, and require high equipment requirements. Therefore, there is a need to develop more economical, rapid, and sensitive platforms for early-stage tumor cell detection. In recent years, biosensors have attracted widespread attention due to their advantages such as fast response, simple operation, strong specificity, low cost, high sensitivity, and ease of miniaturization. Among them, colorimetric biosensors have gained widespread application due to their simplicity, practicality, direct readout, low cost, and easy operation. Standard colorimetric assays typically use enzymes such as horseradish peroxidase, glucose oxidase, alkaline phosphatase, and other proteases to catalyze the colorimetric reaction. However, the high cost, difficulty in purification, high temperature requirements, and high volatility of enzymes significantly limit their practical application. In contrast, artificial nanozymes with the activity of natural enzymes are effective alternatives to natural enzymes. Compared with traditional enzymes, artificial nanozymes offer advantages such as simple preparation, low cost, and good stability.

[0004] In this study, MnCoZDH and Au NPs were loaded onto the surface of MXenes to synthesize a multifunctional composite nanomaterial, MXenes@MnCoZDH@Au NPs. This composite exhibits excellent peroxidase properties, capable of oxidizing 3,3',5,5'-tetramethylbenzidine in the presence of hydrogen peroxide, turning it blue. Furthermore, the gold nanoparticles in the MXenes@MnCoZDH@Au NPs composite effectively bind to aptamers, making it an excellent carrier for aptamer immobilization. Based on this, a colorimetric biosensor was developed using the lung cancer cell line A549 as a model for sensitive detection of lung cancer tumor cells. Fe3O4@PDA NPs functionalized with EGFR nucleic acid aptamers were used as capture probes. Carboxylated EGFR aptamers were modified on the Fe3O4@PDA NPs surface using EDC and NHS to specifically screen and enrich tumor cells. Furthermore, the MXenes@MnCoZDH@Au-aptamer composite specifically binds to captured tumor cells. The highly efficient peroxidase properties of the MXenes@MnCoZDH@Au NPs complex were exploited to catalyze the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) for quantitative detection of A549 cells. This cell sensor exhibits excellent sensitivity and stability, and holds great promise for application in tumor cell detection. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a biosensor that can effectively measure tumor cells. The present invention also provides a method for preparing a biosensor comprising MXenes@MnCoZDH@Au NPs.

[0006] In order to achieve the above object, the present invention discloses the following technical solutions: The first aspect of the present invention discloses a biosensor based on MXenes@MnCoZDH@Au NPs, comprising a labeling probe and a capture probe; The multifunctional composite nanomaterial MXenes@MnCoZDH@Au NPs is used as a labeling probe, which is used for signal amplification in the colorimetric sensor to detect circulating tumor cells. Magnetic beads coated with dopamine functionalized with nucleic acid aptamers were used as capture probes for screening and enriching circulating tumor cells.

[0007] The labeled probe is used in combination with an aptamer that specifically binds to tumor cells.

[0008] The aptamers that specifically bind to tumor cells include EpCAM targeting aptamers and Vimentin aptamers.

[0009] The second aspect of the present invention discloses a method for preparing the multifunctional composite nanomaterial MXenes@MnCoZDH@Au NPs, which comprises the following steps: S1. Synthesis of Ti3C2MXenes nanosheets: Immerse titanium aluminum carbide powder in HF solution and stir at room temperature; after etching, wash continuously with deionized water until the pH is > 6; centrifuge at high speed and then vacuum dry to obtain multilayer Ti3C2MXenes nanosheet powder; then mix the m-MXenes powder with DMSO, ultrasonicate at room temperature for 18 hours, centrifuge at high speed, wash and vacuum dry to obtain single-layer or multilayer Ti3C2MXenes nanosheet powder; S2. Synthesis of MXenes@MnCo-ZIF-67: f-MXenes, Mn(NO₃)₂·6H₂O, and Co(NO₃)₂·6H₂O were dispersed in methanol under magnetic stirring. A methanol solution containing 2-methylimidazole was then added to the mixture and stirred. The mixture was allowed to stand, centrifuged, and then washed. Vacuum drying afforded a blue-purple MXenes@MnCo-ZIF-67 powder. S3. Synthesis of MXenes@MnCoZDH: MXenes@NiCo-ZIF-67 was dissolved in potassium hydroxide solution and stirred continuously at room temperature. The mixed solution was then subjected to high pressure reaction and centrifuged. After washing, the pH value was adjusted to 7. Black MXenes@MnCo-ZIF-67 powder was obtained after vacuum drying. S4. Synthesis of MXenes@MnCoZDH@Au NPs: First, chloroauric acid (tetrahydrate) was dissolved in methanol solution to prepare 0.1 g / mL chloroauric acid methanol solution. Then, MXenes@MnCoZDH powder was dispersed in the methanol solution and the prepared chloroauric acid methanol solution was added, and magnetic stirring was performed at room temperature. Then, NaBH4 methanol solution was added to the above mixture, and magnetic stirring was continued at room temperature. After stirring, the mixed solution was centrifuged and the precipitate was collected. After washing, it was vacuum-dried to obtain MXenes@MnCoZDH@Au NPs powder.

[0010] Furthermore, the labeled probe MXenes@MnCoZDH@Au-Apt was used in combination with an aptamer that specifically binds to tumor cells. EpCAM With MXenes@MnCoZDH@Au-Apt VimentinThe synthesis method is as follows: first, the thiol-modified aptamer is dispersed in Tris-HCl buffer. After activation, the aptamer is mixed with MXenes@MnCoZDH@Au NPs, incubated and centrifuged, and then washed with PBS to obtain a mixed solution. Then, MCH solution is added to the above mixed solution. After incubation, it is washed with PBS solution and resuspended in 10 mM PBS (pH=7.4) and placed at 4°C for use.

[0011] The nucleic acid aptamer comprises an EGFR aptamer, and the EGFR aptamer is modified with a carboxyl group; The dopamine-coated magnetic beads are Fe3O4@PDA NPs.

[0012] The specific preparation method of Fe3O4@PDA NPs is as follows: Anhydrous ferric chloride and sodium citrate dehydrate are added to ethylene glycol and stirred, and sodium acetate is added after stirring and continued to stir to obtain a mixture; the resulting mixture is then reacted at high temperature, and the synthesized Fe3O4 nanoparticles are purified. After washing and drying with ethanol and ultrapure water, the obtained Fe3O4 nanoparticles are dispersed in a Tris-HCl solution containing dopamine, and after stirring, a product containing Fe3O4@PDA NPs is formed; the synthesized product is separated and collected using a magnet, and after washing and drying, Fe3O4@PDA NPs are obtained.

[0013] The third aspect of the present invention discloses a colorimetric biosensor constructed using a capture probe and a labeling probe, and the specific method is as follows: The capture probe was added to the cell suspension at different concentrations, vortexed and incubated at room temperature, then the cell suspension was magnetically separated, the supernatant was discarded, and the sediment was resuspended in PBS solution; the labeled probe was then added, reacted at room temperature, the solution containing the labeled probe was magnetically separated, the supernatant was discarded, the sediment was washed with PBS solution to remove unbound labeled probe, and resuspended in PBS solution; then the specific oligonucleotide sequence P1 was added, reacted at room temperature and magnetically separated again, the supernatant was collected, and the supernatant was subjected to a colorimetric reaction.

[0014] As an embodiment of the present invention, the specific operation of the colorimetric reaction is as follows: Add 200 µL of 5 mM 3,3',5,5'-tetramethylbenzidine (TMB) solution, 200 µL of 10 mM hydrogen peroxide (H2O2), and 400 µL of 0.2 M acetic acid-sodium acetate buffer (pH = 3.5) to the harvested supernatant. Incubate in the dark for 8 minutes. Transfer the blue solution (150 µL) to a 96-well plate and measure the absorbance at 652 nm using a microplate reader or perform RGB detection analysis using a smartphone app.

[0015] The logarithmic value of the absorbance signal detected by the biosensor based on MXenes@MnCoZDH@Au NPs and the tumor cell concentration is 10-10 6 The linear relationship was observed in the range of cells / mL, and the linear regression equation was ΔA = 0.0550 log c + 0.3178, with a correlation coefficient of 0.9975, where ΔA was defined as the difference in absorbance between the presence and absence of tumor cells, and ΔA = A − A0, where A is the absorbance in the presence of tumor cells, A0 is the absorbance in the absence of tumor cells, the unit is au, and c is the concentration unit of tumor cells, the unit is cells / mL.

[0016] The application of the MXenes@MnCoZDH@Au NPs-based biosensor in the preparation of tumor cell detection products.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) MnCoZDH and Au NPs were loaded onto the surface of MXenes to synthesize a multifunctional composite nanomaterial, MXenes@MnCoZDH@Au NPs. This composite has good peroxidase properties and can oxidize 3,3',5,5'-tetramethylbenzidine to turn it blue in the presence of hydrogen peroxide. In addition, the gold nanoparticles in the MXenes@MnCoZDH@Au NPs composite can effectively bind to the aptamer and serve as a good carrier for immobilizing the aptamer.

[0018] (2) The present invention verifies that MXenes@MnCoZDH@Au NPs have peroxidase-like activity and can oxidize TMB to oxTMB in the presence of hydrogen peroxide. At the same time, the affinity constant of MXenes@MnCoZDH@Au NPs is calculated to be 0.043 mM, which is lower than that of the traditional and widely used horseradish peroxidase, indicating that it has a high affinity for the substrate TMB.

[0019] (3) The present invention also optimizes the experimental conditions and determines the linear relationship between cell concentration and absorbance value under the conditions of the optimal concentration of EGFR nucleic acid aptamer in the capture probe and the ratio of manganese ions and cobalt ions in the labeling probe. The linear range of the constructed sensor for detecting tumor cells is 10-10 6 cells / mL, with a detection limit of 2 cells / mL. The constructed sensor demonstrated good specificity, reproducibility, and reproducibility. In clinical sample testing, the spiked recoveries of different cell concentrations ranged from 99.73% to 105.40%, with sample standard deviations less than 2.51%. These results demonstrate that the cell-based sensor has great potential for application in clinical diagnostics.

[0020] (4) The detection results of tumor cells can assist in diagnosing the disease progression and survival rate of cancer patients, monitoring treatment response and disease recurrence risk, which has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1A is a transmission electron microscope image of the Fe3O4NPs of the present invention; FIG1B is a transmission electron microscope image of the Fe3O4@PDANPs of the present invention.

[0022] Figure 2 is the X-ray diffraction patterns of Fe3O4NPs and Fe3O4@PDANPs of the present invention.

[0023] Figure 3A -E are the scanning electron microscope images of MAX, m-MXenes, f-MXenes, MnCo-ZIF-67 and MXenes@MnCoZDH of the present invention, respectively.

[0024] Figure 4 AD are the X-ray diffraction patterns of MAX, m-MXenes, f-MXenes, MnCo-ZIF-67, MXenes@MnCo-ZIF-67 and MXenes@MnCoZDH of the present invention, respectively.

[0025] FIG5 is an X-ray photoelectron spectroscopy characterization of MXenes@MnCoZDH of the present invention.

[0026] Figure 6A is the elemental mapping image of the MXenes@MnCoZDH@Au NPs of the present invention; Figure 6B is the EDS characterization of the MXenes@MnCoZDH@Au NPs of the present invention.

[0027] FIG7A is a cyclic voltammetry curve of the colorimetric biosensor of the present invention; FIG7B is an electrochemical impedance spectroscopy curve of the colorimetric biosensor of the present invention.

[0028] FIG8 is the ultraviolet absorption spectrum curve of MXenes@MnCoZDH@Au NPs of the present invention.

[0029] In Figure 9, A is the absorbance of TMB color development catalyzed by MXenes@MnCoZDH@Au NPs of different volumes of the present invention; Figure 9 Figure 9B is the time kinetic curve of the MXenes@MnCoZDH@Au NPs of the present invention; Figure 9C is the absorbance of the MXenes@MnCoZDH@Au NPs of the present invention catalyzing TMB color development at different temperatures; Figure 9D is the absorbance of the MXenes@MnCoZDH@Au NPs of the present invention catalyzing TMB color development at different pH values.

[0030] Figures 10 and 11 are enzyme kinetic analysis diagrams of MXenes@MnCoZDH@Au NPs of the present invention.

[0031] FIG12A shows the optimization of the EGFR aptamer concentration in the biosensor capture probe of the present invention; FIG12B and C show the optimization of the ratio of cobalt to manganese ions in the biosensor labeling probe of the present invention.

[0032] Figures A and C in Figure 13 are the standard curves of the smartphone-assisted biosensor detection platform of the present invention and the detection of tumor cells at different concentrations; Figure 13 B is the standard curve of the absorbance change when the biosensor of the present invention detects tumor cells at different concentrations.

[0033] Figure 14 FIG14A is an experimental verification of the specificity of the sensor of the present invention; FIG14B is an experimental verification of the reproducibility of the sensor of the present invention; and FIG14C is an experimental verification of the stability of the sensor of the present invention.

[0034] Figure 15 This is a flow chart for constructing the sensor of the present invention. DETAILED DESCRIPTION

[0035] The technical solutions of the present invention are described in detail below with reference to the accompanying drawings and examples, but the present invention is not limited to the scope of the examples. Experimental methods and techniques in the following examples where specific conditions are not specified are generally performed in accordance with conventional conditions in the art or the conditions recommended by the manufacturer.

[0036] Example 1 A method for preparing a biosensor based on MXenes@MnCoZDH@Au NPs comprises the following steps: 1. Synthesis of capture probe: (1) Synthesis of Fe3O4@PDA NPs First, 0.325 g of anhydrous ferric chloride and 0.2 g of sodium citrate dehydrate were added to 20 mL of ethylene glycol under mechanical stirring. The mixture was vigorously stirred for 1 hour, followed by the addition of 1.2 g of sodium acetate and stirring for an additional 30 minutes. The resulting mixture was then transferred to a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 200°C for 12 hours. After the autoclave cooled to room temperature, the synthesized Fe₃O₄ nanoparticles were purified, washed three times with ethanol and ultrapure water, and dried under vacuum for 12 hours. Forty mg of the resulting Fe₃O₄ nanoparticles were then dispersed in 20 mL of a 10 mM Tris-HCl solution (pH 8.5) containing 2 mg / mL dopamine and stirred for an additional 8 hours to form Fe₃O₄@PDA NPs. The synthesized product was separated and collected using a magnet. Finally, the Fe₃O₄@PDA NPs were washed several times with ultrapure water and dried under vacuum for further use.

[0037] (2) Capture probe Fe3O4@PDA-Apt EGFR Synthesis First, 20 μL of 25 μmol / L carboxyl-modified EGFR aptamer (Apt EGFR ) was added to 250 μL of an aqueous solution containing 400 mM EDC and 100 mM NHS and activated at room temperature for 1 h. Then, 1 mL of a 2 mg / mL Fe₃O₄@PDA NP suspension was added. After reacting at room temperature for 3 h, the EGFR aptamer-functionalized Fe₃O₄@PDA NPs were collected by magnetic separation. Finally, the NPs were washed three times with 10 mM PBS (pH = 7.4) and dispersed in 1 mL of 10 mM PBS (pH = 7.4) and stored at 4°C until use.

[0038] 2. Synthesis of labeled probes: (1) Synthesis of Ti3C2MXenes nanosheets First, 1 g of titanium aluminum carbide (Ti3AlC2, MAX) powder was immersed in 10 mL of 40% HF solution and stirred at room temperature for 24 h. After etching, the mixture was continuously washed with deionized water until the pH reached >6, centrifuged at 5500 rpm for 5 minutes, and dried in vacuum at 60°C to obtain multilayered Ti3C2 MXene nanosheet powders (m-MXenes). Next, 200 mg of m-MXene powder was mixed with 20 mL of DMSO and sonicated at room temperature for 18 hours. The mixture was then centrifuged at 4000 rpm for 5 minutes and repeatedly washed with deionized water. Finally, the mixture was vacuum dried overnight to obtain single-layer or few-layer Ti3C2 MXene nanosheet powders (f-MXenes) and stored at 4°C in the dark.

[0039] (2) Synthesis of MXenes@MnCo-ZIF-67 Under magnetic stirring, 16 mg of f-MXenes, 0.8 mmol of Mn(NO₃)₂·6H₂O, and 0.8 mmol of Co(NO₃)₂·6H₂O were dispersed in 20 mL of methanol. A 20 mL methanol solution containing 1.0592 g of 2-methylimidazole was then added to the mixture and stirred for 30 minutes. After 24 hours of stagnation, the mixture was centrifuged and washed several times with methanol and water. Finally, it was dried under vacuum at 60°C for 12 hours to obtain a blue-purple powder. The synthesis of MnCo-ZIF-67 was similar to that of MXenes@MnCo-ZIF-67, but without the addition of MXenes.

[0040] (3) Synthesis of MXenes@MnCoZDH 100 mg of MXenes@NiCo-ZIF-67 was dissolved in 300 mL of 2 M potassium hydroxide solution and stirred at room temperature for 3 h. The mixed solution was then transferred to a Teflon-lined stainless steel autoclave and reacted at 120°C for 3 h. The mixture was then centrifuged, washed with deionized water, and the pH was adjusted to 7. Finally, the mixture was vacuum-dried at 60°C for 12 h to obtain a black powder, which was stored at 4°C in the dark.

[0041] (4) Synthesis of MXenes@MnCoZDH@Au NPs First, 1 g of chloroauric acid (tetrahydrate) was dissolved in 10 mL of methanol to prepare a 0.1 g / mL chloroauric acid-methanol solution. Then, 50 mg of MXenes@MnCoZDH powder was dispersed in 10 mL of methanol and 1 mL of 0.1 g / mL chloroauric acid-methanol solution was added. The mixture was magnetically stirred at room temperature for 20 minutes. Then, 2 mL of 0.01 M NaBH₄ methanol solution was added to the mixture, and magnetic stirring was continued at room temperature for 20 minutes. Finally, the mixture was centrifuged and the precipitate was collected. After washing five times with methanol, the precipitate was dried in a vacuum oven at 60°C and stored at 4°C in the dark.

[0042] (5) Labeled probe MXenes@MnCoZDH@Au-Apt EpCAM With MXenes@MnCoZDH@Au-Apt Vimentin Synthesis: First, the thiol-modified EpCAM aptamer (Apt EpCAM ) were dispersed in 10 mM Tris-HCl buffer (containing 10 mM TCEP, pH = 7.4) and activated at 37 °C for 1 h to inhibit the formation of disulfide bonds and promote the binding of Au-S bonds. Then, 6 μL of Apt EpCAM The mixture was mixed with 6 μL of MXenes@MnCoZDH@Au NPs, incubated at 37 °C for 2 h, centrifuged and washed three times with 10 mM PBS (pH = 7.4) to remove unbound Apt. EpCAM Finally, to reduce nonspecific binding, 6 μL of 2 mM MCH solution was added to the above mixture, incubated at 37 °C for 30 minutes, washed three times with 10 mM PBS (pH = 7.4), and resuspended in 10 mM PBS (pH = 7.4) and stored at 4 °C for later use. Vimentin Synthesis and characterization of MXenes@MnCoZDH@Au-Apt EpCAM The preparation method is similar to that of EpCAM Replaced with thiol-modified Vimentin aptamer (Apt Vimentin )

[0043] 3. Construction of colorimetric biosensor: 50 μL of capture probe Fe3O4@PDA-Apt EGFRAdd to the cell suspension of different concentrations, vortex thoroughly 5 times, and incubate at room temperature for 20 minutes. Then, place the test tube containing the above mixture on a magnetic stand for 5 minutes for magnetic separation, discard the supernatant, and resuspend the sediment in 1 mL, 10 mM PBS solution (pH = 7.4). Next, add 20 μL of labeled probe (MXenes@MnCoZDH@Au-Apt EpCAM With MXenes@MnCoZDH@Au-Apt Vimentin Add 10µL of each to the mixture, react at room temperature for 30 minutes, and place the tube containing the mixture on a magnetic rack for another 5 minutes for magnetic separation. Discard the supernatant, wash the pellet three times with 10 mM PBS (pH 7.4) to remove unbound labeled probe, and resuspend it in 1 mL of 10 mM PBS (pH 7.4). Specific oligonucleotide sequence P1 is then added, react at room temperature for 30 minutes, and place the tube containing the mixture on a magnetic rack for another 5 minutes for magnetic separation. Collect the supernatant. For the colorimetric reaction, add 200 µL of 5 mM 3,3',5,5'-tetramethylbenzidine (TMB) solution, 200 µL of 10 mM hydrogen peroxide (H2O2), and 400 µL of 0.2 M acetic acid-sodium acetate buffer (pH = 3.5) to the supernatant. Incubate in the dark for 8 minutes. Transfer the blue solution (150 µL) to a 96-well plate and measure the absorbance at 652 nm using a microplate reader or perform RGB detection analysis using a smartphone app.

[0044] Example 2. Characterization of Fe3O4@PDA NPs The Fe3O4@PDA NPs synthesized in Example 1 were characterized by TEM and XRD. Figure 1 and 2 As shown. Figure 1 As shown in Figure A, the Fe3O4NPs are well-shaped spherical particles with a size of approximately 220 nm. Figure 1 Figure B shows a typical transmission electron microscopy image of the core-shell structure of Fe3O4@PDA NPs. The dark Fe3O4 core is clearly coated with a gray polydopamine (PDA) shell, and the thickness of the PDA shell is about 30 nm. In addition, X-ray diffraction (XRD) analysis ( Figure 2 ) showed that Fe3O4NPs had a typical cubic structure, and PDA encapsulation did not change the crystal structure of Fe3O4.

[0045] Example 3. SEM characterization of MXenes@MnCoZDH The MXenes@MnCoZDH synthesized in Example 1 was characterized by SEM. Figure 3A -E. Shows the SEM morphology of MAX, m-MXenes, f-MXenes, MnCo-ZIF-67 and MXenes@MnCoZDH. From the SEM image of m-MXenes ( Figure 3B ) It can be seen that after selective etching of the Al layer in HF solution, a typical accordion-like structure is formed, and the resulting material consists of two-dimensional (2D) nanosheets. m-MXenes are further processed into f-MXenes by ultrasonic exfoliation in DMSO, as shown in Figure 3C As shown in Figure 3, the number of layers is significantly reduced, showing a thinner morphology, and partial single-layer MXene nanosheets are observed on the side. Figure 3D It shows that MnCo-ZIF-67 has a regular cubic structure, but there is a serious aggregation phenomenon. From the SEM image of MXenes@MnCoZDH ( Figure 3E ), it can be seen that the f-MXenes are almost completely covered by the smaller MnCoZDH nanoparticles, indicating the successful synthesis of the composite nanomaterial MXenes@MnCoZDH.

[0046] Example 4. X-ray diffraction characterization of MXenes@MnCoZDH XRD characterization was performed on the MXenes@MnCoZDH synthesized in Example 1, and the results were as follows: Figure 4 As shown. Figure 4 It can be seen from A in the middle that in the MXenes after exfoliation, the (104) crystal plane corresponding to the diffraction peak of the original Ti3AlC2 at 39.0° has disappeared, indicating that the Al layer in Ti3AlC2 has been removed. In addition, the (002) diffraction peak of Ti3AlC2 moves from 9.5° to 8.8°. According to Bragg's Law, it can be inferred that its interlayer spacing has increased. Compared with m-MXenes, the (002) diffraction peak of f-MXenes further moves to 5.2°, indicating that its interlayer spacing has further increased, and the results are in line with expectations. The diffraction peaks of the prepared ZIF-67 are completely consistent with its standard XRD diffraction peak (CCDC: 671074), indicating that ZIF-67 with a cubic structure ( Figure 4 At the same time, the XRD pattern of MnCo-ZIF-67 is basically consistent with that of ZIF-67, indicating that MnCo-ZIF-67 retains the structural framework of ZIF-67, while Mn 2+ The introduction of Mn did not destroy its framework structure, which may be due to 2+ and Co2+ The ionic radius of MXenes@MnCo-ZIF-67 is almost the same. Figure 4 Figure C) shows almost all the characteristic diffraction peaks of f-MXenes and MnCo-ZIF-67. After alkali treatment, the (002) diffraction peak of MXenes in the composite material still exists. At the same time, the other diffraction peaks are basically consistent with the characteristic peaks of Mn (OH) 2 and Co (OH) 2 (JCPDS 02-1072, JCPDS 01-0357). Figure 4 As shown in Figure D, the diffraction peaks at 19.1°, 31.3°, 36.8°, 50.3°, 55.6°, 59.1°, and 65.1° correspond to the (001), (100), (101), (102), (110), (003), and (200) crystal planes of Mn(OH)2, respectively; while the diffraction peaks at 36.8°, 38.9°, 50.6°, 61.7°, and 68.4° correspond to the (101), (002), (102), (111), and (200) crystal planes of Co(OH)2, respectively. These results indicate that the composite nanomaterial MXenes@MnCoZDH has been successfully synthesized.

[0047] Example 5. X-ray Photoelectron Spectroscopy Characterization of MXenes@MnCoZDH XPS characterization of MXenes@MnCoZDH synthesized in Example 1 was performed, and the results were as follows: Figure 5 shown. Figure 5 Figure A shows the full XPS spectrum of the composite material, proving the presence of Mn, Co, F, O, Ti, N and C elements in the composite material. Figure 5 In B), the pair of peaks at 642.2 eV and 653.9 eV belong to Mn 2p 3 / 2 and Mn 2p 1 / 2 , corresponding to Mn in manganese hydroxide 2+ valence state, indicating the presence of Mn(OH)2 in the composite material. Similarly, in the XPS spectrum of Co 2p ( Figure 5 In (C), the peaks at 780.2 eV and 795.4 eV correspond to Co 2p 3 / 2 and Co 2p 1 / 2 The spin-orbit peak of Co(OH)2 2+ The valence states of the composite materials are consistent, indicating the presence of Co(OH)2. Therefore, the above results show that MnCoZDH coexists in the composite materials. In addition, the XPS spectrum of Ti 2p ( Figure 5 In (D), the peaks centered at 457.9 eV and 463.4 eV correspond to Ti 3+ Ti 2p3 / 2 and Ti 2p 1 / 2 The peaks at 458.6 eV and 464.5 eV are attributed to the Ti 2p of the Ti-O bond. 3 / 2 and Ti 2p 1 / 2 The existence of Ti-O bonds is mainly due to the termination of the Ti surface by -O or -OH groups after the surface aluminum is etched away by HF solution. These results indicate the presence of MXenes in the composite material.

[0048] Example 6. Characterization of MXenes@MnCoZDH@Au NPs The elemental mapping and EDS characterization of the MXenes@MnCoZDH@Au NPs synthesized in Example 1 were performed. Figure 6 The distribution of each component in the composite nanomaterial MXenes@MnCoZDH@Au NPs was characterized by elemental mapping images, as shown in Figure 6 As shown in Figure A, Co, Mn and Au elements are evenly distributed around the MXenes matrix. In addition, from the EDS image ( Figure 6 Similar results were also observed in Figure 5B), which further demonstrated the successful synthesis of the composite nanomaterial MXenes@MnCoZDH@Au NPs.

[0049] Example 7: Electrochemical Characterization of Colorimetric Biosensor The colorimetric biosensor constructed in Example 1 was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Figure 7 To investigate the effects of the various construction steps, CV and EIS experiments were performed in a [Fe(CN)6] 3- / 4- In solution. Figure 7 As shown in Figure 1, the bare GCE electrode exhibits a distinct reversible redox peak (curve a). When the electrode surface is modified with the MXenesn@MnCoZDH@AuNPs composite nanomaterial, the current intensity is significantly reduced (curve b). As the nucleic acid aptamer binds to the Au NPs through the Au-S bond, the current intensity further decreases. This is because the negatively charged phosphate backbone of the DNA molecule restricts the [Fe(CN)6] 3- / 4- Diffusion to the electrode surface (curve c). Then, after the addition of MCH, the nonspecific binding sites are blocked, resulting in a further decrease in current intensity (curve d). When A549 cells bind to the aptamer on the electrode surface, the lipid bilayer and proteins on the cell surface are negatively charged, which inhibits the [Fe(CN)6] 3- / 4- Diffusion to the electrode surface, so the current signal is further weakened (curve e). EIS ( Figure 7Middle (B) and CV experimental results showed similar trends, verifying the successful construction of the proposed colorimetric biosensor.

[0050] Example 8. Analysis of peroxidase activity of MXenes@MnCoZDH@Au NPs The peroxidase-like activity of MXenes@MnCoZDH@Au NPs synthesized in Example 1 was analyzed and characterized. This study simulated the peroxidase activity of MXenes@MnCoZDH@Au NPs by catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). Figure 8 Ultraviolet-visible spectroscopy (UV-vis) in the presence of hydrogen peroxide (H2O2) shows that MXenes@MnCo-ZIF-67 has the ability to catalyze the oxidation of TMB, accompanied by a color change. The colorless TMB is oxidized to blue oxTMB, which has a significant absorption peak at 652 nm, indicating that MXenes@MnCo-ZIF-67 has peroxidase (POD)-like activity. In addition, the POD-like activity of MnCo-ZIF-67, MXenes@MnCo-ZIF-67, and MXenes@MnCoZDH composites was evaluated by measuring the change in absorbance at 652 nm. The absorbance value of MXenes@MnCo-ZIF-67 at 652 nm is significantly higher than that of MnCo-ZIF-67, which may be due to the fact that the addition of MXenes reduces the agglomeration of MnCo-ZIF-67, improves its stability, and thus enhances its POD-like activity. At the same time, after MXenes@MnCo-ZIF-67 is treated with alkali to become MXenes@MnCoZDH, the absorbance of MXenes@MnCoZDH at 652 nm is further enhanced. The MnCoZDH obtained by alkali treatment is smaller in size, and more MnCo-ZIF-67 is anchored on the surface of MXenes, further enhancing the POD-like activity of the composite material. After the surface of MXenes@MnCoZDH is modified with Au NPs, since Au NPs also have peroxidase-like activity, the peroxidase-like activity of the MXenes@MnCoZDH@Au NPs composite material is further enhanced, so its absorbance value at 652 nm is also significantly increased ( Figure 8 ).

[0051] In the presence of TMB and H2O2, different volumes of MXenes@MnCoZDH@Au NPs solution were added. As the volume of MXenes@MnCoZDH@Au NPs solution increased, the absorbance at 652 nm also increased continuously ( Figure 9 Middle A). Figure 9 Figure B shows the time-dependent kinetics of the TMB oxidation reaction catalyzed by MXenes@MnCoZDH@Au NPs in the presence of H₂O₂. Over time, TMB is gradually converted into the product oxTMB. The time-dependent kinetics curve shows a gradual upward trend and levels off after 6 minutes of reaction. Therefore, 6 minutes was selected as the optimal colorimetric reaction time. Figure 9 Figure C shows the catalytic efficiency of MXenes@MnCoZDH@Au NPs at different temperatures. In the range of 20°C to 45°C, the absorbance values ​​are not much different, and the catalytic activity of MXenes@MnCoZDH@Au NPs remains high, indicating that it has good thermal stability in this temperature range and can effectively catalyze the TMB oxidation reaction. However, existing studies have shown that when the temperature is higher than 37°C, the catalytic activity of horseradish peroxidase used in traditional enzymatic reactions will be significantly reduced or even disappear. This result shows that compared with the traditional and widely used horseradish peroxidase, the artificially synthesized nanozyme MXenes@MnCoZDH@Au NPs has better thermal stability. Figure 9 As shown in Figure D, the absorbance at pH 2.0~6.0 was detected. When pH = 3.0, the POD activity of MXenes@MnCoZDH@Au NPs composite nanomaterials was the highest, so pH = 3.0 was selected as the optimal pH value. Figure 10 and 11 As shown in Figure 2, the apparent steady-state kinetics experiment was used to analyze the catalytic activity of MXenes@MnCoZDH@Au NPs. m The calculated value is 0.043 mM, which is lower than the traditional and widely used horseradish peroxidase (0.434 mM). m The lower the value, the higher its affinity for the substrate. This result confirms that MXenes@MnCoZDH@Au NPs have a high affinity for TMB, that is, MXenes@MnCoZDH@Au NPs have excellent oxidase mimetic activity.

[0052] Example 9: Optimization of sensor test conditions The optimal concentration of EGFR aptamer in the capture probe was explored by detecting the changes in absorbance values ​​when the concentration of EGFR aptamer was 0.175μM, 0.200μM, 0.225μM, 0.250μM, 0.275μM and 0.300 μM. Figure 12 Figure A shows that the absorbance reaches a maximum when the EGFR aptamer concentration is 0.250 μM. Therefore, 0.250 μM is the optimal concentration of the EGFR aptamer in the capture probe.

[0053] In order to ensure that the synthesized MXenesn@MnCoZDH@Au NPs have the best pseudoenzyme activity, the ratio of manganese and cobalt ions was optimized. During this process, the total molar number of Mn and Co was kept at 1.6 mmol, while the molar ratio of Mn and Co ions was adjusted. The molar number of Co ions was 0.4 mmol, 0.6 mmol, 0.8 mmol, 1 mmol, 1.2 mmol and 1.4 mmol, respectively, and the corresponding number of Mn ions was 1.2 mmol (named MnCo bi-1), 1 mmol (named MnCo bi-2), 0.8 mmol (named MnCo bi-3), 0.6 mmol (named MnCo bi-4), 0.4 mmol (named MnCo bi-5) and 0.2 mmol (named MnCo bi-6). The effects of different ratios of manganese and cobalt ions on absorbance were compared. Figure 12 As shown in Figures B and C, the absorbance reached its maximum when 1.2 mmol of Co ions and 0.4 mmol of Mn ions (designated as MnCo bi-5) were used. Therefore, 1.2 mmol of Co ions and 0.4 mmol of Mn ions (MnCo bi-5) were used as the optimal ratio of cobalt and manganese ions in the labeled probe.

[0054] Example 10, establishment of standard curve and determination of detection limit The standard curve was established and the detection limit was determined for the biosensor synthesized in Example 1. The experimental results are as follows: Figure 13 As shown in Figure B, as the number of tumor cells increases, the absorbance of the solution also increases accordingly. This is because the increase in tumor cells can be recognized by more MXenes@MnCoZDH@Au NPs complexes, and MXenes@MnCoZDH@Au NPs catalyze TMB color development, resulting in an increase in absorbance. 6Within the cell / mL range, the difference in absorbance detected by the sensor is linearly correlated with the logarithm of the cell concentration. The standard curve is ΔA = 0.0550 log c + 0.3178, with a correlation coefficient of 0.9975. Based on a signal-to-noise ratio of 3, the detection limit of this method is calculated to be 2 cells / mL.

[0055] Based on the RGB analysis technology of smartphones, a platform for monitoring color changes was established through a color picker (color selector). It can capture and record on-site detection images, and then extract the RGB values ​​in the images, thereby establishing a relationship between the RGB values ​​and the logarithm of cell concentration. Through this platform, color changes can be accurately quantified and effective detection can be performed at different cell concentrations. Specifically, Figure 13 As shown in Figures A and C, a series of color change images at different cell concentrations were taken using a smartphone, and the green / blue (G / B) values ​​were converted. Further data analysis yielded a linear regression equation y = 0.0418x + 1.1353 (R² = 0.9934), where x represents the logarithm of cell concentration. The R² value indicates that the equation fits well. In addition, the detection range is 10-10 6 cells / mL, which is consistent with the regression equation of the UV-visible spectrophotometer, further verifying the high accuracy and operability of the smartphone platform in detecting CTCs. This shows that the smartphone-assisted biosensor platform not only has real-time detection capabilities, but also can efficiently and accurately assess CTC concentrations under various on-site testing conditions.

[0056] Example 11: Verification of the specificity of the biosensor For the biosensor synthesized in Example 1, the specificity of the sensor was evaluated by detecting the absorbance values ​​of different cells. Figure 14 As shown in Figure A, the absorbance values ​​of SW620 cells and HGC-27 cells are low and almost negligible, while A549 cells and mixed cells show higher absorbance values. These results indicate that the constructed cell sensor has good selectivity.

[0057] Example 12: Verification of the reproducibility of the biosensor For the biosensor synthesized in Example 1, in order to evaluate the repeatability of the sensor detection signal, 10 independently constructed colorimetric biosensors were used to detect the concentration of 10 4 A549 cells with a concentration of 10 cells / mL were tested, and the absorbance of each sensor was recorded ( Figure 14 (B). The relative standard deviation of the absorbance values ​​of different batches of sensors was 4.10%, indicating that the reproducibility of the colorimetric biosensor was satisfactory.

[0058] Example 13: Verification of the stability of the biosensor For the biosensor synthesized in Example 1, in order to study the stability of the sensor, the constructed colorimetric biosensor was stored at 4°C and the absorbance value was measured every five days to study its stability. Figure 14 Center C shows that after 5 days of storage, the sensor's signal value still retained approximately 99% of its initial value. After 10 days of storage, the signal value dropped to 97% of the initial value. After 15, 20, and 25 days of storage, the signal values ​​remained at 94%, 90%, and 86% of the initial value, respectively. These results demonstrate the excellent stability of the constructed colorimetric biosensor.

[0059] Example 14: Verification of the detection of biological samples by the biosensor synthesized in Example 1 For the biosensor synthesized in Example 1, the feasibility of the sensor in detecting actual samples was evaluated by using a spike recovery experiment. 4 , 5×10 4 , 1×10 5 , 5×10 5 and 1×10 6 cells / mL) A549 cells. The sensor was used to detect spiked samples, and the obtained values ​​were compared with the theoretical values. As shown in Table 1, the spiked recoveries at different cell concentrations ranged from 99.73% to 105.40%, with standard deviations of less than 2.51%. These results demonstrate that this cell-based sensor has great potential for application in clinical diagnostics.

[0060] Table 1 Spike recovery experiment (n=3) sample <![CDATA[Addition amount (×10 4 cells mL − 1 )]]> <![CDATA[Spiked measurement value (×10 4 cells mL − 1 )]]> Recovery rate (%) RSD(%) 1 1.00 1.00 99.73 1.47 2 5.00 5.22 104.39 2.51 3 10.00 10.36 103.57 2.25 4 50.00 50.60 101.20 1.41 5 100.00 105.40 105.40 2.08 The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and also fall within the scope of protection of the present invention and are included in the scope of protection of the present invention.

Claims

1. A biosensor based on MXenes@MnCoZDH@Au NPs, characterized in that: including labeling probes and capture probes; The multifunctional composite nanomaterial MXenes@MnCoZDH@Au NPs is used as a labeling probe, which is used for signal amplification in the colorimetric sensor to detect circulating tumor cells. Magnetic beads coated with dopamine functionalized with nucleic acid aptamers were used as capture probes for screening and enriching circulating tumor cells.

2. The biosensor based on MXenes@MnCoZDH@Au NPs according to claim 1, characterized in that: The labeled probe is used in combination with an aptamer that specifically binds to tumor cells.

3. The biosensor based on MXenes@MnCoZDH@Au NPs according to claim 2, characterized in that: The aptamers that specifically bind to tumor cells include EpCAM targeting aptamers and Vimentin aptamers.

4. The biosensor based on MXenes@MnCoZDH@Au NPs according to claim 3, characterized in that: The preparation method of the multifunctional composite nanomaterial MXenes@MnCoZDH@Au NPs is as follows: S1. Synthesis of Ti3C2 MXenes nanosheets: Titanium aluminum carbide powder was immersed in an HF solution and stirred at room temperature; after etching, it was continuously washed with deionized water until the pH was > 6; after high-speed centrifugation, it was vacuum-dried to obtain multilayer Ti3C2 MXenes nanosheet powder; then, the m-MXenes powder was mixed with DMSO, ultrasonicated at room temperature for 18 hours, and then high-speed centrifuged, washed, and vacuum-dried to obtain single-layer or multilayer Ti3C2 MXenes nanosheet powder; S2. Synthesis of MXenes@MnCo-ZIF-67: f-MXenes, Mn(NO3)2·6H2O, and Co(NO3)2·6H2O were dispersed in methanol under magnetic stirring. A methanol solution containing 2-methylimidazole was then added to the mixture and stirred. The mixture was allowed to stand, centrifuged, and then washed. Vacuum drying gave blue-purple MXenes@MnCo-ZIF-67 powder; S3. Synthesis of MXenes@MnCoZDH: MXenes@NiCo-ZIF-67 was dissolved in potassium hydroxide solution and stirred continuously at room temperature. The mixed solution was then subjected to high pressure reaction and centrifuged. After washing, the pH value was adjusted to 7. Black MXenes@MnCo-ZIF-67 powder was obtained after vacuum drying. S4. Synthesis of MXenes@MnCoZDH@Au NPs: First, chloroauric acid (tetrahydrate) was dissolved in methanol solution to prepare a 0.1 g / mL chloroauric acid methanol solution. Then, MXenes@MnCoZDH powder was dispersed in the methanol solution and the prepared chloroauric acid methanol solution was added, and magnetic stirring was performed at room temperature. Then, NaBH4 methanol solution was added to the above mixture, and magnetic stirring was continued at room temperature. After stirring, the mixed solution was centrifuged and the precipitate was collected. After washing, it was vacuum-dried to obtain MXenes@MnCoZDH@Au NPs powder.

5. The biosensor based on MXenes@MnCoZDH@Au NPs according to claim 4, characterized in that: Labeled probe MXenes@MnCoZDH@Au-Apt used in combination with an aptamer that specifically binds to tumor cells EpCAM With MXenes@MnCoZDH@Au-Apt Vimentin The synthesis method is as follows: first, the thiol-modified aptamer is dispersed in Tris-HCl buffer. After activation, the aptamer is mixed with MXenes@MnCoZDH@Au NPs, incubated and centrifuged, and then washed with PBS to obtain a mixed solution. Then, MCH solution is added to the above mixed solution. After incubation, it is washed with PBS solution and resuspended in 10 mM PBS (pH=7.4) and placed at 4°C for use.

6. The MXenes@MnCoZDH@Au NPs-based biosensor according to claim 1, wherein: The nucleic acid aptamer includes an EGFR aptamer, and the EGFR aptamer is modified with a carboxyl group; The dopamine-coated magnetic beads are Fe3O4@PDA NPs.

7. The biosensor based on MXenes@MnCoZDH@Au NPs according to claim 6, characterized in that: The specific preparation method of Fe3O4@PDA NPs is as follows: Anhydrous ferric chloride and sodium citrate dehydrate are added to ethylene glycol and stirred, and sodium acetate is added after stirring and continued to stir to obtain a mixture; the resulting mixture is then reacted at high temperature, and the synthesized Fe3O4 nanoparticles are purified. After washing and drying with ethanol and ultrapure water, the obtained Fe3O4 nanoparticles are dispersed in a Tris-HCl solution containing dopamine, and after stirring, a product containing Fe3O4@PDA NPs is formed; the synthesized product is separated and collected using a magnet, and after washing and drying, Fe3O4@PDA NPs are obtained.

8. The MXenes@MnCoZDH@Au NPs-based biosensor according to claim 1, wherein: The capture probe and the labeling probe are used to construct a colorimetric biosensor. The specific method is as follows: The capture probe was added to the cell suspension at different concentrations, vortexed and incubated at room temperature. The cell suspension was then magnetically separated, the supernatant was discarded, and the sediment was resuspended in PBS solution. Then add the labeled probe, react at room temperature, perform magnetic separation on the solution containing the labeled probe, discard the supernatant, wash the sediment with PBS solution to remove the unbound labeled probe, and resuspend it in PBS solution; then add the specific oligonucleotide sequence P1, react at room temperature and perform magnetic separation again, collect the supernatant, and perform colorimetric reaction on the supernatant.

9. The biosensor based on MXenes@MnCoZDH@Au NPs according to claim 8, characterized in that: The logarithmic value of the absorbance signal detected by the biosensor based on MXenes@MnCoZDH@Au NPs and the tumor cell concentration is 10-10 6 The linear relationship was observed in the range of cells / mL, and the linear regression equation was ΔA = 0.0550 log c + 0.3178, with a correlation coefficient of 0.9975, where ΔA was defined as the difference in absorbance between the presence and absence of tumor cells, and ΔA = A − A0, where A is the absorbance in the presence of tumor cells, A0 is the absorbance in the absence of tumor cells, the unit is au, and c is the concentration unit of tumor cells, the unit is cells / mL.

10. Use of the MXenes@MnCoZDH@Au NPs-based biosensor according to any one of claims 1 to 9 in the preparation of a tumor cell detection product.

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