Gene nanosensor, and preparation method therefor and use thereof

Abstract

A gene nanosensor, and a preparation method therefor and the use thereof. The preparation method comprises: mixing MWCNTs with PDMS, and subjecting same to a vacuum treatment to obtain an MWCNT-PDMS c

Description

[0001] The present invention relates to the field of biological detection and analysis technology, specifically to a nano-genesensor, and a preparation method therefor and the use thereof. BACKGROUND

[0002] Any discussion of the prior art throughout the specification should not be taken as an admission that such prior art is widely known or forms part of the common general knowledge in the art.

[0003] According to the inventor's research, existing electrochemical biosensors can be used for the detection of microRNA-21 (miR-21). Furthermore, modifying carbon nanotubes (CNT) can enhance the sensitivity, selectivity, speed, and accuracy of electrochemical biosensors in detecting miR-21. However, currently, CNT are primarily randomly coated on the electrode surface, leading to CNT aggregation, bundling, and twisting. Electron transfer can only occur along the length of the CNT, so the tortuous and overlapping conductive pathways of the CNT reduce the efficiency of electron diffusion. Moreover, the existing methods for arranging CNT result in unstable sensors and disconnect the electrodes from the CNT over time, thereby limiting the detection performance of CNT-modified electrochemical biosensors, which needs further improvement. SUMMARY

[0004] To address the shortcomings of the prior art, the objective of the present invention is to provide a nano-genesensor, and a preparation method therefor and use thereof. The nano-genesensor provided by the present invention is used for detecting miR-21, featuring a wide linear range and an extremely low detection limit.

[0005] To achieve the above obj ective, the technical solutions of the present invention are as follows:

[0006] on the one hand, a method for preparing a nano-genesensor involves mixing multi-walled carbon nanotubes (MWCNT) with polydimethylsiloxane (PDMS), then vacuum treating to obtain an MWCNT-PDMS composite material. The MWCNT-PDMS composite material is then mixed with a curing agent and cured to obtain a bare electrode. The bare electrode and a suspension of the MWCNT are placed in an electric field for electrical treatment, causing the MWCNT to be embedded orderly along the direction of the electric field onto the bare electrode, thus obtaining a modified electrode. The carboxyl groups at the ends of the MWCNT on the modified electrode are modified to thiol groups, and gold nanoparticles are connected to the ends of the MWCNT on the modified electrode via Au-S bonds. Then, capture probes containing thiol groups are connected to the gold nanoparticles on the ends of the MWCNT on the modified electrode via Au-S bonds, thus obtaining the nanogenesensor. The capture probe has a structure complementary to the sequence of miR-21 for hybridization.

[0007] On the other hand, a nano-genesensor is obtained by the above method.

[0008] In the third aspect, an application of the above nano-genesensor in detecting miR-21 or in the manufacture of a system for detecting miR-21.

[0009] In the fourth aspect, a miR-21 detection kit comprises the above nano-genesensor and a buffer solution, wherein the buffer solution contains [Fe(CN)6]3~ 4\

[0010] The present invention uses an MWCNT-PDMS composite material to prepare a bare electrode with a surface of uneven micro-sphere structure, which increases the effective surface area of the MWCNT. The electric field treatment indicates that the MWCNT can be densely and highly orderly embedded into the self-supporting carbon nanotube electrode (i.e. the above-mentioned bare electrode) formed by the MWCNT-PDMS composite material, greatly enhancing the active sites of the electrode. By modifying the carboxyl groups at the ends of the MWCNT to thiol groups, gold nanoparticles can be connected to the electrode via Au-S bonds, and then capture probes can be connected via Au-S bonds, thus obtaining the nano-genesensor.

[0011] Research indicates that the nano-genesensor prepared by the present invention exhibits excellent selectivity, reproducibility, and stability in the electrochemical detection of miR-21. At the same time, it also shows high sensitivity in miR-21 detection, with a linear range of 0.01 fM to 100 pM and a detection limit as low as 0.0012 fM. Additionally, it can be used for the high-sensitivity measurement of miR-21 in human serum samples. BRIEF DESCRIPTION OF DRAWINGS

[0012] The accompanying drawings to the specification, which form part of the present invention, are used to provide a further understanding of the present invention, and the illustrative examples of the present invention and the description thereof are used to explain the present invention and are not unduly limiting the present invention.

[0013] FIG. 1 illustrates the manufacturing process and physical characteristics of free-standing-MWCNT under an electric field in an embodiment of the present invention; (A) a schematic diagram of MWCNTs rotating and migrating under an electric field; (B) a statistical analysis of the length of exposed MWCNTs in MWCNT-coated electrodes (MWCNT drop-coated onto MWCNT-PDMS bare electrodes) and free-standing-MWCNT electrodes (MWCNT treated with an electric field), with the length of carbon nanotubes calculated using ImageJ software; (C) an experimental setup for manufacturing self-supporting MWCNTs using an electric field; (D) ATR-FTIR spectra showing the infrared absorption differences between bare electrodes (MWCNT-PDMS electrodes), MWCNT-coated electrodes, and free-standing MWCNT electrodes (electric field-treated); (E) SEM images of the bare electrode (MWCNT-PDMS electrode); (F) SEM images of the MWCNT-coated electrode; (G) SEM images of the free-standing MWCNT electrode (electric field-treated); (H) the motion state of carbon nanotubes at 0 seconds under an AC electric field (E = 8 kVm’1, 10 kHz); (I) the motion state of carbon nanotubes at 1 minute under an AC electric field (E = 8 kVm’1, 10 kHz); (J) the motion state of carbon nanotubes at 5 minutes under an AC electric field (E = 8 kVnr1, 10 kHz).

[0014] FIG. 2 illustrates the enhancement of electrochemical performance and optimization of electric field conditions for self-supporting MWCNT electrodes in an embodiment of the present invention; (A) a comparison of CV (cyclic voltammetry) responses in 10 mM PBS containing 5 mM [Fe(CN)6]3~4~ for bare electrodes (MWCNT-PDMS electrodes), MWCNT-coated electrodes, and free-standing MWCNT electrodes (electric field-treated); (B) a comparison of EIS (electrochemical impedance spectroscopy) responses in 10 mM PBS containing 5 mM [Fe(CN)6]3' / 4‘ for bare electrodes (MWCNT-PDMS electrodes), MWCNT-coated electrodes, and free-standing MWCNT electrodes (electric field-treated); (C) optimization of electric field treatment time ranging from 1 to 10 minutes (frequency = 10 kHz); the oxidation peak current increases and saturates at 5 minutes; (D) optimization of electric field frequency ranging from 0.1 Hz to 100 kHz (t = 5 minutes), with the oxidation peak current improving and saturating at 10 kHz; error bars represent the standard deviation of three parallel experiments.

[0015] FIG. 3 illustrates a schematic diagram of the manufacturing process of the nano-genesensor and the electrochemical characterization during its manufacturing process in an embodiment of the present invention; (A) a schematic diagram of the miR-21 nano-genesensor modification process based on a free-standing electrode: (a) bare electrode (MWCNT-PDMS electrode), (b) MWCNT-coated electrode, (c) self-supporting MWCNT electrode (electric field-treated), (d) free-standing MWCNT electrode / NHS-EDC, (e) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs, (f) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs / capture probe / MCH, (g) freestanding MWCNT electrode / NHS-EDC / L-cysteine / AuNPs / capture probe / MCH / miR-21; (B) CV responses in 5 mM [Fe(CN)(,];" + of (a) bare electrode (MWCNT-PDMS electrode), (b) free-standing MWCNT electrode (treated with an electric field), (c) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs, (d) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs / capture probe / MCH, (e) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs / capture probe / MCH / miR-21; (C) EIS responses in 5 mM [Fe(C\)6]!'4' of (a) bare electrode (MWCNT-PDMS electrode), (b) free-standing MWCNT electrode (treated with an electric field), (c) freestanding MWCNT electrode / NHS-EDC / L-cysteine / AuNPs, (d) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs / capture probe / MCH, (e) free-standing MWCNT electrode / NHS-EDC / L-cysteine / AuNPs / capture probe / MCH / miR-21.

[0016] FIG. 4 illustrates the electrochemical response and calibration curves of the nano-genesensor for detecting miR-21 in an embodiment of the present invention; (A) DPV response of the nanogenesensor to different concentrations of target miR-21 in 10 mM PBS containing 5 mM [Fe(CN)e]3' / 4'; (B) linear calibration plot between the current (AI = I-Io, I being the oxidation peak current, Io being the baseline: dashed line) and the logarithm of miR-21 concentration; results are presented as the average of three independent experiments; error bars represent standard deviation.

[0017] FIG. 5 illustrates the selectivity, reproducibility, and stability characterization of the nanogenesensor for target miR-21 in an embodiment of the present invention; (A) DPV results for blank solution, non-complementary RNA-1 (10 nM), non-complementary RNA-2 (10 nM), target miR-21 (100 pM), and a mixture of three miRNAs; (B) DPV response currents for six different biosensors used to detect 100 pM miR-21; (C) DPV measured every five days for electrodes incubated with 100 pM miR-21; error bars represent the standard deviation of three measurements. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] It should be noted that the following detailed descriptions are all illustrative and intended to provide further clarification of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0019] It should be noted that the terminology used here is for the purpose of describing particular embodiments only and is not intended to be limiting of the exemplary embodiments of the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0020] A typical embodiment of the present invention provides a method for preparing a nanogenesensor. The method involves mixing MWCNT with PDMS, then vacuum treating to obtain an MWCNT-PDMS composite material. The MWCNT-PDMS composite material is then mixed with a curing agent and cured to obtain a bare electrode. The bare electrode and a suspension of MWCNT are placed in an electric field for electrical treatment, causing the MWCNT to be embedded orderly along the direction of the electric field onto the bare electrode, thus obtaining a modified electrode. The carboxyl groups at the ends of the MWCNT on the modified electrode are modified to thiol groups, and gold nanoparticles are connected to the ends of the MWCNT on the modified electrode via Au-S bonds. Then, capture probes containing thiol groups are connected to the gold nanoparticles on the ends of the MWCNT on the modified electrode via Au-S bonds, thus obtaining the nanogenesensor. The capture probe has a structure complementary to the sequence of miR-21 for hybridization.

[0021] In some embodiments, using a dry mixing method to mix MWCNT with PDMS. More specifically, the dry mixing method comprises rotating at a speed of 100-200 rmin’1, and dispersing for 12-36 hours.

[0022] In some embodiments, a mass percentage of MWCNT in the MWCNT-PDMS composite material of 5-10 wt%.

[0023] In some embodiments, the vacuum treating is conducted at an absolute pressure of 50-100 Pa for 20-40 minutes.

[0024] In some embodiments, the curing agent is 5-15% of the mass of the MWCNT-PDMS composite material.

[0025] In some embodiments, the curing agent is used to cure PDMS.

[0026] In some embodiments, the suspension of MWCNT having a concentration of 0.8-1.2 wt.%.

[0027] In some embodiments, during the electrical treatment, the bare electrode serves as one electrode of the electric field. During the electrical treatment, an AC voltage of 35-45 V and 8-12 kHz is applied. The duration of the electrical treatment is 3-7 minutes.

[0028] In some embodiments, modifying the carboxyl groups at the ends of the MWCNT on the modified electrode to thiol groups through an amide reaction using compounds containing thiol and amino groups. Such compounds containing thiol and amino groups include, for example, thiol-containing amino acids (e.g., L-cysteine, etc.). The process of the amide reaction can involve first activating with NHS (N-hydroxy sulfosuccinimide) and EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and then reacting with compounds containing thiol and amino groups.

[0029] In some embodiments, the modified electrode is first activated with NHS and EDC, and then gold nanoparticles are electrodeposited onto the ends of the MWCNT on the modified electrode, the dielectric solution for electrodeposition including chloroauric acid and compounds containing thiol and amino groups. This method avoids disrupting the high orderliness of the MWCNT during the gold nanoparticle deposition process, thereby ensuring the electrochemical performance of the electrode and, consequently, the detection performance of the nano-genesensor.

[0030] In some embodiments, the surface of the modified electrode with connected gold nanoparticles is coated with a solution containing capture probes containing thiol groups for selfassembly, such that the capture probes containing thiol groups are connected to the gold nanoparticles on the surface of the modified electrode via Au-S bonds.

[0031] In some embodiments, after connecting the capture probes, the modified electrode is immersed in a 6-mercapto-l-hexanol solution for incubation to eliminate nonspecific binding.

[0032] Another embodiment of the present invention provides a nano-genesensor obtained by the above method.

[0033] A third embodiment of the present invention provides an application of the above nanogenesensor in detecting miR-21 or in the manufacture of a system for detecting miR-21.

[0034] The system for detecting miR-21 includes an electrochemical workstation, with the nano genesensor serving as the working electrode connected to the electrochemical workstation.

[0035] A fourth embodiment of the present invention provides a miR-21 detection kit, including the above nano-genesensor and a buffer solution, wherein the buffer solution contains [Fe(CN)6]3' 4'.

[0036] The buffer solution is a phosphate-buffered saline (PBS) solution.

[0037] To enable those skilled in the art to understand the technical solutions of the present invention more clearly, the following specific examples are described in detail.

[0038] Example

[0039] MWCNTs and PDMS with a weight ratio of 8 wt.% were dispersed at a speed of 150 rmin’ 1 for 24 hours using the dry mixing method to obtain the MWCNT-PDMS composite material. The MWCNT-PDMS composite material was then vacuum treated at an absolute pressure of approximately 80 Pa for 30 minutes. Subsequently, a PDMS curing agent was added to the MWCNT-PDMS composite material at a weight ratio of 1:10, and the mixture was stirred for 2 minutes to obtain a slurry of the MWCNT-PDMS composite material. Using a 3D-printed mold, a bare MWCNT-PDMS electrode with a radius of 1.5 mm and a thickness of 0.5 mm was fabricated.

[0040] The process of fabricating the bare MWCNT-PDMS electrode involved fixing the mold onto glass and filling the mold cavity with the MWCNT-PDMS composites. Carbon fibers were embedded in the MWCNT-PDMS prior to curing to serve as wires for connecting to the electrochemical workstation during measurements. The MWCNT-PDMS bare electrode was then cured in an oven at 70°C for 2 hours.

[0041] MWCNT (0.05 g) was dispersed into n-decane (5 g) using an ultrasonic cell crusher to form an MWCNT suspension. To embed the carbon nanotubes into the CNT-PDMS bare electrode in the direction of the electric field, the MWCNT suspension was poured into a custom electric field application mold, with the CNT-PDMS bare electrode and an aluminum electrode spaced 50 mm apart serving as the two electrodes of the electric field, as shown in FIG. 1. An AC voltage of 40 V and 10 kHz was applied to the MWCNT suspension for 5 minutes to obtain the modified electrode, referred to as the free-standingMWCNT electrode (self-supporting MWCNT electrode). During the voltage application, the solution remained liquid, with no significant change in viscosity.

[0042] The Free-standing-MWCNT electrode was immersed in a carboxyl activation solution (50 mM NHS and EDC diluted in PBS) for activation for 1 hour.

[0043] The Free-standing-MWCNT electrode was then immersed in a dielectric solution (comprising 1.0 mMHAuCk, 0.5 MH2SO4, and 0.1 mM L-cysteine), and gold nanoparticles (AuNPs) were electrodeposited onto the Free-standing-MWCNT electrode using chronoamperometry (CA) at a constant potential of -0.8 V for 500 seconds.

[0044] 8 pL of the capture probe was coated onto the surface of the Free-standing-MWCNT electrode with connected gold nanoparticles. The capture probe (5-SH-C6-UCAACAUCAGUCUGAUAAGCUA-3', see Seql) was connected to the electrode surface via Au-S bonds formed between the thiol groups at the 5’ end of the capture probe and the AuNPs through a self-assembly process at room temperature. The resulting biosensor was washed with 10 mM PBS to remove any unbound probes. Additionally, to eliminate any nonspecific binding, the prepared nanocomposite was incubated in 1.0 mM MCH (6-mercapto-l-hexanol) for 30 minutes, resulting in the nano-genesensor, as shown in FIG. 3 A.

[0045] Electrochemical measurements of the nano-genesensor:

[0046] cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) measurements were performed in a solution of 10 mM PBS (pH 7.4) containing 5 mM [Fe(CN)6]3‘4’. CV curves were recorded at a scan rate of 50 mV / s, and EIS spectra were collected over a frequency range of 10 kHz to 0.01 Hz. Meanwhile, DPV measurements were conducted with a pulse period of 0.5 s, a pulse width of 0.05 s, and a pulse amplitude of 25 mV.

[0047] To visually observe the dynamic rotation and migration process of MWCNTs under an electric field, the process was simulated under a microscope (magnification of 40x). The experimental setup and the schematic diagram of MWCNT migration are shown in FIGs. 1A and IC. 2 wt.% MWCNTs were randomly dispersed in n-decane in an initially random orientation (FIG. 1H). After applying the AC electric field for 1 minute, the MWCNTs were forced to rotate and migrate along the direction of the electric field under the microscope (FIG. II). The electrode closer to the MWCNT exerted a stronger Coulomb force on the MWCNTs compared to the other electrode, causing the MWCNTs to move toward the closer electrode. Parallel and independent MWCNTs were densely embedded into the electrode (FIG. 1 J).

[0048] The surface morphology and microstructure of the CNT-PDMS electrodes, both untreated and treated with an electric field, were characterized using scanning electron microscopy (SEM, Hitachi SU8100). FIG. IF shows that most MWCNTs were embedded in PDMS without electric field treatment, with exposed lengths ranging from 30 nm to 100 nm (blue histogram in FIG. IB). In contrast, SEM imaging of the electric field-treated MWCNTs (FIG. 1G) demonstrated that MWCNTs were densely embedded in the CNT-PDMS electrode surface and maintained a parallel, independent - 8 - state, with exposed carbon nanotube lengths ranging from 400 nm to 1 um. This provided faster conductive pathways and more reaction sites. Additionally, SEM images proved that after treatment with n-decane (used to dissolve PDMS), the CNT-PDMS electrode surface exhibited an uneven micro-sphere structure. The SEM imaging of the CNT-PDMS electrode without n-decane (FIG. IE) showed a relatively smooth surface with short exposed MWCNTs, thus increasing the effective surface area of embedded MWCNTs.

[0049] To verify the electrochemical performance of self-supporting MWCNT enhanced by an electric field, CV and EIS measurements of the three electrodes in [Fe(CN)6]3' / 4‘ were performed (FIG. 2). The bare electrode showed no obvious [Fe(CN)6]3' / 4‘ redox peaks (FIG. 2A, a), while the MWCNT-coated electrode exhibited clear redox peaks (FIG. 2A, b). The redox peak current difference was 125 pA, and the redox peak potential difference was 270 mV (FIG. 2A, b). The redox peak current difference of the free-standing-MWCNT electrode further increased to 360 pA(FIG. 2 A, c), three times that of the MWCNT-coated electrode and 150 times that of the bare electrode. Additionally, the peak potential difference decreased to 160 mV. The downward trend of electron transfer resistance (Ret) in FIG. 2B was consistent with the CV results. These results indicated that self-supporting MWCNTs had more active catalytic sites, thus enhancing the electrochemical performance of the electrodes. These results also matched the increase in the exposed MWCNT length on the electrode surface observed in SEM images (FIG. IB).

[0050] The electrochemical CV and EIS measurements of each manufacturing step of the nanogenesensor are shown in FIGs. 3B and 3C. The bare electrode and the independent MWCNT electrode exhibited similar electrochemical behaviors (FIG. 3B, a). In the next step, AuNPs were bonded with the thiol groups (-SH) in L-cysteine via Au-S bonds, and the amino groups (-NH2) in L-cysteine reacted with carboxylated MWCNTs via acylation. This significantly increased the oxidation peak current of [Fe(CN)6]3’ / 4‘ by 40% (FIG. 3B, c), indicating that AuNPs were well deposited on the independent CNT electrode. The optimal reduction time for AuNPs was 500 seconds. Subsequently, thiolated miR-21 capture probes were fixed to the AuNPs on the electrode via gold-thiol bonds. Correspondingly, the oxidation peak current of [Fe(CN)6]3' / 4‘ decreased by 48%, and the peak potential difference (AEp = Epa-Epc) increased by 130 mV (FIG. 3B, d). This result was likely due to the negatively charged phosphate groups on the miR-21 capture probe, which hindered the diffusion of [Fe(CN)6] vto the electrode surface. Then, MCH was added to saturate the Au-S bonds to block the blank sites of AuNPs, completing the manufacturing of the nano-genesensor. Due to the insulating and non-electroactive properties of MCH, the oxidation peak current significantly decreased. After binding miR-21 to the nano-genesensor, the peak current notably decreased (1 fM miR-21, e). This was because the formation of base pairing (capture probe / miR-21) hindered the diffusion of [Fe(CN)6]3' 4' to the electrode surface. The Ret values in the EIS curves (FIG. 3C) exhibited an opposite trend, demonstrating the successful manufacturing of the nano-genesensor in this example.

[0051] To evaluate the performance of the nano-genesensor in detecting miR-21, different concentrations of miR-21 were measured using DPV. FIG. 4 A shows the DPV response of the nanogenesensor after capturing miR-21 at concentrations ranging from 0.01 fM to 100 pM. The oxidation peak current decreased with increasing miR-21 concentration, due to the formation of base pairing (capture probe / miR-21) hindering electron flow. FIG. 4B is a linear calibration plot between the current value (AI = I-Io, I being the oxidation peak current, Io being the baseline: dashed line) and the logarithm of miR-21 concentration, where R2= 0.99215, with three repetitions. The extremely low detection limit of 0.0012 fM (S / N=3) and the wide linear range of 0.01 fM to 100 pM were achieved.

[0052] To check the selectivity of the nano-genesensor prepared in this example, mismatch RNA (non-complementary RNA-1: 5'-UUGUACUACACAAAAGUACUG-3', see Seq_2; non-complementary RNA-2: 5'-GUAAGGCAUCUGACCGAAGGCA-3', see Seq_3) and target miR-21 (5'-UAGCUUAUCAGACUGAUGUUGA-3', see Seq_4) were detected. FIG. 5A shows the voltammograms of four types of RNA, including target miR-21, non-complementary RNA-1, non-complementary RNA-2, and a mixture of the three miRNAs. The current value (AI = I-Io, I being the oxidation peak current, Io being the baseline: red dashed line) in the presence of 100 pM target miR-21 was much lower than that of the other RNAs, indicating excellent selectivity of the nanogenesensor for target miR-21. Furthermore, the reproducibility of the nano-genesensor was also studied. Six nano-genesensors were used to detect the DPV response to 100 pM miR-21. The relative standard deviation (RSD) was 3.7% (<5%), as shown in FIG. 5B. After storage for 25 days (FIG. 5C), the AI value of the electrode with 100 pM miR-21 was about 80% of the initial response, indicating good reproducibility and stability of the nano-genesensor prepared in this example.

[0053] Real biological samples (human serum) were diluted 100 times with 10 mM PBS, and then different concentrations of miR-21 (0.01 fM, 0.1 fM, 0.1 pM, 10 pM, 100 pM) were added. Table 1 shows the recovery (detected / added miRNA-21 concentration), ranging from 95.34% to 108.63%, and the corresponding RSD (relative standard deviation bars represent the standard deviation of three replicate experiments), ranging from 3.71% to 5.12%, indicating that miR-21 can be detected in complex biological samples.

[0054] Table 1: Analysis of miRNA-21 in PBS solution containing 1% human serum Sample Added (pM) Detected (pM) Recovery rate (%) Relative standard deviation (%) 1 0 / 0.5 0.54 108.63 4.95 1 / 0 2 2.13 106.26 3.71 Serum sample 10 9.53 95.34 5.12

[0055] The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principles of the present invention should be included within the scope of the present invention's protection.

Claims

1. A method for preparing a gene nanosensor, characterized by mixing multi-walled carbon nanotubes (MWCNT) with polydimethylsiloxane (PDMS), then vacuum treating to obtain an MWCNT-PDMS composite material, mixing the MWCNT-PDMS composite material with a curing agent and curing to obtain a bare electrode, placing the bare electrode and a suspension of MWCNT in an electric field for electrical treatment, during the electrical treatment, the bare electrode serves as one electrode for the electric field causing the MWCNT to be embedded orderly along the direction of the electric field onto the bare electrode to obtain a modified electrode, modifying carboxyl groups at the ends of the MWCNT on the modified electrode to thiol groups, connecting gold nanoparticles to the ends of the MWCNT on the modified electrode via Au-S bonds, and then connecting capture probes containing thiol groups to the gold nanoparticles on the ends of the multi-walled carbon nanotubes on the modified electrode via Au-S bonds to obtain the gene nanosensor; the capture probe has a structure complementary to the sequence of microRNA-21 (miR-21) for hybridization.

2. The method according to claim 1, characterized by using a dry mixing method to mix r\iv M MWCNT with PDMS.1 3. The method according to claim 2, characterized by mixing MWCNT with PDMS at arotational speed of 100-200 r min'1 for 12-36 hours.

4. The method according to claim 1, characterized in that a mass percentage of MWCNT in the MWCNT-PDMS composite material is 5-10 wt.%.

5. The method according to claim 1, characterized in that the vacuum treating is conducted at an absolute pressure of 50-100 Pa for 20-40 minutes.

6. The method according to claim 1, characterized by the curing agent being 5-15% of the mass of the MWCNT-PDMS composite material.

7. The method according to claim 1, characterized in that the curing agent is PDMS.

8. The method according to claim 1, characterized by the suspension of MWCNT having a concentration of 0.8-1.2 wt.%.

9. The method according to claim 1, characterized in that during the electrical treatment, an AC voltage of 35-45V and 8-12 kHz is applied.

10. The method according to claim 9, characterized in that a duration for the electrical treatment is 3-7 minutes.

11. The method according to claim 1, characterized by modifying the carboxyl groups at theends of the MWCNT on the modified electrode to thiol groups through an amide reaction usingcompounds containing thiol and amino groups.

12. The method according to claim 1, characterized in that the modified electrode is firstactivated with NHS and EDC, and then gold nanoparticles are electrodeposited onto the ends of the MWCNT on the modified electrode, a dielectric solution for electrodeposition including chloroauric acid and compounds containing thiol and amino groups.

13. The method according to claim 1, characterized in that a surface of the modified electrode with connected gold nanoparticles is coated with a solution containing capture probes containing thiolgroups for self-assembly, such that the capture probes containing thiol groups are connected to the gold nanoparticles on the surface of the modified electrode via Au-S bonds.

14. The method according to claim 1, characterized in that after connecting the capture probes, the modified electrode is immersed in a 6-mercapto-l-hexanol solution for incubation.

15. A gene nanosensor characterized by being obtained by the method according to any one ofclaims 1 to 14.

16. An application of the gene nanosensor according to claim 15 in detecting miR-21 or in themanufacture of a system for detecting miR-21.

17. A miR-21 detection kit, characterized by comprising the gene nanosensor according to claim15 and a buffer solution, wherein the buffer solution contains [Fe(CN)6]3’ / 4‘.

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