Construction method, product and application of integrated electrochemical nano device

Abstract

The invention discloses a preparation method of an integrated electrochemical nano device as well as a product and application of the integrated electrochemical nano device, and belongs to the field of bioelectricity analytical chemistry. According to the invention, a capture probe is modified in the tip of a glass nano pipette; the method comprises the following steps: modifying an Aptamer / HCOF / DOX composite probe on the inner wall of a nano pipette; and transferring the Aptamer / HCOF / DOX composite probe into a nano pipette to obtain the integrated electrochemical nano device. According to the integrated electrochemical nano device disclosed by the invention, real-time monitoring and drug effect synchronous evaluation of the single-cell miRNA-155 can be realized in single-step operation. According to the invention, the potential of an integrated electrochemical nano device in tracking and analyzing microRNA for screening novel antitumor drugs is highlighted.

Description

Technical Field

[0001] The present invention relates to the field of bioelectroanalytical chemistry, and specifically to a construction method of an integrated electrochemical nanodevice, its products, and applications, and in particular to a construction method of an integrated electrochemical nanodevice, its products, and applications in single-cell microRNA detection and drug evaluation. Background Art

[0002] MicroRNA (miRNA) is a class of short, non-coding RNAs typically consisting of approximately 21-23 nucleotides. Aberrant miRNA expression is closely associated with tumorigenesis and progression. For example, overexpression of certain miRNAs in tumor cells promotes cancer cell proliferation and metastasis, while underexpression of other miRNAs leads to loss of tumor suppressor gene function. Due to cellular heterogeneity, quantitative analysis of miRNAs within single cells is crucial for tumor diagnosis, mechanistic understanding, and drug development. Over the past few decades, numerous studies have focused on single-cell miRNA analysis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), and microfluidic platforms remain widely used. While qRT-PCR offers high sensitivity, it requires cell lysis and RNA amplification, which reduces spatial resolution and compromises cellular integrity. FISH, on the other hand, is limited in quantitative accuracy by low throughput and background noise caused by nonspecific probe binding. While microfluidic systems can isolate single cells, they rely on complex instrumentation and struggle to achieve dynamic, real-time miRNA monitoring. Furthermore, emerging technologies such as single-cell sequencing are limited by high cost, lengthy workflows, and the difficulty in detecting low-abundance miRNAs. These limitations collectively hinder the study of cellular heterogeneity and transient miRNA expression patterns in natural microenvironments. In contrast, nanopipette-based platforms, with their unique advantages of minimal invasiveness and high spatiotemporal resolution, have become transformative tools for single-cell miRNA analysis. Their detection modes are categorized into faradaic and non-faradaic modes. The faradaic mode relies on electron transfer reactions at the electrode surface, sensitively quantifying redox-active substances (such as neurotransmitters and reactive oxygen species) via measurable faradaic currents. The non-faradaic mode enables sensitive detection of non-electroactive targets by monitoring ionic current fluctuations (without requiring redox reactions). For example, Zhao et al. developed a biomimetic transmembrane nanochannel for dynamic quantification of endogenous glutathione (GSH) in single cells using ionic current rectification (ICR). This channel was constructed by coupling cationic silicon-substituted rhodamine (SiRh) to the inner surface of a glass nanopipette. Furthermore, nanopores combining faradaic and non-faradaic detection can simultaneously induce a photocurrent "signal on" and an ionic current "signal off." Xu et al. proposed a concept of aggregation-enhanced electrochemistry (AEE) within confined nanopores. For example, by aggregating CdS quantum dots (QDs) in a nanopipette in response to reactive oxygen species (ROS), they achieved synchronous analysis of ROS in single cells.

[0003] In addition to detection, nanopipette can also be used as a drug delivery channel. For example, nanotools targeting miR-21 can precisely deliver a fixed dose of miR-21 inhibitor to a single HeLa cancer cell and selectively respond to cytoplasmic caspase-3 through sensitive photoelectrochemical means. In addition, in a double-barreled nanopipette, one tube is used for electroosmotic cytoplasmic protein delivery, and the other tube is used for ion assessment of post-delivery effects. After injecting DJ-1 protein through the delivery tube, the upregulation of antioxidant proteins can protect neural PC-12 cells from phorbol ester-induced oxidative stress. This conclusion was reached through targeted analysis of cytoplasmic hydrogen peroxide by the detection tube. However, existing detection and drug delivery require step-by-step operations. The long-term residence of the nanopipette in a single cell will interfere with and damage the cell, and the drug molecules have a short intracellular retention time due to cellular efflux, making it difficult to study the physiological changes of cells under different drug action times. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing a pH-responsive integrated electrochemical nanodevice.

[0005] Another technical problem to be solved by the present invention is to provide an integrated electrochemical nanodevice prepared by the preparation method.

[0006] The final technical problem to be solved by the present invention is to provide the application of the integrated electrochemical nanodevice in single-cell microRNA quantitative detection and / or drug evaluation.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a method for preparing an integrated electrochemical nanodevice, comprising the following steps:

[0008] (1) The capture probe is modified inside the tip of a glass nanopipette;

[0009] (2) Preparation of Aptamer / HCOF / DOX composite probe: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde were used as monomers to react with mSiO2 nanoparticles to form mSiO2@COF complex, which was then etched and dissolved with NaOH solution to obtain HCOF. Finally, the HCOF was loaded with the anticancer drug doxorubicin, and the HCOF / DOX suspension was slowly added to the aptamer and vigorously stirred overnight to obtain the Aptamer / HCOF / DOX composite probe.

[0010] (3) The Aptamer / HCOF / DOX composite probe was transferred into a nanopipette to obtain an integrated electrochemical nanodevice.

[0011] The glass nanopipette in step (1) is drawn, and the tip of the drawn capillary is coated with a gold layer by magnetron sputtering;

[0012] Wherein, the capture probe sequence in step (1) is: SH-GGGGATATTTTCGGGGATAGTGCT, and the concentration of the capture probe is 50-200 nM.

[0013] Wherein, the pore size of the nanopipette in step (1) is 180-200 nm.

[0014] The specific steps in step (1) are as follows: mixing the capture probe solution with the TCEP solution and then backfilling the obtained CP solution into the tip of the nanopipette to react with the gold layer on the inner surface.

[0015] The concentration of the TCEP solution is 5-25 mM.

[0016] Wherein, the aptamer described in step (2) is miRNA-155-Aptamer, and its sequence is ACCCCUAUCACGAUUAGCAUUAA-NH2.

[0017] Wherein, the concentration of the HCOF / DOX suspension in step (2) is 0.1-0.5 mg / mL, and the concentration of the aptamer is 1-5 μM.

[0018] Wherein, the concentration of the Aptamer / HCOF / DOX in step (3) is 1 to 5 μM.

[0019] The present invention also includes an integrated electrochemical nanodevice prepared by the preparation method.

[0020] The present invention also includes the use of the integrated electrochemical nanodevice in single-cell microRNA quantitative detection and / or drug evaluation.

[0021] Wherein, the microRNA is miRNA-155.

[0022] Specifically, in terms of solution detection, the integrated electrochemical nanodevice of the present invention is used to detect the content of miRNA-155 in the following steps: using a dual Ag / AgCl electrode system, the working electrode is inserted into the integrated electrochemical nanodevice, and the reference electrode is placed in a bath containing PBS and a gradient concentration of miRNA-155 (pH = 7.4). Next, the integrated electrochemical nanodevice and the electrode are placed in a solution of miRNA-155 (PBS buffer, pH = 7.4) with different concentrations. A voltage of -1V is applied to the working electrode for 60 seconds to drive miRNA-155 into the nanopore. Then, the nanopipette is left to stand for 10 minutes to allow the target in the solution to hybridize, the current-voltage (IV) curve is recorded, and the current value at -1V is substituted into the standard curve to obtain the target concentration value.

[0023] Furthermore, the miRNA-155 to be detected was dissolved in PBS to prepare a series of miRNA-155 solutions. The prepared integrated electrochemical nanodevice was then used to electrochemically detect miRNA-155 using a homemade two-electrode system. The IV curves of the different miRNA-155 concentrations were measured using a picoammeter. Finally, a standard equation was fitted using the miRNA-155 concentration and the corresponding ion current value at -1V. The current signal value measured in subsequent experiments was substituted into the standard equation to calculate the miRNA-155 concentration. When the integrated electrochemical nanodevice was exposed to miRNA-155, the strong binding ability of Aptamer to miRNA-155 caused the Aptamer / HCOF / DOX to detach from the nanopipette and be injected into the cell via -1V electroosmosis for 60s. The change in ion current can be used to quantify the miRNA-155 level.

[0024] Specifically, for single-cell detection, one electrode in the nanopipette is inserted into the cell as a working electrode, while the other electrode is immersed in the culture medium as a reference electrode. After the working electrode selects a cell, the nanopipette is micromanipulated using an upconversion motorized fluorescence microscope until the tip of the nanopipette is inserted into the cell. This process is observed using an imaging system, and a -1V DC voltage is applied for sampling. The nanopipette is left to stand for 10 minutes to allow the intracellular target to hybridize within the nanopipette, and the current-voltage curve is recorded.

[0025] Furthermore, the Ag / AgCl wire was placed in the nanopipette and in the bath respectively. The functionalized nanopipette was fixed by the MM-500-R dimensional micromanipulator holder mounted on the inverted microscope, and its insertion into the single cell membrane (Z-axis displacement range 0 to -2μm) was precisely controlled to connect to a picoammeter for current measurement. The Ag / AgCl wire in the functionalized nanopipette and in the bath was connected to a picoammeter to monitor the current in real time. The initial potential was maintained at +200mV to prevent the backflow of extracellular fluid. During intracellular sampling and electrochemical characterization, when the functionalized nanopipette just contacted the target cell membrane, the point where the nanopipette contacted the cell membrane was marked as the displacement zero point, and then the position of the functionalized nanopipette on the Z axis was lowered from 0 to -2μm. After insertion, the potential was adjusted to -1.0V for 20 to 30s to extract the cytoplasmic sol into the functionalized nanopipette. The nanopipette was removed from the cells and culture medium and placed in a beaker containing PBS as the supporting electrolyte. Ag / AgCl wires were placed inside the nanopipette and in the PBS solution, respectively, serving as the working and reference electrodes. A picoammeter was used to apply a sweep voltage from -1.0 V to +1.0 V at a scan rate of 50 mV s⁻¹, and current-voltage (IV) curves were recorded for electrochemical analysis of intracellular biomolecules.

[0026] The actual single cell is a breast cancer cell with a high miRNA-155 content, and the miRNA-155 content of different cell lines is detected by the integrated electrochemical nanodevice.

[0027] The HCOF can be loaded with different drugs for tumor cell therapy. The miRN-155 current signal changes can be measured through the specific channels of a functionalized glass nanopipette, making it suitable for testing different cell lines and universally applicable.

[0028] The mechanism of this invention is that when the aptamer / HCOF / DOX composite probe binds to the target miRNA-155, the stronger binding force between the aptamer and miRNA-155 causes the aptamer / HCOF / DOX to detach from the nanopipette and be injected into the cell through electroosmosis. Real-time changes in miRNA-155 levels can be quantified by recording changes in the ionic current. Furthermore, in the acidic microenvironment of the tumor, the pH-responsive cleavage of the HCOF structure promotes the controlled release of DOX, enabling dynamic assessment of drug effects at the single-cell level. The device can distinguish drug response differences between normal cells and tumor cells at the single-cell level, significantly reducing off-target cellular toxicity.

[0029] Beneficial effects: Compared with the existing technology, the present invention has the following advantages: The present invention has developed an innovative electrochemical nanodevice that can realize real-time monitoring of single-cell miRNA-155 and simultaneous evaluation of drug efficacy in a single-step operation. The present invention integrates the diagnostic and therapeutic functions into an ultra-small nanopipette, realizing the integration of diagnosis and treatment. It can approach or insert a single living cell almost harmlessly for non-destructive detection and diagnosis while carrying out treatment in a timely manner according to the specific situation of the cell, thereby improving the timeliness of treatment and being able to achieve diagnosis and treatment at the single-cell level. Compared with traditional methods, the positioning and treatment of diseased cells are more accurate, which can effectively avoid "overtreatment" and damage to surrounding normal cells. Moreover, the present invention can be customized according to the size, shape, surface chemical properties and functional molecules carried by the nanotubes, different disease types and treatment needs, so as to adapt to various complex single-cell diagnosis and treatment scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the preparation process of Aptamer / HCOF / DOX of the present invention;

[0031] Figure 2 Schematic diagram of the preparation process of Aptamer / HCOF / DOX functionalized nanopipette for miRNA-155 detection;

[0032] Figure 3 a is the current-voltage (IV) curve of each modification step of the functionalized nanopipette and the IV curve in response to 10 nM miRNA-155; Figure 3 b is the Nyquist plot of the ITO glass electrode after each modification step; Figure 3 c Ionic currents of 30 independently prepared functionalized nanopipettes at -1 V and +1 V.

[0033] Figure 4 a is the optimization of capture probe concentration, Figure 4 b is different concentrations of capture probe and I -1V The relationship between Figure 4 c is the IV curve of the functionalized nanopipette in response to 100 pM miRNA-155 at different incubation times;

[0034] Figure 5 a is the IV curve of the functionalized nanopipette for different concentrations of miRNA-155; Figure 5 b is the calibration curve of I-lgC, where I is the current value at -1 V and C is the miRNA-155 concentration; Figure 5c Selectivity study of 1 nM miRNA-155 and various interfering substances including PBS, miRNA-21 (2 μM), miRNA-10b (2 μM), and miRNA-144 (2 μM);

[0035] Figure 6 a is a bright field image of the functionalized nanopipette penetrating different positions of a single MDA-MB-231, scale bar: 25 μm; Figure 6 Below a is the current corresponding to -1V at different positions; Figure 6 b Bright field images and current (-1.0 V) box plots of three different cell lines, scale bar: 25 μm; Figure 6 c is the quantitative analysis of miRNA-155 expression level by functionalized nanopipette and real time PCR;

[0036] Figure 7 Schematic diagram of the integrated electrochemical nanodevice for single-cell miRNA-155 detection and drug evaluation;

[0037] Figure 8 (a) Changes in ionic current at -1.0 V after incubation of MDA-MB-231 cells with Aptamer / HCOF / DOX / miRNA-155 complex for 0, 1.5, 3, 4.5 and 5 hours (n=5); (b) Upper part: fluorescence image of a single MDA-MB-231 cell stained with H33342, scale bar 10 μm; lower part: corresponding fluorescence intensity value; (c) Changes in ionic current at -1.0 V after MDA-MB-231 cells were incubated with Aptamer / HCOF / DOX / miRNA155 complex and Aptamer / HCOF / TAM / miRNA-155 complex for 5 hours, respectively (n=10). DETAILED DESCRIPTION

[0038] The present invention is further described below through specific embodiments and drawings. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the principles of the present invention, and these should also be regarded as falling within the scope of protection of the present invention.

[0039] Reagents and instruments used in this experiment:

[0040] 1,3,5-Tris(4-aminophenyl)benzene (TPB) (Shanghai Bidex Pharmaceutical Co., Ltd.); 2,5-dimethoxyterephthalaldehyde (DMTP) (Shanghai Bidex Pharmaceutical Co., Ltd.); sodium hydroxide (NaOH) (Sinopharm Chemical Reagent Co., Ltd.); hydrochloric acid (Sinopharm Chemical Reagent Co., Ltd.); polyvinylpyrrolidone-K30 (PVP-K30) (Shanghai Yuanye Biotechnology Co., Ltd.); potassium chloride (Sinopharm Chemical Reagent Co., Ltd.); potassium ferrocyanide (K3[Fe(CN)6]) (Sinopharm Chemical Reagent Co., Ltd.); potassium ferrocyanide (K4[Fe(CN)6]·3H2O) (Sinopharm Chemical Reagent Co., Ltd.); polyethyleneimine (PEI, MW600) (Shanghai MacLean Biochemical Technology Co., Ltd.). Glass nanopipette (Sutter Instruments, USA); DMEM medium (Jiangsu KeyGen Biotech Co., Ltd.); trypsin digestion solution (Jiangsu KeyGen Biotech Co., Ltd.); phosphate-buffered saline (PBS) (Jiangsu KeyGen Biotech Co., Ltd.); Hoechst 33342 (Jiangsu KeyGen Biotech Co., Ltd.); cell viability assay kit Calcein-AM and propidium iodide (PI) (Jiangsu KeyGen Biotech Co., Ltd.); tamoxifen (TAM) (Shanghai Aladdin Reagent Co., Ltd.); doxorubicin (DOX) (Shanghai Aladdin Reagent Co., Ltd.); tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Shanghai Beyotime Biotechnology Co., Ltd.).

[0041] Table 1. Oligonucleotide sequences used in the present invention

[0042]

[0043] Note: The sequences used in the experiment were synthesized by Shanghai Bioengineering Co., Ltd. and purified by HPLC.

[0044] The following items were used: a heat-collecting constant-temperature heating magnetic stirrer (model DF-101S, Gongyi Yuhua Instrument Co., Ltd.); a water bath stirrer (model DF-101SA-H, Nanjing Kerr Instrument Equipment Co., Ltd.); an air drying oven (model 876–1, Shanghai Pudong Yuexin Scientific Instrument Factory); an electronic balance (model FA2004, Shanghai Hengping Instrument Factory); a transmission electron microscope (TEM) (model JEM-2100, JEOL Ltd.); an upconversion motorized inverted fluorescence microscope (model NIB1000, Ningbo Yongxin Optical Co., Ltd.); a high-speed refrigerated centrifuge (model SORVALL ST16R, manufactured by Thermo-Fisher Scientific, USA); and a confocal laser scanning microscope (CLSM) (model FV 3000, Olympus Corporation, Japan); ultraviolet-visible spectrometer (UV) (model UV-2600, Shimadzu Enterprise Management (China) Co., Ltd.); Fourier transform infrared spectrometer (model IRTRACER-100, Shimadzu Corporation, Japan); EMCCD camera (model iXon Life, Oxford Instruments Andor); scanning electron microscope (SEM) (model FEI Inspect F50, JEOL Ltd.).

[0045] Example 1 Preparation of Aptamer / HCOF / DOX

[0046] 1. Synthesis of mSiO2 nanoparticles

[0047] Dissolve 200mg of CTAB (cetyltrimethylammonium bromide) in 25mL of deionized water, add 7mL of ethanol and 50μL of diethanolamine, and stir at 60°C for 30 minutes. Then add 2mL of TEOS (ethyl silicate), continue stirring at 60°C for 2 hours, centrifuge at 12,000rpm for 12 hours, and wash with ethanol and deionized water to obtain mSiO2. Reflux at 78°C for 24 hours (using 25mL of a solution of hydrochloric acid and ethanol in a ratio of 1:9). Wash twice with deionized water and ethanol, then vacuum dry overnight.

[0048] 2. Preparation of Aptamer / HCOF / DOX

[0049] First, 40 mg of mSiO2 nanoparticles and 100 mg of PEI (polyethyleneimine (MW600) 99%, Shanghai MacLean Biochemical Technology Co., Ltd.) were dissolved in 5.0 mL of deionized water to obtain a PEI aqueous solution and an mSiO2 solution, respectively. The PEI aqueous solution was gradually added dropwise to the mSiO2 solution while stirring, and the mixture was stirred at room temperature for 1.5 hours. The mixture was centrifuged at 12,000 rpm for 12 minutes, and washed twice with deionized water to obtain mSiO2-PEI. 140 mg of mSiO2-PEI and 160 mg of PVP-K30 were dissolved in 80 mL of anhydrous acetonitrile and ultrasonically dispersed for 30 minutes. Then, 19.2 mg of DMTP and 16.0 mg of TPB were added to form a homogeneous solution. 200 μL of acetic acid was then added to the mixture, stirred at room temperature for 4 hours, and 800 μL of acetic acid was added. The mixture was heated at 80°C for 13 hours to prepare mSiO2@COF, which was then isolated and purified by multiple washings with tetrahydrofuran. After removing mSiO2 from the mSiO2@COF, the prepared mSiO2@COF was dispersed in 4M NaOH solution (50 mL), stirred at room temperature for 12 h, and finally centrifuged at 12,000 rpm for 12 min before freeze-drying to obtain HCOF. The prepared HCOF (5 mg) was dispersed in a DOX solution (5 mg DOX dissolved in 10 mL water), stirred at room temperature for 24 h, collected by centrifugation, and washed to remove residual DOX. A composite probe consisting of miRNA-155-aptamer and DOX@HCOF was prepared. A DOX@HCOF suspension (1.0 mg in 50 μL water) was slowly added to 256 μL of miRNA-155-aptamer solution (100 μM), and the solution was vigorously stirred overnight. After brief sonication and further incubation, 10 μL of TCEP solution (20 mM) was added to the solution and allowed to stand at room temperature for 30 min to eliminate disulfide bonds between DNA strands. The mixed solution was backfilled into the tip of the functionalized nanopipette and incubated at room temperature for 12 hours. The nanopore of the functionalized nanopipette was then rinsed with water and placed in a vacuum chamber for 10 minutes. This process was repeated three times to reduce nonspecific adsorption. The nanopore probe was stored in a refrigerator at 4°C until use. Figure 1 A schematic diagram of the preparation process of Aptamer / HCOF / DOX is shown.

[0050] Example 2 Preparation of miRNA-155 responsive nanopipette

[0051] Borosilicate glass tubes (1.00 mm outer diameter, 0.78 mm inner diameter) were used as starting materials. They were thoroughly cleaned with piranha solution for 30 minutes, rinsed thoroughly with ultrapure water and ethanol, and dried under nitrogen (N2) gas. Nanotubes with a pore diameter of 190 nm were produced using a P-2000 laser puller. The pulling parameters were as follows: HEAT (temperature) = 750°C, FIL (tension) = 4, VEL (velocity) = 31, DEL (delay) = 120, and PUL (pulse control) = 170. The tip of the drawn nanotube was sprayed with gold at a 30° angle. The sample was dried at 80°C for 30 minutes to obtain a gold-coated nanopore. Prior to surface modification of the nanopipette, a Tris-HCl / KCl solution (20.0 mM Tris-HCl, 50.0 mM KCl, pH 7.4) containing 200 nM capture probe (CP) was dissolved in water, heated at 95°C for 5 minutes, and then cooled to room temperature. The CP solution was mixed with an equal volume of 1 mM TCEP solution (1:1 volume ratio) and allowed to stabilize at room temperature for 1 hour to remove disulfide bonds in the CP. 7.5 μL of the 200 nM CP solution was backfilled into the nanopipette tip and allowed to react with the inner gold layer for 12 hours (4°C). Subsequently, 400 μL of a mixture of 1.0 μM Aptamer / HCOF / DOX and 20 μL of 10.0 mM TCEP was backfilled into the nanopipette tip to react with the CP. After rinsing with PBS (pH 7.4), the nanopipette was placed in a vacuum chamber for 10 minutes, repeated three times. To demonstrate the reliability of the functionalized nanopipette modification method, we replaced the bare glass nanotube with a clean ITO glass electrode. First, the ITO glass was cut into 1 cm × 3 cm strips. The ITO glass was then ultrasonically cleaned with deionized water, ethanol, and acetone until the surface was clean and dried in a 60°C oven. The ultrasonic cleaning sequence was deionized water, anhydrous ethanol, acetone, anhydrous ethanol, and deionized water, with each ultrasonic cleaning at a power of 300 W and a duration of 10 minutes. The ITO glass surface was modified with a gold layer and Aptamer / HCOF / DOX using the same method. Electrochemical impedance spectroscopy measurements were performed using 10 mM K3[Fe(CN)6] / K4[Fe(CN)6] (volume ratio 1:1) as the redox medium and 0.1 mol / L KCl as the electrolyte. The electrochemical impedance spectroscopy (EIS) test of ITO glass was performed using a CH760D electrochemical workstation manufactured by Shanghai Chenhua Instrument Co., Ltd. In the impedance test, ITO glass was used as the working electrode, the reference electrode was an Ag / AgCl electrode, and the counter electrode was a Pt electrode. Figure 2 A schematic diagram of the preparation process of the Aptamer / HCOF / DOX functionalized nanopipette is shown.

[0052] according to Figure 2The nanopipette was modified step by step as shown, and the current-voltage (IV) curve of each modification step was obtained by electrochemical linear sweep voltammetry (LSV) ( Figure 3 a), and the process was also verified by electrochemical impedance spectroscopy (EIS) on gold-coated conductive glass ( Figure 3 b), the experimental observation is that the diameter of the semicircle corresponding to the charge transfer resistance (Rct) in the EIS spectrum increases with the gradual modification process, which is consistent with the trend of increasing charge transfer resistance during the layer-by-layer construction of the nanopipette surface, confirming the possibility of successful surface modification. 30 independently prepared nanopipettes were tested, and 6 nanopipettes with large deviations were discarded. The measured I -1V / I +1V The relative standard deviation (RSD) was 2.77%, confirming the good preparation repeatability ( Figure 3 c). The above results indicate the successful construction of the Aptamer / HCOF / DOX functionalized nanopipette and its potential in detecting miRNA-155.

[0053] Example 3 Detection of miRNA-155 in solution

[0054] 1. Optimization of capture probe (CP) concentration

[0055] For solution-based assays, we first optimized the capture probe concentration. Tris-HCl / KCl solutions containing the capture probe (CP) were prepared at concentrations of 50 nM, 100 nM, 200 nM, and 300 nM and stirred thoroughly to ensure uniform mixing. Each solution was then heated at 95°C for 5 minutes. After cooling to room temperature, the solution was mixed with a 1 mM TCEP solution at a 1:1 volume ratio and allowed to stand at room temperature for 1 hour to remove disulfide bonds from the CP solution. Next, multiple nanopipettes were prepared and 7.5 μL of each CP solution at different concentrations was backfilled into the tip of each pipette. The solution reacted with the gold layer on the inner surface of the pipette at 4°C for 12 hours, with multiple replicates for each concentration to enhance the accuracy and reliability of the experimental results. After the reaction, the Aptamer / HCOF / DOX-modified nanopipettes were rinsed with PBS to remove unbound CP and impurities and then placed in a vacuum chamber for 10 minutes. This vacuum treatment step was repeated three times. Finally, two Ag / AgCl electrodes were inserted into the nanopipette and PBS solution (pH 7.4) as the working electrode and reference electrode, respectively. A sweep voltage ranging from -1.0 V to +1.0 V was applied between the Ag / AgCl wire inside the nanopipette and the Ag / AgCl wire in the bath solution at a scan rate of 50 mV s. -1 , record the current-voltage (IV) curve. The results are as follows Figure 4 a and Figure 4 As shown in Figure b, when the capture probe concentration exceeds 200 nM, the current at -1 V remains basically unchanged. Therefore, the concentration of the capture probe (CP) is 200 nM, which together with the Aptamer / HCOF / DOX-modified nanopipette forms the optimal integrated electrochemical nanodevice.

[0056] 2. Optimization of incubation time

[0057] The same steps were used to optimize the reaction time of the capture probe 200 nM Aptamer / HCOF / DOX modified nanopipette and miRNA-155. The PBS solution was replaced with a PBS bath containing 100 pM miRNA-155. The incubation times were 0 min, 3 min, 5 min, 10 min, and 15 min, respectively. The results are shown in Figure 4 c. When the reaction time exceeds 10 minutes, the current at -1 V remains basically unchanged. To avoid the impact of excessive detection time on cell activity in subsequent cell experiments, we selected 10 minutes as the optimal reaction time, and therefore all subsequent detection experiments adopted this condition.

[0058] 3. Detection of miRNA-155 solutions containing different concentrations or replacing them with interfering substances

[0059] Two Ag / AgCl electrodes were inserted into the Aptamer / HCOF / DOX functionalized nanopipette prepared in Example 2 and the bath solution (PBS and different concentrations of miRNA-155 (1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM), pH = 7.4) as working electrode and reference electrode respectively. A -1.0V voltage was applied between the Ag / AgCl wire inside the nanopipette and the Ag / AgCl wire in the bath solution for 60s to drive the solution containing miRNA-155 into the nanopipette. Then, the nanopipette was allowed to stand for 10 minutes to allow the target substance miRNA-155 to hybridize with aptamer / HCOF / DOX. During this process, the current-voltage curve was recorded. The results are shown in FIG. Figure 5 a shows, Figure 5 aThe current response at -1 V increased with the increase of miRNA-155 concentration (1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM), and the linear regression equation was I = -4.829logC-78.35 (R 2 =0.993), and the detection limit was 0.623fM( Figure 5 b).

[0060] Subsequently, by replacing miRNA-155 with interfering substances (PBS, miRNA-21, miRNA-10b, miRNA-144), selectivity studies were conducted and it was found that only miRNA-155 could cause significant changes in ion current, verifying the selective response of the method ( Figure 5 c), therefore, the subsequent interference of other miRNAs in the cell on the detection can be ignored.

[0061] Example 4 Determination of intracellular miRNA-155 and dynamic evaluation of drug effects at the single-cell level

[0062] The MM-500-R three-dimensional micromanipulator (Rayward / China) mounted on the inverted microscope holds the functionalized nanopipette for precise control of the insertion of the functionalized nanopipette into the observation of single cells. The Ag / AgCl wires were placed in the functionalized nanopipette prepared in Example 2 and in the culture medium, respectively. A picoammeter was connected for current measurement. Before collection, the potential was maintained at +200 mV to prevent the extracellular solution from entering the nanopipette. When the nanopipette just touched the target cell membrane, the first position was taken as 0, and then the position of the nanopipette on the Z axis was lowered from 0 to -2 μm. After insertion, the potential was adjusted to -1.0 V for 20 s to extract the cytoplasmic sol into the nanopipette. The scanning voltage range was -1.0 V to +1.0 V, and the scanning rate was 50 mV s -1 , recording the current-voltage (IV) curve. By combining an upconversion microscope with a micromanipulator, we used the functionalized nanopipette to sample different sites of MDA-MB-231 (a highly invasive and metastatic breast cancer cell line), and the measured current values ​​were similar in amplitude ( Figure 6 a). Subsequently, we detected the expression level of miRNA-155 in different cell lines, including MDA-MB-231, MCF-7 (a breast cancer cell line with almost no metastatic ability), and MCF-10A cells (normal breast epithelial cells), with 20 independent cells tested in each experiment. Figure 6 As shown in Figure 2b, the statistical average current values ​​of MDA-MB-231, MCF-7, and MCF-10A cells were -30.72±1.64nA, -28.44±0.65nA, and -25.79±0.50nA, respectively. The calculated relative expression levels of miRNA-155 in the three cell lines were approximately 165.5pM, 55.60pM, and 16.05pM, respectively. This result is consistent with the content ratio reported in the literature and the real-time PCR detection data ( Figure 6 c). This consistency validates the accuracy of the method for quantifying miRNA-155 expression. Figure 7Schematic diagram of an integrated electrochemical nanodevice for single-cell miRNA-155 detection and drug evaluation. Upon exposure to miRNA-155, specific recognition of miRNA-155 with the aptamer results in the release of the aptamer / hollow covalent organic framework / doxorubicin (Aptamer / HCOF / DOX) complex from the functional nanopipette, leading to changes in ionic current. Furthermore, in the acidic tumor microenvironment, the pH-sensitive HCOF nanoparticles disintegrate and release the drug doxorubicin (DOX), enabling dynamic evaluation of drug effects at the single-cell level.

[0063] The released Aptamer / HCOF / DOX / miRNA-155 complex was injected into the cells by electroosmosis by applying a voltage of +1V. For tumor cells, the acidic environment causes HCOF to cleave, thereby releasing DOX and inducing cell apoptosis. The current value of MDA-MB-231 cells treated with a single electroosmotic treatment was monitored at fixed time intervals of 1.5 hours. It was found that the current value gradually decreased and reached a steady state at 4.5 hours ( Figure 8 a). In addition, based on the changes in intracellular miRNA-155 concentration (by Figure 5 The curve b shows the change of miRNA-155 concentration from 165.5 pM to 4.44 pM). The potential of nanopipette to evaluate drug effects was studied using H33342-stained MDA-MB-231 cells. Figure 8 As shown in b, the fluorescence gradually increased from 0 hours to 4.5 hours, indicating that the cell activity decreased and entered the early apoptosis stage. The current reduction and cell staining results showed that the concentration of miRNA-155 triggered by the drug showed an overall down-regulation effect, and cell death occurred. The integrated electrochemical nanodevice constructed by the present invention is also suitable for the evaluation of other drugs. It is only necessary to replace the doxorubicin (DOX) in the Aptamer / HCOF / DOX nanoparticles with the target drug. Tamoxifen (TAM) is one of the most effective drugs for the treatment of breast cancer and can induce apoptosis in breast cancer cells. When TAM replaces the DOX loaded on HCOF and the nanopipette is modified using the same method described in the experimental section, Figure 8 The statistical results of 10 single cells in (c) showed that the average tamoxifen-induced current decreased from -30.89 nA to -25.96 nA, corresponding to a decrease of approximately 129.8 pM in miR-155 concentration. Compared with TAM drugs, DOX has a better therapeutic effect on MDA-MB-231 cells.

Claims

1. A method for preparing an integrated electrochemical nanodevice, characterized in that: The following steps are involved: (1) The capture probe is modified inside the glass nanopipette tip; (2) Preparation of Aptamer / HCOF / DOX composite probe: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde were used as monomers to react with mSiO2 nanoparticles to form mSiO2@COF complex, and then NaOH solution was added to etch and dissolve to obtain HCOF. Finally, HCOF was loaded with the anticancer drug doxorubicin, and the HCOF / DOX suspension was slowly added to the aptamer and vigorously stirred overnight to obtain the Aptamer / HCOF / DOX composite probe; (3) The Aptamer / HCOF / DOX composite probe was transferred into a nanopipette to obtain an integrated electrochemical nanodevice.

2. The method for preparing an integrated electrochemical nanodevice according to claim 1, characterized in that: The capture probe sequence in step (1) is SH-GGGGATATTTTCGGGGATAGTGCT, and the concentration of the capture probe is 50-300 nM.

3. The method for preparing an integrated electrochemical nanodevice according to claim 1, characterized in that: The pore size of the nanopipette in step (1) is 180-250 nm.

4. The method for preparing an integrated electrochemical nanodevice according to claim 1, characterized in that: The specific steps in step (1) are as follows: after mixing the capture probe solution with the TCEP solution, the obtained CP solution is backfilled into the tip of the nanopipette to react with the gold layer on the inner surface.

5. The method for preparing an integrated electrochemical nanodevice according to claim 1, characterized in that: The aptamer described in step (2) is miRNA-155-Aptamer, and its sequence is ACCCCUAUCACGAUUAGCAUUAA-NH2.

6. The method for preparing an integrated electrochemical nanodevice according to claim 1, characterized in that: The concentration of the HCOF / DOX suspension in step (2) is 0.2-1.0 mg / mL, and the concentration of the aptamer is 1.0-5.0 μM.

7. The method for preparing an integrated electrochemical nanodevice according to claim 1, characterized in that: The concentration of the Aptamer / HCOF / DOX in step (3) is 1-10 μM.

8. An integrated electrochemical nanodevice prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the integrated electrochemical nanodevice according to claim 8 in single-cell microRNA quantitative detection and / or drug evaluation.

10. The use according to claim 9, characterized in that The microRNA is miRNA-155.

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