An ultrasensitive electrochemical sensor based on target-induced MOF growth strategy and its construction and application

Abstract

The present invention discloses an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy and its construction and application. The construction of the ultrasensitive electrochemical sensor is carried out according to the following steps: activation of a bare gold electrode; pretreatment of streptavidin magnetic beads; specific capture of the target; extension of miRNA poly A; assembly of a metal organic framework electrode; and differential pulse voltammetry detection. The present invention does not require the addition of primers or specific enzymes, and can be directly added to the reaction system. E. coli Poly(A) polymerase can directly polymerize a poly(A) sequence at the end of miRNA-21, promote the adsorption of miRNA-21 to the electrode surface, induce the growth of MOF, and thus achieve ultra-sensitive detection of miRNA-21.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and in particular to an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy and the construction and application thereof. Background Art

[0002] Lung cancer is a malignant tumor with the second highest morbidity and mortality rate worldwide, second only to breast cancer, and poses a serious threat to human health. Histologically, lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the predominant type of lung cancer, accounting for approximately 85% of all lung cancer cases. It includes adenocarcinoma (AD), squamous cell carcinoma (SCC), and large cell lung cancer (LCC). Due to the lack of typical early symptoms, NSCLC patients are diagnosed in the middle or late stages, missing the optimal surgical opportunity. This results in a five-year survival rate of only 5%-15%, compared to 50%-70% for patients diagnosed early and undergoing surgical resection. Therefore, improving the efficiency of early diagnosis of NSCLC is crucial for improving patient prognosis. However, current routine screening programs are insufficient for early diagnosis of NSCLC and need to be improved. For example, conventional CT imaging methods, such as conventional-dose CT screening, lack the ability to differentiate benign from malignant pulmonary nodules, and low-dose CT suffers from a high false-positive rate.

[0003] MicroRNA (miRNA) is a small, endogenous noncoding RNA, typically 18-22 nucleotides in length, that regulates the expression of complementary mRNAs and plays a crucial role in gene translation. Loss or reduction of their corresponding proteins can lead to disease. Studies have shown that aberrant miRNA expression is associated with a variety of diseases, particularly tumors, including neuroblastoma, pituitary adenoma, thyroid cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, colorectal cancer, cervical cancer, and leukemia. These miRNAs are located at tumor-associated fragile sites on the genome. In addition to aberrant expression in tissues, they can also be released into the bloodstream. Recent studies have demonstrated that serum miRNAs may serve as ideal biomarkers for noninvasive cancer diagnosis. However, accurate detection and quantitative analysis of miRNAs in blood remain challenging due to the following factors: 1) the short sequences of miRNA molecules and the high sequence similarity between their members; and 2) low miRNA levels in the human body. Therefore, developing a highly sensitive and specific miRNA quantitative analysis method to detect miRNA in the blood and monitor the trend of changes in miRNA types and quantities is of great significance for real-time monitoring of tumor dynamics, evaluating treatment effects, and achieving individualized treatment.

[0004] To date, researchers have developed a variety of methods for detecting miRNAs, including Northern blot analysis using radiolabeled probes, microarray-based

[18] and polymerase chain reaction (PCR)-based methods, liquid phase single-cell assays, in situ hybridization, and high-throughput sequencing. Although Northern blot analysis is currently a relatively reliable testing technology, it has problems such as low sensitivity, low throughput, the need for a large amount of RNA samples, and the easy degradation of RNA during the experiment. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) requires RNA to be reversed into cDNA for amplification and detection. It performs well in terms of sensitivity, specificity, and reproducibility, but it is more prone to problems during the reverse transcription process and fluorescence readout requires expensive instruments and fluorescent labels, and is easily interfered by background fluorescence. The core technology of microarray analysis is the hybridization of the target and the probe, and quantitative analysis of miRNA by detecting fluorescence intensity. This method can achieve high throughput and analyze the entire human genome at one time, but its reproducibility and accuracy are relatively poor, and it is generally used for preliminary screening. In summary, although there are many miRNA detection methods, they generally have the following limitations: (1) low detection sensitivity and accuracy; (2) cumbersome, complex, and time-consuming operation steps; (3) some detection methods require the use of corresponding kits and instruments, which are expensive and costly.

[0005] Electrochemical biosensors have attracted widespread attention in the field of miRNA detection due to their advantages such as high specificity, low detection limit, low cost, and high sensitivity. Electrochemical biosensors are self-integrated devices that combine the sensitivity of electroanalytical methods with the selectivity of biological components, providing quantitative or semi-quantitative analytical information through direct spatial contact between the biorecognition element and the electrochemical transduction element. Typical DNA hybridization-based electrochemical biosensors have been developed for the detection of miRNAs, with detection ranges ranging from picomolar (pM) to nanomolar (nM). The modified electrochemical biosensor has increased the detection sensitivity to 10 fM by improving the adsorption efficiency between miRNA and gold electrode. However, due to the extremely low content of circulating miRNA in body fluids, this method still cannot meet the actual needs of clinical detection.

[0006] To improve the sensitivity and accuracy of sensor detection, researchers have developed a variety of signal amplification strategies using methods and technologies to modify electrodes with various functional materials, such as nanomaterials. Among these functional materials, metal-organic frameworks (MOFs) have garnered widespread attention from researchers across the board due to their inherent advantages. MOFs are a novel porous material formed by the self-assembly of inorganic metal ions and organic ligands containing nitrogen or oxygen. They offer advantages such as adjustable porosity and pore size, large specific surface area, and stable chemical and mechanical properties. MOFs have a wide range of applications. They can be used for gas storage and separation. For example, in 2003, Yaghi's research group discovered that MOF-5 can store H2. In the field of catalysis, MOFs can adjust the pore size and channel size of MOFs by adjusting the metal nodes and organic ligands, thereby adjusting their catalytic ability. They can also be modified and regulated after synthesis, and their catalytic activity can be enhanced by hybridization with other materials. The tunable pore size and structure of MOFs and their derivatives give them the ability to load bioactive macromolecules, drugs, and nanomaterials such as AuNPs and PtNPs. MOFs are also frequently used in the research field of electrochemical biosensors. For example, Hu et al. constructed an electroactive bimetallic MOF material using Ni+ and Co2+ for the detection of miRNA-126. Li et al. constructed a MOF@Pt@MOF nanozyme with catalase-like properties using MIL-88 and PtNPs. They generated a cascade amplification signal through a primer exchange reaction and used it to detect the content of exosomal miRNA-21 in breast cancer patients and healthy subjects.

[0007] Based on this, this project intends to develop an electrochemical signal transduction mode based on the target-induced MOFs growth strategy, combined with magnetic bead separation and poly A sequence extension technology of RNA molecules, to construct a new ultra-sensitive electrochemical sensor for the detection of lung cancer-related miRNA-21, providing a safe, reliable, rapid and sensitive detection method for the early clinical screening and diagnosis of NSCLC. Summary of the Invention

[0008] The purpose of the present invention is to provide an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy and its construction and application.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy comprises the following steps:

[0011] (1) A bare gold electrode was polished with alumina powder, ultrasonically cleaned in ddH2O, anhydrous ethanol, and ddH2O in sequence, and dried with nitrogen. The electrode was then placed in a 0.5 M sulfuric acid solution and subjected to cyclic voltammetry scanning at a scan rate of 0.1 V / s between -0.2 and +1.6 V. After the cyclic voltammetry curve stabilized, the electrode was removed and rinsed with ddH2O, and dried with nitrogen to obtain an activated gold electrode.

[0012] (2) Vortex 20 μl of 10 mg / ml streptavidin magnetic bead solution for 20 seconds to obtain a uniformly dispersed magnetic bead dispersion; remove 20 μl of the magnetic bead dispersion, discard the supernatant after magnetic separation, and wash the magnetic beads twice with Buffer I; then add 80 μl of Buffer I and 20 μl of 10 μM biotinylated capture probe solution to the magnetic beads, vortex for 30 minutes, discard the supernatant after magnetic separation, and wash the magnetic beads three times with Buffer I; then add 10 μl of 5×SSC solution to the magnetic beads to obtain a pretreated magnetic bead resuspension;

[0013] (3) Take 10 μl of the magnetic bead resuspension solution pretreated in step (2), add 10 μl of the target sequence, rotate and mix for 15 min, discard the supernatant after magnetic separation, wash the precipitate twice with Buffer I, and then add 7.5 μl of RNase-free water to the precipitate, heat it at 95°C for 2 min, and immediately magnetically separate it to collect the supernatant containing the target sequence;

[0014] (4) Mix 1 μl 10× E. coli Poly(A) Polymerase Reaction Buffer, 1 μl 10 mM ATP, 1 μl E. coli Poly(A) Polymerase, and 7.5 μl of the supernatant containing the target sequence collected in step (3), incubate at 37°C for 10 min, and add 20 μl 5× SSC solution to obtain a target solution with a polyA tail;

[0015] (5) Pipette 5 μl of the polyA tail target solution obtained in step (4) and drop it onto the gold electrode activated in step (1), and incubate at room temperature for 10 min; then drop 3 μl of 40 mM zinc nitrate solution onto the electrode, and then drop 3 μl of 160 mM 2-methylimidazole solution, and incubate at room temperature for 30 min. Rinse the electrode surface with ddH2O and blow dry with nitrogen to obtain a MOF assembled electrode;

[0016] (6) placing the MOF assembled electrode obtained in step (5) in an electrochemical detection solution and scanning by differential pulse voltammetry;

[0017] Wherein, the sequence of the biotinylated capture probe is: 5'-TTCAACATCAGTCTGATAAGCTATTT-3'-biotin;

[0018] The formula of Buffer I is: 10mM Tri-HCl, 1mM EDTA, 1M NaCl, 0.01% to 0.1% Tween-20, pH = 7.5;

[0019] The formula of the 5×SSC solution is: 0.75 M NaCl, 0.075 M sodium citrate, pH = 7;

[0020] The sequence of the target is: 5′-UAGCUUAUCAGACUGAUGUUGA-3′;

[0021] The formula of the electrochemical detection solution is: 2.5 mM potassium ferrocyanide, 2.5 mM potassium ferrocyanide, 0.1 M KCl, pH = 7;

[0022] The conditions for the differential pulse voltammetry scan were: pulse width 0.05 s, sensitivity 1e -5 , starting potential -0.1V, maximum potential 0.5V.

[0023] An ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy constructed using the above-mentioned construction method.

[0024] The above-mentioned ultrasensitive electrochemical sensor based on the target-induced MOF growth strategy is used in the preparation of products for detecting miR-21.

[0025] The detection principle of the present invention is:

[0026] First, the target miRNA-21 was specifically captured by the capture probe on the surface of streptavidin magnetic beads. After release by thermal denaturation, the 3' end of miRNA-21 was extended with a poly(A) sequence using E. coli Poly(A) polymerase. The poly(A) tail was then fixed to the electrode surface by the specific binding effect between the poly(A) tail and the gold electrode (AuE). By dripping the precursor solution of ZIF-8 type MOFs on the electrode surface, the poly(A) extension product of miRNA-21 was used as the nucleation site to induce the in situ growth of ZIF-8 on the AuE, thereby hindering the electron transfer on the electrode surface and generating an "attenuated" electrochemical detection signal, thus achieving ultra-sensitive detection of miRNA-21.

[0027] The significant advantages of the present invention are:

[0028] (1) Electrochemical technology itself has many advantages such as simplicity, low cost, fast response and sensitivity.

[0029] (2) Through the self-assembly process of MOFs on the miRNA-modified electrode, a multifunctional sensing interface was constructed. Nanostructured MOFs can perform multiple tasks, such as encapsulating the target to form a closed body, reducing ion collisions, and forming attenuated signals.

[0030] (3) The MOFs signal amplification strategy can sensitively detect microRNAs with extremely low levels in body fluids.

[0031] (4) Without the addition of primers or specific enzymes, E. coli Poly(A) polymerase can be directly added to the reaction system to directly polymerize a poly(A) sequence at the end of miRNA-21, promote the adsorption of miRNA-21 to the electrode surface, and induce the growth of MOF, thereby achieving ultrasensitive detection of miRNA-21. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A: Electrode characterization; a: activated gold electrode, b: MOF-assembled electrode. B: Polyacrylamide gel electrophoresis analysis of poly(A) tail extension; lane a: marker; lane b: sample ②; lane c: sample ③; lane d: sample ①.

[0033] Figure 2 : DPV signals before (A) and after (B) extension of poly A. a: activated gold electrode, b: without target, c: with target, d: without target + MOF, e: with target + MOF.

[0034] Figure 3 : (A) DPV signals corresponding to different concentrations of miRNA-21 (0, 0.01, 0.1, 1, 10, 100, 1000 fM), (B) calibration curve corresponding to DPV current signal and logarithm of miRNA-21 concentration.

[0035] Figure 4 : Peak currents of different miRNAs.

[0036] Figure 5 : RT-PCR and sensor detection of miRNA-21 expression levels in total miRNA samples of cancer cell lines.

[0037] Figure 6 : RT-PCR (left) and sensor (right) detection of miRNA-21 expression levels in total miRNA samples in human serum. DETAILED DESCRIPTION

[0038] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0039] In the following examples, the biotinylated probe is: 5'-TTCAACATCAGTCTGATAAGCTATTT-3'-biotin.

[0040] In the following examples, the miRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3'.

[0041] In the following examples, the miRNA-122 is: 5'-UGGAGUGUGACAAUGGUGUUUG-3'.

[0042] In the following examples, the miRNA-141 is: 5'-UAACACUGUCUGGUAAAGAUGG-3'.

[0043] In the following examples, the miRNA-199 is: 5'-ACAGUAGUCUGCACAUUGGUUA-3'.

[0044] In the following examples, the miRNA-210 is: 5'-AGCCCCUGCCCACCGCACACUG-3'.

[0045] In the following examples, the Buffer I formula is: 10 mM Tri-HCl, 1 mM EDTA, 1 M NaCl, 0.01% to 0.1% Tween-20, pH = 7.5.

[0046] In the following examples, the 5×SSC solution has a formula of: 0.75 M NaCl, 0.075 M sodium citrate, and a pH of 7.

[0047] Example 1

[0048] The construction of an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy involves the following steps:

[0049] (1) Activation of gold electrodes

[0050] The bare gold electrode was polished with 0.3 μm and 0.05 μm alumina powder in sequence, and then ultrasonically cleaned in ddH2O, anhydrous ethanol, and ddH2O for 3 minutes, and blown dry with nitrogen. The electrode was then placed in a sulfuric acid solution (0.5 M) and cyclic voltammetry was performed at a scan rate of 0.1 V / s between -0.2 and +1.6 V to clean the electrode until the cyclic voltammetry curve was stable. The electrode was then taken out and rinsed with ultrapure water, and blown dry with nitrogen to obtain an activated gold electrode.

[0051] (2) Pretreatment of magnetic beads

[0052] Place 20 μl of streptavidin magnetic bead solution (10 mg / ml) on a vortex oscillator and oscillate for 20 seconds to obtain a uniformly dispersed magnetic bead dispersion; remove 20 μl of the magnetic bead dispersion, separate it on a magnetic rack, discard the supernatant, and wash the magnetic beads twice with Buffer I; then add 80 μl of Buffer I and 20 μl of biotinylated probe (10 μM) to the magnetic beads, oscillate on a vortex oscillator for 30 minutes, separate it on a magnetic rack, discard the supernatant, and wash the magnetic beads three times with Buffer I; then add 10 μl of 5×SSC solution to the magnetic beads to resuspend them to obtain the pretreated magnetic bead resuspension.

[0053] (3) Capturing specific miRNA-21

[0054] Take 10 μl of the magnetic bead resuspension solution pretreated in step (2), add 10 μl of the miRNA-21 sequence solution, rotate and mix for 15 minutes, separate on a magnetic rack, discard the supernatant, wash the precipitate twice with Buffer I, and then add 7.5 μl of RNase-free water to the precipitate to resuspend it, then heat it at 95°C for 2 minutes, immediately separate on a magnetic rack, and collect the supernatant containing the miRNA-21 sequence.

[0055] (4) miRNA poly A extension

[0056] Mix 1 μl 10×E.coli Poly(A) Polymerase Reaction Buffer, 1 μl ATP (10 mM), 1 μl E.coli Poly(A) Polymerase, and 7.5 μl supernatant containing the miRNA-21 sequence collected in step (3), incubate at 37°C for 10 min, and then add 20 μl 5×SSC solution to obtain a miR-21 solution with a polyA tail.

[0057] (5) Assembly of MOF

[0058] 5 μl of the polyA-tailed miR-21 solution obtained in step (4) was transferred and dropped onto the gold electrode activated in step (1), and incubated at room temperature for 10 min. Then, 3 μl of zinc nitrate solution (40 mM) was added to the electrode, followed by 3 μl of 2-methylimidazole solution (160 mM), and incubated at room temperature for 30 min. The electrode surface was rinsed with ddH2O and dried with nitrogen to obtain a MOF-assembled electrode.

[0059] (6) Measurement

[0060] Electrochemical impedance spectroscopy detection uses a three-electrode system, including a working electrode, a counter electrode, and a reference electrode. The MOF assembly electrode obtained in step (5) is used as the working electrode, the platinum electrode is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode. The test solution formula is: 2.5mM potassium ferrocyanide, 2.5mM potassium ferrocyanide, 0.1M KCl, pH = 7, and the test parameters are: After the computer and the electrochemical workstation instrument are successfully connected, click Control-OpenCircuitPotential on the computer software page - then a number will appear, remember this number, return to Setup-IMPAC-then select the parameters: enitE (V) = the number just remembered, HighFreq (Hz) = 1e +5 ,LowFreq(Hz)=0.1。

[0061] Differential pulse voltammetry detection uses a three-electrode system, including a working electrode, a counter electrode, and a reference electrode. The MOF assembly electrode obtained in step (5) is the working electrode, the platinum electrode is the counter electrode, and the Ag / AgCl electrode is the reference electrode. The test solution formula is: 2.5mM potassium ferrocyanide, 2.5mM potassium ferrocyanide, 0.1M KCl, pH = 7. The test parameters are: the electrode is placed in the test solution with PulseWidth (s) = 0.05, Sensitivity (A / V) = 1e -5 The voltammetric scan was performed at a scan rate of InitE(V)=-0.1, HighE(V)=0.5 potential.

[0062] Cyclic voltammetry detection uses a three-electrode system, including a working electrode, a counter electrode, and a reference electrode. The MOF assembly electrode obtained in step (5) is the working electrode, the platinum electrode is the counter electrode, and the Ag / AgCl electrode is the reference electrode. The test solution formula is: 2.5 mM potassium ferrocyanide, 2.5 mM potassium ferrocyanide, 0.1 M KCl, pH = 7, and the test parameters are: lnitE (V) = -0.2, HighE (V) = 0.6, LowE (V) = -0.2, Sensitivity (A / V) = 1e -5 .

[0063] Example 2

[0064] Characterization of MOF assembled electrodes, the steps are as follows:

[0065] (1) Same as step (1) in Example 1.

[0066] (2) Add 3 μl of zinc nitrate solution (40 mM) to the gold electrode activated in step (1), and then add 3 μl of 2-methylimidazole solution (160 mM). Incubate at room temperature for 30 min, rinse the electrode surface with ddH2O, and blow dry with nitrogen to obtain a MOF assembled electrode.

[0067] (3) The activated gold electrode and the MOF assembled electrode obtained in step (2) were subjected to cyclic voltammetry detection respectively, and the detection conditions were the same as those in step (6) of Example 1.

[0068] like Figure 1 As shown in Figure A, due to the poor conductivity of MOF, the peak current of the MOF-assembled electrode (curve b) is significantly lower than that of the activated gold electrode (curve a). Therefore, it is preliminarily verified that MOF was successfully assembled on the gold electrode.

[0069] Example 3

[0070] Characterization of poly A sequence extension, steps are as follows:

[0071] Prepare sample ①: 5 μL 1 μM miRNA-21, 0.7 μL Ecolibuffer, 0.7 μL ATP, and 0.7 μL Ecoli polyAmerase. Mix well and incubate at 37°C for 10 min. Then add 1 μL 6× Loading buffer and 10× nucleic acid dye. Prepare sample ②: 5 μL 1 μM miRNA-21, 1 μL 6× Loading buffer, and 10× nucleic acid dye and mix well. Prepare sample ③: 0.7 μL Ecolibuffer, 0.7 μL ATP, 0.7 μL Ecoli polyAmerase, 1 μL 6× Loading buffer, and 10× nucleic acid dye and mix well. Perform polyacrylamide gel electrophoresis.

[0072] like Figure 1 As shown in B, miRNA-21 of about 20 bp in size can be clearly observed in lane b; in lane c, since there is no miRNA-21, the A sequence cannot be polymerized even in the presence of poly A enzyme, so there is no migration band in lane c; in lane d, a band with a slower migration speed than that in lane b is produced. This is because under the conditions of 37°C, Ecolibuffer, ATP and miRNA-21, poly A enzyme polymerizes the A sequence at the end of miRNA-21. Since the molecular weight of miRNA-21 increases after the poly A tail is extended, the migration band is only near the sample well, proving that the extension of miRNA-21 is successful.

[0073] Example 4

[0074] To verify the effect of poly(A) sequence extension on the efficiency of miRNA adsorption on AUE, the steps are as follows:

[0075] (1) Same as step (1) in Example 1.

[0076] (2) Same as step (2) in Example 1.

[0077] (3) Take 10 μl of the magnetic bead resuspension solution pretreated in step (2), add 10 μl of miRNA-21 sequence solution (0 nM or 2 nM), rotate and mix for 15 min, separate on a magnetic rack, discard the supernatant, wash the precipitate twice with Buffer I, and then add 7.5 μl of RNase-free water to the precipitate to resuspend it, then heat it at 95°C for 2 min, immediately separate on a magnetic rack, and collect the supernatant.

[0078] (4) Mix 1 μl of 10× E. coli Poly (A) Polymerase Reaction Buffer, 1 μl of ATP (10 mM), 1 μl of E. coli Poly (A) Polymerase, and 7.5 μl of the supernatant collected in step (3), incubate at 37°C for 10 min, and then add 20 μl of 5× SSC solution to obtain a resuspension.

[0079] (5) Bare electrode: The gold electrode activated in step (1) is used as the working electrode.

[0080] Without poly A extension + with / without target: 5 μl of the supernatant collected in step (3) was added dropwise to the gold electrode activated in step (1), incubated at room temperature for 10 min, rinsed the electrode surface with ddH2O, and dried with nitrogen to obtain a working electrode.

[0081] With poly A extension + with / without target: 5 μl of the resuspension obtained in step (4) was added dropwise to the gold electrode activated in step (1), incubated at room temperature for 10 min, rinsed the electrode surface with ddH2O, and dried with nitrogen to obtain a working electrode.

[0082] Without poly A extension + with / without target + MOF: 5 μl of the supernatant collected in step (3) was added dropwise to the gold electrode activated in step (1), and incubated at room temperature for 10 min. Then, 3 μl of zinc nitrate solution (40 mM) was added dropwise to the electrode, followed by 3 μl of 2-methylimidazole solution (160 mM), and incubated at room temperature for 30 min. The electrode surface was rinsed with ddH2O and dried with nitrogen to obtain a working electrode.

[0083] With poly A extension + with / without target + MOF: 5 μl of the resuspension obtained in step (4) was added dropwise to the gold electrode activated in step (1), and incubated at room temperature for 10 min. Then, 3 μl of zinc nitrate solution (40 mM) was added dropwise to the electrode, followed by 3 μl of 2-methylimidazole solution (160 mM), and incubated at room temperature for 30 min. The electrode surface was rinsed with ddH2O and dried with nitrogen to obtain a working electrode.

[0084] (6) The working electrode, the platinum wire electrode, and the Ag / AgCl electrode constitute a three-electrode system and perform measurement (differential pulse voltammetry). The detection conditions are the same as those in step (6) of Example 1.

[0085] from Figure 2 As shown in A, when there is no poly A extension, the signal values ​​of the bare electrode and with / without miRNA-21 are similar. Figure 2 As shown in Figure B, when poly(A) tails are added, the signal values ​​of the bare electrode and those without miRNA-21 are similar, while the signal with miRNA is 28.36% lower than that of the bare electrode. After MOF assembly, the current signal of miRNA-21 without poly(A) tails is reduced by 45.7%, while that of miRNA-21 with poly(A) tails is reduced by 96.4%. This is because the presence of miRNA-21 provides nucleation sites for MOF growth, which facilitates its growth. However, the absence of a poly(A) tail prevents efficient adsorption of miRNA-21 on the electrode, significantly reducing the MOF effect. Adenine has the highest affinity of all nucleic acid bases, so modifying miRNA-21 with a poly(A) tail promotes rapid and efficient adsorption of miRNA-21 to the gold electrode surface, facilitating subsequent detection.

[0086] Example 5

[0087] The DPV test was performed on miRNA-21 at different concentrations. The steps are as follows:

[0088] (1) Same as step (1) in Example 1.

[0089] (2) Same as step (2) in Example 1.

[0090] (3) Same as step (3) in Example 1.

[0091] (4) Same as step (4) in Example 1.

[0092] (5) Same as step (5) in Example 1.

[0093] (6) Same as step (6) in Example 1.

[0094] like Figure 3 As shown in Figure A, as the concentration of miRNA-21 increases, the DPV signal value decreases, and the rate of change increases. This is because after the target miRNA-21 is adsorbed on the gold electrode, the presence of the latter will cause the Coulomb repulsion of ferrocyanide ions away from the electrode surface, resulting in a lower Faradaic current relative to the bare electrode. Figure 3 B is based on Figure 3 The change rate calculated from the DPV signal of A showed a good linear relationship when the concentration range of miRNA-21 was 0.01fM~1000fM.

[0095] Example 6

[0096] To evaluate the specificity of the capture probe used in this sensor in selectively isolating the target miRNA-21, different miRNA types (miR-122, miR-141, miR-199, miR-210, and miR-21) were detected and the obtained DPV signals were compared with those of NoT. In this study, the concentration of all interfering miRNAs was set at 100 fM, while the concentration of the target miRNA-21 was set at 10 fM. The steps were as follows:

[0097] (1) Same as step (1) in Example 1.

[0098] (2) Same as step (2) in Example 1.

[0099] (3) Take 10 μl of the magnetic bead resuspension solution pretreated in step (2), add 10 μl of miRNA solution, rotate and mix for 15 minutes, separate on a magnetic rack, discard the supernatant, wash the precipitate twice with Buffer I, and then add 7.5 μl of RNase-free water to the precipitate to resuspend it, then heat it at 95°C for 2 minutes, immediately separate on a magnetic rack, and collect the supernatant.

[0100] (4) Mix 1 μl of 10× E. coli Poly (A) Polymerase Reaction Buffer, 1 μl of ATP (10 mM), 1 μl of E. coli Poly (A) Polymerase, and 7.5 μl of the supernatant collected in step (3), incubate at 37°C for 10 min, and then add 20 μl of 5× SSC solution to obtain a resuspension.

[0101] (5) 5 μl of the resuspension obtained in step (4) was added dropwise to the gold electrode activated in step (1), and incubated at room temperature for 10 min. Then, 3 μl of zinc nitrate solution (40 mM) was added dropwise to the electrode, followed by 3 μl of 2-methylimidazole solution (160 mM), and incubated at room temperature for 30 min. The electrode surface was rinsed with ddH2O and dried with nitrogen to obtain a MOF assembled electrode.

[0102] (6) Same as step (6) in Example 1.

[0103] Depend on Figure 4 It can be seen that the current signal of the constructed electrochemical biosensor for interfering miRNA is similar to the background signal of NoT, while the target miRNA-21 stimulates a weak current signal. This is because the probe used is perfectly complementary to the target miRNA-21 and can capture miRNA-21. Therefore, miRNA-21 can be assembled on the electrode to become the nucleation site of MOF. MOF encapsulates miRNA-21 inside, reducing the collision between nucleic acids and ions in the electrolyte solution, thus generating a weaker current.

[0104] Example 7

[0105] Measure miRNA-21 in cell biological samples.

[0106] After human embryonic kidney cells (293T), human cervical cancer cells (HeLa), human breast cancer cells (MCF-7), and human breast cancer cells (MDA-MB-231) were cultured to a certain density, the cells were washed, digested, and the RNA in the cells was extracted using the Tiangen RNApreppure culture cell / bacterial total RNA extraction kit, and the RNA concentration was tested using a microplate reader. RT-PCR experiments were performed according to the instructions of the Takara PrimeScript RTreagent Kit with gDNA Eraser (Perfect Real Time). At the same time, the sensor was used to perform experiments with reference to the method provided in Example 1. Except that the target added in step (3) was the extracted cellular miRNA-21, the remaining steps were the same as in Example 1 to investigate the performance of the biosensor in this study.

[0107] According to previous studies, miRNA-21 is expressed at higher levels in cancer cells (such as HeLa, MDA-MB-231, and MCF-7) and at lower levels in normal human cells (such as HEK293T). The results of the present invention show that the relative expression levels of miRNA-21 in different cell lines obtained by RT-PCR analysis are consistent with previous reports; the present invention further uses the proposed sensor to detect the expression level of miRNA-21. Figure 5 As can be seen, the relative expression levels of miRNA-21 in these cell lines, as reflected by the rate of change of the current signal, are consistent with RT-PCR analysis and previous research results in the literature. This result demonstrates that the proposed sensor can be successfully applied to measure the expression levels of miRNA-21 in different cell lines.

[0108] Example 8

[0109] The miRNA was extracted from serum samples of normal subjects and patients with non-small cell lung cancer (NSCLC) using the Xinhai Gene Whole Blood miRNA Extraction Kit, and the miRNA concentration was measured using a microplate reader. The extracted miRNA was converted into cDNA according to the instructions of the Takara PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) for RT-PCR. Electrochemical sensor detection was also performed according to the method provided in Example 1. Except that the target added in step (3) was miRNA-21 extracted from the patient's serum, the remaining steps were the same as in Example 1.

[0110] The results are as follows Figure 6 As shown, the biosensor constructed by the present invention is consistent with the results of RT-qPCR. The results show that the biosensor can be used as an alternative method for miRNA detection and has great potential for clinical application.

[0111] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy, characterized by: The following steps are involved: (1) Polish the bare gold electrode with alumina powder, clean it ultrasonically in ddH2O, anhydrous ethanol, and ddH2O, and blow dry it with nitrogen; The electrode was then placed in a 0.5 M sulfuric acid solution and subjected to cyclic voltammetry scanning at a scan rate of 0.1 V / s between -0.2 and +1.6 V. After the cyclic voltammetry curve stabilized, the electrode was removed and rinsed with ddH2O and dried with nitrogen to obtain an activated gold electrode. (2) Vortex 20µl of 10mg / ml streptavidin magnetic bead solution for 20s to obtain a uniformly dispersed magnetic bead dispersion; remove 20µl of magnetic bead dispersion, discard the supernatant after magnetic separation, and wash the magnetic beads twice with Buffer I; then add 80µl Buffer I and 20µl of 10µM biotinylated capture probe solution to the magnetic beads, vortex for 30min, discard the supernatant after magnetic separation, and wash the magnetic beads three times with Buffer I; then add 10µl of 5×SSC solution to the magnetic beads to obtain a pretreated magnetic bead resuspension; (3) Take 10µl of the magnetic bead resuspension solution pretreated in step (2), add 10µl of the target sequence, rotate and mix for 15 minutes, discard the supernatant after magnetic separation, wash the precipitate twice with Buffer I, and then add 7.5µl of RNase-free water to the precipitate, heat it at 95℃ for 2 minutes, and immediately perform magnetic separation to collect the supernatant containing the target sequence; (4) Add 1µl 10× E. coli Poly(A) Polymerase Reaction Buffer, 1µl 10mM ATP, 1µl E. coli Poly(A) Polymerase and 7.5µl of the supernatant containing the target sequence collected in step (3) were mixed evenly, incubated at 37°C for 10 min, and 20µl of 5×SSC solution was added to obtain a target solution with a polyA tail; (5) Pipette 5µl of the polyA tail target solution obtained in step (4) and drop it onto the gold electrode activated in step (1), and incubate at room temperature for 10 min. Then, drop 3µl of 40mM zinc nitrate solution onto the electrode, and then drop 3µl of 160mM 2-methylimidazole solution onto the electrode, and incubate at room temperature for 30 min. Rinse the electrode surface with ddH2O and blow dry with nitrogen to obtain a MOF assembled electrode. (6) placing the MOF assembled electrode obtained in step (5) in an electrochemical detection solution and scanning by differential pulse voltammetry; Wherein, the sequence of the biotinylated capture probe is: 5'-TTCAACATCAGTCTGATAAGCTATTT-3'-biotin.

2. The method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy according to claim 1, characterized in that: The formula of Buffer I is: 10 mM Tri-HCl, 1 mM EDTA, 1 M NaCl, 0.01%~0.1% Tween-20, pH=7.

5.

3. The method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy according to claim 1, characterized in that: The formula of the 5×SSC solution is: 0.75 M NaCl, 0.075 M sodium citrate, pH=7.

4. The method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy according to claim 1, characterized in that: The sequence of the target is: 5'-UAGCUUAUCAGACUGAUGUUGA-3'.

5. The method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy according to claim 1, characterized in that: The formula of the electrochemical detection solution is: 2.5 mM potassium ferrocyanide, 2.5 mM potassium ferrocyanide, 0.1 M KCl; pH=7.

6. The method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy according to claim 1, characterized in that: The conditions for the differential pulse voltammetry scan were: pulse width 0.05 s, sensitivity 1e -5 , starting potential -0.1V, maximum potential 0.5V.

7. An ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy constructed by the construction method according to claim 1.

8. Use of the ultrasensitive electrochemical sensor according to claim 7 in preparing a product for detecting miRNA-21.

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