CN116698944A - Ultrasensitive electrochemical sensor based on target-induced MOF growth strategy and construction and application thereof - Google Patents
Ultrasensitive electrochemical sensor based on target-induced MOF growth strategy and construction and application thereof Download PDFInfo
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Abstract
The invention discloses an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy, and construction and application thereof. The construction of the ultrasensitive electrochemical sensor is carried out according to the following steps: activating a bare gold electrode; pretreatment of streptavidin magnetic beads; specifically capturing a target; extension of miRNA poly A; assembling a metal organic frame electrode; differential pulse voltammetry detection. The invention does not need to add primer and specific enzyme, and is directly added into a reaction systemE.coliPoly (A) polymerase can directly polymerize Poly A sequence at the tail end of miRNA-21, promote the adsorption of miRNA-21 and the electrode surface, induce the growth of MOF, and realize the ultrasensitive detection of miRNA-21.
Description
Technical Field
The invention relates to the technical field of electrochemical sensors, in particular to an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy, and construction and application thereof.
Background
Lung cancer is a malignant tumor with worldwide morbidity and mortality second to breast cancer, severely compromising human health. Histologically, lung cancer can be classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Among them, NSCLC is a major type of lung cancer, accounting for about 85% of all lung cancers, including Adenocarcinoma (AD), squamous Cell Carcinoma (SCC), and large cell lung cancer (LCC). Because NSCLC lacks typical early symptoms, patients are diagnosed at mid-to late stages, losing the best chance of surgery, resulting in a 5-year survival rate of only 5% -15%, whereas NSCLC patients diagnosed early and surgically resected have a 5-year survival rate as high as 50% -70%. Therefore, improving the early diagnosis efficiency of NSCLC plays an important role in improving the prognosis of patients, but the current conventional screening projects have insufficient capability for early diagnosis of NSCLC, such as the conventional dose CT screening in imaging methods and the capability of identifying benign and malignant lung nodules, and the low dose CT has the defects of higher false positive rate and the like.
MicroRNA (miRNA) is an endogenous non-coding small RNA, generally 18-22 nucleotides in length, which can regulate the expression of complementary mRNA, and plays an important role in gene translation, and the deletion or reduction of corresponding proteins leads to the occurrence of diseases. Studies have shown that aberrant expression of miRNAs is associated with a variety of diseases, particularly tumors such as neuroblastoma, pituitary adenoma, thyroid cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, colorectal cancer, cervical cancer, leukemia, and the like, and that these miRNAs are located genomically at fragile sites (fragilesites) associated with tumors. In addition to the presence of abnormal expression in tissues, it can also be released into the blood. Recent researches indicate that serum miRNA can be used as an ideal marker for noninvasive diagnosis of tumors. However, the accurate detection and quantitative analysis of miRNA in blood still has challenges at present, because: 1) The miRNA molecule sequence is short and the sequence similarity among all members is high, and 2) the content of miRNA in human body is low. Therefore, the development of the quantitative analysis method of miRNA with high sensitivity and high specificity to detect miRNA in blood and monitor the trend of variation of the type and the quantity of the miRNA has great significance for monitoring tumor dynamics and evaluating treatment effect in real time and realizing individual treatment.
By now, researchers have developed a variety of methods for detecting mirnas, including northern blot analysis with radiolabeled probes, based on microarrays [18] And based on a polymeraseMethods of chain reaction (PCR), liquid phase single cell assays, in situ hybridization, and high throughput sequencing. Although the RNA blot analysis is a reliable detection technology at present, the RNA blot analysis has the problems of low sensitivity, low flux, large amount of RNA samples required for detection, easy degradation of RNA in the experimental process and the like; real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) needs to reverse RNA into cDNA and then carry out amplification detection, has good sensitivity, specificity and reproducibility, but is easy to cause problems in the reverse transcription process, needs expensive instruments and fluorescent markers for fluorescent reading, and is easy to be interfered by background fluorescence; the core technology of microarray analysis is hybridization of a target object and a probe, and miRNA is quantitatively analyzed by detecting fluorescence intensity. The method can realize high-throughput and one-time analysis of the whole genome of a human, but has relatively poor reproducibility and accuracy, and is generally used for primary screening. In summary, although there are many methods for detecting mirnas, there are generally the following limitations: (1) low detection sensitivity and accuracy; (2) cumbersome, complex and time-consuming steps of operation; (3) The partial detection mode needs to use a corresponding kit and instrument, and has high price, high cost and the like.
Electrochemical biosensors have been receiving wide attention in the field of miRNAs detection because of their high specificity, low detection limit, low cost, high sensitivity, and the like. Electrochemical biosensors are self-integrated devices that combine the sensitivity of electroanalytical methods with the selectivity of biological components to provide quantitative or semi-quantitative analytical information through direct spatial contact of a biological recognition element with an electrochemical transduction element. Typical electrochemical biosensors based on DNA hybridization have been developed for the detection of miRNAs, ranging from picomolar (pM) to nanomolar (nM). The modified electrochemical biosensor improves the detection sensitivity to 10fM by improving the adsorption efficiency between miRNA and gold electrodes, but the method still cannot meet the actual requirements of clinical detection because the content of the circulating miRNA in body fluid is extremely low.
In order to improve the detection sensitivity and accuracy of the sensor, researchers adopt different functional materials such as nanometer materials to modify the method and technology of the electrodeVarious signal amplification strategies have been developed. Among the functional materials, metal-organic frameworks (Metal-OrganicFrameworks, MOFs) have received extensive attention from various groups of researchers since their own market. MOFs is a novel porous material formed by inorganic metal ions and organic ligands containing nitrogen or oxygen in a self-assembly mode, and has the advantages of adjustable porosity, adjustable pore diameter, large specific surface area, stable chemical and mechanical properties and the like. MOFs are widely used in gas storage and separation, as found by the Yaghi research group 2003 in which MOF-5 stores H 2 The method comprises the steps of carrying out a first treatment on the surface of the MOFs in the catalytic field can be prepared by adjusting the aperture and channel size of MOFs through adjusting metal nodes and organic ligands, adjusting the catalytic capacity of MOFs, and can also be prepared by modifying and adjusting after synthesis, and the MOFs are hybridized with other materials to improve the catalytic activity of MOFs; the tunability of the pore size and structure of MOFs and derivatives thereof endows the MOFs and derivatives thereof with the capability of loading substances such as bioactive macromolecules, drugs and nanomaterials such as AuNPs, ptNPs and the like. MOFs are also commonly used in the field of electrochemical biosensor research: the bimetal MOF material with electric activity is constructed by utilizing Ni+ and Co2+ as Hu and the like and is used for detecting miRNA-126; li and the like construct MOF@Pt@MOF nano enzyme with a catalase-like effect by using MIL-88 and PtNPs, and generate cascade amplification signals through a primer exchange reaction, so as to detect the content of exosome miRNA-21 in breast cancer patients and healthy subjects.
Based on the above, the subject aims to develop an electrochemical signal transduction mode based on a target-induced MOFs (metal-organic frameworks) growth strategy, and combines a magnetic bead separation and RNA (ribonucleic acid) molecule poly-A sequence extension technology to construct an ultrasensitive novel electrochemical sensor for detecting lung cancer related miRNA-21, so that a safe, reliable, rapid and sensitive detection method is provided for early clinical screening and diagnosis of NSCLC.
Disclosure of Invention
The invention aims to provide an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy, and construction and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for constructing an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy, comprising the steps of:
(1) Polishing bare gold electrode with alumina powder, sequentially at ddH 2 O, absolute hexanol, ddH 2 Ultrasonic cleaning in O and drying with nitrogen; then the electrode is placed in 0.5M sulfuric acid solution to conduct cyclic voltammetry scanning between-0.2 and +1.6V potential at a scanning rate of 0.1V/s until the cyclic voltammetry curve is stable, and the electrode is taken out for ddH 2 O is washed clean, and nitrogen is dried to obtain an activated gold electrode;
(2) Vortex oscillating 20 mu l of 10mg/ml streptavidin magnetic bead solution for 20s to obtain uniformly dispersed magnetic bead dispersion liquid; transferring 20 μl of the magnetic bead dispersion, magnetically separating, discarding supernatant, and washing the magnetic beads with BufferI for 2 times; then 80. Mu.l of BufferI and 20. Mu.l of 10. Mu.M biotinylated capture probe solution are added to the magnetic beads, vortex shaking is carried out for 30min, the supernatant is discarded after magnetic separation, and the magnetic beads are washed 3 times by using Buffer I; then 10 mul of 5 XSSC solution is added into the magnetic beads to obtain pretreated magnetic bead heavy suspension;
(3) Taking 10 mu l of the magnetic bead heavy suspension pretreated in the step (2), adding 10 mu l of target sequence, rotating and mixing for 15min, performing magnetic separation, discarding supernatant, washing precipitate with BufferI for 2 times, adding 7.5 mu l of Rnase-freewater into the precipitate, heating at 95 ℃ for 2min, performing magnetic separation immediately, and collecting supernatant containing the target sequence;
(4) Uniformly mixing 1 mu l of 10 XE.coliPoy (A) Polymerase reaction buffer, 1 mu l of 10mM TP, 1 mu l of E.coliPoy (A) Polymerase and 7.5 mu l of the supernatant containing the target sequence collected in the step (3), incubating at 37 ℃ for 10min, and adding 20 mu l of 5 XSSC solution to obtain a solution with a polyA tail target;
(5) Transferring 5 μl of the target solution with polyA tail obtained in the step (4), dripping onto the gold electrode activated in the step (1), and incubating for 10min at room temperature; then 3. Mu.l of 40mM zinc nitrate solution and 3. Mu.l of 160mM 2-methylimidazole solution were added dropwise to the electrode, incubated at room temperature for 30min, ddH 2 O flushing the surface of the electrode, and drying with nitrogen to obtain an MOF assembled electrode;
(6) Placing the MOF assembly electrode obtained in the step (5) in an electrochemical detection liquid, and scanning by using a differential pulse voltammetry;
wherein the sequence of the biotinylated capture probe is: 5'-TTCAACATCAGTCTGATAAGCTATTT-3' -biotin;
the formula of the BufferI is as follows: 10mM Tris-HCl, 1mM EDTA, 1M NaCl, 0.01% -0.1% Tween-20, pH=7.5;
the formula of the 5 XSSC solution is as follows: 0.75MNaCl, 0.075M sodium citrate, ph=7;
the sequence of the target is as follows: 5'-UAGCUUAUCAGACUGAUGUUGA-3';
the formula of the electrochemical detection liquid is as follows: 2.5mM potassium ferricyanide, 2.5mM potassium ferrocyanide, 0.1m kcl, ph=7;
the conditions of the differential pulse voltammetry scanning are as follows: pulse width 0.05s, sensitivity 1e -5 The initial potential is-0.1V, and the highest potential is 0.5V.
An ultrasensitive electrochemical sensor based on target-induced MOF growth strategy constructed by the construction method.
The application of the ultrasensitive electrochemical sensor based on the target-induced MOF growth strategy in the preparation of the miR-21 detection product is provided.
The detection principle of the invention is as follows:
firstly, specifically capturing target miRNA-21 through a capture probe on the surface of a streptavidin magnetic bead, performing thermal denaturation release, and then performing poly-A sequence extension on the 3' end of the miRNA-21 by using E.colipoly (A) polymerase, and fixing the poly-A tail on the surface of an electrode by utilizing the specific combination effect of the poly-A tail and a gold electrode (AuE); by dripping a precursor solution of ZIF-8 MOFs on the surface of the electrode, taking a poly-A extension product of miRNA-21 as a nucleation site, inducing the in-situ growth of ZIF-8 on AuE, further blocking the electron transfer on the surface of the electrode, generating an 'attenuation' -type electrochemical detection signal, and realizing the ultra-sensitive detection of miRNA-21.
The invention has the remarkable advantages that:
(1) The electrochemical technology has the advantages of simplicity, low cost, quick response, sensitivity and the like.
(2) Through the self-assembly process of MOFs on the miRNA modified electrode, a multifunctional sensing interface is constructed, and the MOFs with the nano structure can complete various tasks, such as packaging target objects in the MOFs to form a closed body, reducing ion collision, forming attenuation signals and the like.
(3) The MOFs signal amplification strategy can sensitively detect microRNA with extremely low content in body fluid.
(4) The E.colipoly (A) polymerase is directly added into a reaction system without adding primers and specific enzymes, so that a poly-A sequence can be directly polymerized at the tail end of the miRNA-21, the adsorption of the miRNA-21 and the electrode surface is promoted, the growth of MOF is induced, and the ultrasensitive detection of the miRNA-21 is realized.
Drawings
Fig. 1: a: characterization of the electrode; a: activating a gold electrode, b: MOF assembled electrodes. B: performing polyacrylamide gel electrophoresis analysis on poly A tail extension; lane a: a Marker; lane b: a sample (2); lane c: a sample (3); lane d: sample (1).
Fig. 2: DPV signal before poly A extension (A) and after poly A extension (B). a: activating a gold electrode, b: no target, c: target object, d: no target + MOF, e: there is a target + MOF.
Fig. 3: (A) DPV signals corresponding to miRNA-21 (0, 0.01, 0.1, 1, 10, 100, 1000 fM) at different concentrations, and (B) a calibration curve corresponding to the logarithm of the concentration of miRNA-21.
Fig. 4: peak currents of different mirnas.
Fig. 5: RT-PCR and sensors detect the expression level of miRNA-21 in the total miRNA sample of the cancer cell line.
Fig. 6: RT-PCR (left) and sensor (right) detect the expression level of miRNA-21 in the total miRNA sample in human serum.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
In the following examples, the biotinylated probes were: 5'-TTCAACATCAGTCTGATAAGCTATTT-3' -biotin.
In the following examples, the miRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3'.
In the following examples, the miRNA-122 is: 5'-UGGAGUGUGACAAUGGUGUUUG-3'.
In the following examples, the miRNA-141 is: 5'-UAACACUGUCUGGUAAAGAUGG-3'.
In the following examples, the miRNA-199 is: 5'-ACAGUAGUCUGCACAUUGGUUA-3'.
In the following examples, the miRNA-210 is: 5'-AGCCCCUGCCCACCGCACACUG-3'.
In the following examples, the BufferI formulation is: 10mM Tris-HCl, 1mM EDTA, 1M NaCl, 0.01% -0.1% Tween-20, pH=7.5.
In the following examples, the 5 XSSC solution formulation was: 0.75M naci, 0.075M sodium citrate, ph=7.
Example 1
The construction of the ultrasensitive electrochemical sensor based on the target induced MOF growth strategy comprises the following steps:
(1) Activation of gold electrodes
The bare gold electrode was polished with 0.3 μm and 0.05 μm alumina powder in sequence, followed by ddH in sequence 2 Ultrasonically cleaning O, absolute hexanol and ddH2O for 3min, and drying with nitrogen; and then placing the electrode in sulfuric acid solution (0.5M), performing cyclic voltammetry scanning between-0.2 and +1.6V potential at a scanning rate of 0.1V/s to clean the electrode until the cyclic voltammetry curve is stable, taking out the electrode, washing the electrode with ultrapure water, and drying with nitrogen to obtain the activated gold electrode.
(2) Pretreatment of magnetic beads
Placing 20 mu l of streptavidin magnetic bead solution (10 mg/ml) on a vortex oscillator to oscillate for 20s to obtain uniformly dispersed magnetic bead dispersion liquid; transferring 20 μl of the magnetic bead dispersion, separating with a magnetic frame, discarding supernatant, and cleaning the magnetic beads with BufferI for 2 times; then 80. Mu.l of BufferI and 20. Mu.l of biotinylation probe (10. Mu.M) are added into the magnetic beads, the mixture is oscillated on a vortex oscillator for 30min, a magnetic frame is separated, the supernatant is discarded, and the magnetic beads are washed 3 times by using BufferI; then, 10. Mu.l of a 5 XSSC solution was added to the magnetic beads to resuspend, thereby obtaining a pretreated magnetic bead resuspension.
(3) Capture-specific miRNA-21
Taking 10 mu l of the magnetic bead heavy suspension pretreated in the step (2), adding 10 mu lmiRNA-21 sequence solution, rotating and mixing for 15min, separating by a magnetic frame, discarding the supernatant, washing the precipitate with BufferI for 2 times, adding 7.5 mu l of Rnase-freewater into the precipitate to resuspension, heating at 95 ℃ for 2min, immediately separating by the magnetic frame, and collecting the supernatant containing the miRNA-21 sequence.
(4) MiRNA poly-A extension
Mu.l of 10 XE.coliPoy (A) Polymerase reaction buffer, 1. Mu.l of ATP (10 mM), 1. Mu.l of E.coliPoy (A) Polymerase, 7.5. Mu.l of the supernatant containing the miRNA-21 sequence collected in the step (3) were mixed uniformly, incubated at 37℃for 10min, and then 20. Mu.l of a 5 XSSC solution was added to obtain a solution having a polyA tail miR-21.
(5) Assembly of MOFs
Transferring 5 μl of the solution with the polyA tail miR-21 obtained in the step (4), dripping the solution onto the gold electrode activated in the step (1), and incubating for 10min at room temperature; then 3. Mu.l of zinc nitrate solution (40 mM) and 3. Mu.l of 2-methylimidazole solution (160 mM) were added dropwise to the electrode, incubated at room temperature for 30min, ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the MOF assembled electrode.
(6) Measurement of
The electrochemical impedance spectrum detection uses a three-electrode system, which comprises a working electrode, a counter electrode and a reference electrode, wherein the MOF assembly electrode obtained in the step (5) is used as the working electrode, the platinum electrode is used as the counter electrode, the Ag/AgCl electrode is used as the reference electrode, and the formula of the detection solution is as follows: 2.5mM potassium ferricyanide, 2.5mM potassium ferrocyanide, 0.1MKCl, pH=7, detection parameters are: after the computer and electrochemical workstation instrument are successfully connected, clicking Control-OpenCircuitPotential-then a number appears on the computer software page, remembering this number, returning to Setup-IMPAC-then selecting parameters: lenitE (V) =the number just remembered, highFreq (Hz) =1e +5 ,LowFreq(Hz)＝0.1。
The differential pulse voltammetry detection uses a three-electrode system, which comprises a working electrode, a counter electrode and a reference electrode, wherein the MOF assembly electrode obtained in the step (5) is used as the working electrode, the platinum electrode is used as the counter electrode, the Ag/AgCl electrode is used as the reference electrode, and the formula of the detection solution is as follows: 2.5mM potassium ferricyanide, 2.5mM potassium ferrocyanide, 0.1MKCl, pH=7, detection parameters are: the electrodes were placed in the detection solution so that PulseWidth(s) =0.05 and sensitivity (A/V) =1e -5 Is of (1)The scan rate was voltammetric scanned between lnitE (V) = -0.1, high E (V) = 0.5 potential.
The cyclic voltammetry detection uses a three-electrode system, which comprises a working electrode, a counter electrode and a reference electrode, wherein the MOF assembly electrode obtained in the step (5) is used as the working electrode, the platinum electrode is used as the counter electrode, the Ag/AgCl electrode is used as the reference electrode, and the formula of the detection solution is as follows: 2.5mM potassium ferricyanide, 2.5mM potassium ferrocyanide, 0.1MKCl, pH=7, detection parameters are: lnitE (V) = -0.2, highe (V) = 0.6, lowe (V) = -0.2, sensitivity (a/V) = 1e -5 。
Example 2
Characterization on MOF assembled electrodes was as follows:
(1) Step (1) was performed as in example 1.
(2) 3. Mu.l of zinc nitrate solution (40 mM) and 3. Mu.l of 2-methylimidazole solution (160 mM) were added dropwise to the gold electrode after the activation in step (1), and incubated at room temperature for 30min, ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the MOF assembled electrode.
(3) And (3) respectively carrying out cyclic voltammetry detection on the activated gold electrode and the MOF assembled electrode obtained in the step (2), wherein the detection conditions are the same as those in the step (6) of the example 1.
As shown in fig. 1A, the peak current drop was significant for the MOF assembled electrode (curve b) compared to the activated gold electrode (curve a) due to the poor conductivity of the MOF. Thus, preliminary verification of MOFs successfully assembled on gold electrodes.
Example 3
Characterization of poly-a sequence extension, the procedure is as follows:
configuration sample (1): mu.L of 1. Mu.M miRNA-21, 0.7. Mu.L of LEcolibuffer, 0.7. Mu.LATP and 0.7. Mu. LEcoli polyAmerse were mixed and incubated at 37℃for 10min, and then 1. Mu.L of 6 XLoadingbuffer and 10 Xnucleic acid dye were added. Configuration sample (2): mu.L of 1. Mu.M miRNA-21, 1. Mu.L of 6 XLoadingbuffer and 10 Xnucleic acid dye were mixed. Configuration sample (3): 0.7. Mu. LEcolibuffer, 0.7. Mu. LATP, 0.7. Mu. LEcolipolyAmerse, 1. Mu.L of 6 XLoadingbuffer, 10 Xnucleic acid dye were mixed. Polyacrylamide gel electrophoresis was performed.
As shown in FIG. 1B, miRNA-21 of about 20bp in size was clearly observed in lane B; in lane c, however, no migration band was present since no miRNA-21 would not polymerize the A sequence even in the presence of the polymerase; in lane d a band was generated with slower migration than lane b, because the poly A enzyme polymerizes the A sequence at the end of miRNA-21 in the presence of miRNA-21 at 37℃ Ecolibuffer, ATP, and since the molecular weight becomes large after extension of the poly A tail by miRNA-21, migration bands were only near the spotted wells, demonstrating successful extension of miRNA-21.
Example 4
The effect of poly (a) sequence extension on the efficiency of miRNA adsorption at AUE was verified as follows:
(1) Step (1) was performed as in example 1.
(2) Step (2) was performed as in example 1.
(3) Taking 10 mu.l of the magnetic bead heavy suspension pretreated in the step (2), adding 10 mu.l of an iRNA-21 sequence solution (0 nM or 2 nM), rotating and mixing for 15min, separating by a magnetic frame, discarding the supernatant, washing the precipitate for 2 times by using BufferI, adding 7.5 mu.l of Rnase-freemaker into the precipitate to resuspension, heating at 95 ℃ for 2min, immediately separating by the magnetic frame, and collecting the supernatant.
(4) Mu.l of 10 XE.coliPoy (A) Polymerase reaction buffer, 1. Mu.l of ATP (10 mM), 1. Mu.l of E.coliPoy (A) Polymerase, 7.5. Mu.l of the supernatant collected in step (3) were mixed uniformly, incubated at 37℃for 10min, and then 20. Mu.l of a 5 XSSC solution was added to obtain a resuspension.
(5) Bare electrode: and (3) taking the gold electrode after the activation in the step (1) as a working electrode.
No poly a extension + presence/absence of target: transferring 5 μl of the supernatant collected in step (3), dripping onto the activated gold electrode in step (1), incubating at room temperature for 10min, and ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the working electrode.
With poly a extension + with/without targets: transferring 5 μl of the heavy suspension obtained in step (4), dripping onto the activated gold electrode in step (1), incubating at room temperature for 10min, and ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the working electrode.
No poly a extension + with/without target + MOF: transferring 5 μl of the supernatant collected in step (3), dripping onto the activated gold electrode in step (1), incubating at room temperature for 10min, and dripping 3 μl of nitric acid onto the electrodeZinc solution (40 mM), 3. Mu.l of 2-methylimidazole solution (160 mM) was added dropwise thereto, and incubated at room temperature for 30min, ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the working electrode.
With poly a extension + with/without target + MOF: transferring 5 μl of the heavy suspension obtained in step (4), dripping onto the gold electrode activated in step (1), incubating at room temperature for 10min, dripping 3 μl of zinc nitrate solution (40 mM) onto the electrode, dripping 3 μl of 2-methylimidazole solution (160 mM), incubating at room temperature for 30min, and ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the working electrode.
(6) The working electrode, the platinum wire electrode and the Ag/AgCl electrode form a three-electrode system, and measurement (differential pulse voltammetry) is carried out, and the detection conditions are the same as in the step (6) of the example 1.
As seen from fig. 2A, the bare electrode was similar to the signal value with/without miRNA-21 when no poly a extension was present. As seen from fig. 2B, when there is extension of poly a, the signal value of the bare electrode is similar to that of no miRNA-21, whereas the signal with miRNA is 28.36% lower than that of the bare electrode. After MOF assembly, the current signal of miRNA-21 not terminally subjected to poly-a extension was reduced by 45.7%, while the current signal of miRNA-21 subjected to poly-a extension was reduced by 96.4%, since the presence of miRNA-21 may provide nucleation sites for MOF, contributing to the growth of MOF. And because no poly A tail is arranged, miRNA-21 cannot be adsorbed on the electrode efficiently, so that the MOF effect is greatly reduced, adenine has the highest affinity in all nucleic acid bases, and therefore, the poly A tail is used for modifying miRNA-21, the rapid and efficient surface adsorption of miRNA-21-gold electrode can be promoted, and the subsequent detection is facilitated.
Example 5
DPV detection is carried out on miRNA-21 with different concentrations, and the steps are as follows:
(1) Step (1) was performed as in example 1.
(2) Step (2) was performed as in example 1.
(3) Step (3) was performed as in example 1.
(4) Step (4) was performed as in example 1.
(5) Step (5) was performed as in example 1.
(6) Step (6) was performed as in example 1.
As shown in fig. 3A, the smaller the DPV signal value, the greater the rate of change, as the miRNA-21 concentration is higher, because the presence of the latter, after adsorption of the target miRNA-21 on the gold electrode, causes coulombic repulsion of ferricyanide ions away from the electrode surface, resulting in a lower faraday current relative to the bare electrode. FIG. 3B shows the change rate calculated from the DPV signal of FIG. 3A, and the DPV signal has a good linear relationship when the concentration of miRNA-21 ranges from 0.01fM to 1000 fM.
Example 6
To assess the specificity of the capture probes used in the sensor in selectively isolating target miRNA-21, different miRNA types (miR-122, miR-141, miR-199, miR-210, miR-21) were detected and the obtained DPV signals were compared to NoT. In this study, the concentrations of all interfering miRNAs were set at 100fM, while the concentration of the target miRNA-21 was set at 10fM, as follows:
(1) Step (1) was performed as in example 1.
(2) Step (2) was performed as in example 1.
(3) Taking 10 mu l of the magnetic bead heavy suspension pretreated in the step (2), adding 10 mu lmiRNA solution, rotating and mixing for 15min, separating by a magnetic frame, discarding supernatant, washing sediment with BufferI for 2 times, adding 7.5 mu l of Rnase-freewater into the sediment for heavy suspension, heating at 95 ℃ for 2min, immediately separating by the magnetic frame, and collecting supernatant.
(4) Mu.l of 10 XE.coliPoy (A) Polymerase reaction buffer, 1. Mu.l of ATP (10 mM), 1. Mu.l of E.coliPoy (A) Polymerase, 7.5. Mu.l of the supernatant collected in step (3) were mixed uniformly, incubated at 37℃for 10min, and then 20. Mu.l of a 5 XSSC solution was added to obtain a resuspension.
(5) Transferring 5 μl of the heavy suspension obtained in step (4), dripping onto the gold electrode activated in step (1), incubating at room temperature for 10min, dripping 3 μl of zinc nitrate solution (40 mM) onto the electrode, dripping 3 μl of 2-methylimidazole solution (160 mM), incubating at room temperature for 30min, and ddH 2 And O flushing the surface of the electrode, and drying with nitrogen to obtain the MOF assembled electrode.
(6) Step (6) was performed as in example 1.
As can be seen from fig. 4, the current signal of the constructed electrochemical biosensor for interfering miRNA is similar to the background signal of the non, and the target miRNA-21 excites a weak current signal, because the probe used is perfectly complementary to the target miRNA-21, and can capture miRNA-21, so that miRNA-21 can be assembled on the electrode to form a nucleation site of MOF, and the MOF encapsulates miRNA-21 in, so that the collision between nucleic acid and ions in the electrolyte solution is reduced, and weak current is generated.
Example 7
And (3) measuring miRNA-21 in the cell biological sample.
After the human embryo kidney cells (293T), the human cervical cancer cells (HeLa), the human breast cancer cells (MCF-7) and the human breast cancer cells (MDA-MB-231) are cultured to a certain density, the cells are washed, digested and the like, RNA in the cells is extracted by adopting a Tiangen RNAprepure cultured cell/bacteria total RNA extraction kit, and the concentration of the RNA is tested by adopting an enzyme-labeled instrument. RT-PCR experiments were performed according to the instructions of TakaraPrimeScriptRTreagentKitwithgDNAEraser (PerfectRealTime). Meanwhile, experiments were performed by using the sensor according to the method provided in example 1, and the rest of the steps were the same as example 1 except that the target added in step (3) was the extracted cellular miRNA-21, so as to investigate the performance of the biosensor in the present study.
According to previous studies, miRNA-21 is reported to be expressed at higher levels in cancer cells (e.g., heLa, MDA-MB-231 and MCF-7) and at lower levels in normal human cells (e.g., HEK 293T). The results of the invention show that the relative expression quantity of miRNA-21 obtained by RT-PCR analysis in different cell lines is consistent with that of the prior report; the present invention further uses the proposed sensor to detect the expression level of miRNA-21, and it can be seen from FIG. 5 that the relative expression level of miRNA-21 in these cell lines reflected by the rate of change of the current signal is consistent with the results of the previous studies in RT-PCR analysis and literature. This result shows that the sensor proposed by the present invention can be successfully applied to the measurement of the expression level of miRNA-21 in different cell lines.
Example 8
And extracting miRNA in serum samples of normal human and non-small cell lung cancer (NSCLC) patients by using a new sea gene whole blood miRNA extraction kit, and testing the miRNA concentration by using an enzyme-labeling instrument. The extracted miRNA was inverted into cDNA according to the instructions of TakaraPrimeScriptRTreagentKitwith gDNAEraser (PerfectRealTime) for RT-PCR experiments. The electrochemical sensor test was performed in the same manner as in example 1, except that the target added in step (3) was miRNA-21 in the extracted patient serum.
The results are shown in FIG. 6, and the biosensor constructed by the present invention is consistent with the results of RT-qPCR. The result shows that the sensor can be used as an alternative method for miRNA detection and has great potential for clinical application.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (8)
1. A construction method of an ultrasensitive electrochemical sensor based on a target-induced MOF growth strategy is characterized by comprising the following steps: the method comprises the following steps:
(1) Polishing bare gold electrode with alumina powder, sequentially at ddH 2 O, absolute hexanol, ddH 2 Ultrasonic cleaning in O and drying with nitrogen; then the electrode is placed in 0.5M sulfuric acid solution to carry out cyclic voltammetry scanning between-0.2 to +1.6V potential at a scanning rate of 0.1V/s until the cyclic voltammetry curve is stable, and the electrode is taken out to use ddH 2 O is washed clean, and nitrogen is dried to obtain an activated gold electrode;
(2) Vortex oscillating 20 mu l of 10mg/ml streptavidin magnetic bead solution for 20s to obtain uniformly dispersed magnetic bead dispersion liquid; transferring 20 mu l of magnetic bead dispersion liquid, performing magnetic separation, discarding supernatant, and cleaning the magnetic beads for 2 times by using Buffer I; adding 80 μl Buffer I and 20 μl10 μM biotinylation capture probe solution into the magnetic beads, carrying out vortex oscillation for 30min, carrying out magnetic separation, discarding the supernatant, and cleaning the magnetic beads for 3 times by using the Buffer I; adding 10 mul of 5 XSSC solution into the magnetic beads to obtain pretreated magnetic bead heavy suspension;
(3) Taking 10 mu l of the magnetic bead heavy suspension pretreated in the step (2), adding 10 mu l of target sequences, rotating and mixing for 15min, performing magnetic separation, discarding supernatant, washing precipitate for 2 times by using Buffer I, adding 7.5 mu l of Rnase-free water into the precipitate, heating at 95 ℃ for 2min, performing magnetic separation immediately, and collecting supernatant containing the target sequences;
(4) 1 μl of 10×E.coli Poly(A) Polymerase Reaction Buffer、1µl 10mM ATP、1µl E.coliUniformly mixing the supernatant containing the target sequence collected in the step (3) of Poly (A) and 7.5 mu l, incubating for 10min at 37 ℃, and adding 20 mu l of 5 XSSC solution to obtain a target solution with a polyA tail;
(5) Transferring 5 mu l of the target solution with the polyA tail obtained in the step (4), dripping the target solution on the gold electrode activated in the step (1), and incubating for 10 minutes at room temperature; then 3 μl of 40mM zinc nitrate solution is dripped on the electrode, 3 μl of 160mM 2-methylimidazole solution is dripped, incubation is carried out at room temperature for 30min, and ddH is carried out 2 O flushing the surface of the electrode, and drying with nitrogen to obtain an MOF assembled electrode;
(6) Placing the MOF assembly electrode obtained in the step (5) in an electrochemical detection liquid, and scanning by using a 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 target-induced MOF growth strategy of claim 1, wherein the method comprises the following steps: the formula of Buffer I is as follows: 10mM Tri-HCl, 1mM EDTA, 1M NaCl, 0.01% -0.1% Tween-20, pH=7.5.
3. The method for constructing an ultrasensitive electrochemical sensor based on target-induced MOF growth strategy of claim 1, wherein the method comprises the following steps: the formula of the 5 XSSC solution is as follows: 0.75M NaCl, 0.075M sodium citrate, ph=7.
4. The method for constructing an ultrasensitive electrochemical sensor based on target-induced MOF growth strategy of claim 1, wherein the method comprises the following steps: the sequence of the target is as follows: 5'-UAGCUUAUCAGACUGAUGUUGA-3'.
5. The method for constructing an ultrasensitive electrochemical sensor based on target-induced MOF growth strategy of claim 1, wherein the method comprises the following steps: the formula of the electrochemical detection liquid is as follows: 2.5mM potassium ferricyanide, 2.5mM potassium ferrocyanide, 0.1M KCl; ph=7.
6. The method for constructing an ultrasensitive electrochemical sensor based on target-induced MOF growth strategy of claim 1, wherein the method comprises the following steps: the conditions of the differential pulse voltammetry scanning are as follows: pulse width 0.05s, sensitivity 1e -5 The initial potential is-0.1V, and the highest potential is 0.5V.
7. An ultrasensitive electrochemical sensor based on target-induced MOF growth strategy constructed by the construction method of claim 1.
8. The use of the ultrasensitive electrochemical sensor of claim 7 in the preparation of a product for detecting miRNA-21.
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