E-DNA sensor based on size exclusion effect of protein monolayer and its application

Abstract

The present invention discloses an E-DNA sensor constructed based on the size exclusion effect of a protein monolayer and its application. The E-DNA sensor comprises an Ag / AgCl reference electrode, a platinum wire counter electrode and a working electrode, wherein the working electrode is obtained by constructing a BSA self-assembled film on the surface of an AuE electrode and modifying a ssDNA probe for miRNA-21. The E-DNA sensor has a wide calibration range of 0.001-100 pM and a low detection limit of 0.48 fM, and can successfully achieve accurate evaluation of miRNA-21 expression levels in cancer cell lines and non-small cell lung cancer (NSCLC) serum samples.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical sensors, and in particular to an E-DNA sensor constructed based on the size exclusion effect of a protein monolayer and applications thereof. Background Art

[0002] Electrochemical analysis methods are an emerging analytical technique with many advantages, such as rapid response, high sensitivity, low cost, simple fabrication, and suitability for large-scale production. Among them, electrochemical DNA (E-DNA) sensors, which are mainly composed of electrodes and specific immobilized DNA probes, are widely used to detect different DNA-binding molecules. With the development of new functional DNA probes such as aptamers, DNAzymes, G-quadruplex, and i-motif configurations, the range of analytes for E-DNA sensors has been greatly expanded, including drug compounds, proteins, metal ions, pollutants, etc., which can be easily detected by E-DNA sensors with suitable DNA probes. In general, the operation of E-DNA sensors depends on the interaction of analytes with immobilized DNA probes, which is accompanied by different predetermined events related to DNA (e.g., hybridization, digestion, cleavage, or conformational changes of DNA chains) and causes changes in electrochemical parameters (e.g., capacitance or conductivity). Unfortunately, the response signal of E-DNA sensors is deeply affected by several parameters of the immobilized DNA probes, such as surface density, conformation, and length. Therefore, the methods for making sensing electrodes vary depending on the specific circumstances of the DNA probe and the analyte. In addition, the immobilization procedure of DNA probes is cumbersome and time-consuming due to the need for multi-step surface modification on electrodes. These shortcomings limit the further application of E-DNA sensors. Therefore, finding a universal and simple method to make sensing electrodes is crucial to promote the practical application of E-DNA sensors. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies in the prior art and to provide an E-DNA sensor constructed based on the size exclusion effect of a protein monolayer and its application.

[0004] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0005] An E-DNA sensor based on the size exclusion effect of a protein monolayer, the E-DNA sensor comprising an Ag / AgCl reference electrode, a platinum wire counter electrode and a working electrode, wherein the working electrode is obtained by constructing a BSA self-assembled film on the surface of an AuE electrode and modifying a ssDNA probe targeting miRNA-21.

[0006] The above ssDNA probe is 5'-TTCAACATCAGTCTGATAAGCTATTT-3'.

[0007] The E-DNA sensor also includes a detection solution, which is prepared by mixing 2 mL of a 10 mM Tris-HCl buffer solution with a pH of 7.4 and 100 μl of a 5 μM hexaammineruthenium solution.

[0008] A method for preparing the above-mentioned working electrode comprises the following steps:

[0009] 1) The AuE electrode was polished to a mirror surface with alumina powder with a diameter of 0.3 μm and 0.05 μm in turn, and then ultrasonically washed with anhydrous ethanol and ddH2O for 3 min each time, and then immersed in a newly prepared piranha solution for 10 min. Subsequently, the electrode was continuously scanned by cyclic voltammetry in the range of -0.3-1.5 V in a 0.5 mol / L sulfuric acid solution for 10 cycles, rinsed with ddH2O, and allowed to stand and dry to obtain the pretreated AuE electrode;

[0010] 2) The pretreated AuE electrode was immersed in a 5 mg / L BSA solution and incubated for 15 min at room temperature to form a BSA self-assembled film on the surface of the gold electrode. The obtained electrode was labeled as BSA / AUE and characterized by electrochemical impedance spectroscopy.

[0011] 3) Prepare a DSN-assisted target recovery amplification reaction system with a total volume of 50 μL: 5 μL of miRNA-21, 5 μL of 1×DSN buffer, 15 μL of 1 μM ssDNA probe, 0.5 μL of 0.5 U / L DSN enzyme, and add ddH2O to 50 μL. Incubate the reaction system at 55°C for 120 minutes, and then heat at 95°C for 30 minutes.

[0012] 4) The reaction solution obtained in step 3) is added dropwise to the surface of the BSA / AUE electrode obtained in step 2), incubated at room temperature for 30 minutes, and then the electrode is washed with PBS buffer to obtain the working electrode.

[0013] The piranha solution is prepared by mixing concentrated H2SO4 and 30% H2O2 by volume in a ratio of 3:1.

[0014] Application of the above-mentioned E-DNA sensor in the detection of miRNA-21.

[0015] A method for detecting miRNA-21 using the above-mentioned E-DNA sensor is specifically: placing a three-electrode system consisting of an Ag / AgCl reference electrode, a platinum wire counter electrode and a working electrode in a detection solution, and detecting using differential pulse voltammetry.

[0016] The detection principle of the present invention is as follows:

[0017] Bovine serum albumin (BSA) can self-assemble on the surface of gold electrode (AuE) to form a self-assembled monolayer (SAM) with holes and horizontal orientation. SAM has a size exclusion effect, which can hinder the adsorption of single-stranded DNA probes (ssDNA probes) on the AuE electrode, but allows smaller DNA fragments to pass through the self-assembled film and adsorb on the surface of the AuE electrode. This adsorption behavior is allowed after the ssDNA probe is digested by DSN enzyme. In the presence of miRNA-21, miRNA-21 hybridizes with the ssDNA probe to form a DNA: RNA heteroduplex. The hybridization of miRNA-21 and ssDNA probe induces DSN enzyme to digest the ssDNA probe in the DNA: RNA heteroduplex, producing a large number of DNA fragments. These DNA fragments, combined with Ru(NH3)6 in the electrolyte, 3+ After binding, an electrochemical signal can be generated.

[0018] The significant advantages of the present invention are:

[0019] The E-DNA sensor based on the size exclusion effect of protein monolayer described in the present invention has a wide calibration range of 0.001~100pM and a low detection limit of 0.48fM, which can successfully achieve accurate evaluation of miRNA-21 expression levels in cancer cell lines and non-small cell lung cancer (NSCLC) serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Schematic diagram of the E-DNA sensor constructed based on the size exclusion effect of protein monolayer.

[0021] Figure 2 : Impedance diagrams of bare gold electrode and BSA modified gold electrode.

[0022] Figure 3 : Polyacrylamide gel electrophoresis analysis of DSN-assisted target recovery system.

[0023] Figure 4 : Response signal and specificity of E-DNA sensor to different concentrations of miRNA-21 standards.

[0024] Figure 5 : Comparison of detection effects of E-DNA sensor and RT-PCR in actual samples. DETAILED DESCRIPTION

[0025] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0026] The experimental materials involved in the present invention are as follows:

[0027] Piranha solution: Concentrated H2SO4 and 30% H2O2 are mixed in a volume ratio of 3:1.

[0028] miRNA-21: 5′-UAGCUUAUCAGACUGAUGUUGA-3′ (U represents uracil).

[0029] miRNA-122: 5′-UGGAGUGUGACAAUGGUGUUUG-3′ (U represents uracil).

[0030] miRNA-141: 5′-UAACACUGUCUGGUAAAGAUGG-3′ (U represents uracil).

[0031] miRNA-199: 5′-ACAGUAGUCUGCACAUUGGUUA-3′ (U represents uracil).

[0032] miRNA-210: 5′-AGCCCCUGCCCACCGCACACUG-3′ (U indicates uracil).

[0033] ssDNA probe: 5′-TTCAACATCAGTCTGATAAGCTATTT-3′.

[0034] 1×DSN buffer, purchased from Evrogen.

[0035] DSN enzyme was purchased from Evrogen.

[0036] Total miRNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0037] The reverse transcription kit was purchased from Takara.

[0038] The principle diagram of the E-DNA sensor based on the size exclusion effect of protein monolayer described in the present invention is as follows: Figure 1As shown. The monolayer (SAM) of bovine serum albumin (BSA) on the gold electrode (AuE) has a size exclusion effect, which plays a gatekeeper role, hindering the adsorption of single-stranded DNA probes (ssDNA probes) on AuE, but allowing smaller DNA fragments to pass through the self-assembled membrane and adsorb on AuE. Based on this, the present invention developed a novel E-DNA sensor to detect miRNA-21 by combining it with a double-stranded specific nuclease (DSN)-assisted target recovery strategy. During the DSN-assisted target recovery process initiated by the target miRNA-21, the ssDNA probes in the solution phase are digested by the DSN enzyme to produce a large number of DNA fragments, which are allowed to adsorb on BSA / AuE and react with Ru(NH3)6] 3+ After binding, an electrochemical signal is generated, thereby realizing the recognition of miRNA-21.

[0039] Example 1 Electrochemical impedance spectroscopy of bare gold electrode and BSA-modified gold electrode

[0040] Step 1: The AuE electrode was polished to a mirror surface with alumina powder with a diameter of 0.3μm and 0.05μm respectively, and then ultrasonically washed with anhydrous ethanol and ddH2O for 3 min each time, and then immersed in a newly prepared piranha solution (a mixture of concentrated H2SO4 and 30% H2O2 in a volume ratio of 3:1) for 10 min. Subsequently, the electrode was continuously scanned by cyclic voltammetry in the range of -0.3-1.5V in a 0.5mol / L sulfuric acid solution for 10 cycles, rinsed with ddH2O, and allowed to stand to dry to obtain the pretreated AuE electrode.

[0041] Step 2: Soak the pretreated AuE electrode in 5 mg / L BSA solution and incubate it at room temperature for 15 minutes to form a BSA self-assembled film on the surface of the gold electrode. The obtained electrode is marked as BSA / AUE and characterized by electrochemical impedance spectroscopy (experimental conditions: set the initial voltage according to the open circuit voltage, set the frequency at the high frequency to 100 kHz, and the frequency at the low frequency to 10 mHz).

[0042] The results are as follows Figure 2 As shown in Figure 2, the assembly process of BSA on the gold electrode was studied using electrochemical impedance spectroscopy (EIS). The bare gold electrode had a low RCT value (charge transfer resistance). After incubation with BSA, the resistance gradually increased, which may be due to the formation of BSA with poor conductivity, which largely hindered the transfer of electrons from the body fluid to the electrode interface.

[0043] Example 2 Analysis of DSN-assisted target recovery system by polyacrylamide gel electrophoresis

[0044] Prepare the DSN-assisted target recovery amplification reaction system according to the table below. Incubate the DSN-assisted target recovery amplification reaction tube at 55°C for 120 minutes, then heat at 95°C for 30 minutes, and characterize the DSN-assisted target recovery amplification reaction by polyacrylamide gel electrophoresis (PAGE).

[0045] Table 1 DSN-assisted target recovery amplification reaction system

[0046]

[0047] The results are as follows Figure 3 As shown, lane 1 is DNA Marker, lane 2 is ssDNA probe, lane 3 is miRNA-21, lane 4 is the mixed product of ssDNA probe and miRNA-21, lane 5 is the mixed product of ssDNA probe and DSN enzyme, and lane 6 is the mixed product of ssDNA probe, miRNA-21 and DSN enzyme; after DSN-assisted target recovery amplification reaction, miRNA-21 can cause the ssDNA probe to be cut into small fragments.

[0048] Example 3 Construction of E-DNA sensor based on size exclusion effect of protein monolayer

[0049] Step 1: The AuE electrode was polished to a mirror surface with alumina powder with a diameter of 0.3μm and 0.05μm respectively, and then ultrasonically washed with anhydrous ethanol and ddH2O for 3 min each time, and then immersed in a newly prepared piranha solution (a mixture of concentrated H2SO4 and 30% H2O2 in a volume ratio of 3:1) for 10 min. Subsequently, the electrode was continuously scanned by cyclic voltammetry in the range of -0.3-1.5V in a 0.5mol / L sulfuric acid solution for 10 cycles, rinsed with ddH2O, and allowed to stand to dry to obtain the pretreated AuE electrode.

[0050] Step 2: Soak the pretreated AuE electrode in 5 mg / L BSA solution and incubate it at room temperature for 15 minutes to form a BSA self-assembled film on the surface of the gold electrode. The obtained electrode is marked as BSA / AUE and characterized by electrochemical impedance spectroscopy (experimental conditions: set the initial voltage according to the open circuit voltage, set the frequency at the high frequency to 100 kHz, and the frequency at the low frequency to 10 mHz).

[0051] Step 3: Prepare a DSN-assisted target recovery amplification reaction system with a total volume of 50 μL: 5 μL 0.1 μM miRNA (miRNA-21, miRNA-122, miRNA-141, miRNA-199 or miRNA-210), 5 μL 1×DSN buffer, 15 μL 1 μM ssDNA probe, 0.5 μL 0.5 U / L DSN enzyme, and add ddH2O to 50 μL. Incubate the reaction system at 55°C for 120 min, and then heat at 95°C for 30 min.

[0052] Step 4: Take 5 μL of the reaction solution obtained in step 3 and add it dropwise to the surface of the BSA / AUE electrode obtained in step 2. Incubate at room temperature for 30 minutes, and then wash the electrode with 1xPBS buffer (pH=7.4) to obtain the working electrode.

[0053] Step 5: Using a platinum wire counter electrode as an auxiliary electrode and an Ag / AgCl electrode as a reference electrode, a three-electrode system sensor is established with the working electrode obtained in step 4, and the sensor is placed in a detection solution for differential pulse voltammetry (DPV) detection (-0.1 to -0.5 potential window, amplitude 50 mV, pulse width 50 ms, pulse period 0.5 s) to obtain the value of the response current. The detection solution is prepared by mixing 2 mL of 10 mM Tris-HCl buffer with a pH of 7.4 and 100 μl of 5 μM hexaamineruthenium solution.

[0054] The changes in the current signal of the E-DNA sensor in response to different concentrations of miRNA-21 are shown in Figure 2. Figure 4 Specifically, in the range of 1 fm to 100 pm, it can be observed that the current signal increases with the increase of miRNA-21 concentration ( Figure 4 A); the current signal was linearly related to the logarithm of the miRNA-21 concentration, and the linear regression equation was I=85.7 lgC+387.9 (I: current signal, C: miRNA-21 concentration), and the correlation coefficient (R 2 ) was 0.9909, and the detection limit (LOD) calculated based on the three-fold signal-to-noise ratio was 0.48 fM ( Figure 4 B); The current signals of the developed E-DNA sensor to other four different interfering RNAs (miRNA-122, miRNA-141, miRNA-199, miRNA-210) are almost the same as the background signals, while a stronger current signal is stimulated to the target miRNA-21 ( Figure 4 C). This result shows that the E-DNA sensor developed by the present invention can detect miRNA-21 sensitively and highly specifically.

[0055] Example 4 Comparison of the detection effects of E-DNA sensor based on the size exclusion effect of protein monolayer and RT-PCR in actual samples

[0056] E-DNA sensor for detecting miRNA-21 based on the size exclusion effect of protein monolayer:

[0057] Step 1: Total miRNA was extracted from cancer cells or human serum using a commercial Total miRNA extraction kit, and the extracted miRNA was quantified using a NanoDrop spectrophotometer;

[0058] Step 2: The AuE electrode was polished to a mirror surface with alumina powder with a diameter of 0.3 μm and 0.05 μm in turn, and then ultrasonically washed with anhydrous ethanol and ddH2O in turn, each for 3 min, and then immersed in a newly prepared piranha solution (concentrated H2SO4 and 30% H2O2 mixed in a volume ratio of 3:1) for 10 min, and then the electrode was continuously scanned by cyclic voltammetry in a 0.5 mol / L sulfuric acid solution in the range of -0.3-1.5 V for 10 cycles, rinsed with ddH2O, and left to dry, thereby obtaining the pretreated AuE electrode;

[0059] Step 3: The pretreated AuE electrode was immersed in a 5 mg / L BSA solution and incubated at room temperature for 15 min to form a BSA self-assembled film on the surface of the gold electrode. The obtained electrode was labeled as BSA / AUE and characterized by electrochemical impedance spectroscopy (experimental conditions: the initial voltage was set according to the open circuit voltage, the frequency at the high frequency was set to 100 KHz, and the frequency at the low frequency was set to 10 mHz);

[0060] Step 4: Prepare a DSN-assisted target recovery amplification reaction system with a total volume of 50 μL: 5 μL of miRNA extracted in step 1, 5 μL of 1×DSN buffer, 15 μL of 1 μM ssDNA probe, 0.5 μL of 0.5 U / L DSN enzyme, and add ddH2O to 50 μL. Incubate the reaction system at 55°C for 120 minutes, and then heat at 95°C for 30 minutes.

[0061] Step 5: Take 5 μL of the reaction solution obtained in step 4 and drop it onto the surface of the BSA / AUE electrode obtained in step 3, incubate at room temperature for 30 minutes, and then wash the electrode with 1xPBS buffer (pH=7.4) to obtain the working electrode;

[0062] Step 6: Using a platinum wire counter electrode as an auxiliary electrode and an Ag / AgCl electrode as a reference electrode, a three-electrode system sensor is established with the working electrode obtained in step 4, and the sensor is placed in a detection solution for differential pulse voltammetry (DPV) detection (-0.1 to -0.5 potential window, amplitude 50 mV, pulse width 50 ms, pulse period 0.5 s) to obtain a response current value. The detection solution is prepared by mixing 2 mL of 10 mM Tris-HCl buffer with a pH of 7.4 and 100 μl of 5 μM hexaamineruthenium solution.

[0063] RT-PCR detection of miRNA-21:

[0064] Step 1: Total miRNA was extracted from cancer cells or human serum using a commercial Total miRNA extraction kit, and the extracted miRNA was quantified using a NanoDrop spectrophotometer;

[0065] Step 2: Reverse the extracted miRNA into cDNA according to the requirements of the reverse transcription kit;

[0066] Step 3: Perform RT-PCR. The total volume of the RT-PCR reaction system is 10 μL: 1 μL of 100 ng / μL template cDNA, 2 μL of 2.5 μM upstream and downstream primers (upstream primer: 5'-ACACTCCAGCTGGGTAGCTTATCAGACTGA-3'; downstream primer: 5'-TGGTGTCGTGGAGTCG-3'), and 5 μL of premixed solution Taq Pro Universal SYBR qPCR Master Mix (Novozymes). The RT-PCR reaction was performed under the following conditions: 40 cycles of denaturation at 95°C for 35 s, annealing at 95°C for 5 s, and extension at 60°C for 34 s, and the reaction was completed after 40 cycles.

[0067] The results are as follows Figure 5 shown. Figure 5 A shows the relative expression levels of miRNA-21 in different cell lines (HEK293T cells, Hela cells, MDA MB 231 cells, MCF7 cells) obtained by RT-PCR analysis. Then, we used the developed E-DNA sensor to detect the expression level of miRNA-21 in different cell lines (HEK293T cells, Hela cells, MDA MB 231 cells, MCF7 cells). Figure 5As shown in Figure 2, the relative expression levels of miRNA-21 in these cell lines reflected by the current signal are consistent with the results obtained by RT-PCR analysis. This result shows that the E-DNA sensor developed by the present invention can be successfully used to detect the expression level of miRNA-21 in different cell lines. At the same time, the present invention applies the E-DNA sensor to the detection of the expression level of miRNA-21 in the serum of patients with non-small cell lung cancer. Figure 5 As shown in C, although miRNA-21 expression was upregulated in most serum samples of NSCLC, the expression level of miRNA-21 was low in two samples. Figure 5 As shown in D, the current signal trend of the E-DNA sensor developed by the present invention for detecting miRNA-21 in the serum samples of these non-small cell lung cancer patients is consistent with the result trend obtained by the RT-PCR method.

[0068] 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. An electrochemical DNA sensor based on the size exclusion effect of protein monolayer, characterized in that: The electrochemical DNA sensor comprises an Ag / AgCl reference electrode, a platinum wire counter electrode and a working electrode, wherein the working electrode is obtained by constructing a BSA self-assembled film on the surface of a gold electrode AuE and modifying a ssDNA probe targeting miRNA-21; The method for preparing the working electrode comprises the following steps: 1) The gold electrode AuE was polished to a mirror surface with alumina powder with a diameter of 0.3 μm and 0.05 μm in turn, and then ultrasonically washed with anhydrous ethanol and ddH2O in turn, each for 3 minutes, and then immersed in a newly prepared piranha solution for 10 minutes, and then the electrode was continuously scanned by cyclic voltammetry in the range of -0.3~1.5V in a 0.5 mol / L sulfuric acid solution for 10 cycles, rinsed with ddH2O, and allowed to stand and dry to obtain a pretreated gold electrode; wherein the piranha solution is prepared by mixing concentrated H2SO4 and 30% H2O2 by volume in a ratio of 3:1; 2) The pretreated gold electrode was immersed in a 5 mg / L BSA solution and incubated for 15 min at room temperature to form a BSA self-assembled film on the surface of the gold electrode. The obtained electrode was labeled as BSA / AuE and characterized by electrochemical impedance spectroscopy. 3) Prepare a DSN-assisted target recovery amplification reaction system with a total volume of 50 μL: 5 μL 0.1 μM miRNA-21, 5 μL DSN buffer, 15 μL 1 μM ssDNA probe, 0.5 μL 0.5 U / L DSN enzyme, and add ddH2O to 50 μL. Incubate the reaction system at 55°C for 120 minutes and then heat at 95°C for 30 minutes. 4) The reaction system obtained in step 3) was added dropwise to the surface of the BSA / AuE electrode obtained in step 2), incubated at room temperature for 30 minutes, and then the electrode was washed with PBS buffer to obtain the working electrode.

2. The electrochemical DNA sensor according to claim 1, characterized in that: The ssDNA probe is 5'-TTCAACATCAGTCTGATAAGCTATTT-3'.

3. The electrochemical DNA sensor according to claim 1, characterized in that: The electrochemical DNA sensor also includes a detection solution, which is prepared by mixing 2 mL of a 10 mM Tris-HCl buffer solution with a pH of 7.4 and 100 μl of a 5 μM hexaammineruthenium solution.

4. The use of the electrochemical DNA sensor according to claim 1 in detecting miRNA-21, characterized in that: The application is for non-disease diagnosis and treatment purposes.

5. A method for detecting miRNA-21 using the electrochemical DNA sensor described in claim 1, characterized in that: A three-electrode system consisting of an Ag / AgCl reference electrode, a platinum wire counter electrode and a working electrode is placed in a detection solution, and detection is performed using differential pulse voltammetry; the method is for non-disease diagnosis and treatment purposes.

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