CN115791924A - E-DNA sensor constructed based on size exclusion effect of protein monolayer and application thereof - Google Patents
E-DNA sensor constructed based on size exclusion effect of protein monolayer and application thereof Download PDFInfo
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Abstract
The invention discloses an E-DNA sensor constructed based on the size exclusion effect of a protein monomolecular layer and application thereof. 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 aiming at miRNA-21. The E-DNA sensor has a wide calibration range of 0.001 to 100pM and a low detection lower limit of 0.48fM, and can successfully realize accurate evaluation of miRNA-21 expression level in cancer cell lines and non-small cell lung cancer (NSCLC) serum samples.
Description
Technical Field
The invention relates to the technical field of electrochemical sensors, in particular to an E-DNA sensor constructed based on the size exclusion effect of a protein monomolecular layer and application thereof.
Background
Electrochemical analysis is an emerging analytical technique with many advantages, such as fast response, high sensitivity, low cost, simplicity of manufacture, and suitability for large-scale production. Among them, electrochemical DNA (E-DNA) sensors, which are composed mainly of electrodes and specific immobilized DNA probes, are widely used to detect different DNA binding molecules. With the development of aptamers, DNAzymes, G-quadruplex and i-motif configurations and other novel functional DNA probes, the range of analytes of the E-DNA sensor is greatly expanded, including pharmaceutical compounds, proteins, metal ions, pollutants and the like, and the E-DNA sensor with the appropriate DNA probes can be used for easily detecting the analytes. In general, the operation of an E-DNA sensor depends on the interaction of the analyte with the immobilized DNA probes, which is accompanied by different predetermined events associated with the DNA (e.g., hybridization, digestion, cleavage, or conformational change of the DNA strands) and causes a change in an electrochemical parameter (e.g., capacitance or conductivity). Unfortunately, the response signal of the E-DNA sensor is deeply influenced by several parameters of the immobilized DNA probe, such as surface density, conformation, length. Therefore, the method of manufacturing the sensing electrode varies depending on the details of the DNA probe and the analyte. In addition, the procedure of immobilizing DNA probes is cumbersome and time-consuming due to the need for multiple surface modifications on the electrodes. These disadvantages limit the further application of E-DNA sensors. Therefore, finding a versatile and simple method to fabricate the sensing electrode is crucial to facilitate practical application of the E-DNA sensor.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides an E-DNA sensor constructed based on the size exclusion effect of a protein monomolecular layer and application thereof.
In order to achieve the purpose, the invention is implemented according to the following technical scheme:
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 (bovine serum albumin) self-assembled membrane on the surface of an AuE electrode and modifying a ssDNA (single-stranded deoxyribonucleic acid) probe aiming at miRNA-21.
The ssDNA probe is 5 'warfarin TTCAACATCAGTCTGATAAGCTATTT-doped 3'.
The E-DNA sensor also comprises a detection solution, wherein the detection solution is prepared by mixing 2mL of Tris-HCl buffer solution with the concentration of 10mM and the pH of 7.4 and 100 mu l of ruthenium hexaammine solution with the concentration of 5 mu M.
A method of making the working electrode described above, comprising the steps of:
1) The AuE electrode was polished to a mirror surface with alumina powders having diameters of 0.3 μm and 0.05 μm in this order, and then with absolute ethanol and ddH in this order 2 Performing ultrasonic washing for 3 min each time, soaking in newly prepared piranha solution for 10min, continuously scanning the electrode in 0.5mol/L sulfuric acid solution at-0.3-1.5V for 10 circles, and performing ddH 2 Rinsing with oxygen, standing and airing to obtain a pretreated AuE electrode;
2) Soaking the pretreated AuE electrode in 5mg/L BSA solution, incubating for 15min at room temperature, forming a BSA self-assembly film on the surface of the gold electrode, marking the obtained electrode as BSA/AUE, and characterizing electrochemical impedance;
3) Preparing a DSN-assisted target recovery amplification reaction system, wherein the total volume of the reaction system is 50 mu L: 5. Mu.L of miRNA-21, 5. Mu.L of 1 XDSN buffer, 15. Mu.L of 1. Mu.M ssDNA probe, 0.5. Mu.L of 0.5U/L DSN enzyme, and supplemental ddH 2 O to 50. Mu.L, incubating the reaction system at 55 ℃ for 120 minutes and then heating at 95 ℃ for 30 minutes;
4) And (3) dropwise adding the reaction liquid obtained in the step 3) onto the surface of the BSA/AUE electrode obtained in the step 2), incubating for 30min at room temperature, and washing the electrode by using PBS (phosphate buffer solution) to obtain the working electrode.
The piranha solution is concentrated H 2 SO 4 And 30% by mass of H 2 O 2 Mixing and preparing according to the volume ratio of 3.
The application of the E-DNA sensor in the aspect of miRNA-21 detection.
A method for detecting miRNA-21 by using the E-DNA sensor specifically comprises the following steps: and (3) placing a three-electrode system consisting of an Ag/AgCl reference electrode, a platinum wire counter electrode and a working electrode in detection liquid, and detecting by adopting a differential pulse voltammetry.
The detection principle of the invention is as follows:
bovine Serum Albumin (BSA) can be self-assembled on the surface of a gold electrode (AuE) to form a self-assembly with pores and horizontal orientationA monomolecular film (SAM) was assembled, which had a size exclusion effect that hindered the adsorption of single-stranded DNA probes (ssDNA probes) on the AuE electrodes, but allowed the smaller size of the DNA fragments to pass through the self-assembled film and adsorb on the AuE electrode surface, which adsorption behavior was allowed after the ssDNA probes were digested by DSN enzyme. In the presence of miRNA-21, miRNA-21 hybridizes to ssDNA probes to form DNA: RNA heteroduplex, hybridization of miRNA-21 to ssDNA probe induces DSN enzymatic digestion of DNA: ssDNA probes in RNA heteroduplexes, producing a large number of DNA fragments. These DNA fragments and Ru (NH) in electrolyte 3 ) 6 ] 3+ After binding, an electrochemical signal can be generated.
The invention has the following remarkable advantages:
the E-DNA sensor based on the size exclusion effect of the protein monomolecular layer has a wide calibration range of 0.001 to 100pM and a low detection lower limit of 0.48fM, and can successfully realize accurate evaluation of miRNA-21 expression level in cancer cell lines and non-small cell lung cancer (NSCLC) serum samples.
Drawings
FIG. 1: schematic diagram of an E-DNA sensor constructed based on the size exclusion effect of a protein monolayer.
FIG. 2: impedance plots of bare gold electrodes and BSA-modified gold electrodes.
FIG. 3: and (3) analyzing the DSN assisted target recovery system by polyacrylamide gel electrophoresis.
FIG. 4 is a schematic view of: response signals and specificity of the E-DNA sensor to miRNA-21 standard substances with different concentrations.
FIG. 5: and comparing the detection effects of the E-DNA sensor and the RT-PCR in the actual sample.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
The experimental materials involved in the invention are as follows:
piranha solution: the concentrated H2SO4 and the H2O2 with the mass fraction of 30 percent are mixed according to the volume ratio of 3.
miRNA-21: 5-.
miRNA-122: 5-.
miRNA-141:5 'UAACACUCUGUGUGUAAAGAUGG) -3' (U represents uracil).
miRNA-199: 5-.
miRNA-210:5' (U represents uracil).
ssDNA probes: 5 'TTCAACATCAGTCTGATAAGCTATTT-doped 3'.
1 XDSN buffer from Evagen.
DSN enzyme, available from Evagen.
Total miRNA extraction kit, purchased from Tiangen Biochemical technology (Beijing) Ltd.
Reverse transcription kit, purchased from Takara.
The schematic diagram of the E-DNA sensor based on the size exclusion effect of the protein monomolecular layer is shown in FIG. 1. The monomolecular film (SAM) of Bovine Serum Albumin (BSA) on the gold electrode (AuE) has a size exclusion effect, which serves as a deterrent, prevents the adsorption of the single-stranded DNA probe (ssDNA probe) on the AuE, but allows the smaller size DNA fragments to pass through the self-assembled film and be adsorbed on the AuE. Based on this, the present invention develops a novel E-DNA sensor to detect miRNA-21 by combining with a double strand specific nuclease (DSN) assisted target recovery strategy. During target miRNA-21 initiated DSN assisted target recovery, solution phase ssDNA probes are digested by DSN enzyme, generating a large number of DNA fragments that are allowed to adsorb onto BSA/AuE, and Ru (NH) 3 ) 6 ] 3+ And generating an electrochemical signal after combination, thereby realizing the identification of miRNA-21.
Example 1 electrochemical impedance characterization of bare gold and BSA modified gold electrodes
Step 1: the AuE electrode was polished to a mirror surface with alumina powders having diameters of 0.3 μm and 0.05 μm in this order, and then with absolute ethanol and ddH in this order 2 Ultrasonic washing with O3 min each time, and then preparing a piranha solution (concentrated H) 2 SO 4 And 30% by mass of H 2 O 2 Mixed according to a volume ratio of 3 2 And (4) rinsing by using oxygen, standing and airing to obtain the pretreated AuE electrode.
And 2, step: soaking the pretreated AuE electrode in a 5mg/L BSA solution, incubating for 15min at room temperature, forming a BSA self-assembled film on the surface of the gold electrode, marking the obtained electrode as BSA/AUE, and characterizing electrochemical impedance (experimental conditions, namely setting initial voltage according to open-circuit voltage, setting the frequency at a high frequency to be 100KHz and setting the frequency at a low frequency to be 10 mHz).
As a result, as shown in fig. 2, the assembly process of BSA on gold electrodes was studied using Electrochemical Impedance Spectroscopy (EIS), and bare gold electrodes have a low RCT value (charge transfer resistance), and after incubation with BSA, the resistance gradually increases, probably because the formation of BSA with poor conductivity largely hinders the transfer of electrons from body fluid to the electrode interface.
Example 2 Polyacrylamide gel electrophoresis analysis of DSN-assisted target recovery System
The DSN-assisted target recovery amplification reaction system was prepared as follows. The DSN-assisted target recovery amplification reaction tube was incubated at 55 ℃ for 120 min and then heated at 95 ℃ for 30min, and the product was characterized by polyacrylamide gel electrophoresis (PAGE).
TABLE 1 DSN-assisted target recovery amplification reaction System
The results are shown in FIG. 3, lane 1 is DNA Marker, lane 2 is ssDNA probe, lane 3 is miRNA-21, lane 4 is the product mixture of ssDNA probe and miRNA-21, lane 5 is the product mixture of ssDNA probe and DSN enzyme, and lane 6 is the product mixture of ssDNA probe, miRNA-21 and DSN enzyme; through the target recovery amplification reaction assisted by the DSN, the miRNA-21 can cause the ssDNA probe to be cut into small fragments by enzyme.
EXAMPLE 3 construction of E-DNA sensor based on the size exclusion effect of protein monolayers
Step 1: the AuE electrode was polished to a mirror surface with alumina powders having diameters of 0.3 μm and 0.05 μm in this order, and then with absolute ethanol and ddH in this order 2 Ultrasonic washing with oxygen for 3 min each time, and mixing with newly prepared piranha solution (concentrated H) 2 SO 4 And 30% by mass of H 2 O 2 Mixed according to a volume ratio of 3 2 And O rinsing, standing and airing to obtain the pretreated AuE electrode.
Step 2: and (2) soaking the pretreated AuE electrode in 5mg/L BSA solution, incubating for 15min at room temperature, forming a BSA self-assembled film on the surface of the gold electrode, marking the obtained electrode as BSA/AUE, and characterizing electrochemical impedance (experimental conditions, namely setting initial voltage according to open-circuit voltage, setting the frequency at a high frequency to be 100KHz and the frequency at a low frequency to be 10 mHz).
And 3, step 3: preparing a DSN-assisted target recovery amplification reaction system, wherein the total volume of the reaction system is 50 mu L: mu.L of 0.1. Mu.M miRNA (miRNA-21, miRNA-122, miRNA-141, miRNA-199, or miRNA-210), 5. Mu.L of 1 XDSN buffer, 15. Mu.L of 1. Mu.M ssDNA probe, 0.5. Mu.L of 0.5U/L DSN enzyme, and supplemental ddH 2 O to 50. Mu.L, the reaction system was incubated at 55 ℃ for 120 minutes and then heated at 95 ℃ for 30 minutes.
And 4, step 4: and (4) dropwise adding 5 mu L of the reaction solution obtained in the step (3) to the surface of the BSA/AUE electrode obtained in the step (2), incubating for 30min at room temperature, and washing the electrode by using 1xPBS buffer solution (pH = 7.4) to obtain the working electrode.
And 5: and (3) taking a platinum wire counter electrode as an auxiliary electrode, taking an Ag/AgCl electrode as a reference electrode, establishing a three-electrode system sensor with the working electrode obtained in the step (4), and placing the sensor in a detection solution for Differential Pulse Voltammetry (DPV) detection (potential window of-0.1 to-0.5, amplitude of 50mV, pulse width of 50ms and pulse period of 0.5 s) to obtain a numerical value of response current, wherein the detection solution is prepared by mixing 2mL of Tris-HCl buffer solution with the concentration of 10mM and the pH of 7.4 and 100 μ l of ruthenium hexamine solution with the concentration of 5 μ M.
The change of the current signal of the E-DNA sensor in response to different concentrations of miRNA-21 is shown in FIG. 4. Specifically, in the range of 1fm to 100pm, an increase in current signal with increasing miRNA-21 concentration was observed (fig. 4A); the current signal is in linear relation with the logarithm of the miRNA-21 concentration, and the linear regression equation is I =85.7 lgC +387.9 (I: current signal, C: miRNA-21 concentration), and the correlation coefficient (R: current signal, C: miRNA-21 concentration) 2 ) 0.9909, limit of detection (LOD) calculated from triple signal-to-noise ratio of 0.48fM (fig. 4B); the developed E-DNA sensor has almost the same current signal as the background signal for the other four different interfering RNAs (miRNA-122, miRNA-141, miRNA-199, miRNA-210), and elicits a stronger current signal for the target miRNA-21 (FIG. 4C). The result shows that the E-DNA sensor developed by the invention can sensitively and highly specifically detect the miRNA-21.
Example 4 comparison of detection effects of E-DNA sensor based on size exclusion effect of protein monolayer and RT-PCR in actual samples
E-DNA sensor based on size exclusion effect of protein monolayer to detect miRNA-21:
step 1: extracting Total miRNA from cancer cells or human serum by using a commercial Total miRNA extraction kit, and quantifying the extracted miRNA by using a NanoDrop spectrophotometer;
step 2: the AuE electrode was polished to a mirror surface with alumina powders having diameters of 0.3 μm and 0.05 μm in this order, and then with absolute ethanol and ddH in this order 2 Ultrasonic washing with O for 3 min each time, and adding newly prepared piranha solution (concentrated H) 2 SO 4 And 30% by mass of H 2 O 2 Mixed according to a volume ratio of 3 2 Rinsing with oxygen, standing and airing to obtain a pretreated AuE electrode;
and 3, step 3: soaking the pretreated AuE electrode in 5mg/L BSA solution, incubating for 15min at room temperature, forming a BSA self-assembled film on the surface of the gold electrode, marking the obtained electrode as BSA/AUE, and characterizing electrochemical impedance (experimental conditions: setting initial voltage according to open-circuit voltage, setting the frequency at a high frequency to be 100KHz and the frequency at a low frequency to be 10 mHz);
and 4, step 4: preparing a DSN-assisted target recovery amplification reaction system, wherein the total volume of the reaction system is 50 mu L: 5 μ L of miRNA extracted in step 1, 5 μ L of 1 XDSN buffer, 15 μ L of 1 μ M ssDNA probe, 0.5 μ L of 0.5U/L DSN enzyme, and supplemental ddH 2 O to 50. Mu.L, incubating the reaction system at 55 ℃ for 120 minutes, and then heating at 95 ℃ for 30 minutes;
and 5: dropwise adding 5 mu L of the reaction solution obtained in the step 4 to the surface of the BSA/AUE electrode obtained in the step 3, incubating for 30min at room temperature, and washing the electrode with 1xPBS buffer solution (pH = 7.4) to obtain the working electrode;
step 6: and (3) taking a platinum wire counter electrode as an auxiliary electrode, taking an Ag/AgCl electrode as a reference electrode, establishing a three-electrode system sensor with the working electrode obtained in the step (4), placing the sensor in a detection solution, and carrying out detection by using Differential Pulse Voltammetry (DPV), wherein the DPV is carried out for (a potential window of-0.1 to-0.5, an amplitude is 50mV, a pulse width is 50ms, and a pulse period is 0.5 s) to obtain a numerical value of response current, and the detection solution is prepared by mixing 2mL of Tris-HCl buffer solution with a concentration of 10mM and a pH of 7.4 and 100 μ l of ruthenium hexamine solution with a concentration of 5 μ M.
Detecting miRNA-21 by RT-PCR:
step 1: extracting Total miRNA from cancer cells or human serum by using a commercial Total miRNA extraction kit, and quantifying the extracted miRNA by using a NanoDrop spectrophotometer;
step 2: inverting the extracted miRNA into cDNA according to the requirements of a reverse transcription kit;
and step 3: performing RT-PCR, wherein the total volume of an RT-PCR reaction system is 10 mu L: mu.L of 100 ng/mu.l template cDNA, 2. Mu.L of each of 2. Mu.L of 2.5. Mu.M upstream and downstream primers (upstream primer: 5-: 40 cycles of denaturation at 95 ℃ for 35s, annealing at 95 ℃ for 5s, extension at 60 ℃ for 34s, and 40 cycles to completion.
The results are shown in FIG. 5. FIG. 5A shows the relative expression levels of miRNA-21 in different cell lines (HEK 293T cells, hela cells, MDA MB 231 cells, MCF7 cells) obtained by RT-PCR analysis. Then, we tested the expression level of miRNA-21 in different cell lines (HEK 293T cells, hela cells, MDA MB 231 cells, MCF7 cells) by using the developed E-DNA sensor. As shown in fig. 5B, the relative expression levels of miRNA-21 in these cell lines reflected by the current signals were consistent with the trend of the results obtained from RT-PCR analysis. The result shows that the E-DNA sensor developed by the invention can be successfully used for detecting the expression level of miRNA-21 in different cell lines. Meanwhile, the E-DNA sensor is applied to the detection of the miRNA-21 expression level in the serum of the patient with the non-small cell lung cancer, as shown in figure 5C, although the miRNA-21 expression of most of the non-small cell lung cancer serum samples is up-regulated, the miRNA-21 expression level of two samples is lower. As shown in FIG. 5D, the trend of the current signal of the E-DNA sensor developed by the present invention for miRNA-21 detection in the serum samples of patients with non-small cell lung cancer is consistent with the trend of the results obtained by the RT-PCR method.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (7)
1. An E-DNA sensor based on the size exclusion effect of a protein monolayer, characterized in that: 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 (bovine serum albumin) self-assembly film on the surface of an AuE electrode and modifying a ssDNA (single stranded deoxyribonucleic acid) probe aiming at miRNA-21.
2. The E-DNA sensor of claim 1, wherein: the ssDNA probe is 5 'warfarin TTCAACATCAGTCTGATAAGCTATTT-3'.
3. The E-DNA sensor of claim 1, wherein: the E-DNA sensor also comprises a detection solution, wherein the detection solution is prepared by mixing 2mL of Tris-HCl buffer solution with the concentration of 10mM and the pH of 7.4 and 100 mu l of hexaammine ruthenium solution with the concentration of 5 mu M.
4. A method of making a working electrode as claimed in claim 1, wherein: the method comprises the following steps:
1) The AuE electrode was polished to a mirror surface with alumina powders having diameters of 0.3 μm and 0.05 μm in this order, and then with absolute ethanol and ddH in this order 2 Performing ultrasonic cleaning for 3 min each time, soaking in newly prepared piranha solution for 10min, continuously scanning the electrode with cyclic voltammetry in 0.5mol/L sulfuric acid solution at-0.3-1.5V for 10 circles, and performing ddH 2 Rinsing with oxygen, standing and airing to obtain a pretreated AuE electrode;
2) Soaking the pretreated AuE electrode in 5mg/L BSA solution, incubating for 15min at room temperature, forming a BSA self-assembly film on the surface of the gold electrode, marking the obtained electrode as BSA/AUE, and characterizing electrochemical impedance;
3) Preparing a DSN-assisted target recovery amplification reaction system, wherein the total volume of the reaction system is 50 mu L: 5. Mu.L of miRNA-21, 5. Mu.L of 1 XDSN buffer, 15. Mu.L of 1. Mu.M ssDNA probe, 0.5. Mu.L of 0.5U/L DSN enzyme, and supplemental ddH 2 O to 50. Mu.L, incubating the reaction system at 55 ℃ for 120 minutes, and then heating at 95 ℃ for 30 minutes;
4) Dropwise adding the reaction liquid obtained in the step 3) to the surface of the BSA/AUE electrode obtained in the step 2), incubating for 30min at room temperature, and washing the electrode with PBS (phosphate buffer solution) to obtain the working electrode.
5. The method of claim 3, wherein: the piranha solution is concentrated H 2 SO 4 And 30% by mass of H 2 O 2 Mixing and preparing according to a volume ratio of 3.
6. Use of the E-DNA sensor of claim 1 for miRNA-21 detection.
7. A method for detecting miRNA-21 using the E-DNA sensor of claim 1, wherein: and (3) placing a three-electrode system consisting of an Ag/AgCl reference electrode, a platinum wire counter electrode and a working electrode in detection liquid, and detecting by adopting a differential pulse voltammetry.
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