A dual signal ratio type biosensor and a preparation method and application thereof

Abstract

The application discloses a double-signal ratio type biosensor and a preparation method and application thereof, and the preparation method comprises the following steps: electrochemically depositing gold nanoparticles on the surface of a pretreated glassy carbon electrode to obtain an Au NPs / GCE electrode; immersing the Au NPs / GCE electrode in a pretreated hairpin probe solution to perform incubation, and then performing post-treatment to obtain an Au NPs / Hp / GCE electrode, namely the double-signal ratio type biosensor. When the double-signal ratio type biosensor prepared by the application is applied to nucleic acid detection, the interference of false positive signals in single-signal detection can be eliminated through the response of electrochemical signals and electrochemiluminescence signals, accurate and reliable detection results can be provided in a complex environment, and high-sensitivity and high-specificity detection of nucleic acids is realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a dual-signal ratio biosensor, its preparation method, and its application. Background Technology

[0002] MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of 18-25 bp, whose main function is post-transcriptional regulation of proteins. Because abnormal expression of miRNAs is closely related to disease development and they are commonly used as cancer biomarkers, accurate detection of miRNAs is essential.

[0003] In recent years, various detection technologies have been used for the sensitive detection of miRNAs, such as traditional Western blotting, qRT-PCR (quantitative real-time PCR), microarray detection, and emerging technologies such as electrochemical (EC), optical, electrochemiluminescence (ECL), and surface-enhanced Raman (SERS) sensing and imaging. However, the aforementioned detection technologies can be affected by false positive signals, thus impacting the detection results.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dual-signal ratio biosensor, its preparation method and application. When the prepared dual-signal ratio biosensor is applied to nucleic acid detection, it can eliminate the interference of false positive signals in single-signal detection through the response of electrochemical signals and electrochemiluminescence signals, provide accurate and reliable detection results in complex environments, and achieve highly sensitive and specific detection of nucleic acids.

[0006] This invention provides the following technical solution: In a first aspect, a method for fabricating a dual-signal ratiometric biosensor is provided, comprising the following steps: Gold nanoparticles were electrochemically deposited on the surface of the pretreated glassy carbon electrode to obtain an Au NPs / GCE electrode. The Au NPs / GCE electrode was immersed in a pretreated hairpin probe solution for incubation, and then post-processed to obtain the Au NPs / Hp / GCE electrode, which is a dual-signal ratiometric biosensor.

[0007] Furthermore, the electrochemical deposition method includes: placing the pretreated glassy carbon electrode in a HAuCl4 solution and electrodepositing for 30 seconds at -0.2V.

[0008] Furthermore, the gold nanoparticles obtained by electrochemical deposition have a particle size of 200~500 nm.

[0009] Furthermore, the pretreatment method of the hairpin probe includes: mixing TCEP and Hp chains in a molar ratio of 1:1, and the ends of the hairpin probe are modified with ferrocene molecules.

[0010] Furthermore, the Au NPs / GCE electrode is immersed in the pretreated hairpin probe solution at a temperature of 37°C for 3-6 hours.

[0011] Furthermore, the post-processing includes rinsing the incubated electrode with PBS buffer, then immersing it in BSA solution, and then rinsing the electrode surface with PBS buffer again.

[0012] In a second aspect, a dual-signal ratio biosensor is provided, which is prepared using the method described in the first aspect.

[0013] Thirdly, an application of the dual-signal ratio biosensor described in the second aspect in nucleic acid detection is provided, comprising the following steps: The target nucleic acid to be detected is mixed with two DNA strands, S1 and S2, in a container containing Mg. 2+ The reaction was carried out in the solution to obtain a solution containing nanozymes; The Au NPs / Hp / GCE electrode was immersed in a solution containing nanozymes, rinsed after immersion, and then incubated in a solution containing heme chloride to obtain the Au NPs / Hp / GCE electrode after reaction. The Au NPs / Hp / GCE electrode after the reaction was used as the working electrode to detect electrochemical and electrochemiluminescence signals. The quantitative detection of the concentration of the target nucleic acid is achieved by using the ratio of the electrochemiluminescence signal to the electrochemical signal.

[0014] Furthermore, the Mg-containing 2+ The concentrations of both DNA strands S1 and S2 in the solution were 0.5 μM, and Mg... 2+ The concentration was 20 mM.

[0015] Furthermore, the concentration of heme chloride in the solution is 0.02 mM, and the electrode is incubated in the solution at a temperature of 37°C for 45-90 min.

[0016] Furthermore, the method for detecting the electrochemical signal includes: using the reacted Au Nps / Hp / GCE electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode, and obtaining the electrochemical signal using square wave voltammetry in 0.1M, pH=8.5 PBS buffer.

[0017] Furthermore, the method for detecting the electrochemiluminescence signal includes: using the reacted Au Nps / Hp / GCE electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode, recording and detecting the electrochemiluminescence signal using an electrochemiluminescence workstation in 0.1M PBS buffer containing 0.1mM luminol and 1mM H2O2 at pH=8.5.

[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention obtains an Au NPs / GCE electrode by electrochemically depositing gold nanoparticles on the surface of a pretreated glassy carbon electrode. The Au NPs / GCE electrode is then immersed in a pretreated hairpin probe solution for incubation, followed by post-treatment to obtain an Au NPs / Hp / GCE electrode. When applied to nucleic acid detection, the target nucleic acid and two DNA strands, S1 and S2, are used in a solution containing Mg. 2+ The reaction is carried out in a solution to obtain a solution of nanozymes containing shearing ability, which realizes the shearing of hairpin probes on the electrode surface and causes changes in the intensity of electrochemical and electrochemiluminescence signals. The ratio of the two signal changes is used to achieve highly sensitive detection of nucleic acids to be tested (such as microRNA-21). (2) The dual-signal ratio biosensor prepared by the present invention can eliminate the interference of false positive signals in single-signal detection through the response of two signals, thereby providing accurate and reliable detection results in complex environments. Attached Figure Description

[0019] Figure 1 This is a scanning electron microscope characterization image of the Au NPs / GCE electrode in Embodiment 1 of the present invention; Figure 2 The changes in ECL and EC signals after microRNA detection using the dual-signal ratio biosensor in Embodiment 2 of this invention; Figure 3 This is a polyacrylamide gel electrophoresis characterization image of the target recognition and enzyme cleavage reaction in Example 3 of the present invention; Figure 4 These are the electrochemical impedance spectroscopy diagrams of the dual-signal ratio biosensor in Example 4 of this invention under different modification stages and reaction conditions; Figure 5The concentration of microRNA-21 in Example 5 of this invention is compared with the ratio of ECL signal to EC signal (I). ECL / I EC The relationship curve. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0021] Example 1

[0022] This embodiment provides a method for fabricating a dual-signal ratio biosensor, the specific steps of which are as follows: Step 1: Preparation of Au NPs / GCE electrode.

[0023] A 3 mm diameter glassy carbon electrode was polished using 1.0 μm and 0.05 μm Al₂O₃ powders, and then ultrasonically cleaned with ethanol and water, respectively. After drying with a bulb syringe, the electrode was electrodeposited in a -0.2 V HAuCl₄ solution for 30 s to obtain a gold nanoparticle-modified glassy carbon electrode (Au NPs / GCE electrode). The gold particles on the electrode surface had a particle size of 200–500 nm. Figure 1 As shown.

[0024] Step 2: Assembly of AuNPs / Hp / GCE electrodes.

[0025] 10 mM TCEP (tris(2-carboxyethyl)phosphine) at a molar ratio of 1:1 was mixed with Hp chains (terminally modified with ferrocene molecules) to pretreat the Hp chains with TCEP, cleaving the S-S bonds. The Au NPs / GCE electrode was then immersed in 50 μL of 1 μM hairpin probe (Hp) solution and incubated at 37°C for 6 h. Subsequently, the electrode was rinsed with PBS buffer (phosphate buffer), and the resulting modified electrode was immersed in 2% wt BSA (bovine serum albumin) solution at 37°C for 1 h to block non-specific active binding sites. Finally, the electrode surface was rinsed with PBS buffer at pH 7.4 to remove unbound material.

[0026] Example 2

[0027] This embodiment provides an application of the dual-signal ratio biosensor described in Embodiment 1 in nucleic acid detection. This embodiment takes microRNA-21 as an example, and the specific steps are as follows: Step 1: Preparation of the reaction solution.

[0028] All DNA strands were dissolved in 20 mM Tris-HCl solution (pH 7.4) containing 1 mM EDTA. After dissolution, all DNA strand solutions were annealed by heating at 85°C for 5 min, with the Hp strand solution gradually cooled to room temperature and the S1 and S2 strands immediately cooled to room temperature.

[0029] First, dissolve hemin (heme chloride) in 1% DMSO (dimethyl sulfoxide) containing 0.2M KCl to prepare a stock solution. Dilute the hemin solution to the desired concentration using a 25mM HEPES solution (pH=7.4) containing 100mM NaCl, 10mM MgCl2, 10mM KCl, 0.05% Triton X-10, and 1% DMSO.

[0030] The working solution was a buffer solution (pH=7.4) containing 100mM NaCl, 20mM MgCl2, 10mM KCl and 20mM Tris.

[0031] Step 2: The reaction on the electrode surface proceeds.

[0032] The Au NPs / Hp / GCE electrode prepared in Example 1 was immersed in a 20 mM Tris-HCl (tris(hydroxymethyl)aminomethane) working solution (pH=7.4) containing 0.5 μM S1, 0.5 μM S2, and different concentrations of miRNA-21 at 37 °C for 90 min. After immersion, the electrode was rinsed with PBS buffer and incubated in a HEPES solution containing 0.02 mM hemin at 37 °C for 60 min to obtain the reacted Au NPs / Hp / GCE electrode.

[0033] Step 3: Detection of electrode signals.

[0034] The Au Nps / Hp / GCE electrode after the reaction was used as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire electrode as the counter electrode. ECL signals were recorded and detected using an electrochemiluminescence workstation in 0.1M PBS buffer (pH=8.5) containing 0.1mM luminol and 1mM H2O2, with a scanning voltage range of 0–0.6V. The above three-electrode system was placed in 0.1M PBS buffer (pH=8.5), with a scanning voltage range of -0.2–0.6V and a frequency of 20Hz, and EC signals were obtained using square wave voltammetry. The concentration of microRNA-21 was detected based on the correlation between the ratio of ECL signals to EC signals and the concentration of microRNA-21.

[0035] Following the steps described above, the concentrations of microRNA-21 were changed to 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM, and ECL and EC signals were detected respectively. Figure 2 As shown, curves a, b, c, d, e, f, g, and h correspond to the aforementioned concentrations of microRNA-21, respectively. Figure 2 It can be seen that as the concentration of miRNA-21 increases, the ECL signal increases significantly, while the EC signal gradually decreases, indicating that in this system, both the EC and ECL signals are concentration-dependent.

[0036] The specific principle is as follows: First, chloroauric acid is used for electrodeposition on the glassy carbon electrode surface to obtain a platform containing a large number of AuNPs. The 3' end of the hairpin chain Hp is modified with ferrocene (Fc) molecules, and the 5' end is modified with a thiol group and designed with a G-quadruplex sequence; simultaneously, an rA cleavage site is added to its loop region, which can be cleaved by MNAzyme (a multicomponent nuclease). A large number of hairpin chains Hp are immobilized on the electrode surface via Au-S chains. Due to the quenching effect of Fc, the ECL signal of luminol in the solution is low, while the EC signal of Fc is strong. When the target microRNA-21 is present, the capture chains S1 and S2 can induce a neighboring hybridization reaction with miRNA-21, forming an S1 / miRNA-21 / S2 biological complex. This complex in Mg 2+ In the presence of [a specific enzyme / organism], MNAzyme forms cleavage sites on *Helicobacter pylori* (Hp). Hp cleavage releases a large amount of Fc-modified single-stranded DNA, while the remaining G-quadruplex strands are immobilized on the electrode surface. MNAzyme, after recognizing and cleaving, can bind to another Hp and initiate the cleavage process, thereby amplifying the signal. At this time, the electrode surface is at K [a specific temperature / temperature]. + With the help of [unclear], the G-quadruplex strand immobilized on the electrode forms a hemin / G-quadruplex structure with hemin. This structure has a catalase-like effect, catalyzing the decomposition of H2O2 to produce reactive oxygen species, thereby enhancing the ECL signal of luminol. Simultaneously, due to the reduction of Fc, the quenching effect of Fc on the luminol ECL signal is weakened, allowing the ECL signal to recover; however, the reduction of Fc also leads to a decrease in the EC signal. Under optimized conditions, the anodic signal intensity of luminol catalyzed by the hemin / G-quadruplex structure increases with increasing microRNA-21 concentration, while the EC signal of Fc decreases. By detecting the ratio of the ECL signal to the EC signal, the concentration of the target microRNA-21 can be detected.

[0037] Example 3

[0038] This embodiment uses polyacrylamide gel electrophoresis and imaging to determine the target recognition and enzyme digestion process.

[0039] Different DNA strands were reacted and mixed in lanes corresponding to the grooves of a freshly prepared 8% polyacrylamide gel. Electrophoresis was then performed for 80 min at 70 V in 1×TBE buffer. After staining with ethidium bromide (EB), images were captured using a biogel imaging system. Lane 1 represents the S2 strand, lane 2 represents the S1 strand, lane 3 represents a mixture of S2 and S1, lane 4 represents the target strand miRNA-21, lane 5 represents a mixture of DNA strands S2, S1, and the target strand, lane 6 represents the Hp strand, lane 7 represents a mixture of S2, S1, and Hp strands, and lane 8 represents a mixture of the target strand, S1 strand, S2 strand, and Hp strand. The concentrations of the target strand, S1, S2, and Hp strands were all 2 μM.

[0040] like Figure 3 As shown, only one bright band appears in lanes 1, 2, and 3, indicating that the S1 and S2 strands migrate to the same position and do not hybridize with each other. When the target analyte miRNA-21 is added to the mixture of S1 and S2, a new bright band appears in lane 5, proving that miRNA-21 can form an S1 / miRNA-21 / S2 complex with S1 and S2 through proximity hybridization. After Hp is added to the mixture of S1 and S2 in lane 7, a new bright band appears, proving that S1 and S2 can bind to Hp. When the target analyte miRNA-21 is mixed with S1, S2, and Hp simultaneously (lane 8), the S1 / S2 / Hp band darkens significantly, and a new bright band appears above it, indicating the formation of the MNAzyme structure. At the same time, two new bands with different migration positions than those in other lanes appear at the bottom, proving that Hp cleavage is successful. This example demonstrates that only proximity hybridization between S1, S2, and miRNA-21 can lead to the assembly of MNAzyme. The cleavage of MNAzyme by *Helicobacter pylori* (Hp) results in a decrease in its electrophoretic band intensity, thus creating a new, faster-migrating band.

[0041] Example 4

[0042] This embodiment provides a method for using 5mM [Fe(CN)6] 3- / 4- Under the condition of redox probes, electrochemical impedance spectroscopy (EIS) was used to verify the stepwise electrode modification process of the biosensor.

[0043] like Figure 4As shown, curve a represents the unmodified GCE electrode, curve b represents the Au NPs / GCE electrode after electrodeposition of Au NPs, curve c represents the electrode after modification of the Au NPs surface with Hp chains, curve d represents the electrode after blocking with BSA, curve e represents the modified electrode after surface Hp is cleaved in the presence of microRNA, and curve f represents the electrode after the formation of hemin / G quadruplexes on the electrode surface.

[0044] The unmodified GCE electrode has a relatively small semicircle diameter in EIS (curve a, Ret = 50 Ω). After electrodeposition of a layer of gold nanoparticles, the electrode surface impedance decreases because the gold nanoparticles, as conductive materials, can accelerate electron transfer. After sequential modification with Hp and BSA, the electron transfer impedance gradually increases from Ret = 750 Ω (curve c) to 3750 Ω (curve d), indicating successful electrode surface modification. In the presence of the target microRNA, Mg... 2+ The MNAzyme enzyme formed by the auxiliary agent cleaves Hp, and the remaining DNA strands on the electrode surface are in K + With the help of hemin, a chair-like structure was formed, increasing the steric hindrance of the electrode surface, and the resistance increased significantly to 4500Ω (curve e). Finally, in the presence of hemin, the formation of the hemin / G-quadriplex structure further hindered electron transfer on the electrode surface, thus significantly increasing the electron transfer impedance by 2250Ω. EIS characterization confirmed the stepwise modification of the electrode and verified the feasibility of this biosensor.

[0045] Example 5

[0046] This embodiment provides an application of the quantitative measurement and analysis process for microRNA-21. Following Example 2, a series of different concentrations of microRNA were detected, and EC and ECL signals were acquired, where the microRNA concentrations were 1, 10, and 10... 2 10 3 10 4 10 5 10 6 fM, the rest of the operations and signal recording process are the same. For example... Figure 5 As shown, the ECL signal intensity increases with increasing microRNA concentration, while the EC signal decreases with increasing microRNA concentration. Plotting the logarithm of the ECL intensity to EC intensity ratio on the ordinate and the logarithm of microRNA concentration on the abscissa reveals that I... ECL / I EC The logarithm of the value is linearly correlated with the logarithm of the microRNA concentration, where the microRNA concentration ranges from 1 fM to 1 nM.

[0047] The electrochemiluminescence biosensor of this invention employs a target-induced proximity hybridization reaction to form an MNAzyme with the substrate chain in Mg 2+ With the assistance of enzyme digestion, a dual signal change strategy of electrochemiluminescence and electrochemistry on the electrode surface was realized. The ratio of the two signals was used to achieve highly sensitive detection of microRNA-21. The linear detection range was 1 fM to nM, and the detection limit was 0.03 fM, providing a new method for the detection of microRNA.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid detection method using a dual-signal ratiometric biosensor, characterized in that, Includes the following steps: Gold nanoparticles were electrochemically deposited on the surface of the pretreated glassy carbon electrode to obtain an Au NPs / GCE electrode. The Au NPs / GCE electrode was immersed in the pretreated hairpin probe solution for incubation, and then post-processed to obtain the Au NPs / Hp / GCE electrode, which is a dual-signal ratiometric biosensor. The 3' end of the hairpin probe is modified with ferrocene molecules, the 5' end is modified with thiol groups and designed with a G-tetramer sequence, and an rA cleavage site is added to the loop region. The target nucleic acid to be detected is mixed with two DNA strands, S1 and S2, in a container containing Mg. 2+ The reaction was carried out in a solution to obtain a solution containing nanozymes; wherein, the biological complex formed by the target nucleic acid and the two DNA strands S1 and S2 was in Mg 2+ MNAzyme is formed in the presence of [the enzyme]. The Au NPs / Hp / GCE electrode was immersed in a solution containing nanozyme. The MNAzyme cleaved the rA cleavage site of the hairpin probe, causing the hairpin probe to cleave and release ferrocene-modified single-stranded DNA. The remaining G-quadruplex strand was fixed on the electrode surface. Rinse after soaking, then place in a potassium-containing solution. + The Au NPs / Hp / GCE electrode was obtained by incubation in a solution of heme chloride; wherein, during incubation, K + With the help of [the agent], the G-tetrachain fixed on the electrode surface forms a hemin / G-tetrachain structure with heme chloride; The Au NPs / Hp / GCE electrode after the reaction was used as the working electrode to detect electrochemical and electrochemiluminescence signals. Among them, the electrochemiluminescence signal was detected in PBS buffer containing luminol and H2O2. The intensity of the anodic electrochemiluminescence signal of luminol catalyzed by the hemin / G-tetramer structure increased with the increase of the concentration of the target nucleic acid, while the electrochemical signal of ferrocene decreased. The quantitative detection of the concentration of the target nucleic acid is achieved by using the ratio of the electrochemiluminescence signal to the electrochemical signal.

2. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The electrochemical deposition method includes: placing a pretreated glassy carbon electrode in a HAuCl4 solution and electrodepositing for 30 seconds at -0.2V.

3. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The gold nanoparticles obtained by electrochemical deposition have a particle size of 200~500 nm.

4. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The pretreatment method of the hairpin probe includes: mixing TCEP and Hp chains in a molar ratio of 1:1, and modifying the ends of the hairpin probe with ferrocene molecules.

5. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The Au NPs / GCE electrode was immersed in the pretreated hairpin probe solution and incubated at 37°C for 3-6 hours.

6. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The post-processing includes rinsing the incubated electrode with PBS buffer, then immersing it in BSA solution, and then rinsing the electrode surface with PBS buffer.

7. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The one containing Mg 2+ The concentrations of both DNA strands S1 and S2 in the solution were 0.5 μM, and Mg... 2+ The concentration was 20 mM.

8. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The K-containing + The concentration of heme chloride in the solution was 0.02 mM. The electrode was incubated in the heme chloride solution at a temperature of 37°C for 45-90 min.

9. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The method for detecting the electrochemical signal includes: using the reacted Au Nps / Hp / GCE electrode as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire electrode as the counter electrode, the electrochemical signal is obtained by square wave voltammetry in 0.1M, pH=8.5 PBS buffer.

10. The nucleic acid detection method using a dual-signal ratio biosensor according to claim 1, characterized in that, The method for detecting electrochemiluminescence signals includes: using the reacted Au Nps / Hp / GCE electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode, recording and detecting the electrochemiluminescence signals using an electrochemiluminescence workstation in 0.1M PBS buffer containing 0.1mM luminol and 1mM H2O2 at pH=8.5.

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