Electrochemical biosensor based on stretched DNA capture probe and preparation method and application thereof

Abstract

The invention discloses an electrochemical biosensor based on a stretched DNA capture probe and a preparation method and application thereof, and the preparation method comprises the following steps: step 1, mixing DNA sequences SH-TPA, SH-TPB, SH-TPC and TPD, then carrying out heating denaturation treatment, and then naturally cooling to form DNATPCB; 2, DNATPCB, a TCEP solution and a hybridization buffer solution are fully reacted and then added to an AuE / NPG electrode, and incubation is carried out; and step 3, after incubation is completed, washing with a PBS solution to obtain an incubation electrode, and immersing the incubation electrode into an MCH solution for reaction to obtain the electrochemical biosensor based on the stretched DNA capture probe. According to the present invention, the hybridization time with miR-21 is significantly shortened, the hybridization efficiency is substantially improved, the wide linear range of 100 aM to 100 nM and the limit of detection (LOD) of 37.97 aM can be achieved within 40 min, and the excellent accuracy and the excellent stability are represented.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an electrochemical biosensor based on a stretched DNA capture probe, and a preparation method and application thereof. Background Art

[0002] MicroRNA (miRNA) is a class of non-coding, single-stranded RNAs approximately 18-24 nt in length. They are widely believed to be closely linked to the development, progression, and prognosis of certain cancers, making rapid and accurate detection crucial in clinical applications. Commonly used miRNA detection methods include real-time quantitative PCR, Northern blot analysis, microarray analysis, and next-generation sequencing. However, these methods require complex procedures, expensive equipment, rigorous laboratory conditions, and are time-consuming. Electrochemical biosensors are suitable for detecting miRNA due to their high sensitivity, good specificity, and simple operation. Currently, electrochemical biosensors for miRNA detection typically employ highly specific DNA capture probes to identify and capture the target miRNA, but this is time-consuming and hinders rapid detection. Therefore, it is necessary to design an electrochemical biosensor for rapid miRNA detection.

[0003] Currently, rapid detection of miRNAs using electrochemical biosensors is difficult to achieve due to the different hybridization kinetics between surfaces and solutions. Forward hybridization rates at interfaces are 10 to 100 times slower than in solution, while reverse melting rates can be tens of times faster. This results in a significant time requirement for miRNA to hybridize with capture probes. Furthermore, the fluctuating disordered conformation of ssDNA restricts the DNA hybridization process, resulting in relatively low hybridization kinetics and binding efficiency.

[0004] Glossary:

[0005] AuE / NPG electrode: For the preparation method, see the paper: F.Yu, Y.Wang, C.Yu, W.Zhang, X.Bai, Sensitive and Specific Y-Shaped Ratio Biosensor for Detecting Serum miR-18a: Potential Early Scanning Tool for Non-Small Cell Lung Cancer, J.Anal.Test.8(2024)237-244.

[0006] Cy3: cyanine fluorophore.

[0007] SH group: sulfhydryl group.

[0008] Hybridization buffer: A denaturing solution containing 50% (v / v) formamide. Summary of the Invention

[0009] The object of the present invention is to provide an electrochemical biosensor based on a stretched DNA capture probe and a preparation method and use thereof, so as to solve the problems raised in the above background technology.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A method for preparing an electrochemical biosensor based on a stretched DNA capture probe comprises the following steps:

[0012] Step 1: DNA sequences SH-TPA, SH-TPB, SH-TPC, and TPD are mixed, then heated for denaturation, and then naturally cooled to form DNATPCB; wherein SH-TPA, SH-TPB, and SH-TPC are all modified with SH groups;

[0013] The DNA sequence of SH-TPA is shown in SEQ ID NO: 1, the DNA sequence of SH-TPB is shown in SEQ ID NO: 2, the DNA sequence of SH-TPC is shown in SEQ ID NO: 3; the DNA sequence of TPD is shown in SEQ ID NO: 3;

[0014] Step 2: After DNATPCB, TCEP solution and hybridization buffer are fully reacted, they are added to the AuE / NPG electrode and incubated;

[0015] Step 3: After the incubation is completed, the incubated electrode is rinsed with PBS solution to obtain the incubated electrode, and the incubated electrode is immersed in MCH solution to react to obtain an electrochemical biosensor based on stretched DNA capture probe: AuE / NPG / TPCP / MCH electrode.

[0016] As a further improvement, SH groups are modified at the head ends of SH-TPA, SH-TPB and SH-TPC in the order of 5'-3'.

[0017] As a further improvement, in step 1, the molar ratio of SH-TPA, SH-TPB, SH-TPC and TPD is 1:1:1:1.

[0018] As a further improvement, the temperature of the heat denaturation treatment is 95° C. and the time is 5 minutes.

[0019] As a further improvement, in step 2, DNATPCB with a concentration of 10 μM, TCEP with a concentration of 10 mM and hybridization buffer are mixed at a volume ratio of 1:1:3 at room temperature for 1 hour to obtain a mixture, and the mixture is added to the AuE / NPG electrode and incubated overnight.

[0020] As a further improvement, in step three, after rinsing with 10 mM PBS, pH 7.4, the electrode was immersed in a 1 mM MCH solution for 60 minutes, thereby obtaining an AuE / NPG / TPCP / MCH electrode.

[0021] An electrochemical biosensor based on a stretched DNA capture probe prepared by the above preparation method.

[0022] A use of an electrochemical biosensor based on a stretched DNA capture probe, wherein the electrochemical biosensor is as described above and is used to prepare a device or a kit for detecting miR-21.

[0023] The advantages of the present invention are as follows:

[0024] The present invention significantly shortens the hybridization time with the target miRNA and greatly improves the hybridization efficiency. In addition, the three ssDNA recognition sites have the same base sequence, which can increase the capture amount of the target miR-21, thereby improving the detection sensitivity. The research results show that the TPCP-based electrochemical biosensor achieves ultra-fast and highly sensitive detection of miR-21, reaching a wide linear range of 100aM to 100nM and a detection limit (LOD) of 37.97aM within 40 minutes. At the same time, the sensor shows excellent accuracy and stability in the detection of miR-21 in serum samples and cell lines. Overall, this biosensor based on a triangular prism structure provides new possibilities for achieving ultra-fast, highly sensitive, highly repeatable and highly accurate detection of miRNA, and is expected to be applied to the early diagnosis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Polyacrylamide gel electrophoresis image.

[0026] Figure 2A Schematic diagram of the ssDNA stretching effect based on the MEF principle.

[0027] Figure 2B is the fluorescence intensity of Cy3 in 10nt, 20nt and 30nt height biosensors.

[0028] Figure 3 SEM images of NPG. A: 50k×, B: 100k×.

[0029] Figure 4A This is the characteristic diagram of the EIS graded electrode.

[0030] Figure 4B This is the characteristic diagram of the CV gradient electrode.

[0031] Figure 5 Performance diagram of the biosensor of the present invention, including feasibility analysis (A), optimization of experimental parameters: TPCP concentration (B), AP-Mb concentration (C), and incubation time of surface hybridization (D), SWV curve (E), and calibration curve of the triangular prism miRNA electrochemical biosensor (F); repeatability (G), selectivity (H), and stability (I) of the electrochemical biosensor of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] 1. Test:

[0034] 1.1 Chemicals, reagents and instruments:

[0035] Ammonium persulfate (APS), potassium hexacyanoferrate trihydrate (K4Fe(CN)6), bisacrylamide (30%), N,N,N',N'-tetramethylethylenediamine (TEMED), GelRed nucleic acid dye, potassium hexacyanoferrate (K3Fe(CN)6), chloroform, potassium hydrogen phosphate (KCl), sodium chloride (NaCl), and RNase-free water were purchased from Shanghai MacLean Biochemical Co., Ltd. (Shanghai, China). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 6-mercaptoethanol (MCH), and 0.1 M phosphate buffer solution (PBS: 1.37 mM NaCl, 26.8 mM KCl, 81.0 mM Na2HPO4, 17.6 mM KH2PO4, pH 7.4) were purchased from Sigma-Aldrich (USA). 12K gold-silver alloy thin film was purchased from Nanjing Yongbo Metal Materials Co., Ltd. (Nanjing, China). All chemically synthesized single-stranded DNA and miRNA were purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Their base sequences are shown in Table 1. The instrument used in this experiment was a CHI 760F electrochemical analyzer (CHI Inc., Shanghai, China) manufactured by Shanghai Chenhua Instrument Co., Ltd. Scanning electron microscopy (SEM) was provided by Regulus 8100 Co., Ltd., Japan (www.phadcalc.com). All solutions were prepared using Millipore deionized water (resistivity 18 megohm cm-1) from a Millipore ultrapure water purification system. Fetal bovine serum (FBS) was purchased from Gibco, USA.

[0036] Table 1. Probe, miRNA, and primer sequences used

[0037]

[0038]

[0039] 1.2 Gel electrophoresis:

[0040] To verify probe hybridization, perform 9% polyacrylamide gel electrophoresis (PAGE) at room temperature for 60 minutes. Hybridized samples (5 μl per well) were electrophoresed in 1× TBE buffer at 100 V for 60 minutes. The gel was then stained with GelRed for 5 minutes and imaged.

[0041] 1.3 Preparation of DNA TPCB:

[0042] The probes SH-TPA, SH-TPB, SH-TPC, and TPD were mixed at a molar ratio of 1:1:1:1 to a concentration of 10 μM, treated at 95°C for 5 minutes to denature them, and then naturally cooled to room temperature to form DNA TPCB, which was stored at 4°C until use.

[0043] 1.4 Preparation of electrochemical biosensor based on stretched DNA triangular prism capture probe:

[0044] The preparation method of bare gold electrode (AuE) and NPG has been previously reported. The AuE / NPG electrode was prepared by drying NPG on AuE and drying it with an infrared lamp

[23] . Then, DNATPCB, TCEP (1 μL, 10 mM) and hybridization buffer (3 μL) were mixed at room temperature for 1 hour. The mixture (5 μL) was added to the AuE / NPG electrode and incubated overnight. After rinsing with PBS (10 mM, pH 7.4), the electrode was immersed in 1 mM MCH solution for 60 minutes to obtain the AuE / NPG / TPCP / MCH electrode.

[0045] 1.5 Performance analysis of biosensors:

[0046] Five microliters of the auxiliary probe, methyl blue (AP-Mb), and a mixture of miR-21 at varying concentrations were added to a gold electrode (AuE) / NPG / TPCP / MCH, followed by incubation at room temperature for 40 minutes. The sensor's performance was evaluated by square wave voltammetry (SWV) in PBS (10 mM, pH = 7.4) with a voltage range of -0.6 V to 0 V, an amplitude of 50 mV, and a frequency of 100 Hz. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were also performed in a potassium ferricyanide solution (5.0 M [Fe(CN)6]3- / 4- in 0.1 M KCl) with a CV scan potential range of -0.2 V to 0.6 V and a scan rate of 0.05 V / s. EIS measurements were performed at frequencies ranging from 0.1 Hz to 100 kHz. The SWV, CV, and EIS curves were analyzed using ORIGIN 2021.

[0047] 1.6 Recovery of miR-21 in serum samples:

[0048] To further evaluate the TPCP biosensor's response to miR-21 in real samples, we performed a recovery test in FBS. Final miR-21 concentrations of 10 fM, 10 pM, and 10 nM were prepared and tested in nuclease-depleted FBS, which was heated at 56°C for 30 minutes and then diluted 10-fold with PBS (10 mM, pH 7.4). Each sample concentration was tested in triplicate, and the signal change was the average of the three results.

[0049] 1.7 Extraction of Total RNA from A549 and H1975 Cells

[0050] A549 and H1975 cells were cultured in 100 mm culture dishes using RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. A humidified atmosphere of 37°C and 5% CO2 was used to extract total RNA from A549 and H1975 cells using the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit (Tiangen Biotechnology (Beijing) Co., Ltd., DP451). Total RNA was eluted with DEPC water, quantified, and stored at -80°C for further use.

[0051] 2. Conclusion

[0052] 2.1 First, we designed a capture probe with four ssDNA strands that hybridize and fold to form a triangular prism DNA structure. It is worth noting that the lengths of the three ssDNA strands of the triangular prism DNA capture probe can be optimized to stretch the top three ssDNA strands, enabling rapid capture of miR-21. This probe is then immobilized on the surface of a nanoporous gold-modified electrode via gold-thiol bonds. The three top strands, each containing three identical ssDNA strands, are used to recognize and capture the target miR-21. In the presence of target miR-21, miR-21 and the Mb-modified auxiliary probe, AP-Mb, are simultaneously recognized and captured by the three ssDNA strands at the top of the triangular prism DNA capture probe, thereby introducing the Mb signal molecule onto the electrode surface. Under electrochemical workstation detection, an electrochemical signal, or Faradaic current, is generated. As miR-21 concentration increases, more AP-Mb is captured on the electrode surface, resulting in an increased electrical signal response. Therefore, miR-21 concentration can be detected by measuring the electrical signal response.

[0053] 2.2PAGE analysis probe hybridization:

[0054] The molecular weight and hybridization status of the probes were evaluated by polyacrylamide gel electrophoresis (PAGE). Figure 1 Lanes 1-3 show the molecular weights of miR-21, AP, and TPA. The three side products formed by the hybridization of TPA, TPB, and TPC are shown in lane 4. Lane 5 shows TPCP, formed by the hybridization of TPA, TPB, TPC, and TPD. When miR-21 and AP-Mb were present simultaneously, they were recognized and captured simultaneously by TPCP, as shown in lane 6. When only AP-Mb was present, no hybridization product was found in lane 7. Therefore, the PAGE results indicate that the TPCP probe was successfully designed and synthesized, and is feasible for miR-21 recognition and capture.

[0055] 2.3 MEF analysis of single-stranded DNA stretching

[0056] Metal-enhanced fluorescence (MEF) is a phenomenon in which fluorescence is enhanced when the fluorescent group is 6-90nm away from the metal nanoparticle. However, at a distance of <5nm, it is quenched and the fluorescence intensity is weakened. Based on this theory, a gold nanoparticle and a fluorescent group were modified at the two vertices of the top of the DNA triangular prism to evaluate the stretching state of 20nt long (about 6.8nm) ssDNA (such as Figure 2A and Figure 2B We modified TPB with cyanine fluorophore (Cy3) to construct a triangular prism structure, and then excited the Cy3 fluorophore. We observed the fluorescence emitted by Cy3 ( Figure 2A After SH was used to bind AuNPs and Cy3 to label TPA and TPB to form a triangular prism structure, the Cy3 fluorophore was excited and the fluorescence intensity increased with the stretching of ssDNA ( Figure 2A b, c, d). The MEF experimental results of the triangular prism structure are shown in Figure 2B As shown, compared to the triangular prism containing only Cy3, the fluorescence intensity of Cy3 in the triangular prisms with a base height of 10 nt and 20 nt is stronger, while the fluorescence of Cy3 in the triangular prism with a base height of 30 nt is partially extinguished. This indicates that the ssDNA stretching effect is best at the top of the triangular prism with a base height of 10 nt.

[0057] Specific operation steps: SH-TPA1, SH-TPA2, and SH-TPA3 were added to 1uM AuNPs respectively, incubated for 2 hours, and then SH-TPA1 / Cy3-TPB1 / TPC1 / TPD in Table 1 were added according to method 1.3;

[0058] SH-TPA2 / Cy3-TPB2 / TPC2 / TPD; SH-TPA3 / Cy3-TPB3 / TPC3 / TPD synthesize 10nt, 20nt, and 30nt DNA triangular prism probes (TPCP), respectively.

[0059] The MEF experimental results of the triangular prism structures with three heights are shown in Figure 2. Figure 2B As shown, compared to the triangular prism containing only Cy3, the fluorescence intensity of Cy3 in the triangular prisms with a base height of 10 nt and 20 nt is stronger, while the fluorescence of Cy3 in the triangular prism with a base height of 30 nt is partially extinguished. This indicates that the ssDNA stretching effect is best at the top of the triangular prism with a base height of 10 nt.

[0060] 2.4 Characteristics of NPG:

[0061] The nanostructure and morphological characteristics of the NPG surface were verified using scanning electron microscopy (SEM). Figure 3As shown, NPG is produced by etching a 12K gold-silver alloy with 65% HNO3. It is rich in uniformly distributed three-dimensional nanoporous structures. Modification on the electrode surface increases the specific surface area of ​​the electrode and provides more binding sites for capture probes, thereby improving sensitivity.

[0062] 3.5 Characteristics of triangular prism capture probes:

[0063] like Figure 4A The electron transfer resistance (Ret) of the bare gold electrode (a) and the NPG electrode (b) was 81Ω and 38Ω, respectively. The Ret of the NPG electrode was significantly reduced due to its high conductivity. Because electron transfer is hindered by negatively charged DNA, the Ret increased to 286Ω, 410Ω, and 513Ω after TPCP, MCH, and miR-21 / AP-Mb assembly, respectively. Figure 4B The AuE / NPG redox peak current was higher than that of the bare gold electrode, but the current decreased after treatment with TPCP and MCH. The current also decreased significantly after capturing miR-21 and AP-Mb. EIS and CV results showed that the triangular prism biosensor was successfully constructed.

[0064] 3.6 Feasibility Analysis of Biosensors:

[0065] like Figure 5 As shown in Figure A, the biosensor prepared on the NPG electrode exhibits a very flat baseline. When AP-Mb alone is present, the background current is very low. In the presence of miR-21, a strong current peak is detected. However, the current detected on the electrode without NPG at the same concentration of miR-21 is much smaller and has a non-flat baseline. Therefore, the TPCP-mediated biosensor is feasible for detecting the target miR-21, and NPG can effectively amplify the current signal.

[0066] 3.7 Optimization of probe concentration:

[0067] At the optimal probe concentration, the time it takes for TPCP on the electrode surface to capture miR-21 and AP-Mb is also a key factor in determining whether rapid detection can be achieved. Therefore, we carefully optimized the time within 90 minutes. Figure 5As shown in the middle BD in the figure, the SWV current gradually increased after 40 minutes of hybridization and then reached a stable state as the incubation time was further extended. Ultimately, 40 minutes was selected as the optimal incubation time. It is worth emphasizing that because the TPCP on the electrode surface has a strong rigid structure and its top recognition threshold ssDNA is in a stretched state, it can easily hybridize with the target miR-21 and form a stable double-stranded structure. Therefore, the electrochemical biosensor constructed with this stretched DNA TPCP can quickly detect miR-21. However, the detection time of this TPCP is slightly longer than that of the sensor constructed with the double tetrahedral probe by Li et al. This may be because the TPCP has three ssDNA capture thresholds on the top, and more target miR-21 needs to be captured to achieve saturation.

[0068] 3.8 Performance analysis of biosensors:

[0069] In order to verify the superiority of TPCP in constructing electrochemical biosensors, it was used to detect different concentrations of miR-21 under optimal conditions. Figure 5 As shown in Figure E, the SWV peak current increased significantly with the increase of miR-21 concentration. The linear regression equations were I = 5.10408logC miR-21 The linear range for miR-21 was 1 fM to 100 nM, with a limit of detection (LOD) of 467.97 aM (LOD = 3.3σ / S, where σ is the standard deviation of the blank and S is the slope of the calibration curve). Compared to other biosensors, as shown in Table 2, the proposed biosensor has an attractively wider linear range and a relatively low limit of detection. It is worth emphasizing that the biosensor of the present invention has a lower limit of detection than sensors constructed with double tetrahedral probes such as Li. This is due to the biosensor's higher electrode surface utilization and the three ssDNA recognition thresholds per TPCP molecule. Therefore, compared to their complex double DNA tetrahedral stretched structures, TPCP has a simpler synthesis process and a lower LOD, making it more suitable for widespread application in miRNA research.

[0070] Table 2 Comparison of multi-capture probe biosensors for miRNA detection

[0071]

[0072]

[0073] 2.9 Recovery detection of miR-21 spiked with 10× FBS:

[0074] To validate the biosensor's application in biological sample detection, the sensor was subjected to spike recovery testing and miR-21 detection in cell lines. The spike recovery test results are shown in Table 3. The recovery rates were 109.80%, 103.47%, and 105.19%, respectively, with RSDs ranging from 3.02% to 7.92%. The spike recovery results demonstrate that the proposed triangular prism biosensor can accurately detect miR-21 in serum samples. Furthermore, miR-21 levels in cell lines A549 and H1975 were tested. The results were 3879.38±381.79 fM and 4117.12±397.83 fM, respectively, with RSDs of 15.17% and 11.03%, respectively, which are nearly consistent with previous test results, indicating that the triangular prism biosensor can accurately detect miR-21 levels in cell lines. In summary, the triangular prism biosensor can accurately detect miR-21 in serum and cell samples and has significant potential for practical miRNA analysis in the medical field.

[0075] Table 3. Content of miR-21 in 10×FBS measured by the biosensor of the present invention

[0076]

[0077] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an electrochemical biosensor based on a stretched DNA capture probe, characterized in that: The steps include: Step 1: DNA sequences SH-TPA, SH-TPB, SH-TPC and TPD are mixed, then heated for denaturation, and then naturally cooled to form DNATPCB; Among them, SH-TPA, SH-TPB and SH-TPC are all modified with SH groups; The DNA sequence of SH-TPA is shown in SEQ ID NO: 1, the DNA sequence of SH-TPB is shown in SEQ ID NO: 2, the DNA sequence of SH-TPC is shown in SEQ ID NO: 3; the DNA sequence of TPD is shown in SEQ ID NO: 3; Step 2: After DNATPCB, TCEP solution and hybridization buffer are fully reacted, they are added to the AuE / NPG electrode and incubated; Step 3: After the incubation is completed, the incubated electrode is rinsed with PBS solution to obtain the incubated electrode, and the incubated electrode is immersed in MCH solution to react to obtain an electrochemical biosensor based on stretched DNA capture probe: AuE / NPG / TPCP / MCH electrode.

2. The method for preparing an electrochemical biosensor based on a stretched DNA capture probe according to claim 1, wherein: In the order of 5'-3', SH groups were modified at the head ends of SH-TPA, SH-TPB and SH-TPC.

3. The method for preparing an electrochemical biosensor based on a stretched DNA capture probe according to claim 1, wherein: In step 1, the molar ratio of SH-TPA, SH-TPB, SH-TPC and TPD is 1:1:1:

1.

4. The method for preparing an electrochemical biosensor based on a stretched DNA capture probe according to claim 1, wherein: The temperature of the heat denaturation treatment is 95° C. and the time is 5 minutes.

5. The method for preparing an electrochemical biosensor based on a stretched DNA capture probe according to claim 1, wherein: In the second step, DNATPCB with a concentration of 10 μM, TCEP with a concentration of 10 mM and hybridization buffer were mixed at a volume ratio of 1:1:3 at room temperature for 1 hour to obtain a mixture, which was then added to the AuE / NPG electrode and incubated overnight.

6. The method for preparing an electrochemical biosensor based on a stretched DNA capture probe according to claim 1, wherein: In the step 3, after rinsing with PBS having a concentration of 10 mM and a pH of 7.4, the electrode was immersed in a 1 mM MCH solution for 60 minutes, thereby obtaining an AuE / NPG / TPCP / MCH electrode.

7. An electrochemical biosensor based on a stretched DNA capture probe prepared by the preparation method according to any one of claims 1 to 6.

8. Use of an electrochemical biosensor based on a stretched DNA capture probe, characterized in that: The electrochemical biosensor as claimed in claim 7 is used to prepare a device or kit for detecting miR-21.

Images