DNA nanosphere electrochemical biosensor based on dumbbell-shaped ring RCA reaction as well as construction method and application of DNA nanosphere electrochemical biosensor

Abstract

The invention discloses a DNA nanosphere electrochemical biosensor based on dumbbell-shaped ring RCA reaction and a construction method and application of the DNA nanosphere electrochemical biosensor based on dumbbell-shaped ring RCA reaction, a target miRNA solution is dropwise added to the surface of an Au-HCP-MCH electrode for incubation, HCP hairpins are opened, the target miRNA is combined with partial fragments of HCP chains, then an RCA reaction solution is dropwise added to the surface of the electrode, and the DNA nanosphere electrochemical biosensor based on dumbbell-shaped ring RCA reaction is obtained. The method comprises the following steps: using HCP as an HCP chain, then using a cohesive end exposed in the HCP chain as a primer chain to trigger RCA of a D ring, spontaneously hybridizing and self-assembling a plurality of repeated complementary sequences in an RCA product to form a three-dimensional DNA nanosphere, inserting an electrochemical indicator into the DNA nanosphere, and detecting the content of target miRNA based on a generated electrochemical signal. The sensor is applied to detection of miRNA in a solution. The method has the characteristics of simplicity in operation, time saving, strong anti-interference capability, good stability and reproducibility, high detection sensitivity, high specificity and the like, shows extremely high accuracy and reliability when detecting miRNA in a human blood sample, and has huge potential in the aspect of clinical diagnosis application based on nucleic acid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterials, and particularly relates to a construction method of a dumbbell-shaped RCA reaction DNA nanosphere electrochemical biosensor and application thereof in detection of clinical blood samples. BACKGROUND

[0002] MicroRNA (miRNA) is a class of non-coding single-stranded RNA molecules with a length of about 18-24 nucleotides, which plays a key role in various biological processes such as cell proliferation, differentiation and apoptosis by regulating gene expression. Abnormal expression of miRNA is closely related to various diseases including cancer. miRNA-21 is abnormally expressed in the serum of various cancer patients, such as lung cancer, breast cancer, pancreatic cancer, liver cancer, cervical cancer and prostate cancer, and it is a recognized carcinogenic miRNA. miRNA is released from cancer cells into blood through exosomes, apoptotic bodies or protein binding. Due to its stability and detectability, blood-derived miRNA has become a key biomarker in cancer research. Detecting blood-derived miRNA as a non-invasive detection method has great potential in early screening. Accurate identification and quantitative analysis of miRNA are of great significance for disease diagnosis, treatment, exploration of its regulation in specific biological pathways and related drug development. However, due to its short sequence, homologous sequence interference and low content in biological samples, accurate identification and reliable analysis of miRNA have been severely hindered.

[0003] At present, various detection methods have been developed to accurately detect the expression level of miRNA. Northern blotting is a commonly used method for detecting miRNA; however, it has some limitations such as complex process, long time-consuming and poor reproducibility. As the gold standard for miRNA detection, quantitative reverse transcription polymerase chain reaction (qRT-PCR) has high detection sensitivity, but its widespread application is limited by challenges such as primer design and high cost. As a high-throughput detection method, microarray technology has high detection efficiency, but is limited to semi-quantitative analysis, complex operation process and high detection cost. In recent years, some isothermal nucleic acid amplification signal technologies such as exponential amplification technology, strand displacement amplification technology (SDA), rolling circle amplification technology (RCA), catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) have attracted widespread attention.

[0004] Currently, biosensors for miRNA detection mainly rely on colorimetric, optical, and electrochemical techniques. Among them, electrochemical biosensors are of great interest due to their ease of operation and cost-effectiveness. Through highly specific interactions between the biological recognition element and the target, electrochemical biosensors produce a suitable electrochemical reading, facilitating the quantitative analysis of the target, either directly or indirectly. When combined with highly sensitive signal amplification techniques, electrochemical sensors have developed into a powerful tool for miRNA detection.

[0005] Figure 1 For a DNA biped walker with dumbbell-wheel structure conversion for nucleic acid detection, on the electrode, a hairpin probe B is fixed on a base probe A through a pH-sensitive DNA triplex structure. When the target appears, it will specifically bind to the specially designed hairpin probe E, triggering its conformational change, and then starting the SDA reaction. Under the synergistic action of polymerase and nicking enzyme, the reaction will generate a large amount of short single-stranded DNA product containing target sequence information. The large amount of product produced by SDA serves as a "key" to start the pre-assembled "DNA wheel" (probe D). After the DNA wheel is activated, its surface will expose a catalytically active DNAzyme. The activated DNA wheel "walks" on the electrode surface through its biped DNAzyme. Every time it walks next to a fixed probe B, the DNAzyme will cut it, releasing the pre-labeled signal molecule on the probe B. The release of the signal molecule leads to a detectable decrease in the electrical signal. A DNA wheel can continuously cut multiple probes B, achieving third-order signal amplification. Finally, by measuring the magnitude of the electrical signal drop, the concentration of the target can be quantitatively analyzed. However, there are still the following shortcomings. (1) Complexity and time consumption of operation: The entire process involves multiple steps (SDA reaction, DNA wheel activation, walking cutting, electrochemical detection), which requires precise control of reaction conditions and time, and may not be suitable for emergency situations that require extremely fast results. (2) Sensitivity to experimental conditions: The reaction efficiency is highly dependent on pH, temperature, enzyme activity, etc. Small fluctuations in conditions can lead to unstable results, requiring higher requirements for operators and experimental environments. (3) Potential interference problems: Various biological molecules (such as proteins, nucleic acids) present in clinical samples may non-specifically adsorb or interfere with complex DNA nanostructures, affecting detection accuracy. (4) Stability and reproducibility challenges: The stability of DNA probes and the walking efficiency of DNA wheels on the electrode surface can affect batch-to-batch and intra-batch reproducibility. (5) High cost: The use of multiple enzymes increases the cost of use. SUMMARY

[0006] In order to reduce the high detection cost in the current miRNA detection technology and simplify the operation process, the purpose of the present application is to construct a three-dimensional DNA nanosphere electrochemical sensor composed of only three DNA chains, which realizes the ultra-sensitive detection of miRNA based on the RCA reaction mediated by dumbbell-shaped DNA ring (D ring).

[0007] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0008] The present application provides a construction method of a DNA nanosphere electrochemical biosensor based on dumbbell-shaped RCA reaction, which specifically comprises the following steps:

[0009] (1) The 5' end of the HCP hairpin is modified by -SH, and then the modified HCP hairpin is fixed on the surface of the Au electrode by AU-S bond to obtain an Au-HCP electrode, and finally the Au-HCP electrode is treated with 6-mercapto-1-hexanol MCH, and the MCH is also fixed on the surface of the Au electrode by AU-S bond to close the remaining bare gold surface, thereby obtaining an Au-HCP-MCH electrode;

[0010] (2) The target miRNA solution is added to the surface of the Au-HCP-MCH electrode for incubation, the HCP hairpin is opened, the target miRNA is combined with the HCP chain part fragment, then the RCA reaction solution is added to the surface of the electrode, the exposed sticky end in the HCP chain triggers the RCA of the D ring as a primer chain, and multiple repeated complementary sequences in the RCA product spontaneously hybridize and self-assemble to form a three-dimensional DNA nanosphere, an electrochemical indicator is loaded in the DNA nanosphere, and the content of the target miRNA is detected based on the generated electrochemical signal;

[0011] The RCA reaction solution contains D ring, phi29 DNA polymerase, dNTP and phi29 reaction buffer without DTT;

[0012] The HCP chain sequence comprises HCP-A region, HCP-B region and HCP-C region arranged in sequence from 5' to 3'; the L-Dumbbell chain sequence comprises L-Dumbbell-X region, L-Dumbbell-Y region and L-Dumbbell-Z region arranged in sequence from 5' to 3'; and the Primer-Dumbbell sequence comprises Primer-Dumbbell-M region sequence and Primer-Dumbbell-N region sequence arranged in sequence from 5' to 3';

[0013] wherein, the sequence of the part close to 5' in L-Dumbbell-X region is base complementary pairing with the sequence of Primer-Dumbbell-M region, the sequence of L-Dumbbell-Z region is base complementary pairing with the sequence of Primer-Dumbbell-N region, forming D-loop structure, the sequence of HCP-B region is base complementary pairing with the target miRNA, and the sequence of HCP-C region is base complementary pairing with the sequence of L-Dumbbell-X region.

[0014] Preferably, the HCP-A region contains 9 T bases, the HCP-B region is the same length as the target miRNA to be detected, the HCP-C region and the L-Dumbbell-X region are the same length, both containing 25-27 bases; the L-Dumbbell-Y region contains 9-10 bases, the L-Dumbbell-Z region and the Primer-Dumbbell-N region are the same length, both containing 15 bases, and the Primer-Dumbbell-M region contains 15 bases.

[0015] Specifically, the target miRNA in step (2) includes but is not limited to one of miRNA-21, miR-155, miRNA-1, miRNA-144 and miRNA-31, preferably miRNA-21 and miR-15. When the target miRNA is miRNA-21, the sequences of HCP, L-Dumbbell and Primer-Dumbbell are 5'-TTT TTT TTT TCA ACA TCA GTC TGA TAA GCT ACC ATG TGT AGA TAG CTT ATC AGA CT-3', 5'-AGT CTG ATA AGC TAT CTA CAC ATG GTA ATG CTA ATC GTG CCA TGT GTA GA-3' and 5'-ATA GCT TAT CAG ACT TCT ACA CAT GGC ACG-3', respectively. When the target miRNA is miR-15, the sequences of HCP, L-Dumbbell and Primer-Dumbbell are 5'-TTT TTT TTT ACC CCT ATC ACG ATT AGC ATT AAC CAT GTG TAG ATT AAT GCT AAT CGT G-3', 5'-CAC GAT TAG CAT TAA TCT ACA CAT GGT AGC TTA TCA GAC TCC ATG TGT AGA-3' and 5'-TTA ATG CTA ATC GTG TCT ACA CAT GGA GTC-3', respectively.

[0016] Specifically, in step (2), the concentration of miRNA in the target miRNA solution is 0~50 nM, preferably 1 pM~1 nM.

[0017] It should be noted that the electrochemical indicator is loaded onto the nanospheres via electrostatic interaction, or embedded in the grooves of the DNA nanospheres, or can be loaded onto the nanospheres in other forms. The electrochemical indicator includes methylene blue (MB), thionine, or toluidine blue. In step (2), the target miRNA content is specifically detected using the current signal generated by the DPV test.

[0018] Specifically, the RCA reaction solution contains 1 μM D loop, 1 U / μL phi29 DNA polymerase, 1 mM dNTP, and 10 μL of 1×phi29 reaction buffer without DTT.

[0019] Specifically, the average height (AH) and average width (AW) of the three-dimensional DNA nanospheres are 15.9 ± 0.8 nm and 68.1 ± 4.0 nm, respectively.

[0020] Specifically, step (2) involves a target miRNA solution, which is a blood sample containing the target miRNA.

[0021] Specifically, the incubation in step (2) is carried out at room temperature.

[0022] A second aspect of the present invention provides an electrochemical biosensor constructed by the above-described construction method.

[0023] The third aspect of this invention provides the application of the above-mentioned electrochemical biosensor in miRNA detection, particularly in the detection of miRNA in blood.

[0024] This invention first develops a three-dimensional DNA nanosphere electrochemical biosensor based on dumbbell-shaped DNA loops (D-loops) and their RCA products for miRNA detection. The target miRNA, a promising non-invasive tumor biomarker, binds to a hairpin trap probe (HCP) and opens its hairpin structure. The exposed sticky ends act as primers to trigger the RCA reaction of the D-loop. During the RCA reaction, using the D-loop as a template, linear DNA containing tandem repeat sequences is rapidly generated under the action of polymerase, ultimately self-assembling into nanospheres. The longer the DNA chain, the more micromolecular molecules (MBs) bind, resulting in signal amplification and achieving ultra-high sensitivity analysis of the target.

[0025] The beneficial effects of this invention are:

[0026] (1) Simple operation and time saving: The entire process only involves the target triggering process, RCA reaction and electrochemical detection. It is simple to operate and relatively short in time, which is suitable for the needs of rapid clinical diagnosis. (2) Sensitivity to experimental conditions: The reaction process has low dependence on external conditions such as pH and temperature. (3) Excellent anti-interference ability: The RCA reaction mediated by dumbbell-shaped DNA loops enables the electrochemical sensor to resist the interference of other components in serum samples, ensuring detection accuracy. (4) Stability and reproducibility challenge: It does not involve random reactions between DNA probes and has good reproducibility. (5) Low cost: The amount of DNA probes and enzymes used is small. (6) High detection sensitivity: The target miRNA can be quantitatively detected and the detection limit can be as low as 29 fM. (7) High detection specificity: Coexisting non-target miRNAs and other biomolecules do not interfere with signal transduction. (8) Strong versatility: The detection of multiple miRNAs can be achieved by adjusting the sequence according to the target miRNA. (9) The three-dimensional DNA nanosphere electrochemical biosensor demonstrated extremely high accuracy and reliability in detecting miRNA in human blood samples, which has great potential in nucleic acid-based clinical diagnostic applications. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the design and signal transmission of a biosensor used for miRNA detection in the prior art.

[0028] Figure 2 A schematic diagram illustrating the design and signal transmission of the three-dimensional DNA nanosphere electrochemical biosensor in this invention.

[0029] Figure 3 The image shows cyclic voltammetry results for different modified electrodes in Example 1.

[0030] Figure 4 The image shows the AC impedance test results for different modified electrodes in Example 1.

[0031] Figure 5 The image shows the DPV current response of the three-dimensional DNA nanosphere electrochemical biosensor to miR-21D in Example 7.

[0032] Figure 6 The images show polyacrylamide gel electrophoresis diagrams of the different components involved in Example 2.

[0033] Figure 7 This is an atomic force microscopy image of the three-dimensional DNA nanospheres prepared in Example 2.

[0034] Figure 8The graphs shown in Example 3 are: DPV current response of the three-dimensional DNA nanosphere electrochemical biosensor to different concentrations of miR-21D (a) and the relationship between the DPV peak current value and the miR-21D concentration (b).

[0035] Figure 9 The images show the DPV current response (a and c) and DPV peak current values ​​(b and d) of the three-dimensional DNA nanosphere electrochemical biosensor for different DNA target probes in Example 4.

[0036] Figure 10 The image shows the electrochemical signal of the three-dimensional DNA nanosphere electrochemical biosensor in Example 5 under different incubation times in a 10% healthy human serum sample.

[0037] Figure 11 The image shows the DPV current response of the three-dimensional DNA nanosphere electrochemical biosensor to MiR-155D in Example 7. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following specific embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. If those skilled in the art make some non-essential adjustments and improvements to the present invention based on the above content, they shall still fall within the protection scope of the present invention.

[0039] Example 1

[0040] A method for constructing a DNA nanosphere electrochemical biosensor based on the dumbbell-shaped ring RCA reaction includes the following steps:

[0041] Synthesis of the D loop: 1 μL of 10 μM DNA template strand (L-Dumbbell) and 1 μL of 10 μM primer-Dumbbell were mixed in 2 μL of 10×T4 DNA ligase buffer. The mixture was adjusted to 16 μL with ultrapure water and heated at 90 °C for 5 minutes, followed by slow cooling to room temperature. Next, 1 μL of T4 DNA ligase (1000 U / μL) was added to the mixture, and the reaction was carried out overnight at 16 °C. Subsequently, 0.5 μL of Exonuclease I (5 U / μL), 0.5 μL of Exonuclease III (5 U / μL), 2 μL of 10×Exonuclease I reaction buffer, and 2 μL of 10×Exonuclease III reaction buffer were added to the mixture, and the reaction was carried out at 37 °C for 4 hours, followed by inactivation at 70 °C for 20 minutes.

[0042] Purification of the D ring: First, add 1 / 10 volume of 3 M NaOAc and 2.5 volumes of anhydrous ethanol to the D ring mixture, incubate at -80°C for 2 hours, and centrifuge (13000 rpm / min, 30 min, 4°C) to remove the supernatant. Then, wash twice with cooled 75% ethanol. The final precipitate is dried under vacuum to remove ethanol and dissolved in 1×TE buffer for subsequent use.

[0043] Electrode modification: A 3 mm diameter gold electrode was first treated with a piranha solution (98% H2SO4: 30% H2O2 = 3:1) for 5 minutes to remove organic contaminants, and then polished with a 0.05 μm alumina slurry to obtain a mirror surface. Finally, the electrode was cleaned with ultrapure water and ethanol for 5 minutes each, and then dried with nitrogen gas for use.

[0044] The 5' end of the HCP hairpin was modified with -SH, and 10 μL of the modified HCP hairpin (SH-HCP) (1 μM) was dropped onto the pretreated clean gold electrode surface and incubated at room temperature for 2 hours. The modified HCP hairpin attached to the gold electrode via AU-S, resulting in an Au-HCP electrode. Free SH-HCP chains were then removed by washing three times with 0.1 M PBS buffer. The Au-HCP electrode was then treated with 10 μL of 1 M MCH for 10 minutes to seal the remaining exposed gold surface, forming a DNA-modified electrode (Au-HCP-MCH electrode) for subsequent miRNA detection.

[0045] Electrochemical detection: A certain concentration of target miR-21D (10 μL) was added to the surface of the Au-HCP-MCH electrode and incubated for 2 hours. The HCP hairpins opened, and miR-21D bound to a partial fragment of the HCP chain. The electrode was then washed with 0.1 M PBS buffer to obtain the Au-HCP-MCH-miR-21D electrode. Next, an RCA reaction solution containing the D loop (1 μM), phi29 DNA polymerase (1 U / μL), dNTPs (1 mM), and 1×phi29 reaction buffer (10 μL) without DTT was added dropwise to the surface of the Au-HCP-MCH-miR-21D electrode and incubated at room temperature for 30 minutes. The exposed sticky ends of the HCP strand then acted as primers to trigger the RCA of the D loop, generating a large number of repeating DNA strands complementary to the D loop, which eventually self-assembled into nanospheres. These nanospheres were then washed with 0.1 M PBS buffer to obtain the Au-HCP-MCH-miR-21D-RCA electrode. Finally, the detection interface was mixed with 10 μL of 1 mM MB for 30 minutes, washed with 0.1 M PBS buffer, and the electrochemical signal was collected. The 1×phi29 reaction buffer without DTT is a solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 10 mM (NH4)2SO4.

[0046] The DNA sequence of the electrochemical sensor is as follows:

[0047] HCP-21D: 5'-TTT TTT TTT TCA ACA TCA GTC TGA TAA GCT ACC ATG TGT AGATAG CTT ATC AGA CT-3' SH-HCP-21D: SH-TTT TTT TTT TCA ACA TCA GTC TGA TAA GCT ACC ATG TGTAGA TAG CTT ATC AGA CT L-Dumbbell-21D: 5'-AGT CTG ATA AGC TAT CTA CAC ATG GTA ATG CTA ATCGTG CCA TGT GTA GA-3' Primer-Dumbbell-21D: 5'-ATA GCT TAT CAG ACT TCT ACA CAT GGC ACG-3' miR-21D: 5'-TAGCTTATCAGACTGATGTTGA-3'.

[0048] The HCP-21D chain sequence is divided into three parts, from 5' to 3': the HCP-A region (sequence: TTT TTT TTT), the HCP-B region (sequence: TCA ACA TCA GTC TGA TAA GCT A), and the HCP-C region (sequence: CC ATG TGT AGA TAGCTT ATC AGA CT). The L-Dumbbell-21D chain sequence is also divided into three parts, from 5' to 3': the L-Dumbbell-X region (sequence: AGT CTG ATA AGC TAT CTA CAC ATG G), the L-Dumbbell-Y region (sequence: TA ATG CTAAT), and the L-Dumbbell-Z region (sequence: C GTG CCA TGT GTA GA). The Primer-Dumbbell-21D sequence is divided into two parts, from 5' to 3': the Primer-Dumbbell-M region sequence (sequence: ATA GCT TAT CAG ACT) and the Primer-Dumbbell-N region sequence (sequence: TCT ACA CAT GGC ACG). Specifically, the portion of the L-Dumbbell-X region closest to 5' (sequence: AGT CTG ATA AGC TAT) is complementary to the Primer-Dumbbell-M region sequence (sequence: ATA GCT TAT CAG ACT), the L-Dumbbell-Z region is complementary to the Primer-Dumbbell-N region sequence, forming a D-ring structure, the HCP-B region is complementary to the miR-21D sequence, and the HCP-C region is complementary to the L-Dumbbell-X region. For HCP-21D, the sequence “A GTC TGA TAA GCT A” in the HCP-A region and the sequence “TAG CTT ATC AGA CT” in the HCP-C region are complementary (i.e., the 18th to 32nd bases near the 5' end are complementary to the 1st to 15th bases near the 3' end), forming a hairpin structure. For L-Dumbbell-21D, the sequence “T CTA CAC ATG G” in the L-Dumbbell-X region and the sequence “CCA TGT GTAGA” in the L-Dumbbell-Z region are complementary, forming a hairpin structure. The selection of complementary bases from the 18th to 32nd bases near the 5' end to the 1st to 15th bases near the 3' end in HCP-21D to form a hairpin structure gives it good specificity for miR-21D.

[0049] The modified electrodes at each stage of Example 1 were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS measurements were performed in the frequency range of 0.1 Hz to 10 kHz, while CV was performed at a scan rate of 0.1 mV / s in the range of -0.2 V to 0.6 V. The working solution for EIS and CV was a 1.0 mM K3Fe(CN)6 / K4Fe(CN)6 (1:1) solution containing 0.1 M KCl.

[0050] Figure 3 To characterize the different modified electrodes using CV. [Fe(CN)6] 3− / 4− The changes in redox peaks reflect the changes in the sensing interface of different modified electrodes. This is similar to the [Fe(CN)6] peaks on a bare Au electrode. 3− / 4− Compared to the redox peaks (black curve), the electrochemical signal of the SH-HCP-immobilized Au electrode is significantly weakened (red curve), which may be due to the negatively charged oligonucleotides reacting with [Fe(CN)6]. 3− / 4− Electrostatic repulsion occurs between them. Subsequently, after MCH occupies the remaining electrode sites, the peak signal further decreases (blue curve). After the addition of miR-21D, miR-21D binds to SH-HCP and opens its hairpin structure, with only a slight change in the current signal observed (green curve). The current signal obtained after the introduction of D loop and phi29 polymerase is the smallest (purple curve), indicating that the RCA reaction proceeded successfully and the three-dimensional DNA nanospheres were successfully assembled.

[0051] Figure 4 Different modified electrodes were characterized using EIS, a technique more sensitive to the surface properties of sensing electrodes. The inset in Figure 4 shows the Randall equivalent circuit of the chemically modified electrode, where Ret, Rs, Zw, and Cdl represent electron transport resistance, solution resistance, Warburg impedance, and double-layer capacitance, respectively. In EIS, [Fe(CN)6] serves as a redox probe. 3− / 4− The semicircular diameter is correlated with Ret, reflecting the modification process of the electrode surface. The results show that the semicircular diameter gradually increases during the continuous modification of the gold electrode, indicating a continuous increase in the electrode interfacial resistance. These EIS results are consistent with CV measurements, confirming the successful construction of the electrochemical biosensor based on three-dimensional DNA nanospheres and the accurate detection of the target miRNA.

[0052] The feasibility of the three-dimensional DNA nanosphere electrochemical biosensor was verified using differential pulse voltammetry (DPV), and the results are as follows: Figure 5As shown, Target represents the target group, which is the three-dimensional DNA nanosphere electrochemical sensor constructed in this embodiment. Blank represents the blank group, in which miR-21D (10 μL, 1 μM) was replaced with the same volume of 1×TAE / Mg during the electrochemical detection process. 2+ The sensor was obtained using a buffer solution. The DPV working solution was phosphate buffer (0.1 M PBS, pH 7.4). The DPV detection parameters were as follows: potential scan range -0.4 V to 0.1 V; pulse period 0.5 s; pulse width 1.1 s; pulse amplitude 50 mV.

[0053] Figure 5 Differential pulse voltammetry (DPV) was used to monitor changes in the electrochemical response in the presence and absence of the target analyte. In the absence of the target analyte (black curve), a weak peak current signal was observed in the blank group, primarily attributed to the adsorption of a small amount of MB on the SH-HCP chain immobilized on the gold electrode. When 10 nM of the target analyte was added, triggering the formation of RCA-based three-dimensional DNA nanospheres, a significant peak current signal was observed in the target analyte group (red curve). Furthermore, the signal-to-noise ratio (SNR) corresponding to the 10 nM target analyte was calculated to be 51. These results demonstrate that the target-triggered electrochemical biosensor based on three-dimensional DNA nanospheres can detect a significantly amplified target analyte signal.

[0054] Example 2

[0055] Construction of DNA nanospheres: HCP chain (1 μL, 10 μM) and miR-21D (1 μL, 10 μM) in 8 μL 1×TAE / Mg 2+ The HCP probe's hairpin structure was opened by incubation at room temperature for 2 hours in buffer solution. Subsequently, it was incubated with RCA reaction solution at room temperature for 30 minutes, followed by inactivation of the enzyme at 75°C for 20 minutes, yielding three-dimensional DNA nanospheres. The self-assembly products triggered by the target probe were characterized by polyacrylamide gel electrophoresis and atomic force microscopy (AFM).

[0056] Polyacrylamide gel electrophoresis was used to test the different components involved in this embodiment and the products of corresponding stages in the preparation of DNA nanospheres. Figure 6The synthesis of the D-ring was analyzed by polyacrylamide gel electrophoresis. Lane 1: L-Dumbbell; Lane 2: D-ring; Lane 3: D-ring + exonuclease I / III. Under the combined action of both, only the D-ring remained. Exonuclease I cleaves single-stranded DNA, and exonuclease III cleaves double-stranded DNA. Lane 3 shows that a clear DNA band remained after the D-ring was broken down by both enzymes, indicating successful D-ring synthesis.

[0057] Figure 6b illustrates the triggering of HCP and the RCA process. Lane 1: HCP; Lane 2: HCP + miRNA; Lane 3: HCP + miRNA + D-loop; Lane 4: HCP + miRNA + RCA. As DNA strands are added stepwise, the electrophoretic mobility gradually decreases from lane 1 to lane 3. After the addition of DNA polymerase (lane 4), the RCA reaction was successfully performed.

[0058] Figure 7. Verification of the structure and size of the three-dimensional DNA nanospheres using AFM images. The AFM image shows nanoscale dot-like structures. The theoretical diameter of the DNA double helix is ​​typically less than 2 nm. Cross-sectional analysis in the right figure shows that the average height (AH) and average width (AW) of the dots are 15.9 ± 0.8 nm and 68.1 ± 4.0 nm, respectively, indicating the formation of a three-dimensional DNA nanosphere structure. The fact that the AH value is much lower than the AW value is mainly due to the well-known tip diffusion effect.

[0059] Example 3: Analysis of the detection capability of a target DNA probe by an electrochemical biosensor based on a dumbbell-shaped ring RCA reaction.

[0060] In this embodiment, except that different concentrations of the target miR-21D (0~50 nM) were dropped onto the Au / HCP / MCH surface during the electrochemical detection process, everything else was the same as in Example 1. Finally, the sensitivity of the electrochemical biosensors constructed with different targets was tested using an electrochemical workstation.

[0061] Figure 8 Figure 8a shows the detection sensitivity analysis of the electrochemical biosensor. As the target concentration increases, the electrochemical signal gradually increases, indicating that more activated HCP chains participate in the subsequent RCA reaction and nanosphere assembly process.

[0062] Figure 8b shows the relationship between the standard calibration curve for miR-21D detection and the miR-21D concentration, along with the corresponding regression equation, where I represents the current intensity and C represents the miR-21D concentration. Within the range of 1 pM to 1 nM, there is a significant linear relationship between the miR-21D concentration and the current intensity, following the regression equation I = 0.99 + 10.25 C, with a correlation coefficient of 0.9935. Based on a signal-to-noise ratio of 3, the detection limit is calculated to be 29 fM.

[0063] Example 4: Analysis of the specificity of the DNA nanosphere electrochemical biosensor to the target DNA probe

[0064] This embodiment is identical to Example 1 except that different DNA target probes are dropped onto the Au / HCP / MCH surface during the electrochemical detection process. Finally, the sensitivity of the electrochemical biosensors constructed with different target DNA probes was tested using an electrochemical workstation. The DNA probe sequences used are as follows:

[0065] Mismatch-1: 5'- TAG CTT ATg AGA CTG ATG TTG A -3';

[0066] Mismatch-2: 5'- TAG CTT ATC AcA CTc ATG TTG A -3';

[0067] Mismatch-3: 5'- TAG CgT Ata AGA CTg ATG TTG A -3';

[0068] miR-429D: 5'- TAA TAC TGT CTG GTA AAA CCG T -3';

[0069] miR-200bD: 5'- TAA TAC TGC CTG GTA ATG ATG A -3';

[0070] Let-7aD: 5'- TGA GGT AGT AGG TTG TAT AGT T -3';

[0071] Let-7dD: 5'- AGA GGT AGT AGG TTG CAT AGT T -3';

[0072] Figure 9a represents the analysis results of the specificity of the DNA nanosphere electrochemical biosensor to the target DNA probe. To verify the specificity of this biosensor, a series of sequences mismatched with the target miR-21D were designed, including Mismatch-1, Mismatch-2, Mismatch-3, miR-429D, miR-200bD, Let-7aD, and Let-7dD. The current signal corresponding to each mismatched sequence is shown in Figure 1. Figure 9 As shown in Figure a. The relative current signal for each mismatch sequence is as follows: Figure 9 As shown in Figure b, the current signal corresponding to Mismatch-1 accounts for only 22.8% of the current signal corresponding to the target miR-21D. The electrochemical signal further decreases with increasing base mismatch count. These results demonstrate that this biodetection method is highly selective for the target miRNA and can distinguish the target miRNA from sequences with only one base mismatch.

[0073] Furthermore, multiple members of the same miRNA family often exhibit high sequence similarity in target cells or blood samples. Therefore, to avoid interference from other members, the detection specificity of this biosensor for other members of the miRNA family was investigated. According to literature, miR-429, miR-200b, let-7a, let-7d, and miR-21 all belong to the miR-200 family. Therefore, DNA mimics were designed for the remaining four miRNAs for subsequent specificity testing. Figure 9 (c and 9d). The results showed that the electrochemical signals induced by the DNA mimics of each non-target miRNA were less than 10% of the target signal, indicating that the biosensor can distinguish the miR-21D target from other miR-200 family members.

[0074] Example 5: Anti-interference test of DNA nanosphere electrochemical biosensor in actual samples.

[0075] A DNA nanosphere electrochemical biosensor was constructed using the method described in Example 1. The target miR-21D at equal concentrations was incubated with 10% healthy human serum samples for 0 minutes, 30 minutes, and 60 minutes, respectively. The incubated miR-21D serum solution was then dropped onto the surface of an Au-HCP-MCH electrode for target detection.

[0076] Figure 10 Its resistance to interference in complex biological environments was studied. For example... Figure 10As shown, the electrochemical signal induced by the target without incubation with human serum was defined as 100%, while the electrochemical signals induced by the target incubated with human serum for 30 minutes and 60 minutes were 98.4% and 89.8%, respectively. Therefore, this biosensor can completely resist the interference of other components in human serum on the signal during the 30-minute target reaction period.

[0077] Example 6: Target Analysis of DNA Nanosphere Electrochemical Biosensor in Real Samples

[0078] This embodiment is identical to Example 1 except that different amounts of the target miR-21D were added to serum samples from healthy individuals, and then the serum solution containing miR-21D was dropped onto the surface of the Au-HCP-MCH electrode. This was to verify the application of the electrochemical biosensor based on three-dimensional DNA nanospheres in target detection of complex clinically relevant biological samples. The concentration of miR-21D in the serum solution was measured using the electrochemical signal generated by the electrochemical biosensor of the three-dimensional DNA nanospheres, and the results are shown in Table 1.

[0079] Table 1

[0080] Figure 6 For recovery testing, the recovery rate of miR-21D in serum samples from healthy individuals ranged from 98.1% to 101.7%. This indicates that the biosensor can be used for target detection in clinical samples.

[0081] Example 7: Universality Analysis of DNA Nanosphere Electrochemical Biosensor

[0082] By modifying the target binding sequence in the hairpin probe, a DNA nanosphere electrochemical biosensor capable of detecting miRNA-155 was constructed according to the method in Example 1. The performance of the newly constructed detection system was evaluated using an electrochemical workstation. The DNA probes used are as follows:

[0083] HCP-155D: 5'-TTT TTT TTT ACC CCT ATC ACG ATT AGC ATT AAC CAT GTG TAGATT AAT GCT AAT CGT G-3' SH-HCP-155 D:SH-TTT TTT TTT ACC CCT ATC ACG ATT AGC ATT AAC CAT GTGTAG ATA ATG CTA ATC GTG-3' L-Dumbbell-155 D: 5'-CAC GAT TAG CAT TAA TCT ACA CAT GGT AGC TTA TCAGAC TCC ATG TGT AGA-3' Primer-Dumbbell-155 D:5'-TTA ATG CTA ATC GTG TCT ACA CAT GGA GTC-3' MiR-155D:5'-TTA ATG CTA ATC GTG ATA GGG GT-3' The HCP-155D chain sequence is divided into three parts, including the HCP-A region (sequence: TTT TTTTTT), HCP-B region (sequence: ACC CCT ATC ACG ATT AGC ATT), and HCP-C region (sequence: AC CAT GTGTAG ATT AAT GCT AAT CGT G) arranged sequentially from 5' to 3'. The L-Dumbbell-155D chain sequence is divided into three parts, from 5' to 3', namely the L-Dumbbell-X region (sequence: CAC GAT TAG CAT TAA TCT ACA CAT GGT), L-Dumbbell-Y region (sequence: AGC TTA TCA), and L-Dumbbell-Z region (sequence: GAC TCC ATG TGT AGA). The Primer-Dumbbell-155D sequence is divided into two parts, from 5' to 3': the Primer-Dumbbell-M region sequence (sequence: TTAATG CTA ATC GTG) and the Primer-Dumbbell-N region sequence (sequence: TCT ACA CAT GGA GTC). Specifically, the portion of the L-Dumbbell-X region closest to 5' (sequence: CAC GAT TAG CAT TAA) is complementary to the Primer-Dumbbell-M region sequence (sequence: TTA ATG CTA ATC GTG), the L-Dumbbell-Z region is complementary to the Primer-Dumbbell-N region sequence, forming a D-ring structure, the HCP-B region is complementary to the miR-155D region, and the HCP-C region is complementary to the L-Dumbbell-X region. For HCP-155D, the sequence “C ACGATT AGC ATT AA” in the HCP-A region pairs complementaryly with the sequence “TT AAT GCT AAT CGT G” in the HCP-C region, forming a hairpin structure. For L-Dumbbell-155D, the sequence “TCT ACA CAT GG” in the L-Dumbbell-X region pairs complementaryly with the sequence “CC ATG TGT AGA” in the L-Dumbbell-Z region, forming a hairpin structure.

[0084] A DNA nanosphere electrochemical biosensor designed to detect miRNA-155 D was developed by altering the miRNA recognition region. Figure 11This study demonstrates the detection of the DNA sequence corresponding to miRNA-155 using a newly constructed DNA nanosphere electrochemical biosensor. The results show that the biosensor exhibits a significant electrochemical signal after the addition of the target miR-155D, indicating its excellent multifunctionality.

[0085] Those skilled in the art should understand that the above description is merely a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction, characterized in that, Specifically comprising the following steps: (1) The 5' end of the HCP hairpin is modified by -SH, and then the modified HCP hairpin is fixed on the surface of the Au electrode by AU-S bond to obtain an Au-HCP electrode. Finally, the Au-HCP electrode is treated with 6-mercapto-1-hexanol MCH, and MCH is also fixed on the surface of the Au electrode by AU-S bond to block the remaining exposed gold surface, thereby obtaining an Au-HCP-MCH electrode; (2) The target miRNA solution is added dropwise to the surface of the Au-HCP-MCH electrode for incubation, the HCP hairpin is opened, the target miRNA is combined with the partial fragment of the HCP chain, then the RCA reaction solution is added dropwise to the surface of the electrode, the exposed sticky end in the HCP chain subsequently triggers the RCA of the D loop as a primer strand, and multiple repeated complementary sequences in the RCA product spontaneously hybridize and self-assemble to form a three-dimensional DNA nanosphere. An electrochemical indicator is loaded in the DNA nanosphere, and the content of the target miRNA is detected based on the generated electrochemical signal; The RCA reaction solution contains a D loop, phi29 DNA polymerase, dNTP and phi29 reaction buffer without DTT; The sequence of the HCP chain comprises an HCP-A region, an HCP-B region and an HCP-C region arranged in sequence from 5' to 3'; The sequence of the Primer-Dumbbell comprises a Primer-Dumbbell-M region sequence and a Primer-Dumbbell-N region sequence arranged in sequence from 5' to 3'; The sequence of the L-Dumbbell-X region is complementary to the sequence of the Primer-Dumbbell-M region, and the sequence of the L-Dumbbell-Z region is complementary to the sequence of the Primer-Dumbbell-N region, forming a D loop structure. The sequence of the HCP-B region is complementary to the sequence of the target miRNA, and the sequence of the HCP-C region is complementary to the sequence of the L-Dumbbell-X region.

2. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, In step (2), the target miRNA is miRNA-21, miR-155, miRNA-1, miRNA-144 or miRNA-31.

3. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, When the target miRNA is miRNA-21, the corresponding HCP, L-Dumbbell and Primer-Dumbbell sequences are 5'-TTT TTT TTT TCA ACA TCA GTC TGA TAA GCT ACC ATG TGT AGA TAG CTT ATC AGA CT-3', 5'-AGT CTG ATA AGC TAT CTA CAC ATG GTA ATG CTA ATC GTG CCA TGT GTA GA-3' and 5'-ATA GCT TAT CAG ACT TCT ACA CAT GGC ACG-3', respectively; When the target miRNA is miR-15, the corresponding HCP, L-Dumbbell and Primer-Dumbbell sequences are 5'-TTT TTT TTT ACC CCT ATC ACG ATT AGC ATT AAC CAT GTG TAG ATT AAT GCT AAT CGT G-3', 5'-CAC GAT TAG CAT TAA TCT ACA CAT GGT AGC TTA TCA GAC TCC ATG TGT AGA-3' and 5'-TTA ATG CTA ATC GTG TCT ACA CAT GGA GTC-3', respectively.

4. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, The miRNA concentration in the target miRNA solution in step (2) is 0-50 nM.

5. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, The electrochemical indicator comprises methylene blue, thionine or toluidine blue.

6. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, The current signal generated by the DPV test in step (2) is used to detect the content of the target miRNA.

7. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, The RCA reaction solution contains 1 μM D-loop, 1 U / μL phi29 DNA polymerase, 1 mM dNTP and 10 μL 1× phi29 reaction buffer without DTT.

8. The method for constructing a DNA nanoball electrochemical biosensor based on dumbbell-shaped loop RCA reaction according to claim 1, wherein, The average height and average width of the three-dimensional DNA nanospheres are 15.9 ± 0.8 nm and 68.1 ± 4.0 nm, respectively.

9. An electrochemical biosensor constructed by the construction method of any one of claims 1-8.

10. The use of the electrochemical biosensor of claim 9 in miRNA detection, in particular, the use in the detection of miRNA in blood.

Images