DNA walker, paper-based miRNA sensor, its fabrication and application

Abstract

This invention discloses a DNA walker, a paper-based miRNA sensor, and their fabrication methods and applications. The fabrication method of the DNA walker includes: thiolation of a first probe using TCEP, followed by incubation with gold nanoparticles to obtain a first probe / gold nanoparticle complex; aging the first probe / gold nanoparticle complex in a first buffer solution, followed by reaction with MCH to obtain gold nanoparticles modified with the first probe; and incubating the first probe-modified gold nanoparticles, a second probe, and the first buffer solution, followed by dispersing the resulting precipitate in a second buffer solution to obtain the DNA walker. The paper-based miRNA sensor prepared by this invention has advantages such as ease of use, simple operation, and no need for large-scale instruments, enabling convenient miRNA detection and showing broad application prospects in the detection field.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to a DNA walker, a paper-based miRNA sensor, and their manufacturing methods and applications. Background Technology

[0002] MicroRNAs (miRNAs) are short, endogenous non-coding RNAs widely found in animals, plants, and microorganisms. They participate in regulating various cellular processes, such as development, differentiation, protein degradation, signal transduction, and stress responses. Therefore, they are important biomarkers for disease diagnosis. Thus, developing highly sensitive and selective miRNA biosensors is of great significance. Currently, methods for miRNA assays mainly rely on traditional nucleic acid detection techniques, including microarrays, quantitative real-time PCR (qRT-PCR), Northern blotting, and next-generation sequencing. While these methods are reliable and sensitive in laboratory settings, they typically involve complex protocols, high costs, and require expensive equipment, making them unsuitable for field testing. To overcome these limitations, developing portable biosensing methods is imperative.

[0003] To improve sensor sensitivity, introducing signal amplification strategies into biosensors is an effective approach. Artificial DNA nanomachines, such as DNA tweezers, DNA motors, DNA switches, and DNA walkers, have become effective alternative biomaterials for designing signal amplification strategies in nucleic acid detection due to their excellent programmability and high stability. DNA walkers, as typical dynamic DNA nanodevices, can autonomously move along specific programmed DNA tracks, aiding in signal transduction and amplification. Typically, a DNA walker consists of three basic components: the walker, the track, and the driving force. The walker is usually a specially designed DNA strand that can be directly attached to the track substrate or assembled with other materials (e.g., gold nanoparticles, magnetic beads, cancer cells, and exosomes) to form a multi-legged walker. The DNA track strand can hybridize with the walker strand and serve as a foothold. The movement of the DNA walker can be driven by some form of energy input, such as enzymatic reactions, strand displacement reactions, and hydrolysis reactions catalyzed by deoxyribozymes. The walking process can be triggered by a target-induced reaction, generating amplified signals and constructing biosensor devices. Summary of the Invention

[0004] The main objective of this invention is to provide a DNA walker, a paper-based miRNA sensor, and their manufacturing methods and applications, in order to overcome the shortcomings of the prior art.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] This invention provides a method for preparing a DNA walker, comprising:

[0007] The first probe was thiolized using TCEP, and then mixed and incubated with gold nanoparticles to obtain a first probe / gold nanoparticle complex.

[0008] The first probe / gold nanoparticle composite was placed in a first buffer solution for aging treatment, and then reacted with 6-mercapto-1-hexanol (denoted as: MCH) to obtain gold nanoparticles modified with the first probe.

[0009] Furthermore, the gold nanoparticles modified with the first probe, the second probe, and the first buffer solution were mixed and incubated, and then the resulting precipitate was dispersed in the second buffer solution to obtain a DNA walker.

[0010] The present invention also provides a DNA walker prepared by the aforementioned preparation method.

[0011] This invention also provides a paper-based miRNA sensor, comprising: a paper substrate modified with probe HP, and a screen-printed electrode serving as a signal reading element, the screen-printed electrode being fixed to the surface of the paper substrate modified with probe HP.

[0012] This invention also provides a method for preparing the aforementioned paper-based miRNA sensor, comprising:

[0013] Paper sheets were incubated in a mixed solution containing NaIO4 and LiCl to obtain paper sheets functionalized with aldehyde groups.

[0014] The probe HP is reacted with the aldehyde-functionalized paper to obtain a paper base modified with the probe HP.

[0015] Furthermore, the paper substrate modified with probe HP is assembled with a screen-printed electrode to obtain a paper-based miRNA sensor.

[0016] This invention also provides a method for detecting miRNA based on a DNA walker, comprising:

[0017] Provided the aforementioned DNA walker and paper-based miRNA sensor;

[0018] Nt.BbvCI, λ-Exo, and a DNA walker were incubated with the target miRNA. The resulting reaction solution was then placed on the paper-based miRNA sensor and incubated at room temperature. Electrochemical detection was then performed, and the specificity of the miRNA was achieved based on the obtained DPV peak current value.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention combines nucleic acid-functionalized paper and commercial SPE to construct a paper-based portable biosensor. This sensor has significant advantages such as simplicity and convenience, and has great practical potential.

[0021] (2) The present invention adopts a dual signal amplification strategy (i.e., target cycling and DNA walking amplification), which improves the detection sensitivity and enables highly sensitive and selective miRNA detection;

[0022] (3) The portable paper-based biosensor described in this invention is a new miRNA sensing method with good application prospects in fields such as early disease diagnosis. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the paper-based portable biosensor based on a DNA walker prepared in Embodiment 1 of the present invention;

[0025] Figure 2 This is the background signal and electrochemical response current diagram of the paper-based portable biosensor prepared in Example 2 of the present invention to miR-21;

[0026] Figure 3 This is an electrochemical response current curve of the paper-based portable biosensor prepared in Example 3 of the present invention to different concentrations of miR-21;

[0027] Figure 4 This is a graph showing the relationship between the paper-based portable biosensor prepared in Example 3 of this invention and the concentration of miR-21;

[0028] Figure 5 This is a linear fitting graph of the paper-based portable biosensor prepared in Example 3 of the present invention and the logarithmic value of miR-21 concentration;

[0029] Figure 6 This is a signal response diagram of the miR-21 paper-based portable biosensor prepared in Example 4 of the present invention to other miRNAs and random DNA sequences. Detailed Implementation

[0030] In view of the deficiencies of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. It mainly uses probe-modified gold nanoparticles (AuNPs) as DNA walkers, hairpin probes fixed on the surface of paper as the path of the DNA walkers, and commercially available screen-printed electrodes (SPEs) as signal readout elements. By amplifying DNA walking triggered by the target analyte, it achieves highly sensitive, portable, and timely on-site detection of the target miR-21, thus expanding the application of nucleic acid-based analysis strategies in the field of on-site timely detection.

[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Specifically, as one aspect of the technical solution of this invention, a method for preparing a DNA walker includes:

[0033] The first probe was thiolized using TCEP, and then mixed and incubated with gold nanoparticles to obtain a first probe / gold nanoparticle complex.

[0034] The first probe / gold nanoparticle composite was placed in a first buffer solution for aging treatment, and then reacted with 6-mercapto-1-hexanol to obtain gold nanoparticles modified with the first probe.

[0035] Furthermore, the gold nanoparticles modified with the first probe, the second probe, and the first buffer solution were mixed and incubated, and then the resulting precipitate was dispersed in the second buffer solution to obtain a DNA walker.

[0036] In some preferred embodiments, the first probe has a sequence as shown in SEQ ID NO.1.

[0037] Specifically, the sequence of the first probe is: 5'-SH-TTT TTT TTT TGT ACT GAG GTA CCT TATCAG ACT-3'.

[0038] In some preferred embodiments, the second probe has a sequence as shown in SEQ ID NO.2.

[0039] Specifically, the sequence of the second probe is: 5'-PO4 3 -TCA ACA TCA GTC TGA TAA GCT A-3'.

[0040] In some preferred embodiments, the first buffer solution comprises 0.1 mol / L NaCl and 10 mmol / L phosphate; the pH of the first buffer solution is 7.0.

[0041] In some preferred embodiments, the second buffer solution comprises 0.25 mol / L NaCl and 10 mmol / L phosphate; the pH of the second buffer solution is 7.4.

[0042] In some preferred embodiments, the molar ratio of the first probe to the second probe is 1:1.

[0043] In some more specific embodiments, the method for preparing the DNA walker includes:

[0044] First, AuNPs were synthesized by reducing HAuCl4 with sodium citrate. A 1 mM HAuCl4 solution (100 mL) was rapidly heated to its boiling point under vigorous stirring. Then, 10 mL of a 38.8 mM sodium citrate solution was rapidly added, and the mixture was kept boiling in a reflux condenser for 30 min. After heating was stopped, the solution was slowly cooled to room temperature and stored at 4 °C. Next, probe 1 (the aforementioned first probe) was chemically immobilized on the surface of gold nanoparticles using Au-S. Before modification, the thiolized probe 1 was treated with 10 mM MTCEP. Then, probe 1 at a final concentration of 1–5 μM was mixed with 1 mL of AuNPs. S The mixture was mixed and incubated for 16 h. Subsequently, the first probe / gold nanoparticle complex was aged in buffer solution 1 (i.e., the aforementioned first buffer solution) for 16 h. After centrifugation and washing to remove excess reagent, 1 mM MCH was added to the reaction solution and reacted for 1 h. This was followed by centrifugation and washing again, and the resulting precipitate was resuspended in 1 mL of buffer solution 1. Finally, 1–5 μM probe 2 (i.e., the aforementioned second probe) was added, and the AuNPs modified with probe 1 were incubated at 37 °C for 1 h. After centrifugation to remove the supernatant, the dsDNA-terminated AuNPs precipitate was resuspended in 1 mL of buffer solution 2 (i.e., the aforementioned second buffer solution), which is the DNA walker solution (also known as the DNA walker).

[0045] Furthermore, the final concentration of probe 1 (the concentration here refers to the concentration in the final reaction solution after mixing) is 1 to 5 μM, preferably 3 μM; the molar ratio of probe 1 to probe 2 is 1:1.

[0046] Another aspect of the present invention provides a DNA walker prepared by the aforementioned preparation method.

[0047] Another aspect of the present invention provides a paper-based miRNA sensor, comprising: a paper substrate modified with a probe HP, and a screen-printed electrode serving as a signal reading element, the screen-printed electrode being fixed to the surface of the paper substrate modified with the probe HP.

[0048] Another aspect of the present invention provides a method for preparing the aforementioned paper-based miRNA sensor, comprising:

[0049] The paper was incubated in a mixed solution containing NaIO4 and LiCl (IO4-oxidation of cellulose hydroxyl groups) to obtain paper with aldehyde functionalized groups.

[0050] The probe HP is reacted with the aldehyde-functionalized paper to obtain a paper base modified with the probe HP.

[0051] Furthermore, the paper substrate modified with probe HP is assembled with a screen-printed electrode to obtain a paper-based miRNA sensor (also referred to as: miRNA detection strip).

[0052] In some preferred embodiments, the concentration of NaIO4 in the mixed solution containing NaIO4 and LiCl is 500–900 mmol / L, and the final concentration of LiCl is 500–900 mmol / L.

[0053] In some preferred embodiments, the probe HP has a sequence as shown in SEQ ID NO.3.

[0054] Specifically, the sequence of the probe HP is: 5'-NH2-TTT TTT TTT TAG TCT GAT AAG GTA CCTCAG TAC CTT-Fc-3'.

[0055] In some preferred embodiments, the preparation method includes: dropping a probe HP solution with a concentration of 0.1–0.3 μmol / L onto the surface of the aldehyde-functionalized paper to carry out a reaction.

[0056] In some more specific embodiments, the preparation method of the paper-based miRNA sensor includes:

[0057] Filter paper was treated with sodium periodate to form aldehyde groups (CHO-) to immobilize HP chains. Briefly, 50 circular sheets of paper were immersed in 50 mL of a mixed solution of 40–60 mM NaIO4 and 500–900 mM LiCl and incubated in the dark at 55 °C for 3 h. After washing three times with Milli-Q water, the CHO-functionalized paper was dried at room temperature. Subsequently, 15 μL of 0.1–0.3 μM HP chains were dropped onto the circular paper at 37 °C for 2 h. The paper was then washed three times with Tris-HCl buffer and ultrapure water, and finally dried at room temperature.

[0058] The aforementioned paper pieces and SPEs are linked together with tape to assemble a paper-based miRNA sensor.

[0059] Furthermore, the final concentration of NaIO4 is 500–900 mM, preferably 700 mM, the final concentration of LiCl is 500–900 mM, preferably 700 mM, and the concentration of HP chain is 0.1–0.3 μM, preferably 0.2 μM.

[0060] Another aspect of the present invention provides a method for detecting miRNA based on a DNA walker, comprising:

[0061] Provided the aforementioned DNA walker and paper-based miRNA sensor;

[0062] Nt.BbvCI, λ-Exo, and a DNA walker were incubated with the target miRNA. The resulting reaction solution was then placed on the paper-based miRNA sensor and incubated at room temperature. Electrochemical detection was then performed, and the specificity of the miRNA was achieved based on the obtained DPV peak current value.

[0063] In some preferred embodiments, the miRNA is miR-21, which has the sequence shown in SEQ ID NO.4.

[0064] Specifically, the sequence of miR-21 is: 5'-UAG CUU AUC AGA CUG AUG UUG A-3'.

[0065] In some preferred embodiments, the concentration of Nt.BbvCI in the mixed solution formed by Nt.BbvCI, λ-Exo and DNA walker is 50-200 U / mL, and the concentration of λ-Exo is 50-200 U / mL.

[0066] In some more specific embodiments, the miRNA detection method includes: adding the target miRNA to a DNA walker solution containing 50–200 U / mL Nt.BbvCI and 50–200 U / mL λ-Exo. The mixture is incubated in a centrifuge tube at 37°C for 2 hours. Subsequently, the reaction solution is transferred from the centrifuge tube to HP-modified paper. After incubation at room temperature for 0.5 hours, the circular paper is folded onto the surface of the SPE for electrochemical detection.

[0067] Furthermore, the concentration of Nt.BbvCI is 50–200 U / mL, preferably 100 U / mL, and the concentration of λ-Exo is 50–200 U / mL, preferably 100 U / mL.

[0068] Specifically, the miRNA detection method based on DNA walkers in this invention can directly detect 1 nM.

[0069] Specifically, the detection limit of miRNA in this invention is approximately 0.25 nM.

[0070] The process principle of this invention is as follows: When the target compound miR-21 is absent, probe 1 strand first hybridizes with a portion of probe 2 to form dsDNA, thus blocking the reaction between probe 1 and probe HP. The 5'-PO4 of probe 2... 3- The single-stranded DNA (ssDNA) structure at the ends effectively prevents digestion by λ-exonuclease (λ-Exo), and this portion of ssDNA can hybridize complementary to the target miR-21. In the presence of miR-21, it forms a blunt-ended dsDNA structure with probe 2. This conformation is recognized by λ-Exo, which hydrolyzes probe 2 to form a single nucleotide, releasing probe 1 and transforming it into the ssDNA conformation. Simultaneously, the target miR-21 is released, enabling it to hybridize further with another probe 2 in the dsDNA of probes 1 and 2, thereby triggering the cyclic digestion of probe 2. This involves target cyclic amplification, forming a large number of probe 1-modified AuNPs, triggering subsequent DNA walking amplification. Specifically, the probe 1-modified AuNPs are dropped onto HP-modified paper, where probe 1 hybridizes with probe HP. This opens HP and forms a recognition site with Nt.BbvCI cleavage enzyme, triggering an Nt.BbvCI-catalyzed cleavage reaction. Probe 1 and a portion of the Fc-labeled HP sequence are released from the paper surface. Simultaneously, some free probe 1 on the AuNP surface participates in the formation of new double strands for further cleavage, promoting the multi-legged walking process. As a result, a large amount of Fc-labeled ssDNA is released from the paper surface, subsequently diffusing and adsorbing onto the carbon nanotube-modified SPE working electrode surface, producing a significantly enhanced electrochemical response, thus enabling convenient electrochemical quantitative detection of the target miR-21.

[0071] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0072] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0073] Example 1: Fabrication of a paper-based miRNA sensor

[0074] ① Synthesis of DNA walking devices: First, AuNPs were synthesized by reducing HAuCl4 with sodium citrate. A 1 mM HAuCl4 solution (100 mL) was rapidly heated to boiling point under vigorous stirring. Then, 10 mL of a 38.8 mM sodium citrate solution was rapidly added, and the mixture was kept boiling in a reflux condenser for 30 min. After heating was stopped, the solution was slowly cooled to room temperature and stored at 4 °C. Second, probe 1 strands were chemically immobilized on the surface of gold nanoparticles via Au-S. Before modification, thiolized probe 1 was treated with 10 mM TCEP. Then, probe 1 at a final concentration of 2 μM was mixed with 1 mL of AuNPs. s Mix and incubate for 16 h. Then, age the probe 1-AuNPs complex in buffer solution 1 for 16 h. After centrifugation and washing to remove excess reagent, add 1 mM MCH to the reaction solution and react for 1 h, followed by centrifugation and washing again. The resulting precipitate is then resuspended in 1 mL of buffer solution 1. Finally, add 2 μM probe 2 strand and incubate with the probe 1-modified AuNPs at 37 °C for 1 h. After centrifugation to remove the supernatant, resuspend the dsDNA-capped AuNPs precipitate in 1 mL of buffer solution 2 to obtain the DNA walker solution.

[0075] ② HP probe modification on paper surface: Filter paper was treated with sodium periodate to immobilize HP chains by forming aldehyde groups (CHO-) through a Schiff base reaction. In short, 50 circular sheets of paper were immersed in a mixed solution of 50 mL of 50 mM NaIO4 and 700 mM LiCl and incubated at 55 °C for 3 h in the dark. After washing three times with Milli-Q water, the CHO-functionalized paper was dried at room temperature. Subsequently, 15 μL of 0.1 μM HP chains were dropped onto the circular paper at 37 °C for 2 h. The paper was then washed three times with Tris-HCl buffer and ultrapure water, and finally dried at room temperature.

[0076] ③ Preparation of paper-based miRNA sensor: The paper sheet and SPE obtained in step ② are connected with tape to assemble a paper-based miRNA sensor.

[0077] Figure 1This is a schematic diagram of the paper-based miRNA sensor prepared in Example 1. The test strip is made of HP-modified paper with screen-printed electrodes. In use, the target analyte miR-21 is added to the DNA walker solution and incubated at 37°C for 2 hours. Then, it is dropped onto the paper strip and incubated at room temperature for 0.5 hours. Finally, the circular paper is folded onto the SPE surface for electrochemical detection.

[0078] Example 2: Study on the specific response of a paper-based miRNA sensor to the target miR-21.

[0079] ① Synthesis of DNA walking devices: First, AuNPs were synthesized by reducing HAuCl4 with sodium citrate. A 1 mM HAuCl4 solution (100 mL) was rapidly heated to boiling point under vigorous stirring. Then, 10 mL of a 38.8 mM sodium citrate solution was rapidly added, and the mixture was kept boiling in a reflux condenser for 30 min. After heating was stopped, the solution was slowly cooled to room temperature and stored at 4 °C. Second, probe 1 strands were chemically immobilized on the surface of gold nanoparticles via Au-S. Before modification, thiolized probe 1 was treated with 10 mM TCEP. Then, probe 1 with a final concentration of 3 μM was combined with 1 mM HAuNPs. s Mix and incubate for 16 h. Then, age the probe 1-AuNPs complex in buffer solution 1 for 16 h. After centrifugation and washing to remove excess reagent, add 1 mmol M MCH to the reaction solution and react for 1 h, followed by centrifugation and washing again. The resulting precipitate is then resuspended in 1 mL buffer solution 1. Finally, add 3 μM probe 2 strand and incubate with the probe 1-modified AuNPs at 37 °C for 1 h. After centrifugation to remove the supernatant, resuspend the dsDNA-capped AuNPs precipitate in 1 mL buffer solution 2 to obtain the DNA walker solution.

[0080] ② HP probe modification on paper surface: Filter paper was treated with sodium periodate to immobilize HP chains by forming aldehyde groups (CHO-) through a Schiff base reaction. In short, 50 circular sheets of paper were immersed in a mixed solution of 50 mM NaIO4 and 700 mM LiCl and incubated at 55 °C for 3 h in the dark. After washing three times with Milli-Q water, the CHO-functionalized paper was dried at room temperature. Subsequently, 15 μL of 0.2 μM HP chains was dropped onto the circular paper at 37 °C for 2 h. The paper was then washed three times with Tris-HCl buffer and ultrapure water, and finally dried at room temperature.

[0081] ③ Preparation of paper-based miRNA sensor: The paper sheet and SPE obtained in step ② are connected with tape to assemble a paper-based miRNA sensor.

[0082] ④ Detection application of target miRNA and samples without target: Equal volumes of target miRNA and ultrapure water were added to two separate aliquots of DNA walker solutions containing 50 U / mL Nt.BbvCI and 50 U / mL λ-Exo. The two mixtures were incubated in centrifuge tubes at 37°C for 2 hours. Subsequently, the reaction solution was transferred from the centrifuge tubes to the paper of a paper-based miRNA sensor. After incubation at room temperature for 0.5 hours, the circular paper was folded onto the SPE surface for electrochemical detection. The measured differential pulse voltammetry (DPV) current is shown in [reference needed]. Figure 2 .

[0083] Figure 2 Curve a in the figure represents the current measured in the sample without miR-21, while curve b represents the current measured in the sample with miR-21. The figure shows that without the target compound, the DPV peak current is close to 0, indicating a relatively low background current for the paper-based miRNA sensor. However, the target compound miR-21 sample showed a higher peak current on the sensor, demonstrating that this paper-based miRNA sensor can achieve sensitive detection of miR-21.

[0084] Example 3: Detection experiment of paper-based miRNA sensor for different concentrations of target miR-21.

[0085] ① Synthesis of DNA walking devices: First, AuNPs were synthesized by reducing HAuCl4 with sodium citrate. A 1 mM HAuCl4 solution (100 mL) was rapidly heated to boiling point under vigorous stirring. Then, 10 mL of a 38.8 mM sodium citrate solution was rapidly added, and the mixture was kept boiling in a reflux condenser for 30 min. After heating was stopped, the solution was slowly cooled to room temperature and stored at 4 °C. Second, probe 1 strands were chemically immobilized on the surface of gold nanoparticles via Au-S. Before modification, thiolized probe 1 was treated with 10 mM TCEP. Then, probe 1 with a final concentration of 3 μM was combined with 1 mM HAuNPs. s Mix and incubate for 16 h. Then, age the probe 1-AuNPs complex in buffer solution 1 for 16 h. After centrifugation and washing to remove excess reagent, add 1 mmol M MCH to the reaction solution and react for 1 h, followed by centrifugation and washing again. The resulting precipitate is then resuspended in 1 mL buffer solution 1. Finally, add 3 μM probe 2 strand and incubate with the probe 1-modified AuNPs at 37 °C for 1 h. After centrifugation to remove the supernatant, resuspend the dsDNA-capped AuNPs precipitate in 1 mL buffer solution 2 to obtain the DNA walker solution.

[0086] ② HP probe modification on paper surface: Filter paper was treated with sodium periodate to immobilize HP chains by forming aldehyde groups (CHO-) through a Schiff base reaction. In short, 50 circular sheets of paper were immersed in a mixed solution of 50 mL of 50 mM NaIO4 and 700 mM LiCl and incubated at 55 °C for 3 h in the dark. After washing three times with Milli-Q water, the CHO-functionalized paper was dried at room temperature. Subsequently, 15 μL of 0.2 μM HP chains was dropped onto the circular paper at 37 °C for 2 h. The paper was then washed three times with Tris-HCl buffer and ultrapure water, and finally dried at room temperature.

[0087] ③ Preparation of paper-based miRNA sensor: The paper sheet and SPE obtained in step ② are connected with tape to assemble a paper-based miRNA sensor.

[0088] ④ Detection application of target miRNAs at different concentrations: Target miRNAs at concentrations of 0, 1, 10, 100, and 1000 nM were added to DNA walker solutions containing 100 U / mL Nt.BbvCI and 100 U / mL λ-Exo, respectively. The mixtures were incubated in centrifuge tubes at 37°C for 2 hours. Subsequently, the reaction solution was transferred from the centrifuge tubes to the paper of a paper-based miRNA sensor. After incubation at room temperature for 0.5 hours, the circular paper was folded onto the surface of the SPE for electrochemical detection. The measured DPV current is shown in [Figure showing DPV current]. Figure 3 The relationship between miR-21 concentration and peak current is shown in [reference needed]. Figure 4 and 5 .

[0089] from Figure 3 As can be seen, the DPV current signal of the prepared paper-based miRNA sensor gradually increases with increasing miR-21 concentration. Although the DPV peak current value is not linearly related to the miR-21 concentration, see... Figure 4 As shown, however, after taking the logarithm of the miR-21 concentration, it was found to have a linear relationship with the DPV peak current value. Figure 5 As shown.

[0090] Example 4: Experiment on the selectivity of paper-based miRNA sensor for different miRNAs and DNA.

[0091] ① Synthesis of DNA walking devices: First, AuNPs were synthesized by reducing HAuCl4 with sodium citrate. A 1 mM HAuCl4 solution (100 mL) was rapidly heated to boiling point with vigorous stirring. Then, 10 mL of a 38.8 mM sodium citrate solution was rapidly added, and the mixture was kept boiling in a reflux condenser for 30 min. After heating was stopped, the solution was slowly cooled to room temperature and stored at 4 °C. Next, probe 1 strands were chemically immobilized on the surface of gold nanoparticles via Au-S. Before modification, thiolized probe 1 was treated with 10 mM TCEP. Then, probe 1 to a final concentration of 3 μM was mixed with 1 mL of AuNPs and incubated for 16 h. Subsequently, the probe 1-AuNPs complex was aged in buffer solution 1 for 16 h. After centrifugation and washing to remove excess reagent, 1 mM MCH was added to the reaction solution and reacted for 1 h, followed by centrifugation and washing again. The resulting precipitate was then resuspended in 1 mL of buffer solution 1. Finally, add 3 μM probe 2 strand and incubate with probe 1-modified AuNPs at 37°C for 1 h. After centrifugation to remove the supernatant, suspend the dsDNA-capped AuNPs precipitate in 1 mL buffer solution 2 to obtain the DNA walker solution.

[0092] ② HP probe modification on paper surface: Filter paper was treated with sodium periodate to immobilize HP chains by forming aldehyde groups (CHO-) through a Schiff base reaction. In short, 50 circular sheets of paper were immersed in a mixed solution of 50 mL of 50 mM NaIO4 and 700 mM LiCl and incubated at 55 °C for 3 h in the dark. After washing three times with Milli-Q water, the CHO-functionalized paper was dried at room temperature. Subsequently, 15 μL of 0.2 μM HP chains was dropped onto the circular paper at 37 °C for 2 h. The paper was then washed three times with Tris-HCl buffer and ultrapure water, and finally dried at room temperature.

[0093] ③ Preparation of paper-based miRNA sensor: The paper sheet and SPE obtained in step ② are connected with tape to assemble a paper-based miRNA sensor.

[0094] ④ Detection selectivity of different miRNAs and DNA: Equal volumes of ultrapure water, 10 μM let-7a, two random sequences (random 1 and 2), and 1 μM miRNA were added to a DNA walker solution containing 100 U / mL Nt.BbvCI and 100 U / mL λ-Exo, respectively. The mixture was incubated in a centrifuge tube at 37°C for 2 h. Subsequently, the reaction solution was transferred from the centrifuge tube to the paper of a paper-based miRNA sensor. After incubation at room temperature for 0.5 h, the circular paper was folded onto the surface of the SPE for electrochemical detection. The measured DPV peak current values ​​are shown in [Figure number missing]. Figure 6 .

[0095] from Figure 6As can be seen, only miR-21 can generate a large DPV current response on the paper-based miRNA sensor. Even if the concentrations of miRNAs and DNA such as let-7a, random 1, and random 2 are ten times that of the target miR-21, they still cannot generate a significant DPV signal response, indicating that the paper-based miRNA sensor prepared in this invention has excellent selectivity.

[0096] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0097] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A non-diagnostic miRNA detection method based on a DNA walker, characterized in that... include: The first probe was thiolated using TCEP, and then incubated with gold nanoparticles to obtain a first probe / gold nanoparticle complex. The first probe / gold nanoparticle complex was aged in a first buffer solution, and then reacted with 6-mercapto-1-hexanol to obtain gold nanoparticles modified with the first probe. The gold nanoparticles modified with the first probe, the second probe, and the first buffer solution were mixed and incubated, and then the resulting precipitate was dispersed in a second buffer solution to prepare a DNA walker. The sequence of the first probe is shown in SEQ ID NO.1; the sequence of the second probe is shown in SEQ ID NO.

2. A paper-based miRNA sensor is provided, comprising a paper substrate modified with a probe HP, and a screen-printed electrode serving as a signal readout element, the screen-printed electrode being fixed to the surface of the paper substrate modified with the probe HP; the sequence of the probe HP is shown in SEQ ID NO.3; Nt.BbvCI, λ-Exo, and a DNA walker were incubated with the target miRNA. The resulting reaction solution was then placed on the paper-based miRNA sensor and incubated at room temperature. Electrochemical detection was then performed, and the specificity of the miRNA was achieved based on the obtained DPV peak current value.

2. The miRNA detection method according to claim 1, characterized in that: The miRNA is miR-21, and the sequence of miR-21 is shown in SEQ ID NO.

4.

3. The miRNA detection method according to claim 1, characterized in that: The concentration of Nt.BbvCI in the mixed solution formed by Nt.BbvCI, λ-Exo and DNA walker is 50~200 U / mL, and the concentration of λ-Exo is 50~200 U / mL.

4. The miRNA detection method according to claim 1, characterized in that: The first buffer solution comprises 0.1 mol / L NaCl and 10 mmol / L phosphate; the pH value of the first buffer solution is 7.

0.

5. The miRNA detection method according to claim 1, characterized in that: The second buffer solution comprises 0.25 mol / L NaCl and 10 mmol / L phosphate; the pH value of the second buffer solution is 7.

4.

6. The miRNA detection method according to claim 1, characterized in that: The molar ratio of the first probe to the second probe is 1:

1.

7. The miRNA detection method according to claim 1, characterized in that, The method for preparing the paper-based miRNA sensor includes: Paper sheets were incubated in a mixed solution containing NaIO4 and LiCl to obtain paper sheets functionalized with aldehyde groups. The probe HP is reacted with the aldehyde-functionalized paper to obtain a paper base modified with the probe HP. Furthermore, the paper substrate modified with probe HP is assembled with a screen-printed electrode to obtain a paper-based miRNA sensor.

8. The miRNA detection method according to claim 7, characterized in that: The concentration of NaIO4 in the mixed solution containing NaIO4 and LiCl is 500~900 mmol / L, and the final concentration of LiCl is 500~900 mmol / L.

9. The miRNA detection method according to claim 7, characterized in that, The preparation method of the paper-based miRNA sensor d includes: dropping a probe HP solution with a concentration of 0.1~0.3 μmol / L onto the surface of the aldehyde-functionalized paper to carry out the reaction.

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