Visual detection of miRNA-10b based on gold nanoparticles (AuNPs) DNA sensor

Abstract

The application provides a DNA sensor based on gold nanoparticles (AuNPs) for visual detection of miRNA-10b. The sensor is obtained by coupling AuNPs-S1 through mercapto between gold nanoparticles (AuNPs) and probe S1; and coupling AuNPs-S2 through mercapto between gold nanoparticles (AuNPs) and probe S2. The combination of the probe and the gold nanoparticles is more firm due to mercapto modification, so that the stability and detection repeatability of the DNA sensor are improved. The aggregation of AuNPs is triggered by hybridization between the target miRNA-10b and the probe, the transition of AuNPs from dispersion to aggregation in the buffer solution is regulated, and the color change from red to blue is visible to the naked eye, so that the rapid and visual detection of the tumor marker miRNA-10b is realized. The linear range is 10 pM-200 nM, and the sensor has excellent stability in serum environments (1%, 10%) and enzymatic environments (5 U / mL DNase and 2 U / mL DNase), and high selectivity. An efficient tool is provided for cancer diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry detection technology, specifically involving the hybridization of target miRNA-10b with a probe to regulate the transition of AuNPs from a dispersed to an aggregated state in a buffer solution, producing a visible color change from red to blue, thereby achieving qualitative and semi-quantitative analysis of miRNA-10b. Background Technology

[0002] Malignant tumors are a major public health problem worldwide. Early diagnosis is key to reducing mortality and improving patient survival, and accurate detection of tumor markers is the core technological support for achieving early diagnosis. MicroRNAs (miRNAs), as typical oncogenic microRNAs, are abnormally highly expressed in various malignant tumor tissues and serums. Their expression levels are closely related to tumor occurrence, development, and prognosis, making them highly promising biomarkers for early tumor diagnosis.

[0003] Currently, miRNA detection mainly relies on molecular biology techniques such as qRT-PCR, gene chips, and next-generation sequencing. While these methods are sensitive and accurate, they generally suffer from limitations such as strong dependence on equipment, cumbersome procedures, long processing times, high costs, and the need for specialized technical personnel. These limitations make them unsuitable for primary healthcare institutions, rapid on-site screening, and point-of-care diagnostic scenarios in resource-limited areas. Therefore, developing a new miRNA detection method that is simple to operate, rapid, intuitive, low-cost, and possesses both excellent sensitivity and specificity is of significant scientific importance and clinical translational value.

[0004] The colorimetric properties of gold nanoparticles (AuNPs) stem from a unique physical phenomenon: localized surface plasmon resonance. Simply put, the free electrons in gold nanoparticles oscillate collectively under illumination of specific wavelengths of light, strongly absorbing green light and surrounding wavelengths. Due to the principle of complementary colors, dispersed particles typically appear red in solution. When the distance, size, shape, or surrounding environment of the particles changes, the absorption peak shifts, and the solution color changes to blue or red. Its core characteristics are mainly reflected in the following aspects: Distance dependence (aggregation color change): This is the most widely used characteristic. When particles aggregate, and the interparticle distance is smaller than the average particle size, plasmon coupling causes a red shift in the absorption peak, changing the solution from red to blue or purple. Lateral chromatography test strips based on this principle can be used for early pregnancy or COVID-19 testing. Size and shape control: By adjusting the synthesis parameters, the visible to near-infrared wavelength range can be covered. Spherical particles change color from orange-red to wine-red and purplish-red as their diameter increases (5-120 nm); rod-shaped particles have two absorption peaks along the horizontal and vertical axes, and their color can be adjusted from red to a very deep near-infrared region by controlling the aspect ratio. Local refractive index sensitivity: The adsorption or binding of biomolecules on the particle surface alters the local dielectric environment, causing a shift in characteristic peaks. This high sensitivity makes it an ideal platform for biosensing. High-temperature thermal effect: Under irradiation with a strong laser at a resonant wavelength, gold nanoparticles can efficiently convert light energy into heat energy, generating localized high temperatures, which can be used for photothermal therapy—that is, irradiating particles enriched at tumor sites with near-infrared light to kill cancer cells through thermotherapy.

[0005] This paper proposes a visual detection method, which offers unique advantages in the POCT field because the results can be directly interpreted by the naked eye without the need for complex signal conversion equipment. Among these, gold nanoparticles (AuNPs) are ideal materials for constructing highly sensitive colorimetric biosensors due to their unique optical properties (surface plasmon resonance effect) and ease of surface functionalization. Based on this, a visual DNA sensor for miRNA-10b is developed, which has clear application prospects for promoting the popularization and convenience of early tumor diagnosis. Summary of the Invention

[0006] A DNA sensor based on gold nanoparticles (AuNPs) was constructed for the visual detection of miRNA-10b, enabling rapid and visual detection of the tumor marker miRNA-10b. A specific DNA sensor was designed to induce AuNP aggregation through hybridization of the target miRNA-10b with a probe. The transition of AuNPs from a dispersed to an aggregated state in a buffer solution was regulated, producing a visible color change from red to blue, thus enabling qualitative and semi-quantitative analysis of miRNA-10b.

[0007] The objective of this invention is achieved through the following technical solution.

[0008] A DNA sensor for the visual detection of the tumor marker miRNA-10b is characterized by the following steps:

[0009] (1) Preparation of TE buffer: Weigh 0.1211 g Tris and 0.0584 g EDTA into a 100 mL beaker, add about 90 mL of ultrapure water to dissolve, then adjust the pH to 8.0 with acetic acid solution, make up to volume in a 100 mL volumetric flask, and store at 4℃.

[0010] (2) Non-denaturing polyacrylamide gel electrophoresis (PAGE) experiment: 4 mL of 30% acrylamide was mixed with 4 mL of ultrapure water and 2 mL of 5×TBE buffer, followed by the addition of 35 μL of ammonium persulfate (APS) and 3.5 μL of tetramethylethylenediamine (TEMED). The mixture was thoroughly stirred and polymerized at 37 °C to form a 12% non-denaturing polyacrylamide gel. The sample was loaded using 6×Tris-glycerol gel loading buffer at a ratio of 1:5, and electrophoresis was performed at 90 V for 70 min. The gel was then stained with Stain-All staining solution and photographed for recording.

[0011] (3) Preparation and synthesis of gold nanoparticles (AuNPs): 50 mL of HAuCl4 (0.01 w / w) solution was heated to boiling under vigorous stirring; 5 mL of 38.8 mM trisodium citrate was quickly added and the mixture was heated for 17 min; heating was stopped and the mixture was cooled to room temperature. The gold nanoparticles were successfully prepared.

[0012] (4) Ligation of DNA with AuNPs: 90 μL (100 μM) DNA1 (S1-SH) was added to 1 μL of 500 mM Tris-HAc (pH 5.2), followed by 1.5 μL of 10 mM TCEP, and incubated at room temperature for 1 h (25℃). The above solution was added to 3 mL of AuNPs solution, and the mixture was magnetically stirred at 600 r / min at 25℃ and stored at room temperature for at least 16 h. The mixture was gently shaken by hand, and 30 μL of 500 mM Tris-HAc (pH 8.2) was added dropwise, followed by 300 μL of 1M NaCl. The mixture was then allowed to stand for 1 day. The mixture was magnetically stirred at 10000 r / min for 20 min, centrifuged to remove the supernatant, and 25 mM Tris-HAc (pH 8.2) was added. The mixture was centrifuged again at 10000 r / min for 10 min to disperse in Tris-HCl (25 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP). KCl (1 mM MgCl2) pH 7.4.

[0013] (5) In vitro ultraviolet measurement experiment: Prepare a series of target standard solutions with concentration gradients, covering the concentration range of 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, and 200 nM, with a buffer solution without the target substance as a blank control (0 pM); take an equal volume of the detection system and mix it with the above target standard solutions of different concentrations, and incubate it at 37°C to allow the detection system to fully react with the target substance; after the reaction is completed, use an ultraviolet-visible spectrophotometer to perform full wavelength scanning of each reaction system, with a scanning range of 300-800 nm, and record the absorption spectrum curves and absorbance (Abs) values ​​at characteristic wavelengths at each concentration.

[0014] (6) Stability analysis:

[0015] Serum stability analysis: The detection system was placed in a buffer solution containing 1% serum (v / v), with a blank control group without serum. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h. The absorption spectrum was scanned in the wavelength range of 300-800 nm using a UV-Vis spectrophotometer. The position, peak shape, and absorbance changes of characteristic absorption peaks were recorded, and the macroscopic state of the solution was observed.

[0016] Enzymatic stability analysis: The enzyme stability of the detection system was investigated using deoxyribonuclease 1 (DNase1) at a concentration of 5 U / mL, with the system without DNase1 serving as a blank control. Samples were taken at the same time points as above, and UV-Vis absorption spectra were scanned to analyze the effect of enzymatic hydrolysis on the system structure. Attached Figure Description

[0017] [ Figure 1 Hybridization of the target miRNA-10b with the probe induces the dissociation of the protective layer on the surface of AuNPs, thereby enabling qualitative and semi-quantitative analysis of miRNA-10b.

[0018] [ Figure 2 Gel electrophoresis verification image of miRNA-10b-induced nucleic acid probe assembly.

[0019] [ Figure 3 Transmission electron microscope (TEM) image.

[0020] [ Figure 4 Zeta potential characterization of gold nanoparticle surface functionalization.

[0021] [ Figure 5 UV-Vis absorption spectroscopy and visualization characterization of target-induced gold nanoparticle assembly.

[0022] [ Figure 6Time-resolved UV-Vis absorption spectroscopy and visualization monitoring of the target analyte-induced assembly system.

[0023] [ Figure 7 UV-Vis absorption spectroscopy characterization of surface functionalization of gold nanoparticles and probe coupling efficiency.

[0024] [ Figure 8 UV-Vis absorption spectra and visualization characterization of miRNA-10b-induced AuNP assembly under different buffer systems.

[0025] [ Figure 9 The aggregation behavior and pH response characteristics of target miRNA-10b-induced gold nanoparticles (AuNPs) were characterized by ultraviolet-visible absorption spectroscopy (UV-Vis) and colorimetry.

[0026] [ Figure 10 Kinetic curves of target-induced gold nanoparticle aggregation at 37℃.

[0027] [ Figure 11 Concentration linear range graph.

[0028] [ Figure 12 Selective analysis.

[0029] [ Figure 13 Stability analysis of 1% serum.

[0030] [ Figure 14 Analysis of the resistance to enzymatic hydrolysis at an enzyme concentration of 5 U / mL.

[0031] [ Figure 15 The probe sequence required in this experiment. Detailed Implementation Plan

[0032] Here, specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention, but do not limit the scope of application or extension of the present invention.

[0033] Example 1: Hybridization of target miRNA-10b with probe induces the aggregation of AuNPs, thereby enabling qualitative and semi-quantitative analysis of miRNA-10b.

[0034] like Figure 1As shown, a DNA sensor based on gold nanoparticles (AuNPs) is used for the visual detection of miRNA-10b, enabling rapid and visual detection of the tumor marker miRNA-10b. Specific DNA probes are designed; hybridization of the target miRNA-10b with the probes induces the aggregation of AuNPs, regulating their aggregation state in a buffer solution and producing a visible color change from red to blue, thereby achieving qualitative and semi-quantitative analysis of miRNA-10b.

[0035] Example 2: Gel electrophoresis verification image of miRNA-10b-induced nucleic acid probe assembly.

[0036] As shown in Figure A, the molecular recognition behavior of nucleic acid assemblies was preliminarily verified using non-denaturing polyacrylamide gel electrophoresis in a TE buffer system at pH 8.0. Figure 2 As shown, lane 1 is the single-stranded probe S1; lane 2 is a mixture of S1 and S2, showing a diffuse band, indicating that the two did not assemble effectively; lanes 3 and 4 are binary systems of S1 and miRNA-10b and S2 and miRNA-10b, respectively, with no significant change in band migration rate; lane 5 is a ternary system of S1, S2 and miRNA-10b, showing a significant lag band, proving that miRNA-10b can trigger specific hybridization and assembly of S1 and S2 to form a high molecular weight complex; lane 6 is the miRNA-10b control.

[0037] As shown in Figure B, the secondary structure of the probe sequence was predicted using the Minimum Free Energy (MFE) algorithm under physiological conditions at 37℃. The results show that the probe can spontaneously fold to form a stable stem-loop structure, comprising a long double-stranded stem region and loop structures at both ends. The secondary structure free energy is -32.06 kcal / mol, with a large absolute negative value, indicating excellent thermodynamic stability. The color bar (Equilibrium Probability) shows that the base pairing probability in the double-stranded stem region is close to 1.0 (red), indicating highly stable pairing; the loop regions have a moderate probability, uniform structural conformation, and no obvious mismatches or unstable regions.

[0038] Secondary structure simulation and gel electrophoresis results corroborate each other, proving that nucleic acid probes can form thermodynamically stable and conformationally uniform stem-loop structures under physiological conditions of 37℃, with high assembly efficiency and single product.

[0039] Example 3: Transmission electron microscope (TEM) image.

[0040] like Figure 3As shown, the left image (AuNPs) shows that the gold nanoparticles are uniformly dispersed spherical with uniform particle size, no obvious aggregation, and good dispersibility; the right image (AuNPs-S1 / S2) shows that the gold nanoparticles have obvious aggregation, forming irregular chains, indicating that the DNA strands (S1 / S2) have been successfully modified and induced to aggregate.

[0041] Example 4: Zeta potential characterization of gold nanoparticle surface functionalization.

[0042] like Figure 4 As shown, the interfacial charge characteristics of gold nanoparticles (AuNPs) and their complexes modified with nucleic acid probes were characterized using Zeta potential analysis. Bare gold nanoparticles (AuNPs) exhibited a weak negative potential and low surface charge density. After functionalization with single-stranded DNA probe S1 (AuNPs-S1), the Zeta potential of the system shifted significantly negatively, indicating that the negatively charged nucleic acid strand was successfully grafted onto the AuNPs surface, increasing the negative charge density on the particle surface. Further modification with probe S2 (AuNPs-S2) further intensified the negative Zeta potential shift, confirming the successful coupling of the S2 strand, and demonstrating a higher enrichment of negative charge on the particle surface with dual probe modification.

[0043] Example 5: UV-Vis absorption spectroscopy and visualization characterization of target-induced gold nanoparticle assembly.

[0044] like Figure 5 As shown, the assembly regulation behavior of miRNA-10b on the AuNPs-S1 / AuNPs-S2 binary system was investigated by combining ultraviolet-visual absorption spectroscopy (UV-Vis) with naked-eye visualization. The mixed system of AuNPs-S1 and AuNPs-S2 (black curve) showed a typical surface plasmon resonance (SPR) absorption peak of gold nanoparticles at 520 nm, indicating that the system was in a well-dispersed state. After the addition of miRNA-10b (red curve), the intensity of the SPR absorption peak at 520 nm decreased significantly and the peak shape broadened, proving that miRNA-10b triggered specific cross-linking and aggregation of AuNPs-S1 and AuNPs-S2 through complementary base pairing. The inset shows that the dispersed AuNPs are wine red (tube 1), and the color changes to blue-purple after aggregation (tube 2), which is consistent with the trend of spectral change, and visually verifies the nanoparticle assembly behavior induced by the target.

[0045] Example 6: Time-resolved UV-Vis absorption spectroscopy and visualization monitoring of target-induced assembly system.

[0046] like Figure 6As shown, time-resolved ultraviolet-visible absorption spectroscopy (UV-Vis) combined with naked-eye visualization was used to track the assembly kinetics triggered by the target material in real time. Within the wavelength range of 300-800 nm, the evolution of the surface plasmon resonance (SPR) absorption peak intensity of gold nanoparticles at 520 nm was monitored over time (9-20 min). With the extension of reaction time, the characteristic absorption peak at 520 nm increased in a time-dependent manner, indicating that the cross-linking assembly reaction of gold nanoparticles induced by the target material continued, and the product concentration accumulated over time. The inset shows that the solution color gradually deepened from light to dark as the reaction progressed, which was highly consistent with the trend of spectral absorption intensity change, visually confirming the time dependence and gradual nature of the assembly reaction.

[0047] Example 7: UV-Vis absorption spectroscopy characterization of gold nanoparticle surface functionalization and probe coupling efficiency.

[0048] like Figure 7 As shown, the upper part shows the SPR absorption spectra of AuNPs-S1 / AuNPs-S2; the bare gold nanoparticles (AuNPs) exhibit a typical surface plasmon resonance (SPR) absorption peak at 520 nm; after functionalization with single-stranded DNA probes S1 and S2 (AuNPs-S1, AuNPs-S2), the SPR absorption peak position did not show a significant red shift, but the absorption intensity changed slightly, indicating that the probes were successfully grafted onto the AuNPs surface and the particles maintained good dispersion and aggregation.

[0049] Example 8: UV-Vis absorption spectra and visualization characterization of miRNA-10b-induced AuNP assembly under different buffer systems.

[0050] like Figure 8As shown, the effects of three buffer systems—PBS-1, PBS-2, and Tris-HCl-2—on the response of the AuNPs-S1 / AuNPs-S2 binary system to miRNA-10b were systematically investigated by combining ultraviolet-visible absorption spectroscopy (UV-Vis) with naked-eye visualization. In each buffer system, the mixture of AuNPs-S1 and AuNPs-S2 exhibited a surface plasmon resonance (SPR) absorption peak at 520 nm for gold nanoparticles. After the addition of miRNA-10b, the SPR absorption peak at 520 nm was significantly reduced and broadened, confirming that miRNA-10b triggered specific cross-linking and aggregation of AuNPs-S1 and AuNPs-S2 through base complementary pairing. The Tris-HCl-2 system showed the most significant change in SPR absorption peak intensity, indicating that this buffer condition is more conducive to nucleic acid hybridization and nanoparticle aggregation, resulting in higher detection sensitivity. The response of the PBS-1 and PBS-2 systems decreased sequentially. The inset shows that the dispersed AuNPs are wine-red, and the color changes to blue-purple after aggregation, consistent with the trend of spectral changes, which visually verifies the nanoparticle assembly behavior induced by the target under different buffer systems.

[0051] Example 9: The aggregation behavior and pH response characteristics of target miRNA-10b-induced gold nanoparticles (AuNPs) were characterized by ultraviolet-visible absorption spectroscopy (UV-Vis) and colorimetry.

[0052] like Figure 9 As shown, after adding miRNA-10b, no obvious color change occurred in the system, and the solution remained the characteristic red color of AuNPs, indicating that the nanoparticles did not aggregate significantly, and the cross-linking reaction required a certain amount of time to complete. After 60 min of incubation, the experimental group solution containing the target miRNA-10b changed from red to blue-purple, while the blank control group without the target remained red, confirming that the target can mediate specific cross-linking and aggregation of AuNPs-S1 and AuNPs-S2 through base complementary pairing. UV-Vis spectroscopy showed that the dispersed AuNPs exhibited a characteristic surface plasmon resonance (SPR) absorption peak at 520 nm. After target-induced aggregation, the SPR absorption peak at 520 nm significantly decreased, and the absorption in the long wavelength region (600-700 nm) increased, consistent with the trend of solution color change. Further investigation into the effect of pH on sensing performance showed that the target-induced AuNP aggregation effect was most significant at pH 8.0, with the largest decrease in the 520 nm absorption peak. pH 7.4 and pH 10.0 showed the greatest effect at pH 10.0. The aggregation degree decreased sequentially under conditions of 7.0. The above results demonstrate that a weakly alkaline environment (pH 8.0) is more conducive to DNA hybridization reaction, which can effectively improve the aggregation efficiency of AuNPs and the sensitivity of the sensing response, providing a basis for the subsequent optimization of the miRNA-10b colorimetric sensing system.

[0053] Example 10: Kinetic curve of target-induced aggregation of gold nanoparticles at 37℃.

[0054] like Figure 10 As shown, the kinetics of target-induced gold nanoparticle (AuNPs) aggregation were monitored at 37℃ and room temperature. In the initial state (0 min), AuNPs were in a dispersed state, exhibiting a characteristic surface plasmon resonance absorption peak at 520 nm, and the solution was red. With prolonged incubation time, the target mediated specific cross-linking and aggregation of AuNPs-S1 and AuNPs-S2, the absorption peak at 520 nm gradually decreased, the absorption in the long wavelength region gradually increased, and the solution color changed from red to blue-purple.

[0055] Example 11: Concentration linear range graph.

[0056] like Figure 11 As shown, the top image displays actual photographs of AuNPs systems at different concentrations of the target analyte, visually illustrating the color change of the system as the target analyte concentration increases. Below, within the target analyte concentration range of 10 pM–200 nM, the system absorbance exhibits a strong negative linear correlation with the target analyte concentration. As the target analyte concentration increases from 0 to 200 nM, the intensity of the characteristic absorption peak at 520 nm decreases significantly with a gradient, while the absorbance in the 650 nm–700 nm band increases synchronously. Furthermore, the spectral curves at each concentration gradient demonstrate good repeatability and stability.

[0057] Example 12: Selectivity analysis.

[0058] like Figure 12 As shown in the left figure (base mismatch specificity): only the perfectly complementary target miRNA-10b can induce significant aggregation of gold nanoparticles (AuNPs), and the SPR absorption peak at 520 nm decays sharply; the signal responses of the single-base mismatch (SM), double-base mismatch (DM), and triple-base mismatch (TM) groups are weak, with no significant difference from the blank control, demonstrating the high specificity of single-base resolution; the right figure (homologous miRNA interference): the target miRNA-10b produces a strong colorimetric response; the homologous sequences miRNA-21, miRNA-155, miRNA-9, and miRNA-141 do not show obvious aggregation, and their spectral curves highly overlap with the blank control, confirming that the system has excellent anti-interference ability against the target; after incubation, only the target group solution changes from red to blue, while the mismatch and interference groups remain red, and the spectral changes are consistent with the macroscopic color, achieving specific discrimination visible to the naked eye.

[0059] Example 13: Stability analysis of 1% serum.

[0060] like Figure 13As shown, to evaluate the applicability of the constructed system in complex biological environments, its long-term stability in 1% serum was investigated. Spectral changes within 0–10 h were detected by ultraviolet-visible absorption spectroscopy (UV-Vis). Under 1% serum conditions, the absorption spectra of the samples completely overlapped within 0–10 h, with no significant shift or attenuation in the position and intensity of the characteristic absorption peaks, and no visible change in solution color. This indicates that the system is structurally stable in low-concentration serum and has not undergone significant degradation or aggregation. These results confirm that the system exhibits excellent time stability in a 1% serum environment.

[0061] Example 14: Analysis of the resistance to enzymatic hydrolysis at an enzyme concentration of 5 U / mL.

[0062] like Figure 14 As shown, the enzyme stability of the detection system was investigated using deoxyribonuclease 1 (DNase1). An enzyme concentration of 2 U / mL was used, with a system without DNase1 serving as a blank control. Samples were taken at the same time points as described above, and UV-Vis absorption spectra were scanned to analyze the effect of enzymatic degradation on the system structure. At an enzyme concentration of 2 U / mL, the detection system maintained stable spectral characteristics, with no significant fluctuations in the absorption curves at each time point, intact peak shapes, and a homogeneous solution state, showing no obvious signs of enzymatic degradation. The results demonstrate that the detection system possesses excellent resistance to enzymatic degradation and can effectively resist the degradation effects of biological enzymes.

[0063] Example 15: The probe sequence required in this experiment.

[0064] like Figure 15 As shown, it includes scaffold helper strands S1 and S2, as well as wild-type miRNA-10b and single / double / triple mutation control sequences; used to construct a miRNA-10b detection control system and adapt it to the assembly of tumor multi-target sensors.

Claims

1. A DNA sensor based on gold nanoparticles (AuNPs) for the visual detection of miRNA-10b, characterized in that... The sensor includes: (1) Gold nanoparticles (AuNPs) are coupled to probe S1 via thiol groups. Gold nanoparticles (AuNPs) are coupled to probe S2 via thiol groups. Thiol modification makes the binding of probes to gold nanoparticles stronger, improving the stability and detection repeatability of DNA sensors. (2) By hybridizing the target miRNA-10b with probes S1 and S2, AuNPs are induced to aggregate, resulting in a visible color change from red to blue, thereby achieving qualitative and semi-quantitative analysis of miRNA-10b. (3) Detectable signal changes include changes in solution color and changes in ultraviolet-visible absorption spectrum.

2. The DNA sensor according to claim 1, characterized in that, The preparation of gold nanoparticles (AuNPs) is achieved through the following steps: (1) 50 mL of HAuCl4 (0.01 w / w) solution was heated to boiling with vigorous stirring; (2) Quickly add 5 mL of 38.8 mM trisodium citrate and continue heating for 17 min; (3) Stop heating and cool to room temperature. The gold nanoparticles have been successfully prepared.

3. The DNA sensor according to claim 1, characterized in that, The ligation of DNA to AuNPs is achieved through the following steps: (1) Add 90 μL (100 μM) DNA1 (S1-SH) to 1 μL 500 mM Tris-HAc (pH 5.2), then add 1.5 μL 10 mM TCEP and incubate at room temperature for 1 h (25℃). (2) Add the above solution to 3 mL of AuNPs solution, stir magnetically at 600 r / min at 25°C, and store at room temperature for at least 16 h; (3) Shake gently by hand, add 30 μL of 500 mM Tris-HAc (pH 8.2), then add 300 μL of 1M NaCl and let stand for 1 day; (4) Stir magnetically at 10,000 r / min for 20 min, centrifuge to remove supernatant, add 25 mM 200 μL Tris-HAc (pH 8.2); centrifuge again at 10,000 r / min for 10 min to disperse in Tris-HCl (25 mM Tris-HCl, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2) pH 7.

4.

4. The conditions for the sensor to detect the target miRNA-10b according to claim 1: incubation at 37°C for 1 h.

5. The sensor according to claim 1 maintains structural stability under the following conditions: (1) Stable in 1% serum solution for at least 10 h; (2) Stable for at least 10 h in an enzyme concentration solution of 5 U / mL; (3) Store stably at 4°C for at least 7 days.

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