Au NPs-MnO2 nano material with ultrathin core-shell structure as well as preparation method and application of Au NPs-MnO2 nano material
An electrochemical biosensor was prepared by catalyzing hairpin self-assembly of CHA and hybridization chain reaction HCR cascade using ultrathin core-shell Au NPs-MnO2 nanomaterials. This solved the sensitivity and specificity problems of existing bladder cancer miRNA-21 detection technologies, enabling low-cost and high-sensitivity early diagnosis of bladder cancer.
Patent Information
- Application Number
- CN202511529675.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-24
- Publication Date
- 2026-01-09
AI Technical Summary
Existing miRNA-21 detection technologies for bladder cancer are expensive, cumbersome to conduct, prone to false positives, and have poor specificity and sensitivity, making it difficult to achieve high-sensitivity early detection.
An electrochemical biosensor was fabricated using ultrathin core-shell Au NPs-MnO2 nanomaterials as the substrate for oxidase-like catalysis, TMB, to generate an electrochemical signal. This signal was then amplified by combining catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) for dual signal amplification. This biosensor was used to detect the bladder cancer biomarker miRNA-21.
It achieves ultra-sensitive detection of miRNA-21 in the urine of bladder cancer patients, with a detection limit as low as 0.194 fM, and has excellent selectivity, stability and reproducibility, making it suitable for large-scale production.
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Figure CN121294619A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of new materials technology, specifically relating to an ultrathin core-shell structure Au NPs-MnO2 nanomaterial, its preparation method, and its application. Background Technology
[0002] According to the 2022 global cancer statistics, bladder cancer ranks 9th in incidence among malignant tumors worldwide, 6th among men, and 13th in mortality. In 2022, there were approximately 614,000 new cases of bladder cancer globally, with about 220,000 deaths. After age-standardized adjustments, the incidence rate for this disease increased to 9.3 per 100,000 for men and 2.4 per 100,000 for women; correspondingly, the mortality rate was 3.1 per 100,000 for men and 0.8 per 100,000 for women. Based on whether the tumor invades the muscle layer, bladder cancer can be divided into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Tis, Ta, and T1 stages belong to NMIBC, while subsequent stages belong to MIBC. NMIBC has a high recurrence rate, requiring long-term follow-up, resulting in high monitoring and treatment costs. The prognosis for advanced MIBC is poor, with a 5-year survival rate of only 15% and a survival period of only 12-14 months. Therefore, early screening for bladder cancer patients is particularly important.
[0003] Currently, the main diagnostic methods for BLCA include cystoscopy, urine cytology, and liquid biopsy. Although cystoscopy remains the gold standard for diagnosing BLCA, it is relatively expensive and can cause physical and psychological trauma to patients, making it unfavorable for early detection. Urine cytology has low sensitivity for low-grade BLCA (LG-BLCA), increasing nursing costs. Liquid biopsy is non-invasive, provides real-time information on disease progression and treatment response, and samples are readily available, non-invasive, and repeatable. It is also inexpensive, widely available, and more acceptable to patients.
[0004] Existing urinary biomarkers are mainly classified into protein, DNA, RNA, and others. Among RNA biomarkers, miRNAs have become a research hotspot due to their high stability, sensitivity, and strong specificity. miRNA-21, in particular, is specific among many bladder cancer biomarkers because it negatively regulates the p53 tumor suppressor gene pathway, leading to cellular dysregulation and inducing cancer cells. Therefore, developing a specific and ultrasensitive detection method for miRNA-21 is crucial for early detection of BLCA. Current research, specifically the 21-year AC study, shows that miRNA-21 concentration increases with BLCA stage, with a cutoff value of 15 ng between BLCA and non-BLCA stages.
[0005] With technological advancements, common techniques for detecting miRNAs in urine samples include real-time quantitative polymerase chain reaction (RT-qPCR), Northern blotting, next-generation sequencing (NGS), and biosensors. However, these techniques suffer from drawbacks such as high cost, cumbersome experiments, susceptibility to false positives, poor specificity and sensitivity, and time consumption. Electrochemical biosensors, on the other hand, have emerged as a promising technology for miRNA detection due to their ease of operation, high sensitivity, low cost, and miniaturization. However, the application of electrochemical biosensors often encounters challenges posed by extremely low miRNA concentrations, making it difficult for traditional detection methods to achieve high sensitivity. Therefore, it is essential to research and develop detection techniques with high sensitivity and specificity to achieve rapid and ultrasensitive miRNA detection. Summary of the Invention
[0006] This invention aims to solve the above-mentioned technical problems and provides an ultrathin core-shell structured Au NPs-MnO2 nanomaterial. The ultrathin core-shell structured Au NPs-MnO2 nanomaterial serves as an oxidase-like substrate to catalyze the generation of electrochemical signals from TMB, and is used to prepare an electrochemical biosensor for detecting the bladder cancer biomarker miRNA-21.
[0007] The technical solution of this invention is as follows: An ultrathin core-shell structured Au NPs-MnO2, characterized in that: the ultrathin core-shell structured Au NPs-MnO2 nanomaterial has Au NPs-PVP as the core and ultrathin manganese dioxide as the shell.
[0008] Preferably, the thickness of the manganese dioxide shell in this invention is 45-55 nm, and the overall particle size of the ultrathin core-shell structure Au NPs-MnO2 nanomaterial (Ult Au NPs-MnO2) is 136.05 ±15.29 nm.
[0009] The ultrathin core-shell structured Ult Au NPs-MnO2 nanomaterials of this invention, by introducing high-valence Mn(IV)-O bonds, can induce charge imbalance and generate unsaturated sites. Furthermore, the ultrathin structure of MnO2 facilitates charge transfer between Au and Mn. Simultaneously, the lower reaction temperature employed in this system regulates nucleation and growth kinetics to obtain more open metal sites, thereby improving the oxidation performance of the Ult Au NPs-MnO2 nanomaterials. Its oxidase mechanism primarily involves the Ult Au NPs-MnO2 nanozyme exerting its oxidase-like activity under the action of O2, catalyzing the substrate TMB to generate ox TMB, while simultaneously producing H2O2 and O2. ·- .
[0010] Preferably, the preparation method of Au NPs-PVP according to the present invention is as follows: using chloroauric acid as raw material, sodium citrate as reducing agent, and polyvinylpyrrolidone as surface ligand, Au NPs-PVP is synthesized; more specifically, the preparation method of Au NPs-PVP according to the present invention is as follows: taking 5-20 mM HAuCl4·3H2O, adding deionized water to a total volume of 100 mL and mixing evenly, raising the temperature to 120-130 ℃, adding 800-900 μL of sodium citrate aqueous solution with a concentration of 0.05-0.2 M, refluxing for 30-45 min, and when the temperature drops to 70-80 ℃, adding 5-15 mL of polyvinylpyrrolidone with a concentration of 0.01-0.05 g / mL, refluxing for 20-40 min, the reaction is completed, and Au NPs-PVP is obtained.
[0011] Preferably, the preparation method of the ultrathin core-shell structured Au NPs-MnO2 nanomaterial of the present invention includes the following steps: using Au NPs-PVP and potassium permanganate as raw materials, an ultrathin manganese dioxide shell layer is generated on the surface of gold nanospheres by hydrothermal method to obtain the ultrathin core-shell structured Au NPs-MnO2; more specifically, the method includes: taking Au NPs-PVP, centrifuging, adding deionized water to the precipitate at a material-to-liquid ratio of 1g:4-6mL to disperse evenly; adding KMnO4 with a concentration of 5-15 mM at a volume ratio of Au NPs-PVP to KMnO4 of 1mL:15-20μL, sonicating for 30-60 min, and then refluxing at 88-92 °C for 30-40 min, according to Au... The NPs-PVP and KMnO4 were mixed in a volume ratio of 1 mL: 5-10 μL, and then KMnO4 with a concentration of 5-15 mM was added. The mixture was refluxed at 78-82 °C for 6-8 h to obtain the ultrathin core-shell structure Au NPs-MnO2.
[0012] The present invention also provides the application of the ultrathin core-shell structure Au NPs-MnO2 nanomaterial in the preparation of an electrochemical biosensor for detecting miRNA, wherein the ultrathin core-shell structure Au NPs-MnO2 nanomaterial serves as an oxidase-like substrate TMB to generate an electrochemical signal.
[0013] Preferably, the electrochemical biosensor of the present invention comprises: an ultrathin core-shell Au NPs-MnO2 nanomaterial; a DNA capture probe P immobilized on the surface of the ultrathin core-shell Au NPs-MnO2 nanomaterial; and a DNA hairpin structure for performing catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR). Specifically, the ultrathin core-shell Au NPs-MnO2 can catalyze the oxidation reaction of the substrate TMB to generate an electrochemical signal. The DNA hairpin structure undergoes a cascade reaction triggered by the target miRNA-21, and the generated double-stranded DNA polymer is loaded on the surface of the ultrathin core-shell Au NPs-MnO2, inhibiting its catalytic activity, thereby achieving ultrasensitive detection of miRNA-21.
[0014] Preferably, the DNA capture probe P of the present invention is a single-stranded DNA pre-fixed on the surface of the ultrathin core-shell structure Au NPs-MnO2 nanomaterial; the DNA hairpin structure includes H1, H2, H3 and H4, wherein H1 and H2 are used to form a catalytic hairpin self-assembly CHA cycle, and H3 and H4 are used to form a hybridization chain reaction HCR.
[0015] A kit comprising the electrochemical biosensor described in this invention for detecting the bladder cancer biomarker miRNA-21.
[0016] This invention utilizes an electrochemical biosensor containing a specific ultrathin core-shell structured Au NPs-MnO2 nanomaterial, which offers simple acquisition, low power consumption, and ultrasensitivity for the specific detection of miRNA-21. The signal is dually amplified through a cascaded catalytic hairpin assembly (CHA) and a hybridization chain reaction (HCR). Furthermore, based on the mediation of the ultrathin core-shell structure Au NPs-MnO2, ultra-high sensitivity detection of miRNA-21 in the urine of bladder cancer patients is achieved, with a detection limit as low as 0.194 fM, and excellent selectivity, stability, and repeatability.
[0017] Compared with the prior art, the beneficial effects of the present invention are at least as follows: 1. The ultrathin core-shell structure Au NPs-MnO2 nanomaterial of the present invention uses gold nanospheres Au NPs-PVP as the core and is then wrapped with an ultrathin layer of manganese dioxide as the shell to form an ultrathin shell structure. The synergistic effect between the ultrathin shell structure and Au and manganese dioxide gives the ultrathin core-shell structure Au NPs-MnO2 nanomaterial extremely strong oxidase-like activity, which directly catalyzes the oxidation reaction of the substrate TMB (3,3',5,5'-tetramethylbenzidine). The oxidized TMB (oxTMB) generates a strong electrochemical signal (current change), which can be detected by electrodes. The degree of TMB oxidation directly determines the strength of the signal. The less TMB is oxidized, the weaker the current signal. The weaker the current signal, the higher the concentration of miRNA-21. By comparing the current signal with a standard curve, the content of miRNA-21 can be calculated with ultrasensitive sensitivity, realizing the early diagnosis of bladder cancer.
[0018] 2. The preparation method of the ultrathin core-shell structured Au NPs-MnO2 nanomaterial of the present invention is simple, convenient to operate, low in cost, and suitable for large-scale production.
[0019] 3. The ultrathin core-shell Au NPs-MnO2 nanomaterial of the present invention, through its special structure (Ult Au NPs-MnO2 nanocore-shell structure), and the DNA capture probe P and DNA molecular machine (CHA+HCR) fixed on the surface of the ultrathin core-shell Au NPs-MnO2 nanomaterial, are used to prepare an electrochemical biosensor by dual signal amplification, thereby enabling ultrasensitive detection of the bladder cancer marker miRNA-21, achieving highly sensitive detection of urinary markers in bladder cancer patients, with a detection limit as low as 0.194 fM. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the preparation of Ult Au NPs-MnO2 nanomaterials.
[0021] Figure 2 The images show TEM images and UV experimental results of the Au NPs-MnO2 solution prepared in Example 1.
[0022] Figure 3 The diagram shows the optimized conditions (pH and material concentration) for the Ultra Au NPs-MnO2 nanomaterials prepared in Example 1.
[0023] Figure 4 This is a diagram illustrating the types of nanoenzymes derived from the Ultra Au NPs-MnO2 nanomaterials prepared in Example 1.
[0024] Figure 5 This is a comparison of the catalytic activity of Au NPs-MnO2 nanomaterials loaded with dsDNA prepared in Example 1.
[0025] Figure 6 The figure shows the experimental results of the catalytic effect of Ultra-MnO2 nanomaterials loaded with dsDNA prepared in Example 1.
[0026] Figure 7 The figures show the experimental results of the dsDNA content loaded on the Ultra Au-MnO2 nanomaterials prepared in Example 1. Figures A and B show the determination of the amount of primer ssDNA P; Figures C and D show the determination of the amount of H1-H2 (CHA reaction).
[0027] Figure 8 The figures show the results of the sensitivity experiments of Au NPs-MnO2 nanomaterials prepared in Example 1 to miRNA-21 based on the CHA reaction and the CHA-HCR cascade reaction. Figures A and B are the results of the CHA reaction, and figures C and D are the results of the CHA-HCR cascade reaction.
[0028] Figure 9 The figure shows the experimental results of selectivity, interference, stability and reproducibility of the Au NPs-MnO2 nanomaterials prepared in Example 1 on miRNA-21. Detailed Implementation
[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and 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.
[0030] The DNA sequences used below are as follows: Capture probe P (SEQ ID No. 1): 5'-AGTCTGAAGCTACATCGTCCAGTTACTTTTTTTTTT-SH-3'; Hairpin H1 (SEQ ID No. 2): 5'-GTAACTGGACGATGTAGCTTCAGACTATCAGACTGATGTTGAGGGGCGACTTGAAACTCAACATCAGTCTGATAAGCTA-3'; Hair clip H2 (SEQ ID No. 3): 5'-GGGGCGACTTGAAACATCAGACTGATGTTGAGTTTCAAGTCGCCCCTCAACATC-3'; Hairpin H3 (SEQ ID No. 4): 5'-GTTTCAAGTCGCCCCGAAGGAGGGGCGACTC-3'; Hairpin H4 (SEQ ID No. 5): 5'-GGGGCGACTTGAAACAGTCGCCCCTCCTTC-3'.
[0031] Example 1: Synthesis of Au NPs-MnO2 nanomaterials Taking a gold sphere with a diameter of 20 nm and an Au-MnO2 sphere with a diameter of 136 nm as an example, the specific steps are as follows: (1) Preparation of Au NPs-PVP: Take 10 mM HAuCl4·3H2O, add deionized water (total volume 100 mL), mix well, and when the temperature rises to 120℃, add 875 μL of sodium citrate aqueous solution (0.1 M), reflux for 30 min (color changes from colorless to wine red), and when the temperature drops to 70℃, add 10 mL of polyvinylpyrrolidone (PVP 8000; 0.02 g / mL). -1 The mixture was refluxed for 30 min to complete the reaction, yielding an Au NPs-PVP solution.
[0032] (2) Preparation of Ult Au NPs-MnO2 nanomaterials: Take 10 mL of Au NPs-PVP solution, centrifuge to remove unreacted ligands, add 20 mL of deionized water and disperse evenly in a three-necked flask; quickly add 180 μL of KMnO4 (10 mM), sonicate for 60 min, then reflux at 90 ℃ for 30 min, add another 80 μL of KMnO4 (10 mM) and reflux at 80 ℃ for 8 h, to obtain Ultra Au NPs-MnO2 with a particle size of about 100 nm and PVP ligands. TEM and EDS mapping results are shown below. Figure 2 Figures A, B, C, and D show that Ult AuNPs-MnO2 exhibits an ultrathin core-shell structure with an average diameter of 136±15 nm and a 15 nm gold core. EDS mapping shows that Au is mainly located in the central region of Ult AuNPs-MnO2, while Mn is distributed on the periphery, confirming that Ult AuNPs-MnO2 has a thin core-shell structure.
[0033] (3) Preparation of Deep Au NPs-MnO2 nanomaterials: Take 1 mL of Au NPs-PVP solution, centrifuge to remove unreacted ligands, add an equal volume of deionized water and disperse evenly in a reaction flask; quickly add 40 μL of KMnO4 (10 mM), sonicate for 1-10 min, and then heat at 90 ℃ for 2 h to obtain Deep Au NPs-MnO2 with a particle size of about 35 nm and PVP ligands. TEM and EDS mapping results are shown below. Figure 2 Figures E, F, G, and H show the core-shell structure of Deep Au NPs-MnO2, with an average diameter of 35 nm and a 9 nm gold core. EDS mapping shows that Au is mainly located in the central region of Deep Au NPs-MnO2, while Mn is distributed on the periphery, confirming that Deep Au NPs-MnO2 has a relatively thick core-shell structure. Experimental Example 1
[0034] 1. Effect of pH on the catalytic effect of Ultra-Au-MnO2 nanomaterials The experiment involved preparing PBS (100 mM NaCl) buffer solutions with different pH values, with pH gradients of 4, 5, 6, 7, 7.4, and 8. At least 5 mL of each pH PBS buffer solution was placed in a centrifuge tube, and 20 μL of Ult Au-MnO2 was added. After mixing thoroughly, 10 μL of TMB (10 mM) solution was added, and the reaction was allowed to proceed for 5 min. DPV was then measured within a voltage range of 0.1–0.6 V.
[0035] like Figure 3 Results A showed that TMB exhibited different peak shapes under different pH conditions. The electron transfer process of TMB occurs in two steps: the first step involves the free radical losing an electron, and the second step involves free radical oxidation. At low pH conditions, the first step is more difficult, leading to a positive shift in the oxidation potential, which partially overlaps with the peak of the second step. In contrast, at higher pH conditions, although TMB may also undergo two-step oxidation, the oxidation product quinone diimide forms an azo compound, resulting in a single peak. To make the detection results more intuitive, this invention selected pH=8 as the buffer solution for subsequent experiments.
[0036] 2. Effect of Ult Au-MnO2 nanomaterial dosage on its catalytic effect In this invention, the catalytic activity was studied in a pH=4 environment where the amino group in the TMB molecule completely lost one electron in performance verification 1.
[0037] The experiment was conducted with Ultra Au-MnO2 dose gradients of 5, 10, 15, 20, and 25 μL. PBS (pH=4; total reaction volume 250 μL) buffer was added to centrifuge tubes, and different volumes of Ultra Au-MnO2 were added and mixed thoroughly. Then, 10 μL of TMB (10 mM) solution was added, and the mixture was reacted for 5 min. Ultraviolet measurements were performed in the wavelength range of 300-700 nm.
[0038] like Figure 3 Results B showed that with the increase of Ultra-MnO2, the degree of oxidation of TMB was greater, and the color change was from light blue to dark blue. When the dosage of Ultra-MnO2 was 20 μL, TMB was completely catalyzed.
[0039] 3. Experiments on the types of active oxygen generated by Ult Au-MnO2 nanomaterials (1) Reactive oxygen species 1 O2 verification The experiment was conducted as follows: 220 μL of PBS (pH=7.4) buffer was placed in a centrifuge tube, 20 μL of Ult Au-MnO2 was added, and then quantitative amounts of the following solutions (blank, low concentration, and high concentration of tryptophan) were added. After mixing well, 10 μL of TMB (10 mM) solution was added, and the reaction was carried out for 5 min. The results were then measured under ultraviolet light in the wavelength range of 300-700 nm.
[0040] like Figure 4 Results A show that the Ult Au-MnO2 nanomaterials can produce during the catalytic process. 1 O2.
[0041] (2) Active O2 ·- Verification The experiment involved taking 220 μL of PBS (pH=7.4) buffer into a 1 mL centrifuge tube, adding 20 μL of UltAu-MnO2, and then adding less than 10 μL of solution (blank, low concentration, and high concentration of p-benzoquinone). After mixing thoroughly, 5 μL of TMB (10 mM) solution was added, and the reaction was allowed to proceed for 5 min. The results were then measured using ultraviolet light in the wavelength range of 300-700 nm.
[0042] like Figure 4 The BC results show that O2 can be generated during the catalytic process of Ult Au-MnO2 nanomaterials. ·- .
[0043] Figure 5 The process of generating reactive oxygen species mainly involves the Ult Au-MnO2 nanozyme, under the action of O2, exerting its oxidase-like function to catalyze the substrate TMB to generate ox TMB, while simultaneously producing H2O2 and O2.·- In the presence of H2O2 in the reaction system, the Ult Au-MnO2 nanomaterials act as a peroxidase-like enzyme, catalyzing the formation of ox TMB from TMB, while simultaneously generating H2O and ·OH.
[0044] 4. Catalytic effect test of Ultra Au-MnO2 and Deep Au-MnO2 nanomaterials loaded with dsDNA The experiment involved verifying the catalytic oxidation effect of double-stranded DNA (dsDNA) on Ultra Au-MnO2 and Deep Au-MnO2 nanomaterials before and after loading them with double-stranded DNA (dsDNA). The catalytic oxidation effect was used to verify the loading capacity of Au-MnO2 nanomaterials of different thicknesses on double-stranded DNA (dsDNA).
[0045] Take 5 μL of P (2 μM) into a centrifuge tube, add 20 μL of Ult Au-MnO2, and then add PBS buffer (pH 7.4) to a total volume of 30 μL. Incubate at room temperature for 1 h to obtain Ult Au-MnO2@P. At the same time, take 5 μL of miRNA-21 (0.5 μM) into a centrifuge tube, add 15 μL of H1 and H2 respectively, and finally adjust the volume to 50 μL with PBS. React at 30℃ for 2 h to obtain H1-H2. Then take 10 μL of H1-H2 solution and add it to a centrifuge tube containing Ult Au-MnO2@P. React at room temperature for 2 h, then add H3 and H4 respectively, adjust the volume to 50 μL with PBS, and react at 25℃ for 1.5 h. After the reaction is complete, Ult Au-MnO2@P-H1-H2-H3-H4 can be obtained. The preparation method of Deep Au-MnO2@P-H1-H2-H3-H4 is the same as that of Ultra Au-MnO2@P-H1-H2-H3-H4.
[0046] Ultra Au-MnO2, Deep Au-MnO2, and the prepared Ultra Au-MnO2@P-H1-H2-H3-H4 and Deep Au-MnO2@P-H1-H2-H3-H4 solutions were respectively placed in centrifuge tubes. Then, PBS (pH=7.4) buffer and 10 μL TMB (10 mM) solution were added, respectively, for a total solution volume of 5 mL. After reacting for 5 min, differential pulse voltammetry (DPV) was used for measurement. Scan voltage: 0.1–0.5 V, pulse amplitude: 0.05 V, pulse width: 0.05 s.
[0047] like Figure 5The results showed that the catalytic amplification effect of Ultra Au-MnO2@P-H1-H2-H3-H4 nanomaterial was higher than that of DeepAu-MnO2@P-H1-H2-H3-H4. These results indicate that dsDNA is more easily adsorbed on the surface of the ultrathin core-shell structure Ultra Au-MnO2, masking more active sites and thus achieving the purpose of signal amplification.
[0048] 5. Catalytic effect test of Ult Au-MnO2 nanomaterials loaded with dsDNA The experiment involved loading single-chain P (Ult Au-MnO2@P), double-chain H1-H2-P (Ult Au-MnO2@P-H1-H2), and double-chain H1-H2-H3-H4-P (Ult Au-MnO2@P-H1-H2-H3-H4) onto Ult Au-MnO2 nanomaterials, and verifying the successful loading of dsDNA by catalytic TMB oxidation.
[0049] Take 5 μL of P (2 μM) into a centrifuge tube, add 20 μL of Ult Au-MnO2, and then add PBS buffer (pH 7.4) to a total volume of 30 μL. Incubate at room temperature for 1 h to obtain Ult Au-MnO2@P. Simultaneously, take 5 μL of miRNA-21 (0.5 μM) into a centrifuge tube, add 15 μL of H1 and 1 μM of H2, and finally adjust the volume to 50 μL with PBS. React at 30 °C for 2 h to obtain H1-H2. Then take 10 μL of H1-H2 solution and add it to a centrifuge tube containing Ult Au-MnO2@P. React at room temperature for 2 h to obtain Ult Au-MnO2@P-H1-H2. Add 4 μL of H3 and 1 μM of H4, and adjust the volume to 50 μL with PBS. React at 25 °C for 1.5 h. After the reaction is complete, Ult Au-MnO2@P is obtained. Au-MnO2@P-H1-H2-H3-H4.
[0050] Ultra-Au-MnO2, Ultra-Au-MnO2@P, Ultra-Au-MnO2@P-H1-H2, and Ultra-Au-MnO2@P-H1-H2-H3-H4 (Ult Au-MnO2 concentration 20 μL) were respectively placed in centrifuge tubes. Then, PBS (pH=7.4) buffer and 10 μL of TMB (10 mM) solution were added, respectively, for a total solution volume of 5 mL. After reacting for 5 min, differential pulse voltammetry (DPV) was used for measurement. Scan voltage: 0.1–0.5 V, pulse amplitude: 0.05 V, pulse width: 0.05 s.
[0051] like Figure 6The results showed that the current signals, from strongest to weakest, were: Au-MnO2@P-H1-H2-H3-H4, Ult Au-MnO2@P-H1-H2, Ult Au-MnO2@P, and Ult Au-MnO2. These results indicate that double-stranded dsDNA was successfully loaded onto UltAu-MnO2 nanomaterials, and the catalytic effect of Ult Au-MnO2 decreased with increasing double-strand length.
[0052] 6. Investigation on the content of dsDNA loaded in Ult Au-MnO2 nanomaterials The experiment involved determining the content of single-chain P (Ult Au-MnO2@P) and double-chain H1-H2-P (Ult Au-MnO2@P-H1-H2) loaded on Ult Au-MnO2 nanomaterials.
[0053] (1) Determination of the content of single-chain P (Ult Au-MnO2@P) Take 0-10 μL of P (2 μM) into centrifuge tubes, add 20 μL of Ult Au-MnO2, and then add PBS buffer (pH 7.4) to a total volume of 30 μL. Incubate at room temperature for 1 h to obtain Ult Au-MnO2@P. At the same time, take 5 μL of miRNA-21 (0.5 μM) into centrifuge tubes, add 15 μL of H1 and H2 respectively, and finally adjust the volume to 50 μL with PBS. React at 30 °C for 2 h to obtain H1-H2. Then take 10 μL of H1-H2 solution and add it to centrifuge tubes containing Ult Au-MnO2@P. React at room temperature for 2 h, then add 4 μL of H3 and H4 (1 μM) respectively, adjust the volume to 50 μL with PBS, and react at 25 °C for 1.5 h. After the reaction is complete, Ult Au-MnO2@P-H1-H2-H3-H4 can be obtained. Take 5 mL of Ult Au-MnO2@P-H1-H2-H3-H4, TMB and PBS solutions respectively, react for 5 min, and then measure using differential pulse voltammetry (DPV).
[0054] like Figure 7 AB results showed that the catalytic effect was complete when 4 μL of P (2 μM) was added.
[0055] (2) Determination of the content of double-chain H1-H2-P (Ult Au-MnO2@P-H1-H2) Take 4 μL of P (2 μM) into a centrifuge tube, add 20 μL of Ult Au-MnO2, and then add PBS buffer to a total volume of 30 μL. Incubate at room temperature for 1 h to obtain Ult Au-MnO2@P. At the same time, take 5 μL of miRNA-21 (0.5 μM) into a centrifuge tube, add 15 μL of H1 and 1 μM of H2, and finally adjust the volume to 50 μL with PBS. React at 30 °C for 2 h to obtain H1-H2. Then take 16 μL of H1-H2 solution and add it to a centrifuge tube containing Ult Au-MnO2@P. React at room temperature for 2 h, then add 4 μL of H3 and 1 μM of H4, and adjust the volume to 50 μL with PBS. React at 25 °C for 1.5 h. After the reaction is complete, Ult Au-MnO2@P-H1-H2-H3-H4 can be obtained. Take 5 mL of Ult Au-MnO2@P-H1-H2-H3-H4, TMB and PBS solutions respectively, react for 5 min, and then measure using differential pulse voltammetry (DPV).
[0056] like Figure 7 CD results showed that the catalytic effect was complete when 12 μL of H1-H2 was added. Experimental Example 2:
[0057] 1. Validation experiments on the sensitivity of Ult Au-MnO2 nanomaterials to miRNA-21 based on CHA reaction and CHA-HCR cascade reaction. (1) Sensitivity verification experiment of Ult Au-MnO2 nanomaterials against miRNA-21 based on CHA reaction The experiment investigated the effect of the amount of miRNA-21 added (0-10 nM, the miRNA-21 content in bladder cancer patients and normal human bodies) on the catalysis of TMB during the synthesis of Ult Au-MnO2@P-H1-H2-H3-H4 nanomaterials.
[0058] Ult Au-MnO2@P-H1-H2 was placed into centrifuge tubes, followed by the addition of PBS (pH=7.4) buffer and 10 μL of TMB (10 mM) solution, for a total solution volume of 5 mL. After reacting for 5 min, differential pulse voltammetry (DPV) was used for measurement. Scan voltage: 0.1-0.5 V, pulse amplitude: 0.05 V, pulse width: 0.05 s.
[0059] like Figure 8The AB results showed three good linear relationships for miRNA-21 concentration in the range of 0-10 nM. Specifically, the detection limit was as low as 10.96 fM in the range of 0-0.01 nM, indicating that the Ult Au-MnO2 nanozyme electrochemical sensor based on CHA signal amplification can effectively cover the miRNA-21 levels in normal individuals and bladder cancer patients, and exhibits good sensitivity for miRNA-21.
[0060] 2. Sensitivity verification experiment of Ult Au-MnO2 nanomaterials against miRNA-21 based on CHA-HCR cascade reaction The experiment investigated the effect of the amount of miRNA-21 added (0-10 nM, the miRNA-21 content in bladder cancer patients and normal human bodies) on the catalysis of TMB during the synthesis of Ult Au-MnO2@P-H1-H2-H3-H4 nanomaterials.
[0061] Ult Au-MnO2@P-H1-H2-H3-H4 was placed into centrifuge tubes, followed by the addition of PBS (pH=7.4) buffer and 10 μL of TMB (10 mM) solution, for a total solution volume of 5 mL. After reacting for 5 min, differential pulse voltammetry (DPV) was used for measurement. Scan voltage: 0.1-0.5 V, pulse amplitude: 0.05 V, pulse width: 0.05 s.
[0062] like Figure 8 The CD results showed three good linear relationships for miRNA-21 concentration in the 0-10 nM range. Specifically, in the 0-0.01 nM range, the detection limit was as low as 0.194 fM, far lower than the detection limit for miRNA-21 based on the CHA reaction. This indicates that the Ult Au-MnO2 nanozyme electrochemical sensor based on CHA-HCR signal amplification can effectively cover the miRNA-21 levels in both normal individuals and bladder cancer patients, demonstrating higher detection sensitivity.
[0063] 3. Verification experiment on the selectivity and interference of Ult Au-MnO2 nanomaterials against miRNA-21 (1) Selectivity test of Ult Au-MnO2@P-H1-H2-H3-H4 nanomaterials for miRNA-21 To investigate the specificity of Ult Au-MnO2 nanomaterials for miRNA-21 based on the CHA-CHR cascade reaction system, this invention selected common miRNAs and small molecules in urine (miRNA-16, miRNA-155, miRNA-125b, uric acid, ascorbic acid, glucose, and urea) for investigation. The experimental procedure was as follows: during the CHA cycle reaction, miRNA-21, miRNA-16, miRNA-155, miRNA-125b, uric acid, ascorbic acid, glucose, and urea were added respectively, with other conditions the same as in 4-(2).
[0064] like Figure 9 Results A showed that the Ult Au-MnO2 nanomaterials, based on the CHA-CHR cascade signal amplification system, exhibited high selectivity for miRNA-21.
[0065] (2) Interference experiment of Ult Au-MnO2@P-H1-H2-H3-H4 nanomaterials on miRNA-21 To investigate the interference of other miRNAs and small molecules in the CHA-CHR cascade reaction system based on Ult Au-MnO2 nanomaterials on the detection of miRNA-21, this invention selected common miRNAs and small molecules in urine (miRNA-16, miRNA-155, miRNA-125b, uric acid, ascorbic acid, glucose, and urea) for investigation. The experimental procedure was as follows: during the CHA cycle reaction, after adding miRNA-21, equal amounts of miRNA-16, miRNA-155, miRNA-125b, uric acid, ascorbic acid, glucose, and urea were added again, with other conditions the same as in Experiment 1, 6-(2).
[0066] like Figure 9 Results B show that the Ult Au-MnO2 nanomaterials have relatively low interference with the detection of miRNA-21 from other miRNAs and small molecules in the CHA-CHR cascade signal amplification system.
[0067] 4. Validation experiment on the stability and reproducibility of miRNA-21 by Ult Au-MnO2 nanomaterials. (1) Investigation on the stability of miRNA-21 by Ult Au-MnO2@P-H1-H2-H3-H4 nanomaterials To investigate the stability of Ultra Au-MnO2 nanomaterials in miRNA-21 detection based on the CHA-CHR cascade reaction system, this invention verifies the stability by observing the catalytic effect of Ultra Au-MnO2@P-H1-H2-H3-H4 nanomaterials on TMB at different time points (0-15d) after synthesis. Other conditions are the same as in Experiment 1, 6-(2).
[0068] like Figure 9 The results showed that the Ult Au-MnO2 nanomaterials, based on the CHA-CHR cascade signal amplification system, exhibited good stability for the detection of miRNA-21.
[0069] (2) Reproducibility study of miRNA-21 using Ult Au-MnO2@P-H1-H2-H3-H4 nanomaterials To investigate the stability of the Ult Au-MnO2 nanomaterial electrode based on the CHA-CHR cascade reaction system for miRNA-21 detection, this invention studied reproducibility by using three Ult Au-MnO2@P-H1-H2-H3-H4 biosensors to detect the same miRNA-21 sample. Other conditions were the same as in Experiment 1, 6-(2).
[0070] like Figure 9 The results showed that the constructed Ultra Au-MnO2@P-H1-H2-H3-H4 biosensor exhibited excellent stability, with standard deviations of 0.78%, 0.9%, and 1.6% for three parallel experiments, indicating that the Ultra Au-MnO2@P-H1-H2-H3-H4 biosensor had good detection reproducibility.
[0071] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.
Claims
1. An ultrathin core-shell structured Au NPs-MnO2 nanomaterial, characterized in that: The ultrathin core-shell structure Au NPs-MnO2 uses Au NPs-PVP as the core and ultrathin manganese dioxide as the shell.
2. The ultrathin core-shell structured Au NPs-MnO2 nanomaterial as described in claim 1, characterized in that: The thickness of the manganese dioxide shell is 45-55 nm, and the overall particle size of the ultrathin core-shell structure Au NPs-MnO2 is 136.05 ±15.29 nm.
3. The ultrathin core-shell structured Au NPs-MnO2 nanomaterial as described in claim 1, characterized in that: The preparation method of Au NPs-PVP is as follows: Au NPs-PVP is synthesized by using chloroauric acid as raw material, sodium citrate as reducing agent, and polyvinylpyrrolidone as surface ligand.
4. The ultrathin core-shell structured Au NPs-MnO2 nanomaterial as described in claim 3, characterized in that: The specific preparation method of Au NPs-PVP is as follows: Take 5-20 mM HAuCl4·3H2O, add deionized water to a total volume of 100 mL and mix well. When the temperature is raised to 120-130 ℃, add 800-900 μL of sodium citrate aqueous solution with a concentration of 0.05-0.2 M, reflux for 30-45 min, and when the temperature drops to 70-80 ℃, add 5-15 mL of polyvinylpyrrolidone with a concentration of 0.01-0.05 g / mL, reflux for 20-40 min, and the reaction is completed to obtain Au NPs-PVP.
5. The method for preparing ultrathin core-shell structured Au NPs-MnO2 nanomaterials as described in any one of claims 1-4, characterized in that: Includes the following steps: Using Au NPs-PVP and potassium permanganate as raw materials, an ultrathin manganese dioxide shell was generated on the surface of gold nanospheres by hydrothermal method, thus obtaining the ultrathin core-shell structure Au NPs-MnO2.
6. The method for preparing ultrathin core-shell structured Au NPs-MnO2 nanomaterials as described in claim 5, characterized in that: Specifically: Au NPs-PVP was taken, centrifuged, and deionized water was added to the precipitate at a ratio of 1g:4-6mL to disperse it evenly. KMnO4 with a concentration of 5-15 mM was added at a ratio of 1mL:15-20μL for Au NPs-PVP to KMnO4, and the mixture was sonicated for 30-60 min. Then, it was refluxed at 88-92 ℃ for 30-40 min. KMnO4 with a concentration of 5-15 mM was added at a ratio of 1mL:5-10μL for Au NPs-PVP to KMnO4, and the mixture was refluxed at 78-82 ℃ for 6-8 h to obtain the ultrathin core-shell structure AuNPs-MnO2.
7. The application of the ultrathin core-shell structured Au NPs-MnO2 nanomaterials as described in any one of claims 1-4 in the preparation of an electrochemical biosensor for detecting miRNA, characterized in that: The ultrathin core-shell structured Au NPs-MnO2 nanomaterials serve as the substrate for oxidase-like catalysis, TMB, to generate electrochemical signals.
8. The application of the ultrathin core-shell structured Au NPs-MnO2 nanomaterial as described in claim 4 in the preparation of an electrochemical biosensor for detecting miRNA, characterized in that: The electrochemical biosensor includes: Ultrathin core-shell structured Au NPs-MnO2 nanomaterials; DNA capture probe P immobilized on the surface of the ultrathin core-shell structured Au NPs-MnO2 nanomaterial; And DNA hairpin structures for catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR).
9. The application as described in claim 8, characterized in that: The DNA capture probe P is a single-stranded DNA strand pre-fixed on the surface of the ultrathin core-shell Au NPs-MnO2 nanomaterial; the DNA hairpin structure includes H1, H2, H3 and H4, wherein H1 and H2 are used to form a catalytic hairpin self-assembly CHA cycle, and H3 and H4 are used to form a hybridization chain reaction HCR.
10. A reagent kit, characterized in that, The invention comprises the electrochemical biosensor of claim 7 for detecting the bladder cancer biomarker miRNA-21.