| 摘要: |
| 目的 构建一种自催化发夹组装无酶信号放大的靶标快速荧光法，用于检测靶标miRNA。方法 首先在金纳米颗粒（AuNP）表面修饰5-羧基荧光素（FAM）标记的DNA发夹探针H1，形成探针AuNP-H1，H1的荧光被AuNP猝灭。加入靶标miRNA会导致AuNP上的H1标记荧光素远离AuNP而重新发射荧光，随后H1与DNA 发夹探针H2发生循环自组装，靶标循环被利用，导致荧光信号放大。将探针AuNP-H1、探针 H2及不同浓度的miRNA-721共反应后，在480 nm激发波长下测定体系的荧光强度。结果 以急性心肌炎生物标志物miRNA-721为模型靶标，在优化的实验条件下使用20 μL探针AuNP-H1、50 μL 3 μmol/L探针 H2（退火缓冲液为20 mmol/L Tris-HCl、100 mmol/L NaCl、5 mmol/L MgCl2，pH 7.4）与50 μL不同浓度（0.1～5 μmol/L）的miRNA-721在30 ℃共反应20 min，发现在520 nm处的相对荧光强度变化值（∆F＝F－F0）与miRNA-721浓度（C）呈良好的线性关系，拟合线性方程为∆F＝29 232×lgC－52 435（R2＝0.991 0）。该荧光法检测限为1.23 nmol/L。在正常人血清中的加标回收率为92.71%～104.02%。一次完整的miRNA分析可以在20 min内完成。结论 该方法可用于生物样品中miRNA-721的检测，为急性心肌炎的早期诊断提供了一种快速检测手段。 |
| 关键词: 荧光 无酶信号放大 催化发夹组装 微RNA-721 金纳米颗粒 |
| DOI：10.16781/j.CN31-2187/R.20230751 |
| 投稿时间：2023-12-19修订日期：2024-02-20 |
| 基金项目:国家自然科学基金面上项目（82273894）. |
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| Rapid fluorescence detection of microRNA-721 through enzyme-free signal amplification by surface-catalyzed hairpin assembly of gold nanoparticle |
| LIU Ying1,XI Xiaoyu2,FENG Jiadong2,LU Feng1,2* |
(1. School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China;
2. Department of Pharmaceutical Analysis, School of Pharmacy, Naval Medical University (Second Military Medical University), Shanghai 200433, China
*Corresponding author) |
| Abstract: |
| Objective To develop a rapid fluorescence assay testing method for microRNA-721 (miRNA-721) detection using catalytic hairpin assembly for signal amplification without the need of enzymes. Methods A DNA hairpin probe H1 labeled with 5-carboxyfluorescein (FAM) was modified on the surface of gold nanoparticle (AuNP) to form probe AuNP-H1, and the fluorescence of H1 was quenched by AuNP. When the target miRNA was introduced, it caused the fluorescein modified in H1 on the AuNP to move away from the AuNP and emit fluorescence. This was followed by cyclic self-assembly between H1 and H2, and the target cycle was used to amplify the fluorescence signal. The fluorescence intensity of the system was measured at 480 nm excitation wavelength after co-reacting with the probe AuNP-H1, probe H2 and different concentrations of miRNA-721. Results When biomarker of acute myocarditis miRNA-721 was used as a model target, 20 μL of probe AuNP-H1 and 50 μL of 3 μmol/L probe H2 (annealing buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 5 mmol/L MgCl2, pH 7.4) were co-reacted with 50 μL of miRNA-721 at various concentrations ranging from 0.1 to 5 μmol/L at 30℃ for 20 min. The change in relative fluorescence intensity at 520 nm (∆F＝F－F0) had a good linear relationship with the concentration of miRNA-721 (C), and the fitted linear equation was ∆F＝29 232×lgC－52 435 (R2＝0.991 0). The detection limit of this fluorescence method was 1.23 nmol/L. The recovery rate in normal human serum ranged from 92.71% to 104.02%. A comprehensive miRNA analysis could be completed in just 20 min. Conclusion The present method can be applied to detect miRNA-721 in biological samples, which providing a rapid diagnostic tool for acute myocarditis. |
| Key words: fluorescence enzyme-free amplification catalytic hairpin assembly microRNA-721 gold nanoparticle |
