基于上转换发光共振能量转移的CRISPR/Cas12a生物传感系统用于HPV16 DNA双信号检测

传统的纯核结构的上转换纳米材料用于生物传感时, 存在表面猝灭效应或者因发光共振能量转移(LRET)效率不高导致灵敏度低等缺点, 对目标物的灵敏检测有一定的局限性. 本文通过多步高温共沉淀, 介导壳层外延成长法, 合成了NaYF₄∶Yb³⁺,Er³⁺(C-UCNPs)核层发光和NaYF₄@NaYF₄∶Yb³⁺,Er³⁺@NaYF₄(CSS-UCNPs)内壳层发光能量限域型上转换纳米材料, 并表征了材料的晶型、 形貌、 表面配体、 元素组成和发光共振能量转移效率. 结果表明, 该材料具有表面猝灭效应低、 发光共振转移效率较高的优势, 随后将其与成簇规律间隔的短回文重复序列及其相关蛋白(CRISPR/Cas)12a-纳米金系统结合, 实现了人乳头瘤病毒DNA(HPV16 DNA)的比色定性和上转换发光定量分析, 检出限为69.8 pmol/L, 双信号检测有效提高了检测结果的准确性. 此外, 本方法不仅特异性强, 还能识别单碱基错配的HPV16 DNA, 提高了DNA片段的容错率.

Dual Signal Detection of HPV16 DNA by CRISPR/Cas12a Biosensing System Based on Upconversion Luminescent Resonance Energy Transfer

Traditional upconversion nanomaterials with pure core structure have some disadvantages such as low sensitivity due to surface quenching effect or low efficiency of luminescence resonance energy transfer（LRET）， the sensitivity of target detection has some limitations. Herein， NaYF₄∶Yb³⁺，Er³⁺（C-UCNPs） core luminescence and NaYF₄@NaYF₄∶Yb³⁺，Er³⁺@NaYF₄（CSS-UCNPs） inner shell layer luminescence energy-limited upconversion nanomaterials were synthesized by a multi-step high-temperature co-precipitation， mediated by a shell layer epitaxial growth method. Meanwhile， the crystalline shape， morphology， surface ligands， elemental composition， and luminescence resonance energy transfer efficiency of the as-prepared materials were characterized and the results demonstrate that the materials processes the advantages of low surface quenching effect and high luminescence resonance transfer efficiency. Subsequently， the nanomaterials were combined with clustered regularly interspaced short palindromic repeats（CRISPR）-CRISPR-associated protein(CRISPR/Cas) 12a-gold nanoparticle system to achieve colorimetric qualitative and upconversion luminescence quantitative analysis of human papillomavirus DNA （HPV16 DNA） with a detection limit of 69.8 pmol/L， and the dual-signal assay effectively improved the accuracy of the detection results. In addition， the method is not only specific， but also recognizes single-base mismatched HPV16 DNA， which greatly improves the fault tolerance of DNA fragments