Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education & Key Disciplines Laboratory of Novel Micro-Nano Devices and System Technology, College of Optoelectronic Engineering, Chongqing University, Chongqing, 400044, China; International R & D Center of Micro-nano Systems and New Materials Technology, Chongqing University, Chongqing, 400044, China.
College of Chemistry and Chemical Engineering, Chongqing University of Science and Technology, Chongqing, 400044, China.
Anal Chim Acta. 2022 Aug 15;1221:340139. doi: 10.1016/j.aca.2022.340139. Epub 2022 Jul 1.
Developing sensitive and miniaturized biosensors for the detection of microRNAs (miRNAs) is highly desirable due to their association with early cancer diagnosis and prognosis. Here, a new microfluidic-based biosensor, combined with multifunctional nanosurface and DSN-assisted target recycle amplification strategy, is designed for the detection of miRNA-21. The design of nanosurface includes gold nanoparticles on porous anodic aluminum oxide (AAO) for surface enhanced Raman scattering (SERS) substrate, Au@Ag core-shell nanoparticles for SERS nanotags and single-stranded DNA (ssDNA) in between for miRNA capture and nanotags immobilization. When the target miRNA is present near the nanosurface, it will be captured by ssDNA via hybridization reaction. Then, triggered by the DSN-assisted target recycle process, the freshly formed DNA/miRNA heteroduplexes are cleaved by DSN enzyme into DNA fragments and single-strand miRNA. The SERS nanotags are also dissociated from the nanosurface, leading to decrease of SERS signal. The cleaved target miRNA can be captured and SERS nanotags are released again in the next cycle, resulting in amplification of detection signal. To improve the accuracy of this biosensor, the functionalized AAO membrane is subdivided into two groups - AAO/Au array linked with encoded core-shell SERS nanotags acting as a reactor and primary detector and AAO/Au@Ag array serving as a collector and secondary detector for the dissociative SERS nanotags from the reactor. The decrease of SERS signal in primary detector and increase of signal in secondary detector ensures the accuracy and it is called dual-SERS detection strategy. The detection of miRNA-21 can be achieved with only 30 μL sample and 10 μL enzyme and a wide linear range of 10 fM∼10 nM is obtained. In addition, the microfluidic dual-SERS detection strategy can greatly reduce the possibility of false positive or false negative in single detection mode and it can be applied to the simultaneous detection of multiple miRNAs via integrating different probes.
由于 microRNAs (miRNAs) 与癌症早期诊断和预后相关，因此开发用于检测 miRNA 的灵敏和微型化生物传感器是非常有必要的。在此，设计了一种新的基于微流控的生物传感器，结合多功能纳米表面和 DSN 辅助目标循环放大策略，用于检测 miRNA-21。纳米表面的设计包括多孔阳极氧化铝 (AAO) 上的金纳米颗粒用于表面增强拉曼散射 (SERS) 基底、Au@Ag 核壳纳米颗粒用于 SERS 纳米标签以及 ssDNA 用于 miRNA 捕获和纳米标签固定。当目标 miRNA 存在于纳米表面附近时，它将通过杂交反应被 ssDNA 捕获。然后，在 DSN 辅助目标循环过程的触发下，DSN 酶将新形成的 DNA/miRNA 异源双链体切割成 DNA 片段和单链 miRNA。SERS 纳米标签也从纳米表面解离，导致 SERS 信号减少。在接下来的循环中，被切割的目标 miRNA 可以被捕获，并且 SERS 纳米标签再次被释放，从而放大检测信号。为了提高该生物传感器的准确性，功能化的 AAO 膜被细分为两组——AAO/Au 阵列与编码的核壳 SERS 纳米标签相连，作为反应器和初级检测器，AAO/Au@Ag 阵列作为从反应器中分离的 SERS 纳米标签的收集器和次级检测器。初级检测器中 SERS 信号的减少和次级检测器中信号的增加确保了准确性，这被称为双 SERS 检测策略。仅需 30 μL 样品和 10 μL 酶即可实现 miRNA-21 的检测，并获得 10 fM∼10 nM 的宽线性范围。此外，微流控双 SERS 检测策略可以大大降低单检测模式中假阳性或假阴性的可能性，并且可以通过整合不同的探针用于多种 miRNAs 的同时检测。
