Exosomal microRNAs (exomiRNAs) have emerged as promising biomarkers for the early clinical diagnosis of osteoporosis. However, their limited abundance and short length in peripheral blood present significant challenges for the accurate detection of exomiRNAs. Herein, we have designed and implemented an efficacious fluorescence-based biosensor for the highly sensitive detection of exomiRNA associated with osteoporosis, leveraging the enhancing 3D DNA walker-induced CRISPR/Cas12a technology. The engineered DNA walker is capable of efficiently transforming target exomiRNA into amplifying DNA strands, thereby enhancing the sensitivity of the developed biosensor. Concurrently, the liberated DNA strands serve as activators to trigger Cas12a trans-cleavage activity, culminating in a significantly amplified fluorescent signal for the highly sensitive detection of exomiRNA-214. Under optimal conditions, the devised technology demonstrated the capacity to detect target exomiRNA-214 at concentrations as low as 20.42 fM, encompassing a wide linear range extending from 50.0 fM to 10.0 nM. Moreover, the fluorescence-based biosensor could accurately differentiate between healthy individuals and osteoporosis patients via the detection of exomiRNA-214, which was in agreement with RT-qPCR results. As such, this biosensing technology offers promise as a valuable tool for the early diagnosis of osteoporosis.

中文翻译：

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增强 3D DNA Walker 诱导的 CRISPR/Cas12a 技术，用于高灵敏度检测与骨质疏松症相关的 ExomicroRNA

外泌体 microRNA (exomiRNA) 已成为骨质疏松症早期临床诊断的有前景的生物标志物。然而，它们在外周血中的丰度有限且长度较短，这对 exomiRNA 的准确检测提出了重大挑战。在此，我们利用增强的 3D DNA walker 诱导的 CRISPR/Cas12a 技术，设计并实现了一种有效的基于荧光的生物传感器，用于高灵敏度检测与骨质疏松症相关的 exomiRNA。工程化的DNA walker能够有效地将目标exomiRNA转化为扩增的DNA链，从而提高所开发的生物传感器的灵敏度。同时，释放的 DNA 链作为激活剂触发 Cas12a反式切割活性，最终产生显着放大的荧光信号，用于高度灵敏地检测 exomiRNA-214。在最佳条件下，所设计的技术证明能够检测浓度低至 20.42 fM 的目标 exomiRNA-214，涵盖从 50.0 fM 到 10.0 nM 的宽线性范围。此外，基于荧光的生物传感器可以通过检测exomiRNA-214准确区分健康个体和骨质疏松症患者，这与RT-qPCR结果一致。因此，这种生物传感技术有望成为骨质疏松症早期诊断的宝贵工具。