我们的初步研究已经确定了血清岩藻糖基化的细胞外囊泡(EV)miR-4732-5p在早期诊断肺腺癌(LUAD)中的潜力,通过岩藻糖捕获策略利用扁豆凝集素(LCA)-磁珠并随后筛选高通量测序和验证实时定量聚合酶链反应(RT-qPCR)。考虑到相对复杂的程序,昂贵的设备,和严格的实验室条件,我们构建了一个电化学生物传感器检测miR-4732-5p。miR-4732-5p在血清中非常低,下降到FM级别,因此,需要通过基于Mg2依赖性DNA酶分裂核酸锁(NAL)循环和杂交链反应(HCR)信号放大的高灵敏度电化学方法进行检测。在这项研究中,通过使用NAL循环与HCR组合的双重扩增反应来实现信号扩增。此外,杂交的DNA链与大量的亚甲基蓝(MB)分子结合以增强信号传导。基于上述战略,我们进一步加强我们的信号放大策略,以提高检测灵敏度和准确性。该测定的实施如下:最初,miR-4732-5p与NAL联合,然后Mg2依赖性DNA酶分裂NAL以释放辅助DNA(S1)链,其随后被固定在电极表面上的捕获探针DNA(C1)链捕获。在此之后,HCR产生了大量的DNA1(H1)和DNA2(H2),和S1链然后通过碱基互补配对与H1和H2链杂交。最后,MB通过π-π堆叠相互作用与H1和H2串联键合,导致在检测到能够通过电极引起MB的氧化还原变化的电势时产生信号电流。此外,我们评估了我们开发的电化学生物传感器测定的性能。结果表明,我们的检测是一种可靠的方法,具有高灵敏度(检测限为2.6×10-17M),优异的特异性,准确性好,再现性,和稳定性。此外,它具有成本效益,需要简单的操作,并且是便携式的,使其适用于血清岩藻糖基化细胞外囊泡miR-4732-5p的检测。最终,这一进展有可能提高早期LUAD患者的诊断效率.
Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π-π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10-17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.