Gao Haiyang, Si Gao, Wang Zhendong, Liu Yanju, Yang Huaixia, Miao Mingsan, Ma Lele
Pharmacy College, Henan University of Chinese Medicine, Zhengzhou, 450046, PR China.
Department of Orthopedic, Peking University Third Hospital, Beijing, 100191, China.
Anal Bioanal Chem. 2025 Jan;417(2):237-250. doi: 10.1007/s00216-024-05660-8. Epub 2024 Nov 23.
Epidermal growth factor receptor (EGFR) mutations play a key role in the development of a variety of cancers. Rapid detection and screening of EGFR mutation types in patients are of great significance for early treatment of patients. In this study, a highly sensitive fluorescent biosensing system based on lanthanide ion-doped multi-type upconversion nanoparticles (UCNPs) combined with polymerization reaction signal amplification was designed and constructed for the simultaneous detection of L858R and 19Del mutations. Two upconversion nanoparticles (NaYF:Yb, Er and NaYF:Yb, Tm) with unique upconversion fluorescence profiles were first prepared using Er and Tm as activators, respectively. Subsequently, the UCNPs were enriched by cDNA complementary hybridization and atom transfer radical polymerization (ATRP) reactions to enhance the signal. Next, the tDNA/cDNA hybrids were cleaved using specific restriction endonucleases to detach UCNPs aggregates from the surface of the magnetic beads. Finally, the fluorescence signal in the supernatant was detected after magnetic separation. The simultaneous quantitative detection of the two EGFR mutations was achieved by analyzing the changes in signal intensity of the characteristic upconversion fluorescence spectra of the two encoded UCNPs at their respective emission peaks. The detection range of the method was from 10 fM to 10 nM, and the detection limits were 2.44 fM for L858R and 2.13 fM for 19Del. The sensing system was able to effectively differentiate between wild-type and other mutation types, and its detection results were consistent with qPCR. The excellent performance of the sensor suggests its promising application in the diagnosis and precision treatment of NSCLC.
表皮生长因子受体（EGFR）突变在多种癌症的发生发展中起关键作用。快速检测和筛查患者的EGFR突变类型对患者的早期治疗具有重要意义。在本研究中，设计并构建了一种基于镧系离子掺杂多型上转换纳米颗粒（UCNPs）结合聚合反应信号放大的高灵敏度荧光生物传感系统，用于同时检测L858R和19Del突变。首先分别以铒（Er）和铥（Tm）作为激活剂制备了两种具有独特上转换荧光光谱的上转换纳米颗粒（NaYF:Yb, Er和NaYF:Yb, Tm）。随后，通过cDNA互补杂交和原子转移自由基聚合（ATRP）反应富集UCNPs以增强信号。接着，使用特异性限制性内切酶切割tDNA/cDNA杂交体，使UCNPs聚集体从磁珠表面脱离。最后，磁分离后检测上清液中的荧光信号。通过分析两种编码UCNPs在其各自发射峰处的特征上转换荧光光谱信号强度的变化，实现了对两种EGFR突变的同时定量检测。该方法的检测范围为10 fM至10 nM，L858R的检测限为2.44 fM，19Del的检测限为2.13 fM。该传感系统能够有效区分野生型和其他突变类型，其检测结果与qPCR一致。该传感器的优异性能表明其在非小细胞肺癌（NSCLC）的诊断和精准治疗中具有广阔的应用前景。
