Enzyme-assisted upconversion fluorescence-encoded biosensing system for simultaneous detection of multiple sites EGFR mutation.

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.