基于现场可编程门阵列的雪崩光电二极管触发式光谱纳米流式检测系统的开发与应用OA

Spectral nano-flow cytometry enables multiparameter characterization of nanoscale biological particles,such as viruses and extracellular vesicles,at single-particle level.However,its performance is limited by the conventional photomultiplier tube(PMT)-triggered electron-multiplying charge-coupled device(EMCCD)exposure strategy,which suffers from a high lower-size detection limit and significant background noise.To address these limitations,a high-sensitivity spectral nano-flow cytometry system based on a field-programmable gate array(FPGA)-controlled single photon counting avalanche photodiode(APD)triggering mechanism was developed in this work.Key innovations included replacing the PMT with a high-quantum-efficiency APD combined with optical spatial filtering to substantially suppress background noise,implementing an FPGA-based high-speed signal processing chain that achieved precise identification and synchronous triggering of APD pulse signals within a 0.2-30 MHz frequency range using equal-precision measurement and threshold comparison algorithms,and optimizing the spectral detection module to enhance fluorescence collection efficiency and spectral resolution.Experimental results demonstrated that the system clearly distinguished a mixture of fluorescent silica beads with molecules of equivalent soluble fluorochrome(MESF)values of 574 and 1438.When detecting Di-8-ANEPPS-labeled red blood cell-derived extracellular vesicles(RBC EVs),the false-trigger rate was reduced from>70%(with PMT triggering)to<10%,with clear baseline separation between target peaks and noise.Furthermore,in experiments involving dual-labeled RBC EVs(Alexa Fluor 488(AF488)/XFD488 Hydroxylamine(XDF488)and Di-8-ANEPPS),the system successfully achieved decoupling of overlapping fluorescence spectra at single-particle level and accurately quantified the fluorescence contribution of each dye.This work provided key technological support for high-throughput,high-precision spectral analysis of weakly fluorescent nanoscale biological particles.