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Viruses are infectious pathogens responsible for a great number of human diseases,such as influenza,diarrhea,and AIDS.On the other hand,viral nanoparticles (VNPs) are finding increasing applications in medicine,biotechnology,and material science by serving as vaccines,gene delivery vehicles,as well as building blocks.Therefore,rapid and sensitive enumeration of VNPs is of great importance in disease assessment and assay development.Traditional plaque assay is the current "gold standard" for the quantification of viruses.Besides time consuming and labor intensive,plaque assay can only be applied to infectious viruses and thus excludes non-infectious VNPs.Although real-time quantitative polymerase chain reaction (qPCR) can be used to estimate virus concentration via gene copy number measurement,it falls short in the quantification of empty capsids.On the basis of a laboratory-built high-sensitivity flow cytometer (HSFCM),we have developed an advanced method that is capable of rapidly detecting and enumerating VNPs.Employing fluorescent nanoparticles as the internal standard,VNP concentration can be measured via single particle enumeration.The concentration of VNPs can be determined in minutes at an analysis speed of 100-200 particles per second.Taking bacteriophages M13,T7 and Lambda as models,the concentrations obtained by the HSFCM agreed well with the results of plaque assay.The most distinct advantage is that this method allows us to analyze the concentration of non-infectious VNPs,such as empty capsids.This is especially beneficial for the assessment of the ratio between empty capsid and intact virus,which is vital to the quality control of vaccine preparation.Besides label-free enumeration based on light scattering detection of single viruses,virus from clinically relevant samples can be accurately detected and enumerated upon nucleic acid staining.This new approach for VNPs enumeration is simple and fast,and broad applications in nanomedicine development and bioscience can be envisioned.