Absolute quantification of particle number concentration using a digital single particle counting system

The accurate determination of the molar concentration or the number concentration of particles in a defined volume is important but challenging. Since particle diversity and heterogeneity cannot be ignored in particle quantification, single particle counting has become quiteimportant. However, most methods require standard samples (calibrators) which are usually difficult to obtain. The authors describe a method for single particle counting that is based on the combination of digital counting and formation of microdroplets in a microchip. By compartmentalizing particles into picoliter droplets, positive droplets encapsulating particles were counted and particle concentrations were calculated by Poisson statistics. The concentration of particles over a wide range (from 5.0x10(3) to 1.8x10(7) particles permL) were accurately determined without the need for using a calibrator. A microdroplet chip including a T-junction channel achieved a 9-fold increase of signal-to-background ratio compared to the traditional flow-focusing chip. This makes the digital counting system a widely applicable tool for quantification of fluorescent particles. Various particles including differently sized fluorescent microspheres and bacteria with large heterogeneity in shape such as Escherichia coli DH5 alpha-pDsRed were accurately quantified by this method.