Ultrasensitive on-column laser-induced fluorescence in capillary electrophoresis using multiparameter confocal detection

Abstract

We report a simple method to efficiently couple on-column, standard Capillary Electrophoresis with Confocal MultiParameter Fluorescence detection (CE-CMPF) using only commercially available components. A molecular collection of 13% and a concentration limit of detection of 1.5 pM fluorescein are achieved in our instrument by gating the arrival time of individual photons in order to reduce the scattering contribution. The proposed scheme allows for amplification-free detection and separation of three different microRNAs from the MCF-7 cell lysate. The limit of detection is approximately 500 times smaller and the separation time is 3 times shorter compared to protocols based on commercial CE instrumentation. Although the optical design can be further improved, it is shown that the current CE-CMPF prototype is already capable of analyzing the microRNA content of single cells. In addition, all CE protocols previously developed for commercial instruments can be performed with our CE-CMPF without modification but with nearly 3 orders of magnitude better limit of detection.