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

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.

Detection of a thousand copies of miRNA without enrichment or modification.

We report direct quantitative analysis of multiple miRNAs with a detection limit of 1000 copies without miRNA enrichment or modification. A 300-fold improvement over the previously published detection limit was achieved by combining capillary electrophoresis with confocal time-resolved fluorescence detection through an embedded capillary interface. The method was used to determine levels of three miRNA biomarkers of breast cancer (miRNA 21, 125b, 145) in a human breast cancer cell line (MCF-7). A 30 pL volume of the cell lysate with approximately a material content of a single cell was sampled for the analysis. MiRNA 21, which is up-regulated in breast cancer, was detected at a level of approximately 12 thousand copies per cells. MiRNAs 125b and 145, which are down-regulated in breast cancer, were below the 1000-copy detection limit. This sensitive method may facilitate the analysis of miRNA in fine-needle-biopsy samples and even in single cells without enrichment or modification of miRNA. Advantageously, the instrumental setup developed here can be reproduced by others as it requires no sophisticated custom-made parts.

Predictive measure of quality of micromixing.

We introduce a predictive measure of micromixing termed quantitative overlap (QO). QO depends on the distribution of reactants throughout the reactor and can be calculated by solving equations of diffusion. We used a bimolecular reaction and a capillary microreactor to experimentally prove that QO is proportional to the product yield.