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.

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

“…The LOD of FITC for this integrated system was 0.5 pM. The pM level LODs could be comparable to those achieved by optimized confocal LIF [13,20] and sheath flow cuvette [11,30] detection systems. The detectable amount Q in of the FITC standard solution was estimated to be 0.8 zmol with a sample injection volume Q v of 1.6 nL under gravity injection.…”

“…It exhibits low background signal by eliminating scattering and autofluorescence originating from the capillary walls . Nevertheless, the detector involving very delicate hydrodynamics, multiplexed sheath flow systems, inherent difficulties with sample collection, capillary exchange, and alignment procedures may be less robust than on‐column mode . However, although it allows for uncomplicated alignment and building, the conventional on‐column detection also comes with an increase of the background signal due to the scattered excitation light, reflection, and refraction light from the capillary wall .…”

Capillary Electrophoresis

“…Mazouchi et al used multiparameter confocal fluorescence detection to reduce the LOD by a factor of 500 when compared to a commercial CE instrument. 87 The arrival times of individual photons are gated to reduce the loss of photons due to scattering. A LOD of 1.5 pM was reported for fluorescein.…”

A Two-Dimensional Affinity Capture and Separation Mini-Platform for the Isolation, Enrichment, and Quantification of Biomarkers and Its Potential Use for Liquid Biopsy

“…Since the introduction of IACE in the early 1990s by our laboratory, Terry Phillips’ laboratory, and others, the technology has increased significantly in popularity, and many applications have been reported using both conventional CE and microchip CE [ 25 , 54 , 55 , 58 , 61 , 62 , 63 , 64 , 66 , 75 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 ]. Furthermore, if IACE is coupled to powerful detectors such as laser-induced fluorescence detectors or mass spectrometers, a significant improvement in limits of detection can been achieved [ 25 , 75 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 ].…”