Automated analysis of individual particles using a commercial capillary electrophoresis system.
ABSTRACT Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.
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ABSTRACT: Miniaturized, portable instrumentation has been gaining popularity in all areas of analytical chemistry. Capillary electrophoresis (CE), due to its main strengths of high separation efficiency, relatively short analysis time and low consumption of chemicals, is a particularly suitable technique for use in portable analytical instrumentation. In line with the general trend in miniaturization in chemistry utilizing microfluidic chips, the main thrust of portable CE (P–CE) systems development is towards chip-based miniaturized CE. Despite this, capillary-based (non-chip) P–CE systems have certain unmatched advantages, especially in the relative simplicity of the regular cylindrical geometry of the CE capillary, maximal volume-to-surface ratio, no need to design and to fabricate a chip, the low costs of capillary compared to chip, and better performance with some detection techniques. This review presents an overview of the state of the art of P–CE and literature relevant to future developments. We pay particular attention to the development and the potential of miniaturization of functional parts for P–CE. These include comp-onents related to sample introduction, separation and detection, which are the key elements in P–CE design. The future of P–CE may be in relatively simple, rugged designs (e.g., using a short piece of capillary fixed to a chip-sized platform on which injection and detection parts can be mounted). Electrochemical detection is well suited for miniaturization, so is probably the most suitable detection technique for P–CE, but optical detection is gaining interest, especially due to miniaturized light sources (e.g., light-emitting diodes).TrAC Trends in Analytical Chemistry 04/2010; 29(4):339-353. DOI:10.1016/j.trac.2009.12.010 · 6.61 Impact Factor
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ABSTRACT: This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 ± 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile–quantile (q–q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial events became too large (>72 for rat liver and >98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q–q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (<55 for rat liver and <81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.Electrophoresis 04/2008; 29(7):1431 - 1440. DOI:10.1002/elps.200700470 · 3.16 Impact Factor