Nantana Nuchtavorn

Recent applications of microchip electrophoresis to biomedical analysis

Many separation methods have been developed for biomedical analysis, including chromatographic (e.g. high performance liquid chromatography (HPLC) and gas chromatography (GC)) and electrophoretic methods (e.g. gel electrophoresis and capillary electrophoresis (CE)). Among these techniques, CE provides advantages in terms of high separation efficiency, simplicity, low sample and solvent volume consumption, short analysis time and applicability to a wide range of biomedically important substances. Microchip electrophoresis (ME) is a miniaturized platform of CE and is now considered as a simpler and more convenient alternative, which has demonstrated potential in analytical chemistry. High-throughput, cost-effective and portable analysis systems can be developed using ME. The current review describes different separation modes and detectors that have been employed in ME to analyze various classes of biomedical analytes (e.g. pharmaceuticals and related substances, nucleic acids, amino acids, peptides, proteins, antibodies and antigens, carbohydrates, cells, cell components and lysates). Recent applications (during 2010-2014) in these areas are presented in tables and some significant findings are highlighted.

Exploring chip-capillary electrophoresis-laser-induced fluorescence field-deployable platform flexibility: Separations of fluorescent dyes by chip-based non-aqueous capillary electrophoresis

Microfluidic chip electrophoresis (chip-CE) is a separation method that is compatible with portable and on-site analysis, however, only few commercial chip-CE systems with laser-induced fluorescence (LIF) and light emitting diode (LED) fluorescence detection are available. They are established for several application tailored methods limited to specific biopolymers (DNA, RNA and proteins), and correspondingly the range of their applications has been limited. In this work we address the lack of commercially available research-type flexible chip-CE platforms by exploring the limits of using an application-tailored system equipped with chips and methods designed for DNA separations as a generic chip-CE platform - this is a very significant issue that has not been widely studied. In the investigated Agilent Bioanalyzer chip-CE system, the fixed components are the Agilent chips and the detection (LIF at 635 nm and LEDIF at 470 nm), while the chemistry (electrolyte) and the programming of all the high voltages are flexible. Using standard DNA chips, we show that a generic CE function of the system is easily possible and we demonstrate an extension of the applicability to non-aqueous CE (NACE). We studied the chip compatibility with organic solvents (i.e. MeOH, ACN, DMF and DMSO) and demonstrated the chip compatibility with DMSO as a non-volatile and non-hazardous solvent with satisfactory stability of migration times over 50h. The generic CE capability is illustrated with separations of fluorescent basic blue dyes methylene blue (MB), toluidine blue (TB), nile blue (NB) and brilliant cresyl blue (BC). Further, the effects of the composition of the background electrolyte (BGE) on the separation were studied, including the contents of water (0-30%) and buffer composition. In background electrolytes containing typically 80 mmol/L ammonium acetate and 870 mmol/L acetic acid in 100% DMSO baseline separation of the dyes were achieved in 40s. Linearity was documented in the range of 5-28 μmol/L, 10-100 μmol/L, 1.56-50 nmol/L and 5-75 nmol/L (r(2) values in the range 0.974-0.999), and limit of detection (LOD) values were 90 nmol/L, 1 μmol/L 1.4 nmol/L, and 2 nmol/L for MB, TB, NB and BC, respectively.

Rapid separations of nile blue stained microorganisms as cationic charged species by chip-CE with LIF

Rapid detection of microorganisms by alternative methods is desirable. Electromigration separation methods have the capability to separate microorganisms according to their charge and size and laser-induced fluorescence (LIF) detection have single‐cell detection capability. In this work, a new combined separation and detection scheme was introduced using chip‐based capillary electrophoresis (chip‐CE) platform with LIF detection. Three microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans were selected as representatives of Gram‐positive bacteria, Gram‐negative bacteria, and fungi. While their cells carry an overall negative charge in neutral to alkaline pH, staining them with nile blue (NB) provided highly sensitive LIF detection with excitation and emission wavelengths at 635 nm and 685 nm, respectively, and at the same time, the overall charge was converted to positive. Electrolyte pH and concentration of polyethylene oxide (PEO) significantly affected the resolution of the microorganisms. Their optimal separation in the 14 mm separation channel was achieved in less than 30 s (Rs > 5.3) in an electrolyte consisting of 3.94 mM Tris, 0.56 mM boric acid, 0.013 mM ethylenediaminetetraacetic acid disodium salt dihydrate (pH 10.5), and 0.025% PEO, with injection/separation voltages of +1000/+1000 V. The separation mechanism is likely employing contributions to the overall cationic charge from both the prevalently anionic membrane proteins and the cationic NB. Importantly, the resulting cationic NB‐stained cells exhibited excellent separation selectivity and efficiency of ∼38000 theoretical plates for rapid separations within 30–40 s. The results indicate the potential of chip‐CE for microbial analysis, which offers separations of a wide range of species with high efficiency, sensitivity, and throughput.

Simultaneous analysis of biologically active pyridines in pharmaceutical formulations by capillary zone electrophoresis.

A capillary zone electrophoretic method using UV detection is developed for the analysis of four biological active pyridines [i.e., nicotine (NIC), cotinine (COT), nicotinic acid (NA), and nicotinamide (NM)]. The separation of the pyridines is achieved in 25 mM sodium dihydrogen phosphate (pH 2.1) using a fused-silica capillary with an effective length of 56 cm and an inner diameter of 50 µm (extended light path), hydrodynamic injection at 50 mbar for 10 s, a temperature of 25°C, applied voltage of 30 kV, and UV detection at 260 nm. These conditions provide baseline separation of all the analytes [resolution (R(s)) > 3.6] in 9.4 min with good linearity (r(2) > 0.998, in ranges of 50-600 µg/mL for NIC, 8-160 µg/mL for NM, and 10-200 µg/mL for COT and NA), precision (relative standard deviation <2.04%), recovery (96.4-101.6%), limits of detection (<3.0 µg/mL), and quantitation (<10 µg/mL). The method is robust upon the alterations of pH of BGE, separating voltage, and injection time [the RSDs of the relative migration time (migration time of the analyte/migration time of the internal standard) and resolution <3.26%]. The method is efficient, reliable, and simple for the routine analysis of NIC, NA, and NM in various products such as gum and tablets and can be applied to determine COT in thermal degradation of NIC gum.