Analytical isotachophoresis of lactate in human serum using dry film photoresist microfluidic chips compatible with a commercially available field-deployable instrument platform.

A dry film resist (DFR) chip compatible with the Agilent Bioanalyzer 2100 was designed and fabricated for use in the analysis of lactate in serum by chip isotachophoresis (ITP). The Agilent Bioanalyzer 2100 is a commercially available field deployable analytical instrument originally developed for the electrophoretic analysis of DNA, RNA and proteins. The DFR chip was designed for the ITP separation of lactate in human serum within 1 min and was made compatible with the Bioanalyzer after packaging in the plastic caddies normally used for the DNA chips. A 20-fold improvement in sensitivity was obtained for the DFR chips in comparison with the standard chips used in earlier work. The limit of detection and limit of quantification for lactate were 24 µM and 80 µM, respectively. This new approach enables the use of commercial platforms like the Agilent Bioanalyzer for new applications including the analysis of small molecules.

Potential of capillary electrophoresis (CE) and chip-CE with dual detection (capacitively-coupled contactless conductivity detection (C4D) and fluorescence detection) for monitoring of nicotine and cotinine derivatization.

Capillary electrophoresis (CE) coupled with capacitively-coupled contactless conductivity (C(4)D) and fluorescence (FD) detectors and chip-CE for monitoring of nicotine and cotinine derivatization was demonstrated. Separation of the substrates and intermediates could be achieved by CE-C(4)D in 7 min (R(s) > 2.7) using 45 mM acetic acid (pH 3.0) and this system was applied to detect the intermediate formation. Final fluorescent products could be analyzed by micellar electrokinetic chromatography (MEKC-FD) in 5 mM borate buffer (pH 9.0) containing 10 mM sodium dodecylsulfate (SDS) (%RSD < 3.00%). Transferring of MEKC-FD to chip-CE allowed for shorter analysis time (2.5 min) and decreased sample consumption. The chip-CE-FD shows good detection and quantitation limits (< 7.5 µM) and precision (%RSD < 2.81%) and was employed to determine nicotine and cotinine in artificial urine. This work reveals the potential of CE and chip-CE with dual detectors as simultaneous, convenient and rapid methods for monitoring pyridine derivatization.

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.

Separation of carboxylic acids in human serum by isotachophoresis using a commercial field-deployable analytical platform combined with in-house glass microfluidic chips.

Portable and field deployable analytical instruments are attractive in many fields including medical diagnostics, where point of care and on-site diagnostics systems capable of providing rapid quantitative results have the potential to vastly improve the productivity and the quality of medical care. Isotachophoresis (ITP) is a well known electrophoretic separation technique previously demonstrated as suitable for miniaturization in microfluidic chip format (chip-ITP). In this work, a purpose-designed ITP chip compatible with a commercial end-used targeted microfluidic system was used to study different injection protocols and to evaluate the effect of the length of the separation channel on the analytical performance. The in-house ITP chips were made from Corning glass and compared to the commercial DNA chip for the ITP separation of anions from the hydrodynamic injection of human serum. Using the in-house ITP chip the isotachophoretic step of lactate from human serum was approximately two times longer. The results of this research suggested that microfluidic ITP with indirect fluorescence detection is a viable technique for separation of organic acids in human serum samples, especially when a chip with suitable design is used.

Isotachophoresis on a chip with indirect fluorescence detection as a field deployable system for analysis of carboxylic acids.

ITP with indirect fluorescence detection (IFD) was introduced three decades ago. Despite this fact, the method has never become widely adopted. The main aim of this work was to utilize the ITP-IFD for the separation of carboxylic acids by using a commercially available, portable, microfluidic chip electrophoresis system. On the 16.8-mm effective length separation channel, a maximum of eight carboxylic acids could be separated, with LOD values in a range from 0.12 to 0.4 mM. The commercial chips used for all experiments have multichannel structures important for analysis of more than one sample per a chip in case of standard use. This multichannel structure was used to investigate the possibility of multiple sample loading for ITP separation. Application of ITP-IFD was investigated for analysis of benzoate in diet soft drinks and the results were in good agreement with results of a CE method.

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 (R(s) > 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.

Chip-based CE for rapid separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans.

Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid - lectin affinity enrichment. After fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470 nm) LED. The separation was completed in 40 s in a microfluidic channel of 14 mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20-mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip-based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35 cm separation length and microfluidic chips with a separation distance of only 14 mm.

Prediction and understanding system peaks in capillary zone electrophoresis.

Introduction of a sample into the separation column (microchip channel) in capillary zone electrophoresis (microchip electrophoresis) will cause a disturbance in the originally uniform composition of the background electrolyte. The disturbance, a system zone, can move in some electrolyte systems along the separation channel and, on reaching the position of the detector, cause a system peak. As shown by the linear theory of electromigration based on linearized continuity equations formulated in matrix form, the mobility of the system zone--the system eigenmobility--can be obtained as the eigenvalue of the matrix. Progress in the theory of electromigration allows us to predict the existence and mobilities of the system zones, even in very complex electrolyte systems consisting of several multivalent weak electrolytes, or in micellar systems (systems with SDS micelles) used for protein sizing in microchips. The theory is implemented in PeakMaster software, which is available as freeware (www.natur.cuni.cz/gas). The linearized theory also predicts background electrolytes having no stationary injection zone (water zone, water gap, water dip, EO zone) or unstable electrolyte systems exhibiting oscillations and creating periodic structures. The oscillating systems have complex system eigenmobilities (eigenvalues of the matrix are complex). This paper reviews the theoretical background of the system peaks (system eigenpeaks) and gives practical hints for their prediction and for preparing background electrolytes not perturbed by the occurrence of system peaks and by excessive peak broadening.