10 000‐fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3‐D numerical simulation with experimental results

Abstract

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10 000. The starting concentration of cTnI was 2.3 μg mL–1 and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL–1. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.

10 000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis

“…47 Briefly, a PMMA microchannel was formed using a hot embossing technique and a surface-modified assisted bonding method. First, a positive pattern of the desired channel structure was formed on a glass substrate using negative photolithography.…”

Recent progress in analytical capillary isotachophoresis

“…The authors proposed the elastic matching method applied to the conductivity profiles as suitable to determine the quasi steady state. Bottenus et al performed a 3D‐numerical simulation of ITP in a microfluidic volume‐coupling PMMA chip device containing two 10 times reductions of cross‐sectional area along the separation path. The simulation results qualitatively agreed with experiments using green fluorescent protein and R‐phycoerythrin which were concentrated by over a factor of 10 000 (leading electrolyte (LE): 20 mM HCl, ethanolamine, 1% w/v PVP, pH 9.5; terminating electrolyte (TE): 60 mM 6‐amino‐n‐hexanoic acid, ethanolamine, 1% w/v PVP, pH 10.0; detection by a fluorescence microscope equipped with a CCD camera).…”

Preconcentration of diluted mixed-species samples following separation and collection in a micro–nanofluidic device

“…Many methods have been proposed for sample preconcentration, including field-amplified sample stacking (FASS), 8 isotachophoresis, [9][10][11][12] electrokinetic trapping, [13][14][15] isoelectric focusing, 16 micellar electrokinetic sweeping, 17 chromatographic preconcentration, 18,19 and membrane a) Author to whom correspondence should be addressed. Electronic mail: rjyang@mail.ncku.edu.tw.…”