Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips

Abstract

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).

Characterization of low viscosity polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips

“…3, we compared relative mobility of two protein samples BSA (66 kDa) and TI (20 kDa) and a small fluorescent dye (Alexa Fluor 647) in different concentrations of polymers indicated in the figure. We saw that when using MC as the separation media and when the concentration of MC was higher than its entanglement point (0.2%), 18 there was a decrease in mobility; however, until 1% the sieving effect was not yet considerable. We also showed that MC and MC-T in term of suppressing EOF and protein retardation were similar.…”

“…In this concentration of polymer solution (0.2%), polymers which we used in this time, PVA, PVP, HPMC, and MC could not entangle with each other. 18,[29][30][31][32] Below entanglement point of polymer solution, the interaction frequency between samples and polymer solutions is important to get a better resolution. In the separation of PVA, the higher EOF which is a counter flow to the electrophoretic mobility increased the separation time and hence, increased the interaction frequency and achieved a better separation of the peaks.…”

“…We also showed that MC and MC-T in term of suppressing EOF and protein retardation were similar. 18 For the other three polymers, we did not expect to have any sieving effect because their concentration was much lower than their TABLE IV. Migration time, and the ratio of FWHM of polymer solutions to PVA solution, theoretical plate number, and resolution in the electropherograms of TI conjugated with Alexa Fluor 647 in different dynamic coatings (Fig.…”

“…6,[14][15][16] Previously our group reported a covalent coating on PMMA microchannels by polyethyleneglycol (PEG) which successfully controlled protein adsorption on surfaces, 6 and dynamic coating by using linear polysaccharides such as cellulose derivatives which have a self-coating ability has been applied to prevent sample adsorption on the surface of PMMA microchips. [17][18][19] As well, we systematically characterized spontaneous adsorption of surfactants and cellulose on PMMA surface using atomic force microscopy (AFM) and infrared external-reflection (IR-ER) spectroscopy. 20 Despite substantial research effort, 21 the understanding of nonspecific adsorption of proteins onto microchannel surfaces remains incomplete.…”

Evaluation of Different Nonspecific Binding Blocking Agents Deposited Inside Poly(methyl methacrylate) Microfluidic Flow-Cells

“…A hybrid dynamic coating of n-dodecyl b-D-altoside (DDM) and methyl cellulose (MC) method has been developed by Dang et al [10] to suppress analytes adsorption inside a PMMA microchannel. Similarly, Mohamadi et al [11] reported a dynamic coating of methylcellulose (MC) and a nonionic detergent (polysorbate 20) onto PMMA channel also for suppressing the protein adsorption. On the other hand, permanent modification of PMMA surface by surface chemistry, mostly with PEG derivatives, has also been used to reduce the non-specific adsorption of analytes into the PMMA surfaces.…”