A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins

Abstract

A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro-column, pH gradient generation, and fraction collection in a single device. Using a sieve micro-valve, anion exchange particles were packed into a microchannel in order to realize a solid-phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro-column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and R-Phycoerythrin (R-PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development.

Keywords: Microfluidics; Protein separation; pH gradient chromatofocusing.

Figure 1

(A) Principle of pH gradient chromatofocusing. (B) Conventional experimental setup for pH gradient chromatofocusing. (C) A microfluidic method to separate proteins based on pH gradient chromatofocusing.

Figure 2

Design of the microfluidic device. The device comprised two layers, one layer for flow channels and another layer for control channels. The overlay CleWin (software used to design photolithographic masks; CleWin 5, Phoenix Software, The Netherlands) design shows the connection of the channels, inlets/outlets, and control ports.

Figure 3

Creation of a microcolumn in a microchannel. (A) Particle packing by sieving particles from particle suspension, and (B) Elution through the microcolumn. 500 μm scale bars are shown.

Figure 4

Parallel mixing reactors for pH gradient generation. (A) Design of the 16 mixing reactors. (B) Process flow in a reactor. (C) Microscope images of loading and mixing blue dye solution and Milli‐Q water. (D) The gradient of concentration of fluorescein (FITC) obtained by Milli‐Q water and loading 100 μM of FITC solution into buffer #1 sites and buffer #2 sites, respectively. The concentration of FITC in each reactor was calculated by volume ratios of the two loading sites (a) and the relationship between the measured fluorescence intensities of FITC and the concentration of FITC were plotted as a standard curve (b).

Figure 5

On‐chip pH gradient chromatofocusing to separate two proteins from the mixture. (A) Preparation of elution buffers with pH gradient in 16 mixing reactors. (B) Microscope images of 16 parallel fractionation reactors acquired by brightfield illumination and fluorescence illumination. The change of pH value of elution buffer solution and obtained fluorescence intensities of the reactors are shown in the graph (1 mm scale bars are shown).