Microfluidic enrichment of small proteins from complex biological mixture on nanoporous silica chip

Abstract

The growing field of miniaturized diagnostics is hindered by a lack of pre-analysis treatments that are capable of processing small sample volumes for the detection of low concentration analytes in a high-throughput manner. This letter presents a novel, highly efficient method for the extraction of low-molecular weight (LMW) proteins from biological fluids, represented by a mixture of standard proteins, using integrated microfluidic systems. We bound a polydimethylsiloxane layer patterned with a microfluidic channel onto a well-defined nanoporous silica substrate. Using rapid, pressure-driven fractionation steps, this system utilizes the size-exclusion properties of the silica nanopores to remove high molecular weight proteins while simultaneously isolating and enriching LMW proteins present in the biological sample. The introduction of the microfluidic component offers important advantages such as high reproducibility, a simple user interface, controlled environment, the ability to process small sample volumes, and precise quantification. This solution streamlines high-throughput proteomics research on many fronts and may find broad acceptance and application in clinical diagnostics and point of care detection.

Figure 1

Integrated microfluidic-MPS system. (a) Schematic of PDMS microfluidic channel on mesoporous silica substrate. (b) Image of single chip with input and output tubing. (c) Image of parallel experimental procedures performed over three devices at the same time.

Figure 2

Characterizations of mesoporous silica thin films prepared with Pluoronic F127. (a) Small angle XRD patterns (0.2°–6°). (b) TEM imaging. (c) N₂ adsorption∕desorption analysis isotherms and pore size distribution (inset).

Figure 3

MALDI MS profiles and statistic analysis. (a) MS spectra of LMW proteins recovery from microfluidic-MPS device. (b) MPS chip and (c) microfluidic channel on bare silicon wafer. (d) Unsupervised hierarchical clustering analysis testing the ability of different settings to enrich LMW proteins (F127: MPS chip; MF F127: microfluidic-MPS device; blank: microfluidic channel on the bare Si wafer). Red indicates peak intensity higher than the median value, green indicates peak intensity lower than the median value, and black represents peak intensity equal to the median values. Each row represents an individual MALDI MS mass peak and each column represents a type of experimental setting. The reproducibility of LMW protein enrichment from (e) microfluidic-MPS chip and (f) MPS chip is investigated by regression curves.