Integrated multifunctional microfluidics for automated proteome analyses.

Proteomics is a challenging field for realizing totally integrated microfluidic systems for complete proteome processing due to several considerations, including the sheer number of different protein types that exist within most proteomes, the large dynamic range associated with these various protein types, and the diverse chemical nature of the proteins comprising a typical proteome. For example, the human proteome is estimated to have >10(6) different components with a dynamic range of >10(10). The typical processing pipeline for proteomics involves the following steps: (1) selection and/or extraction of the particular proteins to be analyzed; (2) multidimensional separation; (3) proteolytic digestion of the protein sample; and (4) mass spectral identification of either intact proteins (top-down proteomics) or peptide fragments generated from proteolytic digestions (bottom-up proteomics). Although a number of intriguing microfluidic devices have been designed, fabricated and evaluated for carrying out the individual processing steps listed above, work toward building fully integrated microfluidic systems for protein analysis has yet to be realized. In this chapter, information will be provided on the nature of proteomic analysis in terms of the challenges associated with the sample type and the microfluidic devices that have been tested to carry out individual processing steps. These include devices such as those for multidimensional electrophoretic separations, solid-phase enzymatic digestions, and solid-phase extractions, all of which have used microfluidics as the functional platform for their implementation. This will be followed by an in-depth review of microfluidic systems, which are defined as units possessing two or more devices assembled into autonomous systems for proteome processing. In addition, information will be provided on the challenges involved in integrating processing steps into a functional system and the approaches adopted for device integration. In this chapter, we will focus exclusively on the front-end processing microfluidic devices and systems for proteome processing, and not on the interface technology of these platforms to mass spectrometry due to the extensive reviews that already exist on these types of interfaces.

Toward point-of-care microchip profiling of proteins.

Profiling of protein biomarkers is powerful for the analysis of complex proteomes altered during the progression of diseases. Lab-on-a-chip technologies can potentially provide the throughput and efficiency required for point-of-care and clinical applications. While initial studies utilized 1D microchip separation techniques, researchers have recently developed novel 2D microchip separation platforms with the ability to profile thousands of proteins more effectively. Despite advancements in lab-on-a-chip technologies, very few reports have demonstrated a point-of-care microchip-based profiling of proteins. In this review, recent progress in 1D and 2D microchip profiling of protein mixtures of a biological sample with potential point-of-care applications are discussed. A selection of recent microchip immunoassay-based techniques is also highlighted.

Ultra-fast two-dimensional microchip electrophoresis using SDS micro-CGE and microemulsion electrokinetic chromatography for protein separations.

A poly(methyl methacrylate) microfluidic chip was used to perform a two-dimensional (2-D) separation of a complex protein mixture in short development times. The separation was performed by combining sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS micro-CGE) with microemulsion electrokinetic chromatography (micro-MEEKC), which were used for the first and second dimensions, respectively. Fluorescently labeled Escherichia coli cytosolic proteins were profiled by this 2-D approach with the results compared to a similar 2-D separation using SDS micro-CGE x micro-MEKC (micelle electrokinetic chromatography). The relatively short column lengths (effective length = 10 mm) for both dimensions were used to achieve separations requiring only 220 s of development time. High spot production rates (131 +/- 11 spots min(-1)) and reasonable peak capacities (481 +/- 18) were generated despite the fact that short columns were used. In addition, the use of mu-MEEKC in the second dimension was found to produce higher peak capacities compared to micro-MEKC (481 +/- 18 for micro-MEEKC and 332 +/- 17 for micro-MEKC) due to the higher plate numbers associated with micro-MEEKC.

Enrichment and detection of Escherichia coli O157:H7 from water samples using an antibody modified microfluidic chip.

Low abundant (<100 cells mL(-1)) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices, each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using benchtop real-time quantitative polymerase chain reaction (PCR), targeting genes which effectively discriminated the O157:H7 serotype from other nonpathogenic bacteria. The recovery of target cells from water samples was determined to be approximately 72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and wastewater samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies.

Generating high peak capacity 2-D maps of complex proteomes using PMMA microchip electrophoresis.

A high peak capacity 2-D protein separation system combining SDS micro-CGE (SDS micro-CGE) with microchip MEKC (micro-MEKC) using a PMMA microfluidic is reported. The utility of the 2-D microchip was demonstrated by generating a 2-D map from a complex biological sample containing a large number of constituent proteins using fetal calf serum (FCS) as the model system. The proteins were labeled with a thiol-reactive AlexaFluor 633 fluorophore (excitation/emission: 633/652 nm) to allow for ultra-sensitive on-chip detection using LIF following the 2-D separation. The high-resolution separation of the proteins was accomplished based on their size in the SDS micro-CGE dimension and their interaction with micelles in the micro-MEKC dimension. A comprehensive 2-D SDS micro-CGE x micro-MEKC separation of the FCS proteins was completed in less than <30 min using this 2-D microchip format, which consisted of 60 mm and 50 mm effective separation lengths for the first and second separation dimensions, respectively. Results obtained from the microchip separation were compared with protein maps acquired using conventional 2-D IEF and SDS-PAGE of a similar FCS sample. The microchip 2-D separation was found to be approximately 60x faster and yielded an average peak capacity of 2600 (+/- 149), nearly three times larger than that obtained using conventional IEF/SDS-PAGE.