Hamed Shadpour

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 μ‐CGE) with microchip MEKC (μ‐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 μ‐CGE dimension and their interaction with micelles in the μ‐MEKC dimension. A comprehensive 2‐D SDS μ‐CGE×μ‐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 ∼60× faster and yielded an average peak capacity of 2600 (±149), nearly three times larger than that obtained using conventional IEF/SDS‐PAGE.

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.

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.

Orthogonal array design for minimizing loss of vitamin B9 in bread

Purpose – The purpose of this paper is to study and minimize loss of vitamin B9 in bread during warming protocol.Design/methodology/approach – In this study, warming intensity, warming time, warming device, and bread storage method were selected as the most effective factors on B9 loss in bread. The variation of B9 in bread and its loss were studied with orthogonal array design (OAD) using the L9 optimization matrix.Findings – With a calculated per cent of contribution (P%) of error of 0.38 per cent and according to the analysis of variance, ANOVA, of the fluorescence data, 86 per cent of B9 was saved by using toaster as the warming device, a bread warming temperature of <200○F and a warming time of <10min. Fluorescence method evaluated warming intensity and warming device as the most powerful factors affecting the B9 concentration in bread with corresponding P% of 42.28 per cent and 41.72 per cent, respectively.Practical implications – In conclusion, heat destroyed significant portion of B9 in bread duri...