American Journal of Science and Engineering, Vol. 2, No.2, 2013 Corresponding Author: Bryant C. Hollins, Email: bhollins@latech.edu 9 Optimization of Oxalyldihydrazide-Immobilized Carbonylated Protein Capture in PMMA Microchips Bryant C. Hollins a , Sawyer D. Stone a , June Feng b , a Department of Biomedical Engineering, Louisiana Tech University, Ruston, LA, USA b Division of Technology, Engineering, and Mathematics, Bossier Parish Community College, Bossier, LA, USA Abstract – Carbonylated proteins are a common marker of in vivo oxidative stress within an organism. We recently reported on capturing oxidized proteins on a PMMA microchannel utilizing oxalyldihydrazide as a novel crosslinker. This study reports on the optimization of the capture methodology. We chose four parameters for optimization. These parameters are interior post density, oxalyldihydrazide concentration, oxalyldihydrazide incubation time, and sample flow rate. Based upon these experimental conditions, we found significant effects on protein capture when oxalyldihydrazide concentration and sample flow rates were altered. We included a COMSOL simulation of fluid flow through the microchannel to explain some of the results we observed. Oxalyldihydrazide incubation time and interior post density had no significance effect on capture efficiency. These optimized parameters reduce the time and sample requirements necessary for the technique and result in a four-fold increase in capture efficiency compared to our previously reported results. Future work includes optimizing the design of the microchip, validating the technique with a complex protein sample, and incorporating on-chip sensing modalities. Key Words – carbonylation, microfluidics, optimization, oxalyldihydrazide, PMMA, protein capture, protein enrichment. I. Introduction Microfluidics refers to the use of fluidic devices on the spatial scale of 1 – 100 μm in any dimension. While this field provides great promise for tackling some of the greatest scientific challenges facing society today, it is still immature. Common applications of microfluidics include miniaturization and automation of common analytical techniques [1- 3], drug screening [4, 5], and drug development [6, 7]. One key advantage for microfluidics is their versatility in applications, particularly since they lend themselves well to surface modification [1, 8]. This advantage puts microfluidics in a position to be a critical component of advances in the medical field [9-11]. The intersection of cell research and microfluidic technology is an area of high interest to researchers. Microfluidic devices have been demonstrated as viable tools for cell culture microenvironment manipulation [12]. Microchannels have been modified with aptamers for the selective capture of prostate-specific membrane antigen (PSMA) [13]. This application was high-throughput with the capability of analyzing 1 mL of solution in less than 30 minutes. Cancer cells have also been captured inside a microchip following surface immobilization of an antibody specific to those cells [14, 15]. Advances in diagnostic systems and implantable biomedical microdevices are enhancing the quality of life for people currently [16]. In the future, it is expected for the applications of BioMEMS to only increase, as technology improves and point-of- care clinical diagnosis becomes possible [17]. By adding renewable power sources, even individuals in developing countries would have access to healthcare advances through the use of BioMEMS [18]. There is a pressing need for developing novel biomarkers for addressing the major health challenges of this century, such as heart disease, cancer, diabetes, and neurodegenerative diseases [19-22]. One such biomarker could be carbonylated proteins [23]. Carbonylation is involved in a wide range of pathological conditions [24, 25]. Carbonylation is a post-translational modification that results in aldehydes being placed on the amino acid backbone of a protein [26, 27]. This modification is the most common form of protein oxidation and is a widely accepted marker of oxidative stress in vivo [28, 29]. There are techniques available for detecting carbonylated proteins in a sample; the most common being the DNPH assay proposed by Levine [30]. The detection can be coupled to proteomics for protein identification [31, 32]. There is great potential for