Bryant Hollins | Louisiana Tech University (original) (raw)

Talks by Bryant Hollins

Protein oxidation is considered to be a sign of oxidative damage. A common form of oxidation is ... more Protein oxidation is considered to be a sign of oxidative damage. A common form of oxidation is carbonylation, an irreversible post-translational modification (PTM) that results in the placement of a carbonyl group on amino acid residues of proteins, particularly lysine, theorenine, and proline. Carbonylation has been implicated in many aging processes and age-related diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Carbonylated proteins have the potential to be used as biomarkers for many diseases, including AD and PD. However, these proteins are found in low abundance in clinical samples, limiting the ways in which they can be used in research. We have recently developed a microfluidic chip for the purpose of enriching these low abundance proteins in a sample. The microfluidic chip is modified to immobilize oxalyldihydrazide on the surface. The affinity of oxalyldihydrazide for carbonyls allows for specific capture of carbonylated proteins in a sample. We have also demonstrated that the capture is selective for these proteins as well. In these experiments, we optimize the capture procedure by investigating the effects of interior chip geometry, the concentration of the crosslinker in the immobilization step, and the flow rate in which proteins are loaded into the chip. We use molecular modeling and fluid dynamics modeling for the purpose of determining point-of-failure for the microfluidic device. The microfluidic device provides a promising platform for the purpose of biomarker discovery in disease research.

Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been... more Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been implicated in aging and many age-related disease states. Protein oxidation is a post-translational modification (PTM) to proteins and can occur in over 35 ways. One form of protein oxidation is carbonylation, an irreversible PTM of proteins where an aldehyde bonds to an amino acid residue, most commonly lysine, proline, theorenine, and arginine. These proteins are used as common markers for oxidative stress within a system. We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. Surface elemental analysis and roughness were examined by X-ray photon spectroscopy (XPS) and atomic force spectroscopy (AFM), respectively. Additionally, fluorescence microscopy was utilized for chemical functional groups mapping to verify the presence of desired crosslinkers. We use in vitro oxidized cytochrome c as a model protein for testing specificity of the methodology for carbonylated proteins. We then mix oxidized cytochrome c with TRITC-BSA to determine selectivity of the method. Capture and elution of the proteins are determined quantitatively. The method demonstrates the potential of using microfluidic platforms for the enrichment of carbonylated proteins.

Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been... more Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been implicated in aging and many age-related disease states. Protein oxidation is a post-translational modification (PTM) to proteins and can occur in over 35 ways. One form of protein oxidation is carbonylation, an irreversible PTM of proteins where an aldehyde bonds to an amino acid residue, most commonly lysine, proline, theorenine, and arginine. These proteins are used as common markers for oxidative stress within a system. We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. Surface elemental analysis and roughness were examined by X-ray photon spectroscopy (XPS) and atomic force spectroscopy (AFM), respectively. Additionally, fluorescence microscopy was utilized for chemical functional groups mapping to verify the presence of desired crosslinkers. We use in vitro oxidized bovine serum albumin as a model protein for testing selectivity and specificity of the methodology for carbonylated proteins. We demonstrate specificity for our protein target, with a device capture efficiency of about 50% for targeted proteins after 30 minutes of continuous loading. Elution of the proteins is confirmed qualitatively. This is the first report to suggest the use of immobilized oxalyldihydrazide on a microchip as an enrichment methodology for low abundance proteins in a sample.

Oxidative stress has been implicated in age-related diseases. A common form of oxidative stress ... more Oxidative stress has been implicated in age-related diseases. A common form of oxidative stress is carbonylation, an irreversible post-translational modification to proteins that can compromise function. The significance of this modification make carbonylated proteins a prime target for proteomic studies; however, they occur in low abundance in clinical samples. We propose a method to preconcentrate these proteins prior to conducting proteomic studies. By activating carboxylic acid on the surface of the PMMA microchip, we can capture proteins that have been oxidized in vitro. We use bovine serum albumin (BSA) as a standard protein for our experiments. A major advantage of this microchip enrichment is the reduction in cost and time needed for sample enrichment. The modularity of the project allows for it to be used for sample preparation for multiple experiments.

Oxidative stress is a known cause of age-related diseases. One of the most common forms of oxida... more Oxidative stress is a known cause of age-related diseases. One of the most common forms of oxidative stress observed in these diseases is carbonylation, an irreverisible post-translational modification of proteins that can affect their function. Because of their role in age-related diseases, carbonylated proteins are a common target for proteomic studies. Often, these proteins are in such low abundance in a clinical sample that they require preconcentration prior to any studies being conducted. The amount of sample required to obtain enough proteins for proteomics is relatively high. We propose a method of enriching proteins in a PMMA microchannel, allowing for the sample size to be greatly reduced. By activating carboxylic acid groups on the surface of PMMA, we can capture proteins that have been oxidized in vitro. The proteins are then eluted from the channel using a formic acid wash. We use bovine serum albumin (BSA) as our standard protein for experiments. The selectivity of the microchannel for carbonylated protein is verified by using standard BSA as a control. We show, qualitatively, the capture and elution of carbonylated proteins from the microchannel.

Surface-Enhanced Raman Spectroscopy (SERS) amplifies Raman laser-stimulated emission of molecules... more Surface-Enhanced Raman Spectroscopy (SERS) amplifies Raman laser-stimulated emission of molecules when they are in close proximity to a metal surface, commonly silver, platinum, and gold. These amplifications are at least six orders of magnitude, thus providing the sensitivity to detect physiologically relevant concentrations of molecules in a biological sample. This enhancement is provided by chemical adsorption, which provides some selectivity due to the chemical bonds involved. In a biological sample, however, one may not be interested in the concentrations of all the molecules present. This problem can be addressed by using the concepts of size and charge exclusion in conjunction with a porous metal electrode acting as a SERS substrate. Size exclusion allows us to focus on receiving spectral information from only certain molecules by restricting the size of the pores present in our substrates. This method lets molecules under the pore diameter to be detected, while rejecting those that are larger than the diameter. Using this electrode, we present a charge to the previously neutral surface of the substrate, varying the charge to enable chemical species to adsorb and desorb at our prompting. Taking advantage of the chemistry of the different molecules present in the sample, we can select molecules for adsorption based on their particulary charge. Applying these concepts, we demonstrate quantitative analysis of several biological molecules typically present in a biological sample at physiological concentrations.

Papers by Bryant Hollins

2016 32nd Southern Biomedical Engineering Conference (SBEC), 2016

The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) m... more The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) may be applied to deliver small molecules to a microfluidic channel. Native PDMS is hydrophobic and often requires surface modifications for biologically relevant applications. Surface modification is not permanent, as the surface reverts to a hydrophobic state via bulk diffusion of monomers to the surface. Likewise, solid substances can be added into PDMS prepolymer mixture prior to curing and these particles can diffuse from the cured polymer bulk to the surface and surrounding fluid media. This characteristic of PDMS has applications for drug delivery to cell culture, cell and analyte labeling, on chip live/dead assays, flow and diffusion visualization, gradient generation, and transport phenomena in microfluidic systems. We use fluorescein to quantify and model this small molecule diffusion out of PDMS thin films and microchannels into fluid flow. The results from microchannel leaching show steady state leaching into the fluid flow over 90 minutes at concentrations around 150 nM. Results from immersion of doped PDMS shows continued leaching of fluorescein from the polymer over 4 days. The results show promise to use PDMS substrates for administering small amounts of substances to microfluidic cell cultures, as well as developing systems for studying cellular behavior with minimal interference.

2016 32nd Southern Biomedical Engineering Conference (SBEC), 2016

Paper microfluidics is an emerging technology that offers a simple and inexpensive alternative to... more Paper microfluidics is an emerging technology that offers a simple and inexpensive alternative to traditional microfluidics. Paper is an attractive medium for microfluidic devices because of its inherent hydrophilicity and low cost. Hydrophobic materials including wax and photoresist are used to pattern the paper. The most common method for making paper microfluidic analytical devices (μPAD) is wax printing, however, this method requires an expensive and specialized printer that is limited to printing documents and channel designs. Our method uses inexpensive materials and tools accessible to most research labs in the US. We utilize 3D printers, a common tool available in many universities because of their versatility. Poly(dimethylsiloxane) (PDMS) wax stamps are used to deposit wax onto paper, forming microfluidic channels. The PDMS stamps are produced with ABS 3D printed molds designed in CAD software. A PDMS stamp is dipped into melted wax and then pressed onto paper much like the process of using a rubber stamp and ink. Once the wax is deposited, the paper is heated, letting the wax penetrate the paper and form hydrophilic channels. This rapid and simple procedure allows researchers to easily produce μPADs with the flexibility of CAD software and 3D printers.

Lab on a Chip, 2012

S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (... more S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (PTM) of cysteine, plays an important role in cellular functions and cell signalling pathways. Nitrosylated proteins are considered as biomarkers of aging and Alzheimer's disease (AD). Microfluidics has been widely used for development of novel tools for separation of protein mixtures. Here we demonstrate two-dimensional micro-electrophoresis (2D μ-CE) separations of nitrosylated proteins from the human colon epithelial adenocarcinoma cells (HT-29) and AD transgenic mice brain tissues. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimensional separations, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection was used with field strength at 200 V cm(-1). After 80 s separation in the first CGE dimension, fractions were successfully transferred to a second MEEKC dimension for a short 10 s separation. We first demonstrate this 2D μ-CE separation by resolving five standard proteins with molecular weight (MW) ranging from 20 to 64 kDa. We also present a high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following menadione (MQ) treatment to induce oxidative stress. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and their age matched controls were also generated. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in biological samples on a microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.

– Carbonylated proteins are a common marker of in vivo oxidative stress within an organism. We re... more – 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 effic...

Proteins are the building blocks of cells in living organisms, and are composed of amino acids. T... more Proteins are the building blocks of cells in living organisms, and are composed of amino acids. The expression of proteins is regulated by the processes of transcription and translation. Proteins undergo post-translational modifications in order to dictate their role physiologically within a cell. Not all post-translational modifications are beneficial for the protein or the cell. One type of post-translational modification, called carbonylation, irreversibly places a carbonyl group onto an amino acid residue, most commonly proline, lysine, arginine, and threonine. This modification can have severe consequences physiologically, including loss of solubility, loss of function, and protein aggregation. Carbonylated proteins have commonly been used as a marker o f oxidative stress. Oxidative stress has been suggested to play a role in many human disease states, such as Alzheimer’s Disease, Amyotrophic Lateral Sclerosis, Parkinson’s Disease, inflammatory diseases, and others. Evidence sh...

Rapid Prototyping Journal

Purpose This paper aims to investigate the applicability of 3D-printed molds to be used as a subs... more Purpose This paper aims to investigate the applicability of 3D-printed molds to be used as a substitute for photolithography in the formation of polymer-based stamps. It proposes leveraging 3D printing as a rapid prototyping tool to be applied to microfluidic fabrication. Design/methodology/approach Different designs are created using computer-aided design (CAD) software and printed via Makerbot 3D printer. The molds serve as negative reliefs for a PDMS stamp. The stamp is used to apply paraffin wax to chromatography paper, creating hydrophobic barriers and hydrophilic channels. The minimum functional channel widths and barrier widths are determined for the method. Findings The method is demonstrated to be effective for bypassing the more cost-prohibitive photolithography approach for rapid paper microdevice fabrication. This approach produces functional channels that can be used for on-chip analytical assays. The minimum functional barrier widths and minimum functional channel widt...

2016 32nd Southern Biomedical Engineering Conference (SBEC), 2016

Fused deposition modeling (FDM) printers are becoming more frequent in everyday use. These types ... more Fused deposition modeling (FDM) printers are becoming more frequent in everyday use. These types of 3D printers are extremely useful for rapid prototyping. Fused deposition modeling printing melts the printing material and extrudes it through a nozzle. The material is laid out in a layer by layer fashion until the object is completed printing. Two common types of filament used in FDM printing are Polylactic Acid (PLA) and Acrylonitrile butadiene styrene (ABS). Some properties that can change the strength of 3D printed piece are things such as infill percentage, layer height, print orientation, extruding temperature, and build speed to name a few. Infill percentage and print orientation were tested to determine the mechanical strength of the material. The infill percentage varied from 20%-100% by increments of 20%. The goal of this project was to analyze the mechanical strength of PLA being printed in various orientations and infill percentages.

Microfluidics provides a promising platform for high throughput screening assays and biomolecule ... more Microfluidics provides a promising platform for high throughput screening assays and biomolecule sensing through capture. We have recently developed an assay for capturing carbonylated proteins, a common marker of oxidative stress, on a surface-modified polymeric microfluidic chip by taking advantage of carbonyl affinity for hydrazide. Optimization of the biomicrofluidic design involves modeling characteristics inside the chip, including fluid flow and molecular binding constants. We use experimentally calculated concentration changes in a flowing protein solution to determine binding constants between oxidized cytochrome c and oxalyldihydrazide. We assume a simple, bimolecule model that has a first-order interaction. We determine binding constants for association and dissociation between carbonyls and oxalyldihydrazide. These constants will guide our future optimization work for improving carbonylated protein capture on this biomicrodevice. This study is the first to model the bind...

Microfluidics provides a promising platform for biomolecule capture. Recent work has shown the fe... more Microfluidics provides a promising platform for biomolecule capture. Recent work has shown the feasibility of microfluidic devices for biomedical applications such as cell capture, angiogenesis promotion, and stem cell culture. Most microfluidic cell devices use rectangular channels. A physiologically relevant concern in microfluidic cell work is the shear stress experienced by the cells in these applications. We model shear stress in microfluidic channels with different cross-sectional areas, including rectangular, tapered, and semi-circular. Fluid flow will be modeled using the physical characteristics of water, the primary solvent used in microfluidic applications. Shear stress is analyzed at the surface of the channel and above the area of the captured cells, up to half the channel depth in each scenario using a Newtonian hydrodynamic shear stress calculation. We determine the maximum fluid velocity possible within each channel without exceeding in vivo shear stresses. Coupled w...

Advances in biopolymers have long been sought to advance fields of biomedical engineering, with p... more Advances in biopolymers have long been sought to advance fields of biomedical engineering, with particular focus in biomicrofluidics and tissue engineering. We recently demonstrated that PDMS, a commonly used polymer in biological applications, can be used to intentionally leach molecules into a sample. In this work, we characterize the diffusion of fluorescein into water from the bulk PDMS, with a focus on calculating the diffusion rate of molecules from the polymer bulk into its surrounding aqueous environment. We look at diffusion from the bulk over a period of days in an attempt to formulate a fluorescein diffusion model from PDMS. Our results can guide future work in tissue engineering application studies, as we establish a framework with fluorescein for determining the properties of molecules capable of diffusion from bulk PDMS. This strategy can be used as a tool for creating self-regulating microfluidic chambers for drug discovery, cell culture, and chemical monitoring applications, as well as a substrate for guiding cell growth and migration in tissue engineering applications.

The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) m... more The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) may be applied to deliver small molecules to a microfluidic channel. Native PDMS is hydrophobic and often requires surface modifications for biologically relevant applications. Surface modification is not permanent, as the surface reverts to a hydrophobic state via bulk diffusion of monomers to the surface. Likewise, solid substances can be added into PDMS prepolymer mixture prior to curing and these particles can diffuse from the cured polymer bulk to the surface and surrounding fluid media. This characteristic of PDMS has applications for drug delivery to cell culture, cell and analyte labeling, on chip live/dead assays, flow and diffusion visualization, gradient generation, and transport phenomena in microfluidic systems. We use fluorescein to quantify and model this small molecule diffusion out of PDMS thin films and microchannels into fluid flow. The results from microchannel leaching show steady state leaching into the fluid flow over 90 minutes at concentrations around 150 nM. Results from immersion of doped PDMS shows continued leaching of fluorescein from the polymer over 4 days. The results show promise to use PDMS substrates for administering small amounts of substances to microfluidic cell cultures, as well as developing systems for studying cellular behavior with minimal interference.

Protein oxidation is considered to be a sign of oxidative damage. A common form of oxidation is ... more Protein oxidation is considered to be a sign of oxidative damage. A common form of oxidation is carbonylation, an irreversible post-translational modification (PTM) that results in the placement of a carbonyl group on amino acid residues of proteins, particularly lysine, theorenine, and proline. Carbonylation has been implicated in many aging processes and age-related diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Carbonylated proteins have the potential to be used as biomarkers for many diseases, including AD and PD. However, these proteins are found in low abundance in clinical samples, limiting the ways in which they can be used in research. We have recently developed a microfluidic chip for the purpose of enriching these low abundance proteins in a sample. The microfluidic chip is modified to immobilize oxalyldihydrazide on the surface. The affinity of oxalyldihydrazide for carbonyls allows for specific capture of carbonylated proteins in a sample. We have also demonstrated that the capture is selective for these proteins as well. In these experiments, we optimize the capture procedure by investigating the effects of interior chip geometry, the concentration of the crosslinker in the immobilization step, and the flow rate in which proteins are loaded into the chip. We use molecular modeling and fluid dynamics modeling for the purpose of determining point-of-failure for the microfluidic device. The microfluidic device provides a promising platform for the purpose of biomarker discovery in disease research.

Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been... more Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been implicated in aging and many age-related disease states. Protein oxidation is a post-translational modification (PTM) to proteins and can occur in over 35 ways. One form of protein oxidation is carbonylation, an irreversible PTM of proteins where an aldehyde bonds to an amino acid residue, most commonly lysine, proline, theorenine, and arginine. These proteins are used as common markers for oxidative stress within a system. We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. Surface elemental analysis and roughness were examined by X-ray photon spectroscopy (XPS) and atomic force spectroscopy (AFM), respectively. Additionally, fluorescence microscopy was utilized for chemical functional groups mapping to verify the presence of desired crosslinkers. We use in vitro oxidized cytochrome c as a model protein for testing specificity of the methodology for carbonylated proteins. We then mix oxidized cytochrome c with TRITC-BSA to determine selectivity of the method. Capture and elution of the proteins are determined quantitatively. The method demonstrates the potential of using microfluidic platforms for the enrichment of carbonylated proteins.

Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been... more Protein oxidation occurs as a result of exposure to oxidative stress. Protein oxidation has been implicated in aging and many age-related disease states. Protein oxidation is a post-translational modification (PTM) to proteins and can occur in over 35 ways. One form of protein oxidation is carbonylation, an irreversible PTM of proteins where an aldehyde bonds to an amino acid residue, most commonly lysine, proline, theorenine, and arginine. These proteins are used as common markers for oxidative stress within a system. We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. Surface elemental analysis and roughness were examined by X-ray photon spectroscopy (XPS) and atomic force spectroscopy (AFM), respectively. Additionally, fluorescence microscopy was utilized for chemical functional groups mapping to verify the presence of desired crosslinkers. We use in vitro oxidized bovine serum albumin as a model protein for testing selectivity and specificity of the methodology for carbonylated proteins. We demonstrate specificity for our protein target, with a device capture efficiency of about 50% for targeted proteins after 30 minutes of continuous loading. Elution of the proteins is confirmed qualitatively. This is the first report to suggest the use of immobilized oxalyldihydrazide on a microchip as an enrichment methodology for low abundance proteins in a sample.

Oxidative stress has been implicated in age-related diseases. A common form of oxidative stress ... more Oxidative stress has been implicated in age-related diseases. A common form of oxidative stress is carbonylation, an irreversible post-translational modification to proteins that can compromise function. The significance of this modification make carbonylated proteins a prime target for proteomic studies; however, they occur in low abundance in clinical samples. We propose a method to preconcentrate these proteins prior to conducting proteomic studies. By activating carboxylic acid on the surface of the PMMA microchip, we can capture proteins that have been oxidized in vitro. We use bovine serum albumin (BSA) as a standard protein for our experiments. A major advantage of this microchip enrichment is the reduction in cost and time needed for sample enrichment. The modularity of the project allows for it to be used for sample preparation for multiple experiments.

Oxidative stress is a known cause of age-related diseases. One of the most common forms of oxida... more Oxidative stress is a known cause of age-related diseases. One of the most common forms of oxidative stress observed in these diseases is carbonylation, an irreverisible post-translational modification of proteins that can affect their function. Because of their role in age-related diseases, carbonylated proteins are a common target for proteomic studies. Often, these proteins are in such low abundance in a clinical sample that they require preconcentration prior to any studies being conducted. The amount of sample required to obtain enough proteins for proteomics is relatively high. We propose a method of enriching proteins in a PMMA microchannel, allowing for the sample size to be greatly reduced. By activating carboxylic acid groups on the surface of PMMA, we can capture proteins that have been oxidized in vitro. The proteins are then eluted from the channel using a formic acid wash. We use bovine serum albumin (BSA) as our standard protein for experiments. The selectivity of the microchannel for carbonylated protein is verified by using standard BSA as a control. We show, qualitatively, the capture and elution of carbonylated proteins from the microchannel.

Surface-Enhanced Raman Spectroscopy (SERS) amplifies Raman laser-stimulated emission of molecules... more Surface-Enhanced Raman Spectroscopy (SERS) amplifies Raman laser-stimulated emission of molecules when they are in close proximity to a metal surface, commonly silver, platinum, and gold. These amplifications are at least six orders of magnitude, thus providing the sensitivity to detect physiologically relevant concentrations of molecules in a biological sample. This enhancement is provided by chemical adsorption, which provides some selectivity due to the chemical bonds involved. In a biological sample, however, one may not be interested in the concentrations of all the molecules present. This problem can be addressed by using the concepts of size and charge exclusion in conjunction with a porous metal electrode acting as a SERS substrate. Size exclusion allows us to focus on receiving spectral information from only certain molecules by restricting the size of the pores present in our substrates. This method lets molecules under the pore diameter to be detected, while rejecting those that are larger than the diameter. Using this electrode, we present a charge to the previously neutral surface of the substrate, varying the charge to enable chemical species to adsorb and desorb at our prompting. Taking advantage of the chemistry of the different molecules present in the sample, we can select molecules for adsorption based on their particulary charge. Applying these concepts, we demonstrate quantitative analysis of several biological molecules typically present in a biological sample at physiological concentrations.

2016 32nd Southern Biomedical Engineering Conference (SBEC), 2016

The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) m... more The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) may be applied to deliver small molecules to a microfluidic channel. Native PDMS is hydrophobic and often requires surface modifications for biologically relevant applications. Surface modification is not permanent, as the surface reverts to a hydrophobic state via bulk diffusion of monomers to the surface. Likewise, solid substances can be added into PDMS prepolymer mixture prior to curing and these particles can diffuse from the cured polymer bulk to the surface and surrounding fluid media. This characteristic of PDMS has applications for drug delivery to cell culture, cell and analyte labeling, on chip live/dead assays, flow and diffusion visualization, gradient generation, and transport phenomena in microfluidic systems. We use fluorescein to quantify and model this small molecule diffusion out of PDMS thin films and microchannels into fluid flow. The results from microchannel leaching show steady state leaching into the fluid flow over 90 minutes at concentrations around 150 nM. Results from immersion of doped PDMS shows continued leaching of fluorescein from the polymer over 4 days. The results show promise to use PDMS substrates for administering small amounts of substances to microfluidic cell cultures, as well as developing systems for studying cellular behavior with minimal interference.

2016 32nd Southern Biomedical Engineering Conference (SBEC), 2016

Paper microfluidics is an emerging technology that offers a simple and inexpensive alternative to... more Paper microfluidics is an emerging technology that offers a simple and inexpensive alternative to traditional microfluidics. Paper is an attractive medium for microfluidic devices because of its inherent hydrophilicity and low cost. Hydrophobic materials including wax and photoresist are used to pattern the paper. The most common method for making paper microfluidic analytical devices (μPAD) is wax printing, however, this method requires an expensive and specialized printer that is limited to printing documents and channel designs. Our method uses inexpensive materials and tools accessible to most research labs in the US. We utilize 3D printers, a common tool available in many universities because of their versatility. Poly(dimethylsiloxane) (PDMS) wax stamps are used to deposit wax onto paper, forming microfluidic channels. The PDMS stamps are produced with ABS 3D printed molds designed in CAD software. A PDMS stamp is dipped into melted wax and then pressed onto paper much like the process of using a rubber stamp and ink. Once the wax is deposited, the paper is heated, letting the wax penetrate the paper and form hydrophilic channels. This rapid and simple procedure allows researchers to easily produce μPADs with the flexibility of CAD software and 3D printers.

Lab on a Chip, 2012

S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (... more S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (PTM) of cysteine, plays an important role in cellular functions and cell signalling pathways. Nitrosylated proteins are considered as biomarkers of aging and Alzheimer's disease (AD). Microfluidics has been widely used for development of novel tools for separation of protein mixtures. Here we demonstrate two-dimensional micro-electrophoresis (2D μ-CE) separations of nitrosylated proteins from the human colon epithelial adenocarcinoma cells (HT-29) and AD transgenic mice brain tissues. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimensional separations, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection was used with field strength at 200 V cm(-1). After 80 s separation in the first CGE dimension, fractions were successfully transferred to a second MEEKC dimension for a short 10 s separation. We first demonstrate this 2D μ-CE separation by resolving five standard proteins with molecular weight (MW) ranging from 20 to 64 kDa. We also present a high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following menadione (MQ) treatment to induce oxidative stress. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and their age matched controls were also generated. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in biological samples on a microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.

– Carbonylated proteins are a common marker of in vivo oxidative stress within an organism. We re... more – 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 effic...

Proteins are the building blocks of cells in living organisms, and are composed of amino acids. T... more Proteins are the building blocks of cells in living organisms, and are composed of amino acids. The expression of proteins is regulated by the processes of transcription and translation. Proteins undergo post-translational modifications in order to dictate their role physiologically within a cell. Not all post-translational modifications are beneficial for the protein or the cell. One type of post-translational modification, called carbonylation, irreversibly places a carbonyl group onto an amino acid residue, most commonly proline, lysine, arginine, and threonine. This modification can have severe consequences physiologically, including loss of solubility, loss of function, and protein aggregation. Carbonylated proteins have commonly been used as a marker o f oxidative stress. Oxidative stress has been suggested to play a role in many human disease states, such as Alzheimer’s Disease, Amyotrophic Lateral Sclerosis, Parkinson’s Disease, inflammatory diseases, and others. Evidence sh...

Rapid Prototyping Journal

Purpose This paper aims to investigate the applicability of 3D-printed molds to be used as a subs... more Purpose This paper aims to investigate the applicability of 3D-printed molds to be used as a substitute for photolithography in the formation of polymer-based stamps. It proposes leveraging 3D printing as a rapid prototyping tool to be applied to microfluidic fabrication. Design/methodology/approach Different designs are created using computer-aided design (CAD) software and printed via Makerbot 3D printer. The molds serve as negative reliefs for a PDMS stamp. The stamp is used to apply paraffin wax to chromatography paper, creating hydrophobic barriers and hydrophilic channels. The minimum functional channel widths and barrier widths are determined for the method. Findings The method is demonstrated to be effective for bypassing the more cost-prohibitive photolithography approach for rapid paper microdevice fabrication. This approach produces functional channels that can be used for on-chip analytical assays. The minimum functional barrier widths and minimum functional channel widt...

2016 32nd Southern Biomedical Engineering Conference (SBEC), 2016

Fused deposition modeling (FDM) printers are becoming more frequent in everyday use. These types ... more Fused deposition modeling (FDM) printers are becoming more frequent in everyday use. These types of 3D printers are extremely useful for rapid prototyping. Fused deposition modeling printing melts the printing material and extrudes it through a nozzle. The material is laid out in a layer by layer fashion until the object is completed printing. Two common types of filament used in FDM printing are Polylactic Acid (PLA) and Acrylonitrile butadiene styrene (ABS). Some properties that can change the strength of 3D printed piece are things such as infill percentage, layer height, print orientation, extruding temperature, and build speed to name a few. Infill percentage and print orientation were tested to determine the mechanical strength of the material. The infill percentage varied from 20%-100% by increments of 20%. The goal of this project was to analyze the mechanical strength of PLA being printed in various orientations and infill percentages.

Microfluidics provides a promising platform for high throughput screening assays and biomolecule ... more Microfluidics provides a promising platform for high throughput screening assays and biomolecule sensing through capture. We have recently developed an assay for capturing carbonylated proteins, a common marker of oxidative stress, on a surface-modified polymeric microfluidic chip by taking advantage of carbonyl affinity for hydrazide. Optimization of the biomicrofluidic design involves modeling characteristics inside the chip, including fluid flow and molecular binding constants. We use experimentally calculated concentration changes in a flowing protein solution to determine binding constants between oxidized cytochrome c and oxalyldihydrazide. We assume a simple, bimolecule model that has a first-order interaction. We determine binding constants for association and dissociation between carbonyls and oxalyldihydrazide. These constants will guide our future optimization work for improving carbonylated protein capture on this biomicrodevice. This study is the first to model the bind...

Microfluidics provides a promising platform for biomolecule capture. Recent work has shown the fe... more Microfluidics provides a promising platform for biomolecule capture. Recent work has shown the feasibility of microfluidic devices for biomedical applications such as cell capture, angiogenesis promotion, and stem cell culture. Most microfluidic cell devices use rectangular channels. A physiologically relevant concern in microfluidic cell work is the shear stress experienced by the cells in these applications. We model shear stress in microfluidic channels with different cross-sectional areas, including rectangular, tapered, and semi-circular. Fluid flow will be modeled using the physical characteristics of water, the primary solvent used in microfluidic applications. Shear stress is analyzed at the surface of the channel and above the area of the captured cells, up to half the channel depth in each scenario using a Newtonian hydrodynamic shear stress calculation. We determine the maximum fluid velocity possible within each channel without exceeding in vivo shear stresses. Coupled w...

Advances in biopolymers have long been sought to advance fields of biomedical engineering, with p... more Advances in biopolymers have long been sought to advance fields of biomedical engineering, with particular focus in biomicrofluidics and tissue engineering. We recently demonstrated that PDMS, a commonly used polymer in biological applications, can be used to intentionally leach molecules into a sample. In this work, we characterize the diffusion of fluorescein into water from the bulk PDMS, with a focus on calculating the diffusion rate of molecules from the polymer bulk into its surrounding aqueous environment. We look at diffusion from the bulk over a period of days in an attempt to formulate a fluorescein diffusion model from PDMS. Our results can guide future work in tissue engineering application studies, as we establish a framework with fluorescein for determining the properties of molecules capable of diffusion from bulk PDMS. This strategy can be used as a tool for creating self-regulating microfluidic chambers for drug discovery, cell culture, and chemical monitoring applications, as well as a substrate for guiding cell growth and migration in tissue engineering applications.

The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) m... more The goal of this study is to show that diffusion of a dopant from poly(dimethylsiloxane) (PDMS) may be applied to deliver small molecules to a microfluidic channel. Native PDMS is hydrophobic and often requires surface modifications for biologically relevant applications. Surface modification is not permanent, as the surface reverts to a hydrophobic state via bulk diffusion of monomers to the surface. Likewise, solid substances can be added into PDMS prepolymer mixture prior to curing and these particles can diffuse from the cured polymer bulk to the surface and surrounding fluid media. This characteristic of PDMS has applications for drug delivery to cell culture, cell and analyte labeling, on chip live/dead assays, flow and diffusion visualization, gradient generation, and transport phenomena in microfluidic systems. We use fluorescein to quantify and model this small molecule diffusion out of PDMS thin films and microchannels into fluid flow. The results from microchannel leaching show steady state leaching into the fluid flow over 90 minutes at concentrations around 150 nM. Results from immersion of doped PDMS shows continued leaching of fluorescein from the polymer over 4 days. The results show promise to use PDMS substrates for administering small amounts of substances to microfluidic cell cultures, as well as developing systems for studying cellular behavior with minimal interference.

Paper microfluidics is an emerging technology that offers a simple and inexpensive alternative to... more Paper microfluidics is an emerging technology that offers a simple and inexpensive alternative to traditional microfluidics. Paper is an attractive medium for microfluidic devices because of its inherent hydrophilicity and low cost. Hydrophobic materials including wax and photoresist are used to pattern the paper. The most common method for making paper microfluidic analytical devices (μPAD) is wax printing; however, this method requires an expensive and specialized printer that is limited to printing documents and channel designs. Our method uses inexpensive materials and tools accessible to most research labs in the US. We utilize 3D printers, a common tool available in many universities because of their versatility. Poly(dimethylsiloxane) (PDMS) wax stamps are used to deposit wax onto paper, forming microfluidic channels. The PDMS stamps are produced with ABS 3D printed molds designed in CAD software. A PDMS stamp is dipped into melted wax and then pressed onto paper much like the process of using a rubber stamp and ink. Once the wax is deposited, the paper is heated, letting the wax penetrate the paper and form hydrophilic channels. This rapid and simple procedure allows researchers to easily produce μPADs with the flexibility of CAD software and 3D printers.

Fused deposition modeling (FDM) printers are becoming more frequent in everyday use. These types ... more Fused deposition modeling (FDM) printers are becoming more frequent in everyday use. These types of 3D printers are extremely useful for rapid prototyping. Fused deposition modeling printing melts the printing material and extrudes it through a nozzle. The material is laid out in a layer by layer fashion until the object is completed printing. Two common types of filament used in FDM printing are Polylactic Acid (PLA) and Acrylonitrile butadiene styrene (ABS). Some properties that can change the strength of 3D printed piece are things such as infill percentage, layer height, print orientation, extruding temperature, and build speed to name a few. Infill percentage and print orientation were tested to determine the mechanical strength of the material. The infill percentage varied from 20%-100% by increments of 20%. The goal of this project was to analyze the mechanical strength of PLA being printed in various orientations and infill percentages.

Carbonylated proteins are a common marker of in vivo oxidative stress within an organism. We rece... more 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.