Nathaniel C. Cady

Nucleic acid purification using microfabricated silicon structures

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.

Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds.

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform.

A simple poly(3,4-ethylene dioxythiophene)/poly(styrene sulfonic acid) transistor for glucose sensing at neutral pH

We demonstrate a simple transistor based on the conducting polymer poly(3,4-ethylene dioxythiophene)/poly(styrene sulfonic acid), capable of sensing glucose in a neutral pH buffer solution by a mechanism involving sensing of hydrogen peroxide.

The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds

Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.

Development of antifouling surfaces to reduce bacterial attachment

It is well documented that bacterial adhesion to surfaces is mediated by the physical and chemical properties of the substrate, as well as the surface characteristics of the organism. Topographical features that limit cell–surface interactions have been shown to reduce surface colonization and biofilm formation. In this study, bacterial attachment to medically relevant materials was evaluated. Our data show that Escherichia coli attachment to glass, silicone, and titanium surfaces was most affected by the surface energy of these materials, as determined by water contact angle. The inherent roughness of the surface, however, was not correlated with cell attachment density. To study the effect of engineered surface roughness on bacterial attachment, topographical features, including arrays of holes and repeating lines/trenches, were formed from silicon wafers and then used as a template to imprint silicone-based polydimethylsiloxane (PDMS). Patterned silicone surfaces were then used in static and microfluidic flow-based experiments to evaluate cellular settlement and attachment. Cell attachment was observed to be strongly dependent upon the topographical features under both static and microfluidic flow conditions. The highest attachment density was observed on flat, un-patterned surfaces, while linear patterned surfaces showed greatly reduced cell attachment. Moreover, surfaces consisting of arrays of holes further reduced cell attachment as compared to linear patterns. These results demonstrate that the size, spacing, and shape of surface features play a significant role in cell–surface attachment and provide insight for the design of surfaces with antifouling properties.

A microchip-based DNA purification and real-time PCR biosensor for bacterial detection

A miniaturized, fully-automated, PCR (polymerase chain reaction)-based detection system has been developed for the rapid detection of bacteria. Monolithic DNA purification/real-time PCR silicon chips were fabricated and tested for their ability to purify and detect the pathogenic bacteria Salmonella typhimurium. Using silica-coated microstructures, nucleic acids could be selectively bound, washed and eluted for subsequent real-time PCR. These microstructures were integrated into a detection microchip containing two distinct regions, one for DNA purification and one for real-time PCR. Using an automated detection platform with integrated microprocessor, pumps, valves, thermocycler and fluorescence detection modules, microchips were used to purify and detect bacterial DNA by real-time PCR amplification using SYBR Green fluorescent dye. As few as 2/spl times/10/sup 3/ S. typhimurium cells could be detected using this system with an average time for detection of 45 min. Detection was augmented by on-chip melting curve analysis capable of differentiating between positive and false-positive results.

Micropatterning of 3D Microenvironments for Living Biosensor Applications.

Micro-scale printing and patterning of living cells has multiple applications including tissue engineering, cell signaling assays, and the fabrication of cell-based biosensors. In this work, a molecular printing instrument, the Bioforce Nano eNabler, was modified to enable micron-scale “quill-pen” based printing of mammalian cells in a 3D hyaluronan/gelatin based hydrogel. Specifically, photo-initiated “thiol-ene” click chemistry was used to couple the thiol groups of thiolated hyaluronan/thiolated gelatin to the alkene groups of 4-arm polyethylene glycol (PEG)-norbornene molecules. Rapid photopolymerization enabled direct printing and controlled curing of living cells within the hydrogel matrix. The resulting hydrogels were biocompatible with human adipose-derived stem cells, NIH-3T3 cells, and mouse embryonic stem cells. The utility of this printing approach was also explored for cell-based biosensors. Micro-printed cells expressing a redox sensitive variant of the green fluorescent protein (roGFP-R12) showed a measurable fluorescent response to addition of oxidizing and then reducing agents. This work represents a novel approach to micron-scale cell patterning, and its potential for living, cell-based biosensors.

Immobilization Mechanisms of Deoxyribonucleic Acid (DNA) to Hafnium Dioxide (HfO2) Surfaces for Biosensing Applications

Immobilization of biomolecular probes to the sensing substrate is a critical step for biosensor fabrication. In this work we investigated the phosphate-dependent, oriented immobilization of DNA to hafnium dioxide surfaces for biosensing applications. Phosphate-dependent immobilization was confirmed on a wide range of hafnium oxide surfaces; however, a second interaction mode was observed on monoclinic hafnium dioxide. On the basis of previous materials studies on these films, DNA immobilization studies, and density functional theory (DFT) modeling, we propose that this secondary interaction is between the exposed nucleobases of single stranded DNA and the surface. The lattice spacing of monoclinic hafnium dioxide matches the base-to-base pitch of DNA. Monoclinic hafnium dioxide is advantageous for nanoelectronic applications, yet because of this secondary DNA immobilization mechanism, it could impede DNA hybridization or cause nonspecific surface intereactions. Nonetheless, DNA immobilization on polycrystalline and amorphous hafnium dioxide is predominately mediated by the terminal phosphate in an oriented manner which is desirable for biosensing applications.

A novel microfluidic device for the in situ optical and mechanical analysis of bacterial biofilms.

Viable methods for bacterial biofilm remediation require a fundamental understanding of biofilm mechanical properties and their dependence on dynamic environmental conditions. Mechanical test data, such as elasticity or adhesion, can be used to perform physical modelling of biofilm behaviour, thus enabling the development of novel remediation strategies. To achieve real-time, dynamic measurements of these properties, a novel microfluidic flowcell device has been designed and fabricated for in situ analysis using atomic force microscopy (AFM). The flowcell consists of microfluidic channels for biofilm establishment that are then converted into an open architecture, laminar flow channel for AFM measurement in a liquid environment. Finite element analysis (FEA) was used to profile fluid conditions within the flowcell during biofilm establishment. Force-mode AFM was used to measure the elastic properties of mature Pseudomonas aeruginosa PAO1 biofilms as well as polyacrylamide hydrogels. Elastic moduli ranging from 0.58 to 2.61kPa were determined for the mature biofilm, which fall within the range of moduli previously reported by optical, rheometric, and microindentation techniques. These results demonstrate the validity of the microfluidic flowcell system as an effective platform for future investigations of biofilm mechanical and morphological response to dynamic environmental conditions.