Gerardo A. Diaz-Quijada

Surface modification of thermoplastics—towards the plastic biochip for high throughput screening devices

Microarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is great interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) Zeonor 1060R and Zeonex E48R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotides were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.

Microfluidic patterning of miniaturized DNA arrays on plastic substrates.

This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two-dimensional microcapillary system (muCS). Fluidic structures were realized through hot-embossing lithography using Versaflex CL30. Like elastomers based on poly(dimethylsiloxane), this thermoplastic block copolymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on muCSs that support arrays comprising up to 2 x 48 spots, each being 45 mum in diameter. Substrates were fabricated from two hard thermoplastic materials, poly(methylmethacrylate) and a polycyclic olefin (e.g., Zeonor 1060R), which were both activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25-32 muM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate-buffered saline, allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at 22, 30, and 40 degrees C to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of the liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual strands when exposed to a specific ssDNA target molecule.

Low Thrombogenicity Coating of Nonwoven PET Fiber Structures for Vascular Grafts

Vascular PET grafts (Dacron) have shown good performance in large vessels (≥ 6 mm) applications. To address the urgent unmet need for small-diameter (2-6 mm) vascular grafts, proprietary high-compliance nonwoven PET fiber structures were modified with various PEG concentrations using PVA as a cross-linking agent, to fabricate non-thrombogenic mechanically compliant vascular grafts. The blood compatibility assays measured through platelet adhesion (SEM and mepacrine dye) and platelet activation (morphological changes, P-selectin secretion, and TXB2 production) demonstrate that functionalization using a 10% PEG solution was sufficient to significantly reduce platelet adhesion/activation close to optimal literature-reported levels observed on carbon-coated ePTFE.

From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

ABSTRACT Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations.

Stretching the Stamp: A Flexible Approach to the Fabrication of Miniaturized DNA Arrays

DNA arrays have emerged as key tools in genomic research to measure relative expression levels of genes among different samples in parallel andwithminimal sample consumption. In addition, they are also becoming popular in other areas such as biomedical diagnostics, cellular analysis, and drug development. DNA arrays typically comprise a set of synthesized gene sequences immobilized on a solid support, which are subjected to hybridization with specific target genes. In most cases, a fluorescence marker such as Cy3 (lex1⁄4 550 nm, lem1⁄4 570 nm) or Cy5 (lex1⁄4 650 nm, lem1⁄4 670 nm) is linked to the target DNA, which allows for the detection of hybridization events throughfluorescence readoutof thearray.ThenumberofDNA probes per surface area is an important criterion for their efficacy: the higher their density, the more information can be extracted from a single experiment. DNA microarrays are commonly fabricated by spotting minute amounts of DNA solutiononto aproper substrate byusing eithermetal pins or microactuated dispensing systems. To this end, the resolution of most commercial spotting techniques is limited to arrays containing 100 to 5000 spots per cm depending on the specificationsof the tools, the conditionsof spotting, and the characteristics of the substrate. Arrays of higher density (e.g., spots 100 nm in diameter or below) have been realized by using cantilever probes, yet reliability and throughput of this technology need to be improved before it may become suitable for production purposes. An alternative fabrication scheme is in situ synthesis, which employs light-sensitive chemistry in conjunction with photolithographic techniques to construct a

Trace amount formaldehyde gas detection for indoor air quality monitoring

Formaldehyde is not only a carcinogenic chemical, but also causes sick building syndrome. Very small amounts of formaldehyde, such as those emitted from building materials and furniture, pose great concerns for human health. A Health Canada guideline, proposed in 2005, set the maximum formaldehyde concentration for long term exposure (8-hours averaged) as 40 ppb (50 μg/m3). This is a low concentration that commercially available formaldehyde sensors have great difficulty to detect both accurately and continuously. In this paper, we report a formaldehyde gas detection system which is capable of pre-concentrating formaldehyde gas using absorbent, and subsequently thermally desorbing the concentrated gas for detection by the electrochemical sensor. Initial results show that the system is able to detect formaldehyde gas at the ppb level, thus making it feasible to detect trace amount of formaldehyde in indoor environments.

Picoliter wells from selective growth of HEK293 cells on chemically modified PDMS surfaces.

In this study, a method for the rapid generation of a variety of bifunctional surfaces that can serve to quickly determine the selective adhesion of HEK293 cells towards different chemical functionalities has been established. Using the information about selective adhesion of HEK293 cells to bifunctional surfaces, we demonstrate the ability to construct stable, high density, and multi-welled surfaces where the mammalian cells form the walls of picoliter volume wells.

Towards Low Cost Disposable High Throughput Screening Devices

Microarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is a high interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) and Zeonor™ 1060R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotide were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.