Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems

An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

<p>Abstract</p> <p>Background</p> <p>Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches.</p> <p>Results</p> <p>In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA) will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay.</p> <p>Conclusions</p> <p>By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at <url>http://www.laurenzi.net</url>.</p

Optoelectronic Detection of DNA Molecules Using an Amorphous Silicon Photodetector

AbstractThis work demonstrates the use of an amorphous silicon (a-Si:H) photodetector to measure the density of covalently-bound DNA molecules tagged with a fluorescent molecule. This device is based on the photoconductivity of a-Si:H in a coplanar electrode configuration. Excitation of a fluorescently-tagged biomolecule with near UV/blue light results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the detection of the presence of the tagged DNA molecules. The design, fabrication and characterization of this integrated a-Si:H-based bio-detector is described. The detection limit of the present device is of the order of 20 pmol/cm2. A surface density of ≤ 30 pmol/cm2 for DNA covalently-bound to an active silica layer was measured with the a-Si:H-based bio-detector.</jats:p

Optoelectronic detection of DNA molecules using an amorphous silicon photodetector

AbstractThis work demonstrates the use of an amorphous silicon (a-Si:H) photodetector to measure the density of covalently-bound DNA molecules tagged with a fluorescent molecule. This device is based on the photoconductivity of a-Si:H in a coplanar electrode configuration. Excitation of a fluorescently-tagged biomolecule with near UV/blue light results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the detection of the presence of the tagged DNA molecules. The design, fabrication and characterization of this integrated a-Si:H-based bio-detector is described. The detection limit of the present device is of the order of 20 pmol/cm2. A surface density of ≤ 30 pmol/cm2 for DNA covalently-bound to an active silica layer was measured with the a-Si:H-based bio-detector.</jats:p

Covalent immobilization of DNA and hybridization on microchips by microsecond electric field pulses

AbstractSingle square voltage pulses were used to enhance by 7 and 9 orders of magnitude the rate of covalent immobilization and hybridization, respectively, of single stranded DNA probes on a chemically functionalized thin film surface (silicon dioxide) using 2 mm size electrodes. These electrodes were scaled down to 20 μm. Photolithography was used to define the electrode voltage line, ground line, and functionalized thin-film area on a plastic substrate (polyimide). At all electrode dimensions, electric field-assisted DNA immobilization and hybridization can be achieved in the microsecond time scale, far faster than the 2 hr or 16 hr needed for immobilization and hybridization, respectively, without the electric field. Pulse conditions optimized with the large-size electrodes (2 mm) were used in the microelectrodes.</jats:p