Enhancement of Adenovirus-Mediated Gene Delivery by Use of an Oligopeptide with Dual Binding Specificity

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Validation of a centrifugal microfluidic sample lysis and homogenization platform for nucleic acid extraction with clinical samples

The applications of microfluidic technologies in medical diagnostics continue to increase, particularly in the field of nucleic acid diagnostics. While much attention has been focused on the development of nucleic acid amplification and detection platforms, sample preparation is often taken for granted or ignored all together. Specifically, little or no consideration is paid to the development of microfluidic systems that efficiently extract nucleic acids from biological samples. Here, a centrifugal microfluidic platform for mechanical sample lysis and homogenization is presented. The system performs sample lysis through a magnetically actuated bead-beating system followed by a centrifugal clarification step. The supernatant is then transferred for extraction using a unique siphon. Several other new microfluidic functions are implemented on this centrifugal platform as well, including sample distribution, a unique hydraulic capillary valve, and self-venting. Additionally, the improved system has features with a small footprint designed specifically for integration with further downstream processing steps. Biological validation of the platform is performed using Bacillus subtilis spores and clinical samples (nasopharyngeal aspirates) for respiratory virus detection. The platform was found to be as efficient as in-tube bead-beating lysis and homogenization for nucleic acid extraction, and capable of processing 4 samples in batch to near PCR-ready products in under 6 min

A low-cost, disposable card for rapid polymerase chain reaction

A low-cost, disposable card for rapid polymerase chain reaction (PCR) was developed in this work. Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated with structured polycarbonate films to form microreactors in a card format. Ice valves [1] were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and investigate the thermal gradient in the reaction chamber in the direction of sample thickness. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 copy of E. coli tuf gene in 27 min.close151

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\u2122 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.Peer reviewed: YesNRC publication: Ye

CD (compact disc)-based DNA hybridization and detection

A DNA hybridization and detection unit was developed for a compact disc (CD) platform. The compact disc was used as the fluidic platform for sample and reagent manipulation using centrifugal force. Chambers for reagent storage and conduits for fluidic functions were replicated from polydimethylsiloxane (PDMS) using an SU-8 master mold fabricated with a 2-level lithography process we developed specially for the microfluidic structures used in this work. For capture probes, we used self-assembled DNA oligonucleotide monolayers (SAMs) on gold pads patterned on glass slides. The PDMS flow cells were aligned with and sealed against glass slides to form the DNA hybridization detection units. Both an enzymatic-labeled fluorescence technique and a bioluminescent approach were used for hybridization detection. An analytical model was introduced to quantitatively predict the accumulation of hybridized targets. The flow-through hybridization units were tested using DNA samples (25-mers) of different concentrations down to 1 pM and passive assays (no flow), using samples of the same concentrations, were performed as controls. At low concentrations, with the same hybridization time, a significantly higher relative fluorescence intensity was observed in both enzymatic and bioluminescent flow-through assays compared to the corresponding passive hybridization assays. Besides the fast hybridization rate, the CD-based method has the potential for enabling highly automated, multiple and self-contained assays for DNA detection

Detection of unamplified target genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids