title: Glass-composite prototyping for flow PCR with in situ DNA analysis.
abstract: In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 microl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 microl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (PhiX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.
authors.affiliation: Institute for Micromanufacturing, Louisiana Tech University, Ruston, LA, USA.
authors.forename: Ilija
authors.initials: I
authors.lastname: Pjescić
authors.forename: Collin
authors.initials: C
authors.lastname: Tranter
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Hindmarsh
authors.forename: Niel D
authors.initials: ND
authors.lastname: Crews
mesh_terms: D004247:DNA / Q000032:analysis* / Q000235:genetics; D005453:Fluorescence; D005820:Genetic Testing; D005898:Glass; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D055585:Physical Phenomena; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D013696:Temperature
chemical_list: D004247:DNA