title: Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations.
abstract: We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.
authors.affiliation: Center for Bio-Modular Multi-Scale Systems, Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Mateusz L
authors.initials: ML
authors.lastname: Hupert
authors.forename: Michael C
authors.initials: MC
authors.lastname: Murphy
authors.forename: Steven A
authors.initials: SA
authors.lastname: Soper
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Cheng
authors.forename: Francis
authors.initials: F
authors.lastname: Barany
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D006824:Hybridization, Genetic; D044085:Microfluidics / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017354:Point Mutation / Q000235:genetics*; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D012826:Silicone Elastomers / Q000737:chemistry
chemical_list: D012826:Silicone Elastomers; D019904:Polymethyl Methacrylate