title: PMMA biosensor for nucleic acids with integrated mixer and electrochemical detection.
abstract: This paper discusses the design, microfabrication and use of an electrochemical biosensor based on a polymer substrate for cost effectiveness and disposability. As model analyte, amplified hsp70 mRNA from Cryptosporidium parvum was chosen. Microfluidic channels were fabricated in poly(methyl methacrylate) (PMMA) using hot embossing with a copper master. The electrochemical transducer, an interdigitated ultramicroelectrode array (IDUA) was also realized directly on the PMMA surface. First, the unstructured PMMA surface was UV functionalized. An 8 min UV treatment resulted in a carboxylic acid density of approximately 8 nmol/cm(2) on the PMMA surface. The surface carboxylic acid groups were then conjugated to cystamine using water-soluble carbodiimide chemistry. Gold (200 nm) was then evaporated onto the thiol-functionalized surface. Using standard photolithography techniques, the IDUA containing 10 microm wide electrodes with 5 microm gaps was then formed followed by a gold etch. The PMMA surface containing the microchannel was subsequently bonded to the PMMA surface containing the IDUA using UV-assisted thermal bonding. The additional UV treatment also served to decrease the water contact angle of the surface from 62.5 degrees +/-0.7 degrees to 48.4 degrees +/-0.2 degrees thus, aiding with the capillary flow in the device. The hsp70 mRNA was isolated from C. parvum oocysts and amplified using nucleic acid sequence-based amplification (NASBA). The amplicon was detected in a sandwich hybridization assay with capture probe-coated superparamagnetic beads and reporter probe-tagged liposomes. The liposomes entrapped potassium ferro/ferrihexacyanide to enable amperometric quantification of the amplicon on the IDUA. Amplified mRNA from only 1 oocyst was detectable with this PMMA biosensor. The final detection device measured approximately 10 mm x 40 mm x 3 mm and contained two detection channels for dual analyses.
authors.affiliation: Department of Biological & Environmental Engineering, Cornell University, Ithaca, NY 14853, United States.
authors.forename: Sam R
authors.initials: SR
authors.lastname: Nugen
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Asiello
authors.forename: John T
authors.initials: JT
authors.lastname: Connelly
authors.forename: Antje J
authors.initials: AJ
authors.lastname: Baeumner
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D017076:Computer-Aided Design; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018511:Systems Integration
chemical_list: D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D019904:Polymethyl Methacrylate