Immobilized molecular beacons: a new strategy using UV-activated poly(methyl methacrylate) surfaces to provide large fluorescence sensitivities for reporting on molecular association events.
We have designed appropriately prepared solid supports consisting of poly(methyl methacrylate) (PMMA) that provide enhanced performance levels for molecular beacons (MBs) that are used for recognizing and reporting on signature DNA sequences in solution. The attachment of primary amine-containing MBs to the PMMA surface was carried out by UV activating the PMMA to produce surface-confined carboxylate groups, which could then be readily coupled to the MBs using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) chemistry. The fluorescence properties of the MBs covalently attached onto this UV-activated PMMA surface were evaluated and compared with the same MBs immobilized onto glass supports. We observed improved limits of detection for the solution complement to the MBs when immobilized onto PMMA, and this was attributed to both the lower autofluorescence levels exhibited by PMMA at the detection wavelengths used and the improved quenching efficiency of the MBs when in their closed hairpin configuration when strapped to a PMMA surface as opposed to glass. As an example of the utility of the PMMA-based immobilization strategies developed for MBs, we report on the analysis of complementary DNAs specific for fruitless (fru) and Ods-site homeobox (OdsH) genes extracted from Drosophila melanogaster fruit flies. The fru gene functions in the central nervous system, where it is necessary for sex determination and male courtship behavior, whereas the OdsH gene is involved in the regulation of transcription.
Situma
Catherine
C
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Moehring
Amanda J
AJ
Noor
Mohamed A F
MA
Soper
Steven A
SA
D000818:Animals; D016587:Antimutagenic Agents / Q000737:chemistry* / Q000528:radiation effects; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism; D044085:Microfluidics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D019904:Polymethyl Methacrylate / Q000737:chemistry* / Q000528:radiation effects; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D016587:Antimutagenic Agents; D015342:DNA Probes; D018076:DNA, Complementary; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; C115884:OdsH protein, Drosophila; D014157:Transcription Factors; C103226:fru protein, Drosophila; D019904:Polymethyl Methacrylate