Enzyme-based colorimetric detection of nucleic acids using peptide nucleic acid-immobilized microwell plates.
The development of label-free or nonlabeling assays for nucleic acids is important in basic biological research and biomedical diagnosis. In this study, we have developed an enzyme-based colorimetric assay for nucleic acids, which combines the robustness of nonlabeling of DNA and RNA samples and the adequate sensitivity of enzymatic reactions. The core of this assay is the use of neutral peptide nucleic acid (PNA) as capture probe and the electrostatic adsorption of horseradish peroxidase (HRP) on hybridized, negatively charged nucleic acids to report the hybridization events, through HRP-catalyzed color reactions of 3,3',5,5'-tetramethylbenzidine and H(2)O(2). The proposed assay has been validated with fully complementary and single base-mismatched DNAs of different chain lengths. The proposed assay has also been validated with total RNA samples extracted from two human cancer cell lines (A 549 lung cancer cell and HeLa cell) for microRNA detection in real samples. Through extensive optimizations of HRP adsorption and nucleic acid hybridization conditions, detection limits of 0.1-0.2 nM for DNA (depending on chain length) and approximately 2 microg of total RNA have been achieved. Surface plasmon resonance spectroscopy has been used to elucidate the HRP adsorption and PNA-nucleic acid hybridizations through real-time measurements and to provide guidance for the development of the colorimetric assay.
Su
Xiaodi
X
Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602. xd-su@imre.a-star.edu.sg
Teh
Huey Fang
HF
Lieu
Xiaohui
X
Gao
Zhiqiang
Z
D001483:Base Sequence; D003124:Colorimetry / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006801:Humans; D008832:Microchemistry; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D012313:RNA / Q000032:analysis; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis; D020349:Surface Plasmon Resonance
D004800:Enzymes, Immobilized; D009696:Nucleic Acids; D020135:Peptide Nucleic Acids; D012313:RNA