title: Ultrasensitive electrochemical DNA assay based on counting of single magnetic nanobeads by a combination of DNA amplification and enzyme amplification.
abstract: An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 degrees C. AP on the AP-p2-DNA-p1-DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 x 10(-16) to 1.0 x 10(-13) mol/L.
authors.affiliation: School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Zhang
authors.forename: Linlin
authors.initials: L
authors.lastname: Li
authors.forename: Lu
authors.initials: L
authors.lastname: Li
authors.forename: Jia
authors.initials: J
authors.lastname: Chen
authors.forename: Guizheng
authors.initials: G
authors.lastname: Zou
authors.forename: Zhikun
authors.initials: Z
authors.lastname: Si
authors.forename: Wenrui
authors.initials: W
authors.lastname: Jin
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry*; D055664:Electrochemical Techniques; D004563:Electrochemistry / Q000379:methods*; D017403:In Situ Hybridization; D008280:Magnetics; D021141:Nucleic Acid Amplification Techniques
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; C036483:T-DNA; D004247:DNA