Enzyme-amplified electrochemical detection of DNA using electrocatalysis of ferrocenyl-tethered dendrimer.
We have developed a sandwich-type enzyme-linked DNA sensor as a new electrochemical method to detect DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of DNA detection as well as a building block to immobilize capture probes. Fc-D was immobilized on a carboxylic acid-terminated self-assembled monolayer (SAM) by covalent coupling of unreacted amine in Fc-D to the acid. Thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bifunctional linker. The target DNA was hybridized with the capture probe, and an extension in the DNA of the target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate the electroactive label, p-aminophenol, from p-aminophenyl phosphate enzymatically. p-Aminophenol diffuses into the Fc-D layer and is then electrocatalytically oxidized by the electronic mediation of the immobilized Fc-D, which leads to a great enhancement in signal. Consequently, the amount of hybridized target can be estimated using the intensity of electrocatalytic current. This DNA sensor exhibits a detection limit of 20 fmol. Our method was also successfully applied to the sequence-selective discrimination between perfectly matched and single-base mismatched target oligonucleotides.
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea.
Eunkyung
E
Kim
Kyuwon
K
Kim
Haesik
H
Yang
Youn Tae
YT
Kim
Juhyoun
J
Kwak
D000469:Alkaline Phosphatase / Q000378:metabolism; D000627:Aminophenols / Q000737:chemistry / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D001360:Avidin / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D019921:Biotinylation / Q000379:methods; D002138:Calibration; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000138:chemical synthesis; D050091:Dendrimers; D004563:Electrochemistry; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D000075163:Metallocenes; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010084:Oxidation-Reduction; D011073:Polyamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared
D000627:Aminophenols; D000814:Aniline Compounds; D003432:Cross-Linking Reagents; D015342:DNA Probes; D050091:Dendrimers; D005296:Ferrous Compounds; D000075163:Metallocenes; D009943:Organophosphorus Compounds; C104700:PAMAM Starburst; D011073:Polyamines; D001360:Avidin; C068798:4-aminophenylphosphate; D006046:Gold; D004247:DNA; D000469:Alkaline Phosphatase; C026729:4-aminophenol; C004998:ferrocene