Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles.
We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months.
Department of Biological Sciences, KAIST, Daejeon, Republic of Korea.
Yunxian
Y
Piao
Zongwen
Z
Jin
Dohoon
D
Lee
Hye-Jin
HJ
Lee
Hyon-Bin
HB
Na
Taeghwan
T
Hyeon
Min-Kyu
MK
Oh
Jungbae
J
Kim
Hak-Sung
HS
Kim
D000363:Agaricales / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis* / Q000276:immunology; D008280:Magnetics; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D037742:Nanotubes, Carbon / Q000737:chemistry / Q000648:ultrastructure; D012680:Sensitivity and Specificity
D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D037742:Nanotubes, Carbon; D014442:Monophenol Monooxygenase