Cited 65 times in

Optimization of phosphatase- and redox cycling-based immunosensors and its application to ultrasensitive detection of troponin I.

Title
 Optimization of phosphatase- and redox cycling-based immunosensors and its application to ultrasensitive detection of troponin I.
Authors
 Md. Rajibul Akanda; Md. Abdul Aziz; Haesik Yang; Sinyoung Kim; Myung Ho Hyun; Vellaiappillai Tamilavan; Kyungmin Jo
Issue Date
2011
Journal Title
 Analytical Chemistry
ISSN
 0003-2700
Citation
 Analytical Chemistry, Vol.83(10) : 3926~3933, 2011
Abstract
The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium-tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl(2), and the optimum conditions for antigen-antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen-antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (L-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (L-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/93234
DOI
10.1021/ac200447b
Appears in Collections:
1. 연구논문 > 1. College of Medicine > Dept. of Laboratory Medicine
Yonsei Authors
사서에게 알리기
  feedback
Link
 http://pubs.acs.org/doi/full/10.1021/ac200447b
Export
RIS (EndNote)
XLS (Excel)
XML

qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse