Ultrasensitive electrochemical immunosensor for HE4 based on rolling circle amplification.
A novel electrochemical immunoassay system for the detection of human epididymis-specific protein 4 (HE4) was developed. A chitosan-titanium carbide (TiC) nanocomposition film was first electrodeposited onto a tin-doped indium oxide (ITO) electrode at a constant potential. Gold (Au) nanoparticles were then electrodeposited on the surface of the chitosan-TiC film by cyclic voltammetry (CV). The capture antibody (anti-HE4) was adsorbed onto the Au and TiC nanoparticles. After a specific sandwich immunoreaction among the capture antibody, HE4, and biotinylated secondary antibody, biotinylated primer DNA was immobilized on the secondary antibody by biotin-streptavidin system. Appropriate amounts of circular template DNA and biotinylated primer DNA were used for rolling circle amplification (RCA) under optimal conditions. The RCA products provided a large number of sites to link DNA detection probes. Doxorubicin hydrochloride intercalated the CG-GC steps between the RCA products and the DNA detection probes, which was monitored by differential pulse voltammetry (DPV) based on the current signal of doxorubicin hydrochloride. With the above-mentioned amplification factors, the current responded to HE4 linearly in the concentration range of 3-300 pM under optimal detection conditions, with a detection limit of 0.06 pM. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical impedance spectroscopy (EIS). This system was successfully employed for the detection of HE4 with good accuracy and renewable ability.
Key Laboratory of Medical Diagnostics of Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China.
Lingsong
L
Lu
Bei
B
Liu
Zhaohui
Z
Zhao
Cuixia
C
Ma
Peng
P
Luo
Chenggui
C
Liu
Guoming
G
Xie
D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D002138:Calibration; D058266:Dielectric Spectroscopy; D055664:Electrochemical Techniques / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D018625:Microscopy, Atomic Force; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000081102:WAP Four-Disulfide Core Domain Protein 2
D011506:Proteins; D000081102:WAP Four-Disulfide Core Domain Protein 2; C569698:WFDC2 protein, human