Backfilling rolling cycle amplification with enzyme-DNA conjugates on antibody for portable electrochemical immunoassay with glucometer readout

被引:38
作者
Ge, Lilin [1 ,2 ]
Li, Bin [1 ,2 ]
Xu, Houxi [1 ]
Pu, Wenyuan [1 ]
Kwok, Hang Fai [2 ]
机构
[1] Nanjing Univ Chinese Med, State Key Lab Cultivat Base TCM Qual & Efficacy, 138 Xianlin Ave, Nanjing 210046, Jiangsu, Peoples R China
[2] Univ Macau, Fac Hlth Sci, Inst Translat Med, Ave Univ, Taipa, Macao, Peoples R China
基金
中国国家自然科学基金;
关键词
Electrochemical immunoassay; Rolling cycle amplification; Glucometer readout; Enzyme-DNA conjugates; Disease-related biomarker; MESOPOROUS SILICA NANOCONTAINERS; PERSONAL GLUCOSE METERS; ALPHA-FETOPROTEIN; ULTRASENSITIVE DETECTION; DISPLACEMENT REACTION; RELEASE; IMMUNOSENSOR; STRATEGY; ANTIGEN; PROTEIN;
D O I
10.1016/j.bios.2019.02.051
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
A simple and feasible electrochemical immunosensing protocol with glucometer readout was designed for the detection of low-abundance disease-related biomarker (alpha-fetoprotein; AFP) on the basis of backfilling rolling cycle amplification (RCA) with invertase-DNA(2) conjugates on the detection antibody. The assay consisted of the immunoreaction, RCA reaction, DNA(2)-invertase hybridization and glucose measurement. Initially, a sandwiched immunoreaction was carried out between anti-AFP capture antibody-coated microplate between nanogold-labeled pAb(2) detection antibody conjugated with DNA). primer (DNA(1)-AuNP-pAb(2)) in the presence of target ATP. Thereafter, the carried primers triggered the RCA reaction in the presence of circular DNA template, polymerase and dNTP, to produce numerous repeated oligonucleotide sequences for hybridization with many invertase-DNA(2) conjugates. The carried invertase molecules accompanying the hybridization reaction hydrolyzed sucrose into glucose, thereby resulting in the amplification of the detectable signal on a handheld personal glucometer (PGM). Under optimum conditions, the developed immunoassay exhibited high sensitivity for the quantitative screening of AFP within a dynamic range of 0.1-100 ng mL(-1) at a low detection limit of 0.087 ng mL(-1) Other biomarkers and proteins did not interfere the signals of this system. In addition, this method was utilized to determine human serum samples containing target AFP, and received well-matched results with the referenced enzyme-linked immunosorbent assay (ELISA) method.
引用
收藏
页码:210 / 216
页数:7
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