Electrochemical mixed aptamer-antibody sandwich assay for mucin protein 16 detection through hybridization chain reactio
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RESEARCH PAPER
Electrochemical mixed aptamer-antibody sandwich assay for mucin protein 16 detection through hybridization chain reaction amplification Lingsong Lu 1
&
Bei Liu 2 & Jianhang Leng 1 & Xiao Ma 1 & Huihui Peng 1
Received: 23 June 2020 / Revised: 20 July 2020 / Accepted: 27 July 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract A mixed aptamer-antibody sandwich assay for the determination of mucin protein 16 (MUC16) was developed based on hybridization chain reaction (HCR) with methylene blue (MB) as an electrochemical indicator. First, MUC16 antibody was adsorbed onto the surface of the Au nanoparticle (AuNP)-modified indium tin oxide (ITO) electrode to effectively capture the target MUC16. After MUC16 was captured by the MUC16 aptamer, an antibody/MUC16/aptamer sandwich structure formed for the highly selective detection of MUC16. The 3′ end of the aptamer was then subjected to HCR with the assistance of auxiliary probes to obtain DNA concatemers. Numerous MB molecules bonded with G bases in the DNA concatemers by immersing the modified ITO electrode into a stirred solution containing MB with KCl. Stepwise changes in the microscopic features of the electrode surface were studied by scanning electron microscopy (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the electrochemical behavior of the different modified electrodes. The oxidation current of MB was detected by differential pulse voltammetry (DPV). Under the optimum conditions, the proposed mixed aptamer-antibody sandwich assay showed wide dynamic range from 0.39 to 200 unit mL−1 with a low detection limit of 0.02 unit mL−1 (S/N ratio = 3). The proposed method showed good accuracy, selectivity, and acceptable reproducibility. Keywords Aptamer . Immunoassay . Hybridization chain reaction . Methylene blue . Mucin protein 16
Introduction The human mucin family member MUC16, known as cancer antigen 125 (CA 125), is a heavily glycosylated protein expressed in low levels by many normal epithelial cells, including ocular, respiratory, and female reproductive tract epithelia [1, 2]. However, the poorly glycosylated form is Lingsong Lu and Bei Liu contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02849-5) contains supplementary material, which is available to authorized users. * Lingsong Lu [email protected] 1
Department of Central Laboratory, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, Zhejiang, China
2
Department of Reproductive Genetics, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, Zhejiang, China
overexpressed by many adenocarcinomas, such as breast and ovarian cancers [3, 4]. Accordingly, MUC16 is an important biomarker used to diagnose and monitor the therapeutic and recurrence of ovarian cancer [5]. Commercial enzyme-linked immunosorbent assay (ELISA) kits with acceptable sensitivity and
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