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A novel and in situ amplified immunoassay strategy with quadruple signal amplification was designed for highly efficient electrochemical detection of low-abundance proteins (carcinoembryonic antigen, CEA, as a model) by using nanogold-functionalized DNAzyme concatamers with redox-active intercalators. To construct such an in situ amplification system, streptavidin-labeled gold nanoparticles (AuNP-SA) were initially used for the labelling of initiator strands (So) and detection antibody (mAb(2)) with a large ratio (mAb(2)-AuNP-S-0), and then two auxiliary DNA strands S-1 and S-2 were designed for in situ propagation of DNAzyme concatamers with the hemin/G-quadruplex format. The quadruple signal amplification was implemented by using the avidinbiotin chemistry, nanogold labels, DNA concatamers, and DNAzymes. In the presence of target CEA, the sandwiched immunocomplex was formed between the immobilized primary antibodies on the electrode and the conjugated detection antibodies on the mAb(2)-AuNP-S-0. The carried So initiator strands could progress a chain reaction of hybridization events between alternating S-1/S-2 DNA strands to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers could be bound to form the hemin/Gquadruplex-based DNAzymes. The formed double-helix DNA polymers could cause the intercalation of numerous electroactive methylene blue molecules. During the electrochemical measurement, the formed DNAzymes could catalyze the reduction of H2O2 in the solution to amplify the electrochemical signal of the intercalated methylene blue. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of 1.0 fg mL(-1) to 20 ng mL(-1) toward CEA standards with a low detection limit of 0.5 fg Intra-assay and inter-assay coefficients of variation (CV) were less than 8.5% and 11.5%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 14 clinical serum specimens between the developed immunoassay and commercialized electrochemiluminescent (ECL) method for detection of CEA.
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ACS APPLIED MATERIALS & INTERFACES
ISSN: 1944-8244
Year: 2013
Issue: 7
Volume: 5
Page: 2773-2781
5 . 9
JCR@2013
8 . 5 0 0
JCR@2023
ESI Discipline: MATERIALS SCIENCE;
JCR Journal Grade:1
CAS Journal Grade:1
Cited Count:
WoS CC Cited Count: 64
SCOPUS Cited Count: 65
ESI Highly Cited Papers on the List: 0 Unfold All
WanFang Cited Count:
Chinese Cited Count:
30 Days PV: 1
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