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  • Title: An enzyme-free electrochemical biosensor for simultaneous detection of two hemophilia A biomarkers: Combining target recycling with quantum dots-encapsulated metal-organic frameworks for signal amplification.
    Author: Rezaei H, Motovali-Bashi M, Radfar S.
    Journal: Anal Chim Acta; 2019 Dec 27; 1092():66-74. PubMed ID: 31708034.
    Abstract:
    A sensitive and selective electrochemical method for simultaneous detection of two hemophilia A-related microRNAs (miR-1246 and miR-4521) was developed. This detection tactic was based on gold nanoparticles (AuNPs), heavy metals quantum dots-encapsulated metal-organic frameworks (QDs@ZIF-8), and catalytic hairpin assembly (CHA) for signal application. Primarily, hairpins H1 and H2 were hybridized with targets miR-1246 (T1) and miR-4521 (T2) for forming H1-T1 and H2-T2 duplex stranded DNAs (dsDNAs) that were able to open the hairpins H3 and H4 for the formation of H1-H3 and H2-H4 dsDNAs. Meanwhile, lots of H1-H3 and H2-H4 dsDNAs were created by releasing the target to take part in the next cycle for signal amplification. And then single stranded fragments of H1-H3 and H2-H4 dsDNAs were utilized for hybridizing the PbS@ZIF-8-S1 and CdS@ZIF-8-S2 in order to amplify the electrochemical signal. The diagnosis of corresponding target miRs using differential pulse voltammetry has been possible by releasing Pb (II) and Cd (II) ions from PbS@ZIF-8 and CdS@ZIF-8 tags by HCI leaching. In this context, encapsulation of heavy metals quantum dots (QDs) was done in zeolitic imidazolate framework-8 (ZIF-8) to form QDs@ZIF-8 muti-core-shell particles by in situ growth of ZIF-8 in the presence of QDs. Since the quantity of QDs tagged to each target miRs grows massively, being resulted from a huge number of QDs that encapsulated in each QDs@ZIF-8 label, the sensitivity of the biosensor using QDs@ZIF-8 particles as signal tags is about 15 times that of a biosensor using QDs as signal tags. Several conditions of determination like incubation time for labeling and capture probe, HCl leaching time, and reaction time of CHA were optimized. Under the optimized conditions, this assay allowed the detection of target miRs in the range of 1 fM to 1  μM with detection limits of 0.19 fM and 0.28 fM for miR-1246 and miR-4521 (S/N = 3). The biosensor can discriminate complementary, 1-base mismatched and non-complementary sequences quite well, according to the catalytic hairpin assembly. Furthermore, the biosensor was utilized efficiently for quick and direct analysis of microRNAs in human serum. Thus, this tactic presents an innovative platform for microRNAs expression profiling in biomedical research and clinical diagnosis.
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