Sensitive fluorometric determination of platelet-derived growth factor BB and avian influenza A virus DNA via dual signal amplification using the hybridization chain reaction and glucose oxidase assisted recycling
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A method is described for fluorometric determination of platelet-derived growth factor BB (PDGF-BB) and avian influenza A (H1N1) virus DNA. It is based on the use of the hybridization chain reaction (HCR) and of glucose oxidase (GOx) assisted dual-recycling amplification. A silver coated glass slide (SCGS) serves as an ideal material for separation. A signal DNA/initiator triggers the HCR and generates a cascade of hybridization to form a nicked double-helix polymer. Upon addition of the analytes (PDGF-BB or H1N1 DNA) and capture DNA immobilized on the SCGS, the nicked double-helix polymer binds on the surface of the SCGS through formation of a [capture DNA/analyte/signal DNA] sandwich structure. The GOx-biotin-streptavidin (SA) complexes were then attached to the nicked double-helix polymer through SA-biotin interaction. After cleavage by DNase I, the bound GOx is transferred into the buffer. Glucose is added and enzymatically oxidized to produce H2O2. The H2O2 formed oxidizes the substrate 3-(p-hydroxyphenyl)-propanoic acid to give a blue fluorescent product (with excitation/emission maxima at 320/416 nm) under the catalysis of horseradish peroxidase. Under optimal conditions, fluorescence increases linearly in the 0.5 to 70 pmol·L−1 PDGF-BB concentration range, and the detection limit is 191 fmol·L−1. For the H1N1 virus DNA, the respective data are 2.5 to 300 pmol·L−1 and 826 fmol·L−1.
KeywordsBiomolecules detection Silver coated glass slide 3-(p-Hydroxyphenyl)-propanoic acid Horseradish peroxidase DNase I
This work is supported by the Innovation Strong School Project of Guangdong education department (No. Q18291), the Non-funded Scientific and Technological Research Projects in Zhanjiang City (No. 2018B01005) and the program for scientific research start-up funds of Guangdong Ocean University (No. R17013).
Compliance with ethical standards
All the experiments are carried out following the relevant laws and institutional guidelines, and ethical standards.
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