We introduce here a novel assay for the detection of platelet-derived growth factor BB (PDGF-BB) via hybridization chain reaction (HCR) based on an aptameric system, where stable DNA monomers assemble only upon exposure to a target PDGF-BB aptamer. In this process, two complementary stable species of biotinylated DNA hairpins coexist in solution until the introduction of initiator aptamer strands triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers. In detail, the aptamer firstly opens the hairpins in the solution, creating long concatemers, and then reacts with the antibody captured PDGF-BB on the well surface. Moreover, several experimental conditions including different PDGF-BB aptamers, the spacer length of the selected aptamer and hairpin, etc. are investigated and optimized. Our results show that the coupling of HCR to aptamer triggers for the amplification detection of PDGF-BB achieves a better performance in the fluorescence detection of PDGF-BB as compared to the traditional antibody-antigen-aptamer assays. Upon modification, the approach presented herein could be extended to detect other types of targets. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment and environmental monitoring.
Herein we report on the development of instantaneous derivatization technology for the homogeneous and simultaneous detection of multiple PCR amplicons specific to the Hepatitis B Virus (HBV) by using three carriers: magnetic beads, polystyrene beads, and thermo-sensitive poly-N-isopropylacrylamide (PNIP). Briefly, PCR amplicons are labeled with digoxin, biotin or FITC via the modified up-stream primers respectively. After PCR amplification, the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with anti-digoxin antibody, streptavidin or anti-FITC antibody in a single vessel, and then each carrier is separated from the others under different conditions based on their physio-chemical attributes. And then direct CL detection proceeds via the instantaneous derivatization reaction between intrinsic guanine nucleobases and 3,4,5-trimethoxylphenylglyoxal (TMPG). This new protocol directly measures the double-stranded DNA and therefore does not require a denaturing step, thus offering an enhanced sensitivity due to the absence of competitive hybridization, i.e., the detection limit had a 20-fold improvement on the conventional PCR measurement. Additionally, by comparison of previous guanine based detection formats, this protocol is easy to be used for the detection of any guanine containing targets without the use of guanine-free or inosine-substituted capture probes. Overall, the proposed technique takes the advantages of sensitivity, high-speed and cost-effectivity, which provides a promising alternative for the analysis of multiple PCR targets in a variety of clinical, environmental, and biodefense fields.
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