A rapid, sensitive and reliable indicator displacement assay (IDA) for specific detection of 2′- and 3′-deoxyadenosine (2′-dAde and 3′-dAde), the latter is also known as cordycepin, was established. The formation of inclusion complex between protonated acridine orange (AOH+) and cucurbit[7]uril (CB7) resulted in the hypochromic shift of fluorescent emission from 530 nm to 512 nm. Addition of cordycepin to the highly fluorescent AOH+/CB7 complex resulted in a unique tripartite AOH+/CB7/dAde complex with diminished fluorescence, and such reduction in emission intensity serves as the basis for our novel sensing system. The detection limits were 11 and 82 μM for 2′- and 3′-deoxyadenosine, respectively. The proposed method also demonstrated high selectivity toward 2′- and 3′-deoxyadenosine, owing to the inability of other deoxynucleosides, nucleosides and nucleotides commonly found in Cordyceps spp. to displace the AOH+ from the AOH+/CB7 complex, which was confirmed by isothermal titration calorimetry (ITC), UV-Visible and proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our method was successfully implemented in the analysis of cordycepin in commercially available Ophiocordyceps and Cordyceps supplements, providing a novel and effective tool for quality assessment of these precious fungi with several health benefits.
Ophiocordyceps and Cordyceps spp. are some of the most popular Chinese herbs because of their bioactive compounds, for example cordycepin that has many pharmacological functions such as anti-tumor, anti-microbes, anti-inflammatory, and immunomodulator. The abundant supply of products in the market has different variations in purity and quality, so the rapid and simple quantitative analysis method to access the quality of Ophiocordyceps and Cordyceps products is urgently required. Indicator displacement assay (IDA) using environmentally sensitive fluorescence (FL) dye can be used for quantitative analysis of the target compound. The concept of this assay is replacing the FL dye in the receptor-dye inclusion complex by the analyte. In this study, screening of host series, such as cucurbits[n]uril (CB[n]) and cyclodextrins as well as the finding of the suitable environmental sensitive FL dyes i.e. esculetin, cascade yellow, nonyl acridine orange (NAO), berberine, and 2-p-toluidinyl naphthalene-6-sulphonate (2,6-TNS) were performed. The FL emission of esculetin, cascade yellow, and NAO either decreased or increased slightly upon addition of all hosts mentioned above. Among FL dyes tested, berberine and 2,6-TNS participated in the inclusion complex with CB7 as suggested by the approximately 500-fold and 4-fold FL increment observed when CB7 was added respectively. Cordycepin showed the highest displacement ability when it was titrated with an inclusion complex of berberine/CB7 and 2,6-TNS/CB7 in pH 4.0. Moreover, in the selectivity assay, cordycepin performed the distinguished displacement ability among the other compounds, suggesting about the high potential for using IDA method as a rapid and simple quality assessment of commercial Ophiocordyceps and Cordyceps product.
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