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.

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of ATP to PPi. This study demonstrates the detection of relative catalytic activity of 3 enzymes—ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), tissue nonspecific alkaline phosphatase (TNAP), and ecto-nucleoside triphosphate diphosphohydrolases (e-NTPDases)—using a single molecular sensor called Kyoto Green. Kyoto Green exhibits excellent performance in sensing the catalytic activity of the commercial representatives of the e-NPPs, TNAP, and e-NTPDases, which are ENPP1, PPase, and apyrase, respectively, in both single-enzyme and multi-enzyme assays. Analysis of SF enzymes in 19 SF samples from human and swine revealed moderate activity of e-NPPs, high activity of e-NTPDases, and low activity of TNAP. Our newly developed method for analysis of multiple enzymatic activities using Kyoto Green in biological SF will assist improvement in accuracy of the CPPD prognosis/diagnosis, which will minimize unnecessary medical procedures.