Abstract
The level of pyrophosphatase (PPase) expression has been suggested as a potential biomarker of various cancers, and its prognostic value has been evaluated in patients suffering from lung cancer, colorectal cancer, and hyperthyroidism. However, the detection of PPase usually needs specific materials that require complicated, time-consuming reactions with restricted linear range and sensitivity, limiting their application in early clinical diagnosis. Herein, we developed a DNAzyme-based biosensor for the detection of PPase. In the presence of PPase, pyrophosphate (PPi) and Cu2+ ions released from the PPi–Cu2+–PPi complex induce the cleavage of the DNAzyme and the corresponding substrate. An apurinic/apyrimidinic (AP) site was elaborately designed within substrates that could encase the fluorophore 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND). The fluorescence of ATMND was initially quenched but restored when the DNAzyme/substrate complex was hydrolyzed with the release of ATMND. In this way, the PPase activity can be estimated by detecting the increased fluorescence of the released ATMND. Under optimized conditions, the activity of PPase could be analyzed at concentrations from 0.5 to 1000 mU, with the lowest detectable concentration being 0.5 mU. This work lays a foundation for developing a DNAzyme-amplified fluorescent biosensor with a high sensitivity, a wide linear range, and single-step operation for use as an easy diagnostic for PPase analysis.
Highlights
Inorganic pyrophosphatase (PPase) is a hydrolytic enzyme that can catalyze the fragmentation of a molecule of pyrophosphate (PPi) into two separate orthophosphate (Pi) ions
The expression levels of PPase have been suggested as a potential biomarker of various cancers, and their prognostic value has been evaluated in patients suffering from lung cancer, colorectal cancer, and hyperthyroidism [3]
The AP site was located at the fourth base from the 5 end of the substrate; its complex could trap ATMND and quench its fluorescence
Summary
Inorganic pyrophosphatase (PPase) is a hydrolytic enzyme that can catalyze the fragmentation of a molecule of pyrophosphate (PPi) into two separate orthophosphate (Pi) ions. In most of these approaches, the specific PPi was chosen as the first reaction substrate based on the native property of PPase and the definition of its activity. From basic oxidation/reduction to powerful DNA polymerizations, were carried out, and the resulting signal was used for PPase activity quantitation These methods need specific materials that require difficult synthesis, complicated procedures for multi-stage reactions, or expensive reagents and instruments. We developed a label-free fluorescent method for the simple and costeffective detection of PPase activity with a large linear range and high sensitivity, as illustrated in Scheme 1. S1c. hSecmheamticarteicprreespernetasetinotnaotifotnheodf ethsiegndeedsisgunbesdtrastue/bDstNraAtez/yDmNeA-bzaysemdeb-iboasesnedsobr ifoosretnhesoflrufoorrestcheenftldueotreecsticoennt deoftePcPtaiosen. of PPase
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