br Introduction As a fundamental metal dependent hydrolytic

Introduction As a fundamental metal-dependent hydrolytic enzyme, inorganic pyrophosphatase (PPase) exhibits a specific catalytic ability, in which one molecule of inorganic pyrophosphate (PPi) can be transformed into two orthophosphate (Pi) ions [1,2]. The process of transformation is extremely exergonic and offers thermodynamic propulsion for several biosynthetic reactions [3,4]. Furthermore, it has been proven that PPase is closely associated with several clinical diseases as well as a variety of biological processes such as phosphorus and carbohydrate metabolism, calcium absorption, bone formation, lipid synthesis and degradation, DNA synthesis, etc. [[5], [6], [7], [8], [9], [10]]. Upon consumption PPase plays a significance role in energy anabolism occurring in all organisms, which consequently results in cell death [11]. Hence, it is of great importance to develop a facile and sensitive strategy for PPase determination. Currently, many diverse approaches have been established for PPase determination including enzymatic, fluorometric, chemiluminescent and colorimetric methods were PPi was used as the substrate [[12], [13], [14], [15], [16], [17]]. Recently, Wang et al. designed an electrochemical method for PPase activity detection based on G-quadruplex-Cu2+ DNAzyme [18]. Whereas, Sun et al. proposed a real-time assay to detect PPase activity using fluorescent gold nanoclusters [19]. Nevertheless, these assays were confined by complicated operations, sophisticated instrumentation and comparatively low sensitivity. G-quadruplexs are composed of accumulating Hoogsteen levels paired G-quartets with a four-strand helical structure, which originates from guanine-rich DNA or RNA sequences [[20], [21], [22], [23]]. Some metal complex probes have been applied for G-quadruplex-based fluorescent detection such as Iridium(III), Cu2+ and so on [[24], [25], [26], [27], [28]]. Interestingly, Thioflavin T (ThT), a commercially available soluble fluorogenic dye, has attracted considerable attention due to its ability to exhibit prominent fluorescence enhanced signaling upon interacting with a G-quadruplex structure [[29], [30], [31], [32], [33]]. Here, we present a label-free assay for highly sensitive and selective detection of PPase based on the fluorescent transformation of G-quadruplex with the help of Cu2+ [34]. The addition of PPase hydrolyzes the PPi from the PPi/Cu2+ complex and generates free Cu2+, leading to fluorescent quenching of G-quadruplex-ThT [[35], [36], [37]]. This signaling mechanism makes it possible to detect PPase by fluorescence spectroscopy.
Experimental
Results and discussion
Conclusions In summary, by taking advantage of the coordination reaction between Cu2+ and PPi and the higher combining capacity of Cu2+ to G-quadruplex, a facile and label-free fluorescent method for the detection of PPase activity with high sensitivity has been successfully developed. The sensing system possessed a linear interval with a scope of 0.5–30 U/L and a 0.48 U/L detection limit. The fluorescent sensing system for PPase activity assay shows several merits such as low cost, good selectivity and convenient operating system without the need of complex procedures. Furthermore, the strategy is able to evaluate the inhibition effect of NaF on PPase activity. This strategy may hold potential applications in PPase-related clinical diagnosis analysis and functional studies.
Acknowledgments This work was supported by State Key Laboratory of Chemo/ Biosensing and Chemometrics, Hunan University (2017006), The Research Innovation Program for Graduates of Central South University (2018zzts384, 2018zzts399).
Introduction Threats to human health caused by viral infectious diseases or cancers still have a tremendous influence on human society [[1], [2], [3], [4], [5]]. The appearance of diseases may be accompanied by changes in many diagnostic indexes, leading to difficulties in early and accurate diagnosis for disease therapy. Compared to single detection, simultaneous detection of multiple biomarkers, such as DNAs, microRNAs, small molecules, and proteins [[6], [7], [8]], is more desirable because of the higher detection efficiency and more accurate diagnosis. However, the complexity of the detection system and the inability to generate unique signals without interference often result in difficulty in simultaneously detecting targets. Thus, the development of highly sensitive and simultaneous detection for multiple biomarkers of critical diseases is challenging.