Quantum Dot Doping-Induced Photoluminescence for Facile, Label-Free, and Sensitive Pyrophosphatase Activity Assay and Inhibitor Screening

Development of simple, convenient, and sensitive assay methods for pyrophosphatase (PPase) activity is of importance, for disease diagnosis and drug discovery. Herein, a simple, rapid, label-free, and sensitive fluorescence sensor for PPase activity assay is developed, using Cu2+ doping-induced quantum dot (QD) photoluminescence as a signal reporter. The Cu2+ doping of ZnSe QD can induce a dopant-dependent emission response, which will be inhibited after the premixing of Cu2+ with pyrophosphate (PPi), to form a Cu2+-PPi complex. Then, the hydrolysis of PPi into phosphate (Pi), specifically catalyzed by PPase, liberates the free Cu2+ to regain the QD doping for the fluorescence response, which is highly dependent on the PPase activity. The PPase can be sensitively and selectively assayed, with a detection limit of 0.1 mU/mL. The developed sensing strategy can be also employed for the PPase inhibitor screening. Thus, the current QD doping-based sensing strategy offers an efficient and promising avenue for Cu2+, PPi, or PPase-related target analysis, and might hold great potential for the further applications in the clinical disease diagnosis.


Introduction
Inorganic pyrophosphatase (PPase), as a ubiquitous hydrolytic enzyme in biological systems, can specifically catalyze the hydrolysis of pyrophosphate (PPi) into orthophosphate (Pi). Such a hydrolysis process is always accompanied by energy release, thus, accommodating the thermodynamic impetus for many biosynthetic reactions [1][2][3]. It, thus, demonstrates a very critical role in a series of important biological processes, for example, carbohydrate and lipid metabolism, DNA synthesis, and other biochemical transformations [4][5][6]. The abnormal level of PPase has been directly connected with several clinical diseases, including hyperthyroidism, colorectal cancer, and lung adenocarcinomas [7][8][9]. It has also served as an important therapeutic target for drug development. The identification of PPase activity is, therefore, of paramount importance for understanding relevant physiological and pathological processes, and also for some disease diagnosis and clinical medicine.
Until now, many methods including radiochemical, enzymatic, and optical, etc. have been proposed for the PPase activity assay [10][11][12]. The optical sensing method is especially attractive, owing to its distinctive advantages, such as simplicity, speed, homogenous, and high sensitivity. The previously reported optical methods for PPase assay could be simply classified into nanomaterials or organic dyes-based types, both of which are usually mediated by Cu 2+ /PPi, to achieve the signal solution (25 µL, 250 mM) was rapidly mixed with zinc acetate (125 µL, 100 mM), GSH (93.75 µL, 200 mM), and NH 4 HCO 3 solution (281.25 µL, 0.2 M, pH 12.3). After vortex agitation for 10 s, the resulting ZnSe QD was obtained. Followed by centrifugation (13,500 rpm, 10 min) via a centrifugal device (3K, Pall Corporation, Ann Arbor, MI, USA), the ZnSe QD was recovered and diluted to the original volume with NH 4 HCO 3 solution (0.2 M, pH 12.3).
To explore the effect of PPi on the fluorescence responses of QD, 50 µL of different concentrations of PPi were first mixed with Cu 2+ solution (50 µL, 200 µM), and then the mixtures were added into the freshly prepared ZnSe QD (100 µL), tris-HCl buffer (700 µL, 50 mM, pH 7.5), and H 2 O (100 µL), for fluorescence measurement.

QD Photoluminescence Regulated by Cu 2+ /PPi
ZnSe QD was obtained with the use of Zn(OAc) 2 and NaHSe as precursors and glutathione, as ligand. As characterized by TEM, the ZnSe QD showed a quasi-spherical shape with a mean diameter of about 3.3 nm ( Figure 1B). The XPS spectra for the ZnSe QD, in the presence of Cu 2+ , or not, were shown in Figure 1C. Upon incubation of ZnSe QD with Cu 2+ , the peak for Cu 2p could be clearly observed, besides the typical peaks for ZnSe QD (Se 3d, C 1s, Zn 2p), suggesting the Cu 2+ incorporation into the ZnSe QD. The fluorescence responses of ZnSe QD doped by Cu 2+ , or not, were shown in Figure 1D. Only an emission response at around 412 nm could be observed for the initial ZnSe QD. After Cu 2+ doping, such an emission response vanished but a new peak, centered at 510 nm, appeared, revealing the Cu 2+ doping-induced QD photoluminescence. Although the corresponding mechanism Nanomaterials 2019, 9, 111 4 of 10 for QD doping needs to be further explored, it could be still regarded as an excellent signal generating and reporting element to probe its potential applications. Furthermore, we explored the fluorescence responses of QD at various Cu 2+ concentrations. It could be seen from Figure 2A that the fluorescence intensity increased gradually with the increasing Cu 2+ concentration from 0 to 100 μM, indicating a high dependency of the fluorescence response of the doped-QD, on the Cu 2+ concentration. Figure 2B shows the corresponding calibration curve. A linear plot could be achieved between the fluorescence intensity and the Cu 2+ concentration (0.5-10 μM), with a correlation coefficient of 0.9947. In the following experiments, 10 μM of Cu 2+ was adopted for the PPi and PPase assay. The effect of PPi on the fluorescence intensity of the QD was probed, by first mixing the PPi (0-120 μM) with 10 μM Cu 2+ , and then adding into the freshly prepared ZnSe QD. The fluorescence intensity of the QD decreased with the increase of the PPi concentration ( Figure 2C). It could be explained that the complex formation between the Cu 2+ and the added PPi, inhibited the Cu 2+ doping for the fluorescence response decrease. The corresponding calibration curve of the fluorescence intensity versus the PPi concentration is shown in Figure 2D. The fluorescence intensity of the QD at 510 nm, decreased sharply with the increase of the PPi concentration from 0 to 20 μM, and reached a plateau after 20 μM. Additionally, a good linear plot for the fluorescence intensity with the PPi concentration (0-20 μM) could be obtained. In the current sensing system, if too much PPi is used, the PPase will hydrolyze the free PPi first, which will not be beneficial for the subsequent PPase activity assay. Thus, the PPi concentration of 20 μM was chosen in the following experiments. Furthermore, we explored the fluorescence responses of QD at various Cu 2+ concentrations. It could be seen from Figure 2A that the fluorescence intensity increased gradually with the increasing Cu 2+ concentration from 0 to 100 µM, indicating a high dependency of the fluorescence response of the doped-QD, on the Cu 2+ concentration. Figure 2B shows the corresponding calibration curve. A linear plot could be achieved between the fluorescence intensity and the Cu 2+ concentration (0.5-10 µM), with a correlation coefficient of 0.9947. In the following experiments, 10 µM of Cu 2+ was adopted for the PPi and PPase assay. The effect of PPi on the fluorescence intensity of the QD was probed, by first mixing the PPi (0-120 µM) with 10 µM Cu 2+ , and then adding into the freshly prepared ZnSe QD. The fluorescence intensity of the QD decreased with the increase of the PPi concentration ( Figure 2C). It could be explained that the complex formation between the Cu 2+ and the added PPi, inhibited the Cu 2+ doping for the fluorescence response decrease. The corresponding calibration curve of the fluorescence intensity versus the PPi concentration is shown in Figure 2D. The fluorescence intensity of the QD at 510 nm, decreased sharply with the increase of the PPi concentration from 0 to 20 µM, and reached a plateau after 20 µM. Additionally, a good linear plot for the fluorescence intensity with the PPi concentration (0-20 µM) could be obtained. In the current sensing system, if too much PPi is used, the PPase will hydrolyze the free PPi first, which will not be beneficial for the subsequent PPase activity assay. Thus, the PPi concentration of 20 µM was chosen in the following experiments.

Optimization of the Experimental Conditions
To verify the detection feasibility of the current sensing strategy toward PPase activity, the corresponding fluorescence spectra, in the presence and absence of PPase, were shown in Figure 3A. The addition of PPase into the premixed Cu 2+ and PPi solution could induce a distinct increase of the fluorescence intensity of QD, at 510 nm, compared with that of no PPase, indicating that the catalytic hydrolysis of PPi into Pi by PPase, liberated the Cu 2+ to regain the QD doping-induced photoluminescence. To achieve the best sensing capability toward the PPase activity, the other experimental conditions, including pH value, reaction temperature, and hydrolysis time, were also optimized. It could be seen from Figure 3B that a maximum signal-to-background ratio could be acquired at a pH 7.5 tris-HCl buffer solution. The reaction temperature would have an effect on the enzymatic process of PPase. It could be seen from Figure 3C that the 37 °C is the suitable temperature for maintaining the PPase activity, and could achieve better performance toward PPase than other tested temperatures. The hydrolysis time between PPi and PPase was also studied ( Figure  3D). The fluorescence intensity of QD at 510 nm, increased with the increase of hydrolysis time and almost reached a plateau value, at 60 min ( Figure 3D). Thus, a 60 min hydrolysis time was employed in the following PPase assay.

Optimization of the Experimental Conditions
To verify the detection feasibility of the current sensing strategy toward PPase activity, the corresponding fluorescence spectra, in the presence and absence of PPase, were shown in Figure 3A. The addition of PPase into the premixed Cu 2+ and PPi solution could induce a distinct increase of the fluorescence intensity of QD, at 510 nm, compared with that of no PPase, indicating that the catalytic hydrolysis of PPi into Pi by PPase, liberated the Cu 2+ to regain the QD doping-induced photoluminescence. To achieve the best sensing capability toward the PPase activity, the other experimental conditions, including pH value, reaction temperature, and hydrolysis time, were also optimized. It could be seen from Figure 3B that a maximum signal-to-background ratio could be acquired at a pH 7.5 tris-HCl buffer solution. The reaction temperature would have an effect on the enzymatic process of PPase. It could be seen from Figure 3C that the 37 • C is the suitable temperature for maintaining the PPase activity, and could achieve better performance toward PPase than other tested temperatures. The hydrolysis time between PPi and PPase was also studied ( Figure 3D). The fluorescence intensity of QD at 510 nm, increased with the increase of hydrolysis time and almost reached a plateau value, at 60 min ( Figure 3D). Thus, a 60 min hydrolysis time was employed in the following PPase assay. Nanomaterials 2019, 9, x FOR PEER REVIEW 6 of 10

Sensing Performance toward PPase Activity
Under the optimized experimental conditions, the PPase activity was tested by the current sensing system. The fluorescence spectra recorded at different PPase concentrations were shown in Figure 4a. A stepwise increase of fluorescence intensity of QD could be observed upon increasing PPase concentrations from 0 to 20 mU/mL, suggesting a PPase concentration-controlled or dependent response manner. Figure 4B shows the fluorescence intensity of QD as a function of the PPase concentration. A good linear relationship of the fluorescence intensity with the PPase concentration, ranged from 0.1 to 2 mU/mL, could be obtained with a regression equation of Y (fluorescence intensity) = 169 + 138X (PPase concentration) and a correlation coefficient of 0.9926. The detection limit toward the PPase was achieved as 0.1 mU/mL, which was compared with the reported methods ( Table 1). The selectivity for the PPase activity of the developed method was also investigated by using other non-specific proteins, including lysozyme, glucose oxidase (GOx), exonuclease I (Exo I), and exonuclease III (Exo III). As shown in Figure 4C, a remarkable fluorescence response could be only observed in the presence of PPase, and also, these control proteins could not interfere with the detection of PPase activity. To further evaluate the potential applications of the currently developed sensor, we challenged the detection toward Cu 2+ , PPi, and PPase spiked in a relatively complex biological matrix (5% diluted fetal bovine serum). The fluorescence responses toward these different species in the diluted serum were all comparable with that in buffer ( Figure 4D), suggesting the applicative potential in the relatively complex biological samples.

Sensing Performance toward PPase Activity
Under the optimized experimental conditions, the PPase activity was tested by the current sensing system. The fluorescence spectra recorded at different PPase concentrations were shown in Figure 4a. A stepwise increase of fluorescence intensity of QD could be observed upon increasing PPase concentrations from 0 to 20 mU/mL, suggesting a PPase concentration-controlled or dependent response manner. Figure 4B shows the fluorescence intensity of QD as a function of the PPase concentration. A good linear relationship of the fluorescence intensity with the PPase concentration, ranged from 0.1 to 2 mU/mL, could be obtained with a regression equation of Y (fluorescence intensity) = 169 + 138X (PPase concentration) and a correlation coefficient of 0.9926. The detection limit toward the PPase was achieved as 0.1 mU/mL, which was compared with the reported methods ( Table 1). The selectivity for the PPase activity of the developed method was also investigated by using other non-specific proteins, including lysozyme, glucose oxidase (GOx), exonuclease I (Exo I), and exonuclease III (Exo III). As shown in Figure 4C, a remarkable fluorescence response could be only observed in the presence of PPase, and also, these control proteins could not interfere with the detection of PPase activity. To further evaluate the potential applications of the currently developed sensor, we challenged the detection toward Cu 2+ , PPi, and PPase spiked in a relatively complex biological matrix (5% diluted fetal bovine serum). The fluorescence responses toward these different species in the diluted serum were all comparable with that in buffer ( Figure 4D), suggesting the applicative potential in the relatively complex biological samples.

PPase Inhibitor Screening
The developed sensing system was also extended for the PPase inhibition evaluation, by using NaF as a typical PPase inhibitor. As shown in Figure 5A, the fluorescence intensity of the QD

PPase Inhibitor Screening
The developed sensing system was also extended for the PPase inhibition evaluation, by using NaF as a typical PPase inhibitor. As shown in Figure 5A, the fluorescence intensity of the QD decreased stepwise, with the concentration increase of added NaF (0-10 mM). Such a trend was especially evident when the NaF concentration was over 10 µM. This strongly indicated the inhibition effect of NaF on the PPase activity. The calibration curve (fluorescence intensity versus the logarithm value of the NaF concentration) showed a typical sigmoidal profile ( Figure 5B). The IC50 value (the inhibitor concentration that can cause 50% inhibition of the enzyme activity) was calculated to be about 58.07 µM, which was basically in accordance with the reported PPase activity assays [13,17]. Thus, the developed sensing system might be used for the screening of potential PPase inhibitors. decreased stepwise, with the concentration increase of added NaF (0-10 mM). Such a trend was especially evident when the NaF concentration was over 10 μM. This strongly indicated the inhibition effect of NaF on the PPase activity. The calibration curve (fluorescence intensity versus the logarithm value of the NaF concentration) showed a typical sigmoidal profile ( Figure 5B). The IC50 value (the inhibitor concentration that can cause 50% inhibition of the enzyme activity) was calculated to be about 58.07 μM, which was basically in accordance with the reported PPase activity assays [13,17]. Thus, the developed sensing system might be used for the screening of potential PPase inhibitors.

Conclusions
In conclusion, a simple, rapid, cost-effective, and sensitive strategy for PPase activity determination was demonstrated for the first time, using quantum dot doping-induced photoluminescence. The Cu 2+ doping of ZnSe QD was first revealed to yield a dopant-specific emission peak. Then, the PPi-as an intermediate species-served for the Cu 2+ constraint, through a strong coordination effect; and its specific hydrolysis into orthophosphate, by the PPase-for the Cu 2+ liberation-in turn, regained the QD doping for the fluorescence response related with the PPase activity. The PPase could be assayed sensitively and selectively with a detection limit of 0.1 mU/mL. The current QD doping-based sensing strategy exhibited several advantages, such as high sensitivity and selectivity, the simple mix-and-detect operation in needless of washing and separation steps, and rapidness (within 60 min). Additionally, it avoided any labelling or modification operation commonly encountered in most QD-based sensing strategy. It could also be efficiently applied for the PPase inhibitors screening. Thus, it opens a convenient and promising avenue for the Cu 2+ -related target detection and might hold a great potential for the further applications in the clinical diagnosis of Cu 2+ , PPi, or PPase-related diseases.

Conclusions
In conclusion, a simple, rapid, cost-effective, and sensitive strategy for PPase activity determination was demonstrated for the first time, using quantum dot doping-induced photoluminescence. The Cu 2+ doping of ZnSe QD was first revealed to yield a dopant-specific emission peak. Then, the PPi-as an intermediate species-served for the Cu 2+ constraint, through a strong coordination effect; and its specific hydrolysis into orthophosphate, by the PPase-for the Cu 2+ liberation-in turn, regained the QD doping for the fluorescence response related with the PPase activity. The PPase could be assayed sensitively and selectively with a detection limit of 0.1 mU/mL. The current QD doping-based sensing strategy exhibited several advantages, such as high sensitivity and selectivity, the simple mix-and-detect operation in needless of washing and separation steps, and rapidness (within 60 min). Additionally, it avoided any labelling or modification operation commonly encountered in most QD-based sensing strategy. It could also be efficiently applied for the PPase inhibitors screening. Thus, it opens a convenient and promising avenue for the Cu 2+ -related target detection and might hold a great potential for the further applications in the clinical diagnosis of Cu 2+ , PPi, or PPase-related diseases.