Yttrium vanadates based ratiometric fluorescence probe for alkaline phosphatase activity sensing

Colloids and Surfaces B: Biointerfaces 185 (2020) 110618

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Yttrium vanadates based ratiometric fluorescence probe for alkaline phosphatase activity sensing

T

Wei Xiao, Fang Liu, Gen-Ping Yan, Wei-Guo Shi, Kang-liang Peng, Xue-qing Yang, Xiao-jing Li, Hou-chuan Yu, Zhi-ying Shi, Hui-Hui Zeng* Jiangxi Key Laboratory of Industrial Ceramics, College of Materials and Chemical Engineering, Pingxiang University, Pingxiang 337055, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Alkaline phosphatase Ratiometric fluorescence probe Carbon dots Lanthanide orthovanadate

Alkaline phosphatase (ALP) is an important biomarker for diagnosis, and the abnormal level of serum ALP is closely related to a variety of diseases. In present work, a ratiometric fluorescence probe based on hybrid nanoparticles CDs@YVO4: Dy3+ nanoparticle is introduced for alkaline phosphatase (ALP) activity determination. The CDs@YVO4: Dy3+ probe is constructed by the carbon dots (CDs) and YVO4: Dy3+ through a simple mixing method, in which the blue emission of CDs at 405 nm acts as the calibrated signal, the green emission of YVO4: Dy3+ at 574 nm decreased with the increasing targets ALP, and used as the output signal. In addition, the Cu2+ and pyrophosphate (PPi) were also employed in this strategy to utilize the excellent fluorescnece quenching efficiency of Cu2+ to the Dy3+ ions emission of CDs@YVO4: Dy3+, as well as the strong affinity of PPi for Cu2+. In the presence of analyts ALP, ALP catalyzes the hydrolysis of PPi, causing the release of Cu2+, resulting in the Dy3+ ions emission quenched, while the CDs emission at 405 nm retained unchanged, based on this, we designed the off-on-off ratiometric fluorescence platform for ALP sensing. The experiment result shows that the ratio of F574/F405 is linear to the concentration of ALP in arange of 0.05∼3000 U/L with a detection limit of 0.04 U/L, which is comparable or better than those reported fluorescence probe, especially the calibrated signal introduction of CDs can eliminate the background interference, improve the accuracy of proposed probe greatly. Furthermore, the discrimination of ALP enzyme inhibitor with the IC50 of 26 μM, and ALP concentration in real human serum sample has also demonstrated the applicability of CDs@YVO4: Dy3+ fluorescence sensor well.

1. Introduction As a widely distributed phosphomonoesterase, alkaline phosphatase (ALP) can nonspecifically catalyze the hydrolysis of phosphate ester groups, and plays a crucial role in regulation of intracellular processes [1–3]. The normal serum alkaline phosphatase level in adults is reported to be about 40 ∼190 U/L, and the ALP level of children and pregnant women is more than 500 U/L [4]. Research has shown that the abnormal level of ALP in serum is directly bound up with miscellaneous diseases such as rickets, hepatitis, even prostate and breast cancers, it usually increases in those patients suffering from rickets, hepatitis, prostatitis and breast cancer, while peoples in chronic nephritis and anemia display a lower serum ALP level, thus ALP general acts as one of important indicator for disease diagnosis [5,6]. So far, great effort has been delivered to develop various strategies for ALP activity determination including electroanalytical, fluorometric and colorimetric assays [7–13]. Among them, the fluorometric assay method has attracted much more interesting due to its outstanding ⁎

advantages of real-time response, noninvasive, as well as high sensitivity [6,14]. Lots of fluorescence probes have also been exploited to detect the ALP activity sensitively, for example, the organic phosphonated fluorescent probe containing a phenolic dihydroxanthene fluorophore [15], CuInS/ZnS quantum dots [16], the polydopamine nanoparticles with MnO2 nanosheet as the fluorescence quencher [14], and Tb-GMP-Eu lanthanide coordination polymer and so on [17]. However, as one of the excellent sensor strategies, till now, only a few ratiometric fluorescence methods have been reported to discriminate the ALP activity [18–20]. It is well known that the ratiometric fluorescence probe is usually composed of two main emission peaks at different wavelength, the ration of two emission peaks intensity varies with the target concentration, and exhibits a linear relationship. Due to the existence of self-calibration signal, the ratiometric fluorescence method can effectively alleviate those affections by the variations in environment, concentration and excitation intensity [21]. Therefore, the development of excellent ratiometric fluorescence probe for the ALP activity identification is very meaningful.

Corresponding author. E-mail address: [email protected] (H.-H. Zeng).

https://doi.org/10.1016/j.colsurfb.2019.110618 Received 6 July 2019; Received in revised form 25 October 2019; Accepted 28 October 2019 Available online 02 November 2019 0927-7765/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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In the past decades, the lanthanide-doped fluorescence nanoparticles have attracted the strong interest of researchers due to their chemical stability and splendid optical property such as the large stokes shift, line emission, and long lifetime [22–25]. So, the lanthanide-doped fluorescence nanoparticles have been widely applied in chemical sensing, biomedical cell imaging, and target therapy etc. For instance, the CeO2: Eu nanoparticles have been exploited for the H2O2 and glucose detection [26]. CaF2: Ce,Tb nanoparticles were developed for the ultrasensitive assay of tumor markers carcinoembryonic antigen (CEA) [27], and ZrO2: Ln3+ nanoparticles were used for the alpha-fetoprotein (AFP) and prostate-specific anti-gen (PSA) detection by the Chen’s group [28]. Yan take advantage of the red emission YVO4: Eu3+ nanoparticles for cell imaging [29,30]. Among these inorganic nanoparticles, the lanthanide orthovanadate has been demonstrated to be one of the best luminescent hosts possessing high efficiency upon electron beam excitation, and being used for the optical communication, field display, and beam displacers due to their excellent thermal and optical properties [31,32]. In particular, the broad transparency (400 to 5000 nm wavelengths) and high birefringence makes YVO4 being an outstanding host material for rare earth ions. Pure YVO4 possesses the intrinsic emission, which arises from the intrinsic luminescence band near 2.8 eV, and it serves as the self-trapped excitons in the form of VO43− molecular complex [33,34]. Up to now, various lanthanide ions such as Eu3+, Dy3+, Tm3+, Nd3+, Er3+, Ho3+, Sm3+, and Pr3+ doped in YVO4 host have been investigated [35,36]. Among them, Dy3+ is reported to give highest luminescence in YVO4, and the YVO4: Dy3+ is a well-known phosphor with high efficiency which emits yellow and blue colors [37]. However, as far as we know, except for the artificial production of light in LED, the application of YVO4: Dy3+ nanoparticle in other field, especially in the chemical sensing and biomedical has not been reported. As an excellent fluorophore, carbon dot has been widely applied to the chemosensor and biosensor field for its superiority such as good photostability, splendid water solubility, and outstanding biocompatibility [38,39]. Utilizing the aggregation-induced quenching of CDs triggered by copper ions and the strong binding capacity of pyrophosphate (PPi) to Cu2+ ions, Feng exploited the CDs-Cu2+-based nanoparticle biosensors for ALP detection [40]. Similarly, a N-doped carbon dots-based off-on probe has also been developed for ALP assay [41]. Li’s group constructed the N-doped carbon dots based fluorescence probe for ALP discrimination, in which p-Nitrophenylphosphate acted as the ALP substrate, and its enzyme catalytic product (p-nitrophenol) as a powerful fluorescence quencher of CDs [19]. In brief, for most CDsbased probes for ALP sensing, the fluorescence of CDs are always quenched by various intermediates. However, as far as we known, multi-signal probe composed of CDs and other luminescence materials, especially those hybrid platforms using CDs as the calibrated signal for biomolecule determination, have nearly not been reported. In this work, we synthesized a novel hybrid nanoparticles CDs@ YVO4: Dy3+, which is composed of the carbon dots (CDs) and YVO4: Dy3+ through a simple mixing method (Scheme 1). The synthesized CDs@YVO4: Dy3+ exhibits both the characteristic emission of CDs and Dy3+ under a single excitation wavelength of 272 nm, with the main emission peaks located on 405 nm, 483 nm, and 574 nm, respectively. In the presence of Cu2+, the fluorescence of YVO4: Dy3+ can be quenched selectively, while the characteristic emission of CDs is insensitive to Cu2+. Utilizing the strong binding capacity of pyrophosphate (PPi) to Cu2+ ions, the Cu2+ can be captured by the PPi, resulting in the fluorescence of YVO4: Dy3+ recover, but the P2O74− groups were hydrolyzed into PO43- ions by the added ALP, and as the increasing ALP, the fluorescence intensity of YVO4: Dy3+ quenched absolutely. Based on this, we designed an off-on-off ratiometric fluorescence probe for ALP sensing. In this strategy, the emission intensity of CDs at 405 nm retained unchanged throughout, acting as the calibrated signal, and the fluorescence intensity of YVO4: Dy3+ at 574 nm decreased with the increasing ALP obviously, which used as the output

signal. The experiment result shows that the ratio of F574/F405 is linear to the concentration of ALP in arange of 0.05∼3000 U/L with a detection limit of 0.04 U/L. Compared with those single wavelength emission ALP fluorescence probes, the calibrated signal introduction of CDs can eliminate the background interference, improve the accuracy of proposed probe greatly. Furthermore, an obvious change of the fluorescence color from green to blue can be conveniently observed by the naked eye under a UV lamp. 2. Experimental Section 2.1. Reagents and chemicals Y(NO3)3·6H2O (> 99.99%), Dy(NO3)3·5H2O (> 99.99%), Polyvinylpyrrolidone (PVP, 55 kDa), alkaline phosphatase (ALP), horseradish peroxidase (HRP), lysozyme (Lys), bovine serum albumin (BSA), thrombin (Thr), protein kinase (CK2), protein kinase (PKA), and cysteine (Cys) were bought from Sigma-Aldrich (USA). Sodium pyrophosphate, sodium orthovanadate (Na3VO4), tris(hydroxymethyl) aminomethane, citric acid, NH3·H2O (28%), hydrochloric acid, and other metallic salts were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All materials were used without further purification. All human plasma samples of normal adults, chronic hepatitis B patients, and nephritis patients with ethical issues were supplied by Jiangxi Provincial People’s Hospital. After centrifugation at 1000 rpm for 5 min, the human serum were then collected and diluted to 1% with ultrapure water. 2.2. Preparation of YVO4:Dy3+ nanoparticles YVO4:Dy3+ nanoparticle was synthesized by the hydrothermal method. Briefly, 2 mL Na3VO4 (100 mM) was dropped gradually into 20 mL mixture solution containing Y(NO3)3 (100 mM, 1.9 mL) and Dy (NO3)3 (100 mM, 0.1 mL) under slowly stirring at room temperature. Then NH3·H2O (28%) added to the above mixture solution to adjust the pH to 9.0, continue stirring for another 10 min. Finally, the suspension were transferred to the Teflon-lined autoclave and heated at 180 °C for 3 h. The obtained nanoparticles were collected by centrifugation (16000 rpm, 20 min), washed with deionized water several times, and dispersed in 100 mL water to form YVO4: Dy3+ (2 mM) stock, which was stored at 4 °C prior to use. 2.3. Preparation of CDs CDs were synthesized by the hydrothermal method. Typically, 0.66 g trisodium citrate and PVP (MW, 10000) 1.92 g were dissolved in deionized water (30 mL) under stirring to form an uniform and transparent solution, then the above solution was transferred to the Teflonlined autoclave chamber and heated to 180 °C for 2 h. The obtained solution containing C-dots was further dialyzed for 24 h (MW, cut off = 1000) to remove the large precipitates. Finally, the purified Cdots were condensed down to 30 mL, and preserved at 4 °C for use. 2.4. Preparation of CDs@YVO4: Dy3+ core-shell nanoparticles 1 mL YVO4: Dy3+ (2 mM) stock were added to 30 mL CDs stock solution. After stirring for 24 h, the mixture was centrifuged at 1000 rpm for 5 min to remove excess unreacted chemicals. The final product was dispersed in 20 mL deionized water, and obtained the CDs@YVO4: Dy3+ stock solution (0.1 mM), preserved at 4 °C for further use. 2.5. Measurement of ALP activity and inhibition ALP detection was carried out by the following route. CDs@YVO4: Dy3+ stock solution (0.1 mM, 3 μL), Cu2+ ions (0.2 mM, 8 μL), 16 μL 2

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Scheme 1. Schematic illustration for the synthesis of CDs@YVO4: Dy3+ and the detection of ALP with CDs@YVO4: Dy3+ as the ratiometric fluorescent sensor.

P2O74- ions (0.4 mM), various concentrations of ALP were orderly added to Tris-HCl buffer (50 mM, pH 7.4, 10 μL), and the total volume was 400 μL. After incubated for 50 min at room temperature, the fluorescence spectra were recorded. For ALP inhibition detection, various concentrations of inhibitor Na3VO4 was mixed with 1000 U/L ALP in Tris-HCl buffer (10 μL, 50 mM, pH 7.4), and incubated at 37 °C for 30 min. Then CDs@YVO4: Dy3+ stock solution (0.1 mM, 3 μL), Cu2+ ions (0.2 mM, 8 μL), 16 μL P2O74− ions (0.4 mM) added into the above solution, the total volume keeps 400 μL, and incubated for another 20 min at 37 °C. Finally, the fluorescence spectra were measured. The ALP of human serum detection was performed by the same procedure. CDs@YVO4: Dy3+ stock solution (0.1 mM, 3 μL), Cu2+ ions (0.2 mM, 8 μL), 16 μL P2O74− ions (0.4 mM), 1% treated human serums (10 μL) and different concentrations ALP solution were added into the Tris-HCl solution (10 μL, 50 mM, pH 7.4), the total volume keeps 400 μL, after incubated for 50 min at 37 °C, the ALP detection was executed.

For a better insight to the surface states information of CDs, XPS measurements were performed. The typical peaks of C 1s (286 eV), N 1s (400 eV), O 1s (532 eV) can be observed clearly in the full range XPS analysis (Fig. 2A) [43–45]. The high-resolved C1 s XPS spectra can be deconvoluted into three peaks at 285.71, 286.25, and 289.69 eV (Fig. 2B), which are assigned to the C 1s states in CeC, CO/CN and CO bonds, respectively [ee]46]. The O1s spectra can be resolved into two peaks at 532.57 and 534.01 eV, corresponding to the C]O, COe, respectively (Fig. 2C) [47]. The two peaks located at 400.73 and 402.42 eV in the N 1s spectrum (Fig. 2D) belong to the CeNC and NCO/ NCee]e3, respectively [48]. These results further confirmed the preparation of CDs with rich carboxyl groups and hydroxyl groups. The FT-IR spectra are employed to validate the conjugation of the carboxyl-modified CDs with YVO4:Dy3+ (Fig. 3A). For CDs (c curve), the high IR absorption bands located at 3432, 2952, 1663, 1429, 1379 cm−1, and 1096 cm−1 can be observed clearly, which are attributed to the OHe stretching vibrations, CHe stretching vibration, C]O stretching mode, NHe bending vibrations, CeN stretching vibrations, and CeO stretching vibration, respectively, meaning that the rich carboxyl groups have been decorated to the CDs surface successfully [49]. These IR absorption bands retained in the spectra of CDs@YVO4: Dy3+ (3430, 2948, 1659, 1435, 1384, and 1089 cm−1, respectively), especially the ν(NHe) bending vibrations, ν(C]O) carbonylic band, and ν(CeO) modes shifted to the lower frequency, suggesting that the COOe groups, hydroxyl groups, and NH2 groups in CDs are both involved in the coordination with YVO4: Dy 3+ [50]. The emission spectra displayed that the fluorescence intensity of CDs was related to the excitation wavelength, when the excitation wavelength was 280 nm, CDs exhibits the strongest blue emission (Fig. 3B). However, with the excitation wavelength ranged from 270 nm to 310 nm, the emission peak of CDs always located at 405 nm, such excitation independent emission behavior of CDs is probably assigned to their uniform size and surface state [43,48], the blue light of CDs can also be seen clearly under the 254 nm UV irradiation (inset of Fig. 3B). The excitation spectra of YVO4: Dy3+ (Fig. S2, Supporting Information) is composed of a strong and broad band around 272 nm upon monitored with 574 nm, which come from the overlap of VO43− absorption and charge transfer transition between Dy3+ and O2- [34]. Excited with 272 nm light, the emission spectra of YVO4: Dy3+ (Fig. S3, Supporting Information) is composed of the characteristic peaks of Dy3+ at 483 nm and 574 nm, corresponding to the 4F9/2→6H15/2 and 4 F9/2→6H13/2 transition of Dy3+, respectively [33]. The bright green emission of YVO4: Dy3+ appears from the photograph under UV lamp

3. Results and discussion 3.1. Characterization of CDs@YVO4: Dy3+ core-shell nanoparticles The as-synthesized YVO4: Dy3+ is of fusiform-like structure in shape with the average size ranging 50∼100 nm (Fig. 1A). The single-crystalline nature of YVO4: Dy3+, and the lattice fringes with d spacing of 0.352 nm (insert of Fig. 1B) are in accordance with the lattice spacing in the (200) planes of tetragonal YVO4 [34]. The result was further confirmed by the X-ray diffraction (XRD), as we can see from Fig. 1C, all the diffraction peaks of YVO4: Dy3+ match well with the No. 01-0701281 JCPDS card [42], suggesting that the tetragonal YVO4: Dy3+ nanoparticles have been synthesized, and the doping of Dy3+ ions didn’t change the host lattice structure. The transmission electronic microscope (TEM) image reveals that the CDs are spherical and monodispersed with the diameters of 5 to 8 nm (Fig. 1D). In view of that the CDs possesses rich functional groups like eOH, COOH, and COee], we postulate that the CDs can self-assemble with YVO4: Dy3+ through simple mixing method, since the oxygen-containing group has high affinity to the metal ions on the surface of YVO4: Dy3+ [25]. The CDs tightly surround the central YVO4: Dy3+ nanoparticle to fabricate the hybrid nanoparticles CDs@YVO4: Dy3+ nanoparticles (Fig. S1, Supporting Information), the thickness of CDs layer is about 15∼18 nm, implying the successful preparation of CDs@YVO4: Dy3+ nanoparticles with the average size ranging 80∼140 nm. 3

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Fig. 1. (A) SEM spectra of YVO4: Dy3+. (B) TEM image of YVO4: Dy3+. Insert shows the high-resolution TEM (HRTEM) image of YVO4: Dy3+ (200 crystal plane) (C) XRD spectra of YVO4: Dy3+. (D) TEM image of CDs. (E) TEM image of CDs@YVO4: Dy3+ nanoparticle.

the captured Cu2+ ions by PPi released, resulting in the quenching of Dy3+ emission again. Based on this, the ‘off-on-off’ strategy was proposed for the ALP detection. To explore the fluorescence quenching mechanism of CDs@YVO4: Dy3+ by Cu2+, the fluorescence of YVO4: Dy3+ nanoparticles and CDs in the presence of Cu2+ ions were measured, respectively. The experiment results exhibited that the fluorescence of YVO4: Dy3+ was quenched thoroughly when 3.75 μM Cu2+ ions added (Fig. S5, Supporting Information), which is slightly better than that quenching efficiency of CDs@YVO4: Dy3+ by Cu2+ (4 μM). It is well known, as one of the transition metal ions, Cu2+ ions usually act as the fluorescence quencher to most of optical probes by the means of either energy or electron transferring to the partially filled d-orbitals of Cu2+ ions [40,51,52]. Also, the light scattering spectrum of CDs@ YVO4: Dy3+ added different concentrations of Cu2+ were measured (Fig. 4B). The LS intensity of CDs@YVO4: Dy3+ increased slowly with the increasing Cu2+, indicating that the distance between CDs@YVO4: Dy3+ nanoparticles changed by Cu2+ ions, in other words, Cu2+ ions caused the aggregation of CDs@YVO4: Dy3+ [53]. Chen et al. argued that the closer distance between Cu2+ and Dy3+ could significantly improve the efficiency of energy transfer between Cu2+ and Dy3+, the similar experiment phenomenon has also been found in the YVO4: Eu3+ nanoparticles [54]. While, for the CDs, due to its optical stability to Cu2+ ions, the fluorescence of CDs cannot be quenched (Fig. S6, Supporting Information). In addition, the ζ-potentials of CDs@YVO4: Dy3+ was studied before and after adding Cu2+, PPi, and ALP (Fig. S7, Supporting Information). The ζ-potential of CDs@YVO4: Dy3+ nanoparticle was measured to be −20.5 mV, which is probably related to the abundant COOe and OeH group on the surface of CDs, and it decreases

(inset of Fig. S3, Supporting Information). In addition, the doping concentration of Dy3+ has also been investigated to optimize the luminescence performance of YVO4: Dy3+ (Fig. S4, Supporting Information). With the increasing Dy3+ concentration, the fluorescence intensity of YVO4: Dy3+ increases till it up to 5%, then it reduced gradually, which is owing to the known concentration quenching effect [32]. Therefore, the optimal doping concentration of Dy3+ was determined to be 5%. The fluorescence emission spectrum of CDs@YVO4: Dy3+ nanoparticles (Fig. 3C) displays the characteristic emission peaks both of CDs and YVO4: Dy3+ and their blue green emission under 254 nm UV lamp can also be observed clearly (inset of Fig. 3C). Moreover, we explored the fluorescence property of CDs@YVO4: Dy3+ after storing for one week and irradiating under UV light for 1 h as well (Fig. 3D), the experiment result exhibits that the fluorescence intensity of CDs@ YVO4: Dy3+ remained almost unchanged, demonstrating its excellent optical stability. 3.2. Sensing mechanism of CDs@YVO4: Dy3+ to ALP As shown in Fig. 4A, the fluorescence of CDs@YVO4: Dy3+ at 574 nm was quenched by Cu2+ ions (4 μM), while the characteristic emission of CDs at 405 nm remained unchanged. The addition of PPi or ALP individually has almost no affection on those characteristic emission both of Dy3+ and CDs, but the added PPi can effectively restore the quenched Dy3+ fluorescence by Cu2+, the fluorescence recovery efficiency of YVO4: Dy3+ was up to 98% in the presence of 16 μM PPi. Considering that the ALP can hydrolyze P2O74− groups into PO43- ions, 4

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Fig. 2. (A) Full range XPS spectra of CDs. (B) The C 1s XPS spectra of samples CDs. (C) The O 1s XPS spectra of samples CDs. (D) The N 1s XPS spectra of samples CDs.

dramatically to −6.2 mV when 4 μM Cu2+ added to. While the addition of PPi made the ζ-potential of CDs@YVO4: Dy3+ reverting to −21.2 mV, since the Cu2+ ions can be capture by PPi efficiently, and finally, the ζ-potential decreased again to −8.2 mV in the presence of ALP [55]. Thus, it can be concluded that the Cu2+ ions triggered the aggregation of CDs@YVO4: Dy3+ due to the high affinity of COOe and OeH groups to metal ions, which made the distance between Cu2+ and Dy3+ close, and ensured the quenching efficiency of CDs@YVO4: Dy3+

nanoparticles by Cu2+ ions though surrounded with a layer of CDs, it also resulted in the significant decreasing of ζ-potential, and the PPi captured Cu2+ successively leads to the increasing ζ-potential. 3.3. Optimization of sensing conditions To obtain the optimal sensing capability, the concentrations of Cu2+ ions, PPi, CDs@YVO4: Dy3+ and temporal dynamics of CDs@YVO4: Fig. 3. (A) FT-IR spectra of YVO4: Dy3+ (a line), CDs@YVO4: Dy3+ (b line), and CDs (c line). (B) FL spectra of CDs under different excitation wavelengths. Inset shows the photograph of CDs under daylight (left) and a 254 nm UV lamp (right), respectively. (C) FL spectra of CDs@YVO4: Dy3+. Inset shows the photograph of CDs@YVO4: Dy3+ under daylight (left) and a 254 nm UV lamp (right), respectively. (D) Fluorescence intensity of CDs@ YVO4: Dy3+ (0.75 μM) at 574 nm peak in different time, Error bars represent standard deviation (SD) (n = 3).

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Fig. 4. (A) FL spectra of CDs@YVO4: Dy3+ (0.75 μM) in the presence of Cu2+, PPi, ALP, Cu2+/ PPi, and Cu2+/ PPi/ ALP in Tris-HCl buffer (50 mM, pH 7.4), λex =272 nm. The concentrations of Cu2+, PPi, and ALP were 4 μM, 16 μM, 2000 U/L, respectively. (B) LS spectra of CDs@YVO4: Dy3+ in the presence of Cu2+ ions with different concentration.

Dy3+ for ALP sensing was investigated. The ratio of fluorescence intensity at 574 nm to that of 405 nm (F574/F405) decrease with the increasing Cu2+ concentration, till it up to 4 μM, the maximum fluorescence quenching efficiency obtained, and then keeps on hold (Fig. S8, Supporting Information). On the contrary, the F574/F405 increased with the increasing PPi concentration, and it reached the maximum value when the PPi concentration was 16 μM. Similarly, the optimization of CDs@YVO4: Dy3+ nanoparticles concentration was measured (Fig. S9, Supporting Information), the results shown that with the increasing CDs@YVO4: Dy3+, the fluorescence recovery of 574 nm increased gradually, when the concentration upped to 0.75 μM, the maximum recovery obtained, and it then decreased. Finally, the temporal dynamics experiment of CDs@YVO4: Dy3+ for ALP detection was executed (Fig. S10, Supporting Information). With the different concentrations of ALP added to, the Dy3+ fluorescence of CDs@YVO4: Dy3+/Cu2+/PPi samples declined in different degree, and slowly reached to a steady value within 50 min (Fig. S11, Supporting Information). Thus, in our present scenario for ALP sensing, the optimal concentration of Cu2+ ions, PPi, and CDs@YVO4: Dy3+ was determined to be 4 μM, 16 μM, and 0.75 μM, respectively, and with the response time of 50 min. We further studied the effects of pH and ionic strength on the fluorescence performance of CDs@YVO4: Dy3+ (Fig. S12 and S13, Supporting Information). The result exhibited that the CDs@YVO4: Dy3+ possesses excellent pH-independency optical stability over a wide range of 3–12, and salt-tolerance performance with ionic strength from 0.1 to 4 M. Both of the pH and ionic strength has no obvious effect on the fluorescence intensity of CDs@YVO4: Dy3+, and the outstanding chemistry, optical stability expands the application range of CDs@ YVO4: Dy3+ efficiently.

the more Na3VO4 introduced, the worse suppression efficiency of ALP activity obtained. Therefore, the fluorescence intensity at 574 nm increased with the increasing concentration of Na3VO4, when the Na3VO4 concentration raised to 200 μM, the fluorescence recovery efficiency of 574 nm was as high as 98%, and the half-maximal inhibitory concentration (IC50) was reckoned to be 26 μM (insert of Fig. 5C). Compared with those reported results, the present approach can not only be used for ALP activity detection, but also ALP inhibitors screen [7,56]. 3.5. Selectivity of assay To study the selectivity of present strategy for ALP sensing, the fluorescence intensity of CDs@YVO4: Dy3+ in the presence of other interferential substances such as HRP, Thr, Lys, BSA, PKA, CK2, and Cys (Fig. 5D) were evaluated. It can be found that the fluorescence intensity of CDs@YVO4: Dy3+ is nearly undisturbed by these materials, demonstrating the favorable selectivity of present probe. Inspired by its high sensitivity and selectivity, the fluorescence probe CDs@YVO4: Dy3+ has been applied to the target ALP detection in real samples. Seven different serum samples from normal adults, chronic hepatitis B patients, and nephritis patients were collected and used for ALP detection through the standard addition method. The ALP concentration of normal adult is calculated to be 102.5 U/L (Table 1), which are consistent with the results from pNPP-based standard chromogenic method [57]. But a much higher and lower ALP concentration occurred in those serum samples from the chronic hepatitis B patients, and nephritis patients, respectively. The good recovery in the range of 98.1% to 106.8% confirms the reliability of this proposed sensing strategy for ALP detection in biological samples as well. 4. Conclusions

3.4. Assay of ALP In summary, we reported the CDs@YVO4: Dy3+ nanoparticles synthesized by a simple mixing method for the ALP sensing in the present work. The fluorescence of CDs@YVO4: Dy3+ is composed of two parts, one is the blue emission at 405 nm from the CDs, and the other is the Dy3+ characteristic emission from YVO4: Dy3+. To construct the ratiometric fluorescence probe for ALP discrimination, the Cu2+ and pyrophosphate were also employed in this strategy, utilizing the excellent fluorescence quenching efficiency of Cu2+ to the Dy3+ ions emission of CDs@YVO4: Dy3+, as well as the strong affinity of PPi for Cu2+. The addition of ALP catalyzes the hydrolysis of PPi, causing the release of Cu2+, resulting in the Dy3+ ions emission quenched, while the CDs emission at 405 nm retained unchanged, based on this, the offon-off ratiometric fluorescence platform was constructed to detect ALP activity. The experiment result shows that the ratio of F574/F405 is linear to the concentration of ALP in the range of 0.05∼3000 U/L, with a detection limit of 0.04 U/L, which is comparable or better than those reported fluorescence probe, especially the calibrated signal introduction of CDs can eliminate the background interference, improve the

Under the optimal conditions, the sensing performance of CDs@ YVO4: Dy3+ probe for ALP detection was evaluated (Fig. 5A). The fluorescence intensity at 574 nm decreased gradually as the increasing ALP concentration, when the ALP concentration was 3000 U/L, it was quenched thoroughly. From Fig. 5B, we can find that in the range of 0.05 ∼ 3000 U/L (insert of Fig. 5B), the ratio of F574/F405 is linear to the concentration of ALP, and the detection limit is 0.04 U/L calculated from 3σ/S, which is comparable with or better than other previously reported assays for the ALP detection (Table S1, Supporting Information). Considering the chemical stability of CDs@YVO4: Dy3+ nanoparticles and the protective effect of the CDs layer surrounded YVO4: Dy3+, which can efficiently prevent the outermost VO43− groups of YVO4: Dy3+ from reacting with those ALP molecules. Furthermore, using this ratiometric fluorescence probe, we explored the depression effect of inhibitor Na3VO4 on ALP activity (Fig. 5C). Since that Na3VO4 can inhibit efficiently the ALP activity, the enzyme substrate PPi cannot be catalyzed hydrolyzed by ALP, leading to the fluorescence restoring, 6

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Fig. 5. (A) FL spectra of CDs@YVO4: Dy3+ (0.75 μM) in the presence of varying concentrations of ALP from 0 to 3000 U/L. The concentrations of ALP were (from top to bottom, U/L): 0.05, 0.5, 1, 10, 50, 100, 500, 1000, 2000, 3000, λex =272 nm. The arrow indicates the signal changes with the increase of ALP concentration. Inset shows the photographs of CDs@YVO4: Dy3+ added to different concentrations of ALP under a 254 nm UV lamp. (B) The Plot of F574/F405 against the concentrations of ALP ranging from 0 to 3000 U/L (where F574 and F405 are the FL intensities of CDs@YVO4: Dy3+ at 574 and 405 nm, respectively). Error bars represent SD (n = 3). (C) FL spectra of CDs@YVO4: Dy3+ (0.75 μM) upon different concentrations of Na3VO4. Insert shows the Plot of the fluorescence recovery of 574 nm peak against the concentrations of Na3VO4 ranging from 0 to 200 μM. (D) Selectivity competition experiments for CDs@ YVO4: Dy3+ (0.75 μM) toward different interferences. All biomolecules were at a concentration of 2500 U/L.

online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110618.

Table 1 Results for detection of ALP from real blood samples. Samples

Added(U/L)

Detected(mean, U/L)

RSD(n = 3,%)

1 2 3 4 5

0 100 200 500 1000 0 0

102.5 209.3 310.9 593.2 1110 401.1 30.5

2.2 2.9 2.0 1.8 2.5 2.0 2.8

6 7

References

Recovery(%)

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106.8 104.2 98.1 100.8

Serum samples were diluted 100-folds. The serum samples of 1–5, 6, and 7 were taken from normal adult, chronic hepatitis B patients, and nephritis patients, respectively. Mean value of three determinations. The detection concentration of normal adults, chronic hepatitis B patients, and nephritis patients is 100.8 U/ L, 400.5 U/L, 32 U/L by the pNPP based standard ELISA method, respectively.

accuracy of proposed probe greatly. Finally, the discrimination of ALP enzyme inhibitor and ALP concentration in real human serum sample has also been measured, demonstrating the applicability of CDs@YVO4: Dy3+ fluorescence sensor well. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (21565019, 21705083 and 21675078), Jiangxi provincial science and technology projects of china (GJJ17132). Appendix A. Supplementary data Supplementary material related to this article can be found, in the 7

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