Reversible Fluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-Time Acid Phosphatase Activity Monitoring.

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A Reversible Fluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-time Acid Phosphatase Activity Monitoring Zhaosheng Qian, Lujing Chai, Qian Zhou, Yuanyuan Huang, Cong Tang, Jianrong Chen, and Hui Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01488 • Publication Date (Web): 26 Jun 2015 Downloaded from http://pubs.acs.org on July 1, 2015

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Analytical Chemistry

A Reversible Fluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-time Acid Phosphatase Activity Monitoring Zhaosheng Qian, Lujing Chai, Qian Zhou, Yuanyuan Huang, Cong Tang, Jianrong Chen and Hui Feng* College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China ABSTRACT: A reversible fluorescence nanoswitch by integrating carbon quantum dots nanoassembly and pyrophosphate ion is developed, and a reliable real-time fluorescent assay for acid phosphatase activity is established on the basis of the fluorescence nanoswitch. Carbon quantum dots abundant in carboxyl groups on the surface, nickel(II) ion and pyrophosphate ion comprise the fluorescent nanoswitch, which operates in the following way: the nanoassembly consisting of carbon quantum dots and nickel ions can be triggered by pyrophosphate ion serving as an external stimulus. At the same time, the fluorescence nanoswitch switches between two fluorescence states ON and OFF accompanying shifts in their physical states aggregation and disaggregation. On the basis of the nanoswitch, the introduction of ACP leads to breakdown of pyrophosphate ions into phosphate ions and resultant fluorescence quenching due to catalytic hydrolysis of ACP towards PPi. Quantitative evaluation of ACP activity in a broad range from 18.2 to 1300 U/L with the detection limit of 5.5 U/L can be achieved in this way, which endows

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the assay with high enough sensitivity for practical detection in human serum and seminal plasma. KEYWORDS: carbon quantum dots, acid phosphatase, fluorescent nanoswitch, pyrophosphate ion, aggregation and disaggregation

n INTRODUCTION Acid phosphatase (ACP) as a hydrolase enzyme is responsible for phosphates scavenging which is widely spread in nature. It is found that in human body an abnormal elevated level of ACP is closely related to many diseases including prostate cancer, Gaucher disease and disorders related to kidneys, veins and bones.1 As a result, ACP is always considered as an important serum marker for related disease2 and a useful prognostic indicator3. Meanwhile, some types of ACPs such as phosphatidic acid phosphatase type 2C4 and tartrate-resistant acid phosphatase5 which can be used as drug targets have caused much attention for drug discovery.6,7 The rapid and convenient analysis of ACP in blood is of particular importance since the level of ACP is used as a preliminary early diagnosis for many diseases. ACP has been determined by several methods including electrochemical oxidation,8 cyclic voltammetry,9 amperometry,10 chromatographic11 and fluorometric approach.12 Due to its high sensitivity, convenience and accessible instrument requirement, fluorescent assay of ACP is regarded as a more desirable method among these methods. Up to date, only few numbers of fluorescent assays for ACP detection have been reported, and fluorometric indicators used in those fluorescent assays were mainly focused on organic dyes and fluorescent polymers.12-15 Two kinds of organic dyes with positive charges as the fluorophore and hexametaphosphate as the substrate were employed to assess ACP activity through aggregation-caused quenching,12,13 but only Guo et al.’s work realized quantitative evaluation of ACP activity/level.12 Atul et al.14


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exploited anionic polyfluorene derivative as the fluorophore with assistance of ferric ions for qualitative assessment of ACP activity. Xie et al.15 used a conjugated polyelectrolyte and pnitrophenyl phosphate to quantitative determination of ACP amounts through a PET quenching mechanism. As a result, most of the preceding assays could not meet the urgent demand for the accurate and convenient assessment of ACP activity/level owing to their non-negligible drawbacks such as complicated synthesis procedures and poor stability for organic dyes, and low sensitivity for those based on fluorescent polymers. Carbon quantum dots (CQDs) as a rising fluorescent nanomaterial have showed outstanding performance in photovoltaic devices, photocatalysis, bioimaging and sensors.16-20 Recent studies showed that CQDs combined a number of key merits from the viewpoint of fluorescent material, including stable light emitting, high quantum yield, good photostability, small size, easy modulation, low toxicity and excellent biocompatibility, and thus are being explored as an alternative for dye-based probe, toxic semiconductor quantum dots and weakly fluorescent polymers. Our pervious work has shown that CQDs as an outstanding fluorophore can be utilized to construct various biosensors for respective and simultaneous detection of DNA and thrombin.21,22 Recently, our group integrated CQDs and metal ions to realize convenient and real-time assays for alkaline phosphatase activity through aggregation/disaggregation of CQDs.23,24 On the basis of the preceding work, we developed a convenient and reversible fluorescent nanoswitch for ACP activity monitoring by integrating carbon quantum dots (CQDs) nanoassembly and pyrophosphate ions (PPi). A nanoswitch is often regarded as a construct which can change its states in response to external chemical, electrochemical or photochemical stimuli.25,26 The majority of pertinent work devoted to constructing DNA-based nanoswitches through hybridization,27 while another new kind of nanoswitches by means of coordination


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between metal ions and molecules/nanomaterials were developed in recent years to achieve controllable operation.28,29 Inspired by the latter nanoswitch, we designed and developed a CQDs-based nanoswitch mediated by nickel ion and triggered by PPi to turn on the fluorescence. Combining with catalytic hydrolysis ability of ACP to PPi, a novel detection strategy for ACP activity on the basis of the nanoswitch was proposed. Different from detection strategy for alkaline phosphatase (ALP), nickel ion was utilized to assemble carbon quantum dots instead of copper ion because copper ion was proven as a strong inhibitor for ACP activity. Nickel ions can heavily assemble carbon quantum dots into bigger nanoassemblies accompanying fluorescence switching off because they are capable to complex with carboxyl group on the surface of CQDs. This nanoassembly can be disaggregated by the introduction of PPi owing to stronger affinity of PPi to nickel ions. Consequently, carbon quantum dots nanoassembly containing nickel ions constitute a fluorescent nanoswitch which can be triggered by PPi. This fluorescent nanoswitch consisting of CQDs, nickel ion and pyrophosphate ion can operate in a reversible way. This fluorescent nanoswitch was further taken advantaged of to assess ACP activity by aid of its capability for catalytic hydrolysis of PPi into Pi.

n EXPERIMENTAL SECTION Materials and Reagents. Triple-distilled water was used throughout the experimental process. Activated

carbon,

nickel(II)

nitrate

(Ni(NO3)2),

sodium

pyrophosphate

(PPi)

and

tris(hydroxymethyl)aminomethane were purchased from Aladdin company (Shanghai, China). Acid phosphatase (ACP, EC 3.1.3.2) from potato, alkaline phosphatase (ALP, EC 3.1.3.1) from bovine intestinal mucosa, acetylcholinesterase (AChE, EC 3.1.1.7), immune globulin G (IgG), bovine serum albumin (BSA) and sodium vanadate were bought from Sigma-Aldrich company (Shanghai, China). Tris(hydroxymethyl)aminomethane buffer solution (Tris-HCl, pH=7.4) was


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prepared by mixing stock solutions of Tris and HCl. All reagents were of analytical grade and without any further purification. Synthesis of carbon quantum dots (CQDs). The detailed preparation procedure was described in our previous paper.23,24 Brief procedure is as follows: The mixture containing activated carbon powder (2.0 g), concentrated H2SO4 (180 mL) and HNO3 (60 mL) was placed in an oil bath and heated at 80 °C for 5 h. The resultant mixture was cooled to room temperature and then diluted with triplex-distilled water (800 mL), and further neutralized with sodium hydroxide. The final dark solution was dialyzed to keep the materials that remained between 1 kDa and 50 kDa dialysis bags. Fluorescence quenching of CQDs by nickel ions. For optimization of incubation time for fluorescence quenching by nickel ions, 40.0 µL of Ni2+ (10.0 mM) was added into 3.0 ml of CQDs (Tris-HCl, pH=7.4) solution. Then, the fluorescence of mixed solution was recorded after different shaking time of 2, 4, 6, 8, 10, 12, 14 and 16 minutes respectively. Under the optimum incubation time, fluorescence quenching of CQDs was assessed with continuous addition of nickel ions. The fluorescence intensity of the mixtures containing CQDs and varying amounts of Ni2+ was monitored using fluorescence spectrometer at the optimal excitation wavelength. For optimization of incubation time for fluorescence recovery by PPi, the fluorescence of CQDs was first quenched by adding 40.0 µL of Ni2+ (10.0 mM) into 3.0 mL of CQDs (Tris-HCl, pH=7.4) solution. Then, 150.0 µL of PPi solution (10.0 mM) was introduced to the above mixture. The fluorescence was recorded after different incubation time of 2, 4, 6, 8, 10 and 12 minutes respectively. Selectivity of the sensing system towards PPi. For the selectivity assessment to PPi, nine kinds of different anions including F–, Cl–, Br–, I–, SO42–, NO3–, Ac–, HCO3– and PO43– were


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selected to evaluate the influence of anions on the fluorescence of CQDs-Ni system. A certain amount of nickel ions were added into 3.00 ml of CQDs solution (3.5 mg/mL), where the concentration of nickel ions was 186.9 µM. A certain amount of each anion (467.3 µM) was then introduced into the above CQDs-Ni system, and then fluorescence spectra of resulting mixtures were monitored at the excitation wavelength of 452 nm. Tris-HCl buffer (pH 7.4) was selected as the buffer for selectivity assessment experiments. Fluorescent ACP assay. The ACP activity was first assessed as extending incubation time with a certain amount of ACP level. The sensing system containing CQDs (Tris-HCl, pH=7.4) solution, Ni2+ (133.33 µM), PPi (500.00 µM), and ACP (600.0 U/L) was monitored by fluorescence spectroscopy every 10 minutes. The fluorescence spectra of the system were recorded from 0 to 60 minutes after the addition of ACP at the excitation wavelength of 452 nm. For the real-time enzyme assays, different levels of ACP with different activity ranging from 10.0 to 980.0 U/L was respectively added into 3.00 ml of CQDs (Tris-HCl, pH=7.4) solution containing Ni2+ (133.3 µM), PPi (500.0 µM). After an incubation time of 60 minutes, the fluorescence spectra of the mixture were recorded respectively. Selectivity of fluorescent ACP assay towards ACP. To evaluate the selectivity of the assay towards ACP, 294.0 µL of each enzyme including ACP, AchE, BSA and IgG (3.0 mg/ml) was added separately into the mixture containing 3.0 mL of CQDs, 60.0 µL of Ni2+ (10.0 mM) and 150 µL PPi (10.0 mM), and then fluorescence spectra of resulting mixtures were monitored at the excitation wavelength of 452 nm after an incubation time of 8.5 h. In order to distinguish ACP from ALP, sodium vanadate as a strong inhibitor for ALP was utilized to inhibit ALP activity. The fluorescence of standard assay solution for ALP and ACP in the presence of sodium


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vadanate (3333.3 µM) was recorded respectively. The incubation time for ACP or ALP with sodium vadanate was one hour. Characterization methods. Transmission electron microscopy (TEM) was conducted on a JEOL-2100F instrument with an accelerating voltage of 200 kV. A Kratos Axis ULTRA X-ray photoelectron spectrometer was used for the X-ray photoelectron spectroscopy analyses. The UV-Vis spectra were recorded on a PerkinElmer Lambda 950 spectrometer. The fluorescence spectra and time-resolved fluorescence decay tests were conducted using an LS 55 PerkinElmer, and an FLS920 Edinburgh Instruments fluorescence spectrophotometer.

Scheme 1. Schematic illustration of detection strategy for ACP activity based on the fluorescent CQDs nanoswitch triggered by PPi.

n RESULTS AND DISCUSSION Principle of ACP activity assay based on fluorescent CQDs nanoswitch. Scheme 1 shows the schematic illustration of detection strategy for ACP activity based on the fluorescent CQDs


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nanoswitch which can be triggered by PPi. In this study, we chose green fluorescent CQDs from chemical oxidation of activated carbon as the fluorophore constituent to construct the nanoswitch. Nickel(II) ion as a molecular glue was selected to aggregate CQDs instead of copper(II) ion which was utilized in our pervious work for a fluorescent assay for alkaline phosphatase because copper ion was found to be a strong inhibitor for ACP. The as-prepared CQDs from chemical oxidation of activated carbon have been proved to possess a vast number of oxygen-containing groups, and these chemical functionalities account for excellent water dispersibility of CQDs while also provide useful functionals to construct the following nanoswitch. The carboxyl group as one of these functionals of CQDs can serve as a good ligand for some metal ions including Ce(III), Cu(II) and Ni(II) which can lead to effective aggregation of CQDs. As a result, nickel ions were utilized to integrate CQDs due to their strong complexation affinity and negligible influence on ACP activity induced by nickel ions. As shown in Scheme 1, the presence of nickel ions triggers severe aggregation of CQDs due to their strong coordination affinity, and thus quenches the fluorescence of CQDs. In this state, CQDs are in the physical aggregation state as a nanoassembly while their fluorescence is switching off. PPi as an external stimulus to generated nanoassembly can convert aggregated CQDs into dispersed CQDs accompanying the formation of more stable Ni-PPi complex. Dispersed CQDs simultaneously exhibit green fluorescence, hence fluorescence signaling is shifted to switching on. On the basis of the nanoswitch, ACP acting as a scissor can effectively and specifically hydrolyze PPi into phosphate ions (Pi) which has a quite small stability constant with Ni(II). Consequently, the addition of ACP into the nanoswitch with ON state would lead to freedom of nickel ions and following re-aggregation of CQDs accompanying fluorescence quenching. In a certain time, varying amounts of ACP would


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lead to different degrees of fluorescence quenching, which can be used to monitoring the activity of ACP. Synthesis and characterization of CQDs. The synthesis and characterization of CQDs were described in our previous paper,23,24 and thus a brief description on CQDs is as follows. Chemical oxidation of activated carbon using concentrated sulfuric and nitric acids was employed to prepare initial CQDs, and the following purification including filtration and dialysis were performed to obtain pure and size-uniformed CQDs. The as-prepared CQDs were further characterized by TEM, XPS and fluorescence spectroscopy respectively. Size distribution of CQDs from TEM images were obtained in the range of 2 – 5 nm, and furthermore crystalline structure of single CQDs was observed from HR-TEM. XPS analysis (Figure S1) suggested that CQDs are composed of carbon and oxygen, and have predominant functionals including carboxyl, hydroxyl and carbonyl group. Element analysis indicated that 25.5 at% of oxygen and 74.5 at% of carbon constitute carbon nanodots. Strong green photoluminescence in water under the UV lamp can be observed for the as-synthesized CQDs. Fluorescence spectra in Figure S2 display that CQDs possess strongest fluorescence emission peak at 518 nm when excited at 446 nm, and show excitation-independent emission characteristic. Assessment of detection strategy for ACP activity based on CQDs nanoswitch. In our previous paper,24 we have assessed the influence of the fluorescence of CQDs by twelve metal cations, and found that copper ion has the strongest quenching ability while the presence of Fe3+, Ni2+, Co2+ and Fe2+ brings apparent impact to the fluorescence intensity with different quenching effect. Among these five metal ions, copper and ferric ions have been reported to be strong inhibitors for ACP activity30,31 and thus are excluded for choice in this work. Further assessment for Ni2+, Co2+ and Fe2+ were carried out as follows. As shown in Figure 1, effective fluorescence


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Figure 1 The changes of fluorescence spectra of CQDs in the presence of different constituents (a) mere CQDs; (b) in the presence of metal ions (133.3 µM); (c) in the presence of metal ions (133.3 µM) and PPi (500.0 µM); (d) in the presence of metal ions (133.3 µM), PPi (500.0 µM) and ACP (980 U/L). Ni2+, Co2+ and Fe2+ are used for A, B, and C respectively.

quenching by Ni2+ and Co2+ can be readily achieved by addition of each metal ions into CQDs solution whereas the presence of Fe2+ in CQDs solution leads to disordered fluorescence spectrum despite the decrease of original fluorescence peak in intensity. The introduction of PPi into the preceding mixtures can result in fluorescence recovery in both intensity and shape for the three metal ions, and greater extent of fluorescence restoration for the mixture containing Ni2+ or Co2+ can be observed than that for Fe2+. Substantial decrease in fluorescence intensity can be observed after the addition of ACP into the preceding mixture containing Ni2+ or Co2+, however, the fluorescence intensity was barely changed for the mixture containing CQDs, Fe2+ and PPi with the addition of ACP, which suggests that Fe2+ has strong inhibition effect on ACP activity while Co2+ and Ni2+ have little impact on it. Furthermore, toxicity of Co2+ is generally regarded much higher than Ni2+ according to the previous report.32 Therefore, nickel(II) ion is the best choice to aggregate CQDs as a molecular glue through balancing their performance and sustainability. As shown in Scheme 1, the detection strategy based on the nanoswitch consisting of CQDs, Ni2+ and PPi for ACP activity was proposed and verified as follows. It can be seen from Figure


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Figure 2 (A) The TEM images of mere CQDs (A), the mixture containing CQDs and Ni(II) (200.0 µM) (B and C), and the mixture containing CQDs, Ni(II) (200.0 µM) and PPi (600.0 µM) (D).

1A that the presence of nickel ions caused mostly fluorescence quenching of CQDs, thus we assume that nickel ions can easily bind to the surface of CQDs through carboxyl group, and the coordination of one nickel ion with several CQDs can result in the severe aggregation of CQDs with each other. The sharp reduction of the fluorescence is originated from severe aggregation of CQDs. In order to verify the proposed mechanism of aggregation caused fluorescence quenching by nickel ions, we compared TEM image of mere CQDs with that of the mixture of CQDs and nickel ions as shown in Figure 2A and 2B. It is clearly illustrated that CQDs are well-dispersed


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ranging from 2 to 5 nm in diameter in the absence of nickel ions, but a vast number of large aggregates of CQDs with around 20 nm in diameter were observed in the presence of nickel ions. High-resolution TEM in Figure 2C displays that the large aggregate is composed of a great number of CQDs. These results provide proofs for severe aggregation of CQDs induced by nickel ions, which contributes to fluorescence quenching of CQDs, i.e., aggregation caused fluorescence quenching mechanism (ACQ). In addition, we observed that continuous addition of PPi anions into the mixture of CQDs and Ni2+ lead to fluorescence recovery to a large extent, and the fluorescence recovery ratio is up to 80% when the concentration of PPi is up to 500.0 µM. It is assumed that the PPi would complex preferentially to nickel ions and thus grab nickel ions from the aggregates between CQDs and nickel ions due to stronger affinity of PPi to nickel ions than the carboxylate groups, and the formation of PPi-Ni complexes would disaggregate the CQDs aggregates and set them free again, which would lead to the fluorescence recovery. The disaggregation of CQDs caused by PPi was confirmed by TEM image in Figure 2D, where CQDs are well-dispersed after the addition of PPi anions. Consequently, the CQDs-based fluorescent nanoswitch can operate in this way: the presence of Ni(II) switches off the fluorescence of CQDs, where they are in dispersed state physically; the following introduction of PPi as an external stimulus can triggers state shift from aggregation state to disaggregation state accompanying switch-on of the fluorescence. To investigate the reversibility of as-built nanoswitch, sequential addition of Ni2+ and PPi into CQDs solution was conducted to achieve signal switching of the system and physical state shifting of CQDs. Figure S4 showed added species dependent character of the CQDs nanoswitch. The fluorescence intensity of the nanoswitch displays reversible changes due to physical state changes through the addition of different species between Ni2+ and PPi. It can run well more than three cycles and shows a good


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Figure 3 (A) The changes of fluorescence spectra of CQDs in the presence of varying amounts of Ni(II). (B) The changes of fluorescence spectra of the mixture containing CQDs and Ni(II) (563.3 µM) in the presence of varying amounts of PPi. (C) The linear fitting curve between fluorescence intensity and concentration of PPi. (D) The selectivity of as-established methods towards PPi. The concentrations of Ni(II) and each anion are 186.9 and 467.3 µM, respectively.

reversibility. Finally, when a certain amount of ACP was introduced into the mixture including CQDs, Ni2+ and PPi after 60 minutes’ incubation, it was observed that the fluorescence intensity decreased to nearly a half of the original value, indicating that PPi was hydrolyzed into phosphate anions under the catalysis of ACP. The reduction of fluorescence intensity is due to re-aggregation of CQDs induced by the free nickel ions from breakdown of PPi-Ni complex in the solution. On the basis of the nanoswitch consisting of CQDs, Ni(II) and PPi, it is feasible to establish real-time assay for ACP activity.


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Quantitative detection of PPi based on CQDs nanoswitch. It has been reported that PPi could be regarded as a potential biomarker for clinic diagnosis and therapy of arthritic disease,33 and are related to some severe medical conditions in term of their levels.34 Thus, quantitative detection of PPi based on CQDs nanoswitch was first performed since PPi as an external stimulus can trigger the nanoswitch. By employment of disaggregation induced enhancement (DIE) followed aggregation caused quenching (ACQ), the rapid and sensitive determination of PPi can be readily realized. The condition optimization including the added amount of nickel ions, and the incubation time for ACQ and DIE were first carried out. The results in Figure S5 indicate that a fast response time can be reached with an equilibrium time of 2 minutes for both ACQ and DIE. Figure 3A shows gradual decrease in fluorescence intensity with the increase of nickel ions in amount, and the fluorescence intensity reaches the minimum when the concentration of nickel ions is up to 563.3 µM. Thus, the concentration of 563.3 µM for nickel ions and 2 minutes incubation time were chosen as the optimal conditions for the following quantitative determination. Under the optimal conditions, the sensing performance of this system for detection of PPi was evaluated by adding varying concentrations of PPi into the preceding CQDs/Ni2+ mixture. The fluorescence response of these systems was recorded by adding different concentrations of PPi in pH 7.4 buffer solutions at room temperature. It can be seen from Figure 3B that the continuous addition of PPi into CQDs-Ni2+ aggregate solution stepwise switches on the fluorescence of the nanoswitch, and nearly complete recovery of the fluorescence is achieved when the concentration of PPi is up to 1300.0 µM. A good linear fitting curve between fluorescence intensity and PPi concentration can be obtained in the range of 8.53 – 700.0 µM. This equation can be expressed as y = 0.4 x + 216.45 where R2 = 0.9997. The detection limit for PPi was determined to be 2.56 µM as estimated form the derived calibration


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Figure 4 (A) The change in fluorescence intensity of sensing system containing CQDs solution, Ni2+ (133.3 µM) and PPi (500.0 µM) as extending the incubation time from 0 to 60 min. (B) Fluorescence spectra of the assay with different units of ACP activity (0.0 – 1300.0 U/L) after 60 min incubation time. (C) The linear fitting curve between fluorescence intensity and ACP level. (D) The selectivity of as-established assay towards ACP. I0 and I represent fluorescence intensities before and after addition of ACP into the assay. Red and green bars represent fluorescence intensity in the absence and presence of sodium vadanate (3333.3 µM) respectively.

curve (≥ 3 standard deviations). Although its detection limit is inferior to that based on CQDsCu2+ system (0.3 µM), its linear scope is much broader than that based on CQDs-Cu2+ system (3.3 – 85.8 µM) in our previous paper.24 To investigate the selectivity of our method towards PPi, nine common anions including F–, Cl–, Br–, I–, SO42–, NO3–, HCO3–, Ac– and PO43– that may


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interfere with PPi sensing were chosen for comparison. As displayed in Figure 3D, separate addition of each anion of chosen anions except phosphate ion into the aqueous dispersion of CQDs containing nickel ions results in a slight decrease in fluorescence intensity, and the introduction of phosphate ion only leads to a slight increase in fluorescence, however, the presence of PPi in the sensing system containing CQDs and Ni(II) leads to a sharp rise of fluorescence intensity. Although the selectivity value (I-I0)/I0 for PPi is more than 3, inferior to that based on CQDs-Cu(II) system,24 it can effectively distinguish PPi from Pi because their large difference in response on the fluorescence of the sensing system. These results clearly indicate that these common anions could not interfere with the PPi detection with our method. Real-time fluorescence assay for ACP activity. On the basis of PPi-triggered fluorescent nanoswitch, ACP activity/level was evaluated in a real-time way using CQDs aqueous solution containing optimum amount of Ni2+ and PPi as the standard assay solution. Because it is well known that ACP shows best catalytic hydrolysis ability to phosphate-containing species at low pH environment such as pH 5,35 we first assessed the feasibility of our assay at pH 5.0 and compared it with that at pH 7.4. As shown in Figure S6, the presence of Ni2+ (200.0 µM) at pH 7.4 leads to more than 70% of fluorescence quenching, and the following addition of PPi (500.0 µM) causes a great fluorescence recovery with the recovery ratio of nearly 80%. However, when pH value of CQDs solution was adjusted to 5.0, the same concentration of Ni2+ only induced 10% of fluorescence quenching to CQDs solution, and no fluorescence recovery can be observed after following introduction of the same concentration of PPi to that at pH 7.4. To attain fluorescence quenching efficiency and recovery ratio at pH 5.0 close to those at pH 7.4, a large amount of Ni2+ (8333.3 µM) or PPi (11500.0 µM) has to be used, which would be time consuming for the following catalytic hydrolysis of PPi into Pi by ACP. Therefore, pH 7.4 was


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chosen to conduct the following detection. The assay solution was monitored by fluorescence emission spectroscopy after the addition of 600.0 U/L of ACP at room temperature. The typical fluorescence spectra were consecutively recorded every 10 minutes as shown in Figure 4A. PPi anions were hydrolyzed into phosphate ions under the catalysis of ACP after the addition of ACP into assay solution, and the breakdown of more and more PPi leads to re-aggregation of CQDs induced by more and more free nickel ions as the increase of time in the presence of ACP. The gradual fluorescence quenching due to catalysis activity of ACP for hydrolysis of PPi as extending the incubation time can be observed, and an equilibrium point can be reached within 60 minute under incubation condition. By balancing the quenching effect and time consumption, 60 minutes’ incubation time was chosen for quantitative determination of ACP activity. The assay was further monitored by fluorescence emission spectroscopy after addition of different units (0.0 – 1300.0 U/L) of ACP after 60 minutes incubation time. Figure 4B displays that the introduction of different ACP level ranging from 0.0 to 1300.0 U/L into the standard assay solution results in a stepwise decline in fluorescence intensity. Surprisingly, it is found that the fluorescence intensity has a good linear relation to ACP level in a broad range of 18.2 – 1300.0 U/L as shown in Figure 4C. The fitting equation can be expressed as y = -0.17 x + 520.3 with R2 = 0.996. The detection limit based on three standard errors is estimated as 5.5 U/L. Up to date, only a few reports based on fluoromentric approach presented quantitative determination of ACP using small organic dyes12,13 or fluorescent polymers14,15 as signal reporter. Three of them utilized concentration unit to present quantitative evaluation and detection limit of ACP (4.9 nM,13 0.18 nM,14 0.5 µg/L15), while only Guo et al’s work employed activity unit to establish their methods for ACP activity with detection limit of 0.05 U/L. Since it is not accessible to convert concentration unit to activity unit of ACP in the preceding three reports, it is hard to


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compare our method with those methods. In comparison with Guo et al.’s assay, the detection limit of our assay is inferior to theirs, however, our assay possesses much broader linear scope and utilizes novel carbon quantum dots as the indicator for the first time. Moreover, it has been reported that normal ACP level in blood serum is 35 - 123 U/L36 and normal value in seminal plasma is up to 30 – 36 U/mL,37 thus the as-established real-time assay is capable of practical detection of ACP level in blood serum and seminal plasma. Furthermore, we evaluated the selectivity of constructed assay for ACP activity monitoring in the presence of other common biological species including bovine serum albumin (BSA), immunoglobulin G (IgG), acetylcholinesterase (AChE) and alkaline phosphatase (ALP). As shown in Figure 4D, the presence of AChE in the standard assay solution leads to a slight increase in fluorescence intensity, and the introduction of BSA and IgG results in a small decline in fluorescence intensity. However, a huge fluorescence quenching can be observed after the addition of ACP or ALP into assay solution, and the selectivity coefficient (I0-I)/I for ACP or ALP is nearly up to 2 while those are no more than 0.2 for the others, suggesting good selectivity for ACP or ALP response. Attempting to distinguish ACP from ALP, sodium vanadate as a strong inhibitor38,39 for ALP was utilized to inhibit ALP activity. As shown in Figure 4D, the assay showed a quite slight positive response to ALP in the presence of sodium vanadate, while an apparent positive response to ACP can be observed. Although the difference in response between ACP and ALP is still not determinative, it represents a forward step to differentiate them because there is no report to effective discriminate ALP or ACP in neutral medium to our best knowledge. This is first time to achieve sensitive detection of ACP activity in a real-time way by employment of carbon quantum dots nanoassembly as a sensing platform, which possesses high enough sensitivity for practical detection of ACP level in blood serum and seminal plasma.


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n CONCLUSION In summary, a real-time fluorometric assay was designed for highly sensitive detection of acid phosphatase based on a reversible fluorescence nanoswitch constructed by carbon quantum dots. Abundance of carboxyl groups on the surface of carbon quantum dots enables severe aggregation caused fluorescence quenching by nickel ions, and the competitive interaction among carboxyl, nickel ions and PPi endows disaggregation induced fluorescence enhancement. The CQDs-based nanoswitch triggered by PPi as an external stimulus can operate in a reversible way and is capable to quantitative determination of PPi. By employment of catalytic hydrolysis of ACP tawards PPi, the introduction of ACP into the nanoswitch enables to achieve quantitative evalution of ACP level with gradual fluorescence quenching as signal readout. To our best knowledge, the present study is the first analytical system based on carbon quantum dots that can quantitatively and sensitively detect ACP activity in real time with a detection limit of 5.5 U/L, which is capable for practical determination of ACP level in human serum and seminal plasma. This novel nanoswitch shifting between aggregation and disaggregation state not only can be used to design new assays for enzyme activity, but also provides an example for smart system with controlled release capability.

n ASSOCIATED CONTENT Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

n AUTHOR INFORMATION Corresponding Author


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*E-mail: [email protected]. Tel: +86-579-82282269. Fax: +86-579-82282269.

n ACKNOWLEDGMENT We are thankful for the support by the National Natural Science Foundation of China (No. 21405142, 21005073 and 21275131) and Zhejiang Province (No. LY13B050001 and LQ13B050002).

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Graphical Abstract

A Reversible Fluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-time Acid Phosphatase Activity Monitoring Zhaosheng Qian, Lujing Chai, Qian Zhou, Cong Tang, Yuanyuan Huang, Jianrong Chen and Hui Feng*

A convenient and highly sensitive fluorescence sensing approach for acid phosphatase (ACP) activity monitoring based on carbon quantum dots (CQDs) nanoswitch was established.


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