A Turn-On Fluorescent Sensor for Selective and ... - ACS Publications

Dec 14, 2016 - of Alkaline Phosphatase Activity with Gold Nanoclusters Based on. Inner Filter Effect ..... the concentration of PNPP increased (0−1 ...
0 downloads 0 Views 4MB Size
Download PDF
Research Article www.acsami.org

A Turn-On Fluorescent Sensor for Selective and Sensitive Detection of Alkaline Phosphatase Activity with Gold Nanoclusters Based on Inner Filter Effect Haijian Liu,† Ming Li,† Yining Xia,*,‡ and Xueqin Ren*,† †

Department of Environmental Science and Engineering, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China ‡ Institute of Quality Standards and Testing Technology for Agro Products of Chinese Academy of Agricultural Sciences, Beijing 100081, China S Supporting Information *

ABSTRACT: In this work, a novel approach for simple and sensitive determination of alkaline phosphatase (ALP) is developed on the basis of an inner filter effect of p-nitrophenylphosphate (PNPP) on the fluorescence of gold nanoclusters (AuNCs). AuNCs with a high quantum yield of 12% were synthesized by one-pot strategy and were directly applied as fluorescent substance. When AuNCs were mixed with PNPP, the fluorescence of the AuNCs was remarkably quenched or was decreased via the inner filter effect since the absorption spectrum of PNPP overlaps well with the excitation spectrum of the AuNCs. While in the presence of ALP, PNPP was catalytically hydrolyzed into p-nitrophenol, which has different absorption characteristics from those of PNPP, resulting in the recovery of the AuNCs fluorescence. Thus, a novel “turn-on” fluorescent sensor for detecting ALP was established with a detection limit as low as 0.002 U/L (signal-to-noise ratio of 3). The turn-on fluorescent sensor exhibits many merits such as high sensitivity, excellent selectivity, and high signal output because of the low background signals. In addition, the developed sensing method was successfully applied to investigate ALP inhibitors and ALP determination in serum samples. A good linear relationship was obtained in the range from 0.02 to 50 U/L, and satisfactory recoveries at four spiking levels of ALP ranged from 95% to 106% with precision below 5%. The very simple sensing approach proposed here should promote the development of fluorescence turn-on chemosensors for chemo/ biodetection. KEYWORDS: alkaline phosphatase, gold nanoclusters, inner filter effect, turn-on, fluorescence

1. INTRODUCTION Alkaline phosphatase (ALP) is an enzyme extensively studied in clinical practice and is widely used as an important biomarker for medical diagnostics. Many diseases are associated with ALP level changes in serum, including bone disease, diabetes, breast and prostatic cancer, and hepatitis.1−4 Therefore, the determination of ALP activity is of vital importance to clinical diagnosis. A variety of techniques have been used to assay ALP activity including isotopic labeling,5 chromatography,6 colorimetry,7,8 chemiluminescence,9 electrochemistry,10−12 and surface-enhanced resonance Raman scattering.13 Among these methods, fluorescence-based methods have drawn significant attention owing to their high sensitivity, cost effectiveness, simplicity, and convenience. Many fluorescent chemosensors have been successfully applied to detect ALP activity on the basis of organic fluorescent dyes,14−17 conjugated polyelectrolytes,18−21 and inorganic semiconductor quantum dots (QDs).22−24 However, these approaches have disadvantages such as the poor photostability and water-solubility of organic fluorescent dyes, the complex synthesis and purification © 2016 American Chemical Society

processes of conjugated polyelectrolytes, and the high toxicity of QDs. Many of these chemosensors have been found not sensitive enough to detect ALP levels in human serum, so their application in bioassays is limited. There is a need to develop sensitive, simple, and nontoxic methods for ALP activity monitoring. Recently, fluorescent noble metal nanoclusters (NCs) have attracted overwhelming attention for their potential application as a new type of fluorophore. Fluorescent noble metal NCs are composed of small numbers of metal atoms exhibiting unique optical properties, low toxicity, good photostability and biocompatibility, large Stokes shifts, and facile preparation methods.25−28 Owing to these interesting features, metal NCs have potential for use in various fields, such as metal ion sensing, small biomolecule detection, and biological labeling and imaging. In addition, metal NCs found application in Received: September 19, 2016 Accepted: December 14, 2016 Published: December 14, 2016 120

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126

Research Article

ACS Applied Materials & Interfaces

Tokyo, Japan). UV−vis absorption spectrum were detected by a UV-2102 spectrophotometer (Unico Instrument Co., Ltd. Shanghai, China). The morphology and size of the AuNCs were characterized by a Tecnai G2 F30 transmission electron microscopy (FEI, USA). Fluorescence lifetime determinations were carried out by utilizing an FLS 920 spectrometer (Edinburgh Instruments, U.K.). 2.2. Chemicals. Chloroauric acid (HAuCl4·3H2O) and sodium borohydride (NaBH4) were purchased from Beijing J&K Co., Ltd. (Beijing, China). PNPP and sodium orthovanadate (Na3VO4) were purchased from Aladdin Industrial Corporation (Shanghai, China). ALP (sensing), galactosidase (Gal), glucose oxidase (GOX), thrombin, pepsin, trypsin, and 11-mercaptoundecanoic acid were obtained from Sigma-Aldrich (Sigma-Aldrich Company). Water was acquired utilizing a Milli-Q system (Millipore, Bedford, MA, USA). In addition, other chemicals were of analytical grade obtained from Sinopharm Chemical Reagents (Beijing, China). 2.3. Preparation of AuNCs. All glassware were soaked with freshly prepared aqua regia for 24 h, were rinsed with distilled water, and were oven-dried before use. The fluorescent AuNCs were prepared according to a modified protocol described previously.39 Briefly, 25 mL HAuCl4 (5 mM) was mixed with 50 mL MUA (10 mM, dissolved in 15 mM NaOH) under vigorous stirring. An additional NaOH (30 mmol) was added to the cloudy mixture of HAuCl4 and MUA until the mixture became clear. Then, a freshly prepared solution of NaBH4 (2 mM, 6.25 mL) was added dropwise. Then, the mixed solution was stirred for 24 h at room temperature. The as-prepared AuNCs were dialyzed using a cutoff dialysis membrane (8−14 kDa) against water for 48 h to remove excess small molecule impurities. The purified AuNCs solution was stored at room temperature prior to use. According to a previous report, 36 the concerntration of AuNCs was determined as 6.15 × 10−5 M, and details are shown in the Supporting Information. 2.4. IFE-Based Fluorescent ALP Activity Assay. The IFE-based fluorescent detection of ALP was performed in accordance with the following procedures. A 50 μL portion of ALP with an activity ranging from 0 to 300 U/L was added to Tris-HCl buffer solution (10 mM, pH 9.0) containing 1 mM PNPP and 0.1 μM MgSO4. The mixture was incubated at 37 °C for 50 min, and then 1 mL AuNCs solution was added. Afterward, the fluorescence intensity was recorded at an excitation wavelength of 330 nm. 2.5. ALP Inhibitor Investigation. To study the application of our established fluorescent approach for ALP inhibitor evaluation, 150 μL of aqueous Na3VO4 solution, having amounts varying from 0 to 960 μM, was added into the ALP mixture system (containing 10 U/L ALP, 1 mM PNPP, and 0.1 μM MgSO4) at 37 °C for 50 min. The mixture was then transferred to 1 mL AuNCs solution, and the fluorescence emission spectra were recorded at an excitation wavelength of 330 nm. 2.6. Human Serum Sample Detection. Human blood samples were collected from healthy adult volunteers at Peking University Third Hospital. The blood samples were pretreated to eliminate any protein interference and to improve the recovery.40 A 3 mL portion of trichloroacetic acid (quality fraction 15%) was added to 1 mL serum to destroy the activity of proteins in the serum and to precipitate them from the solution. The mixture was centrifuged at 10 000 rpm for 10 min after vigorous shaking for 15 min. The supernatant was

detecting various enzymes in enzymatic systems including glucose oxidase (GOX),29 pyrophosphatase,30 and alkaline phosphatase (ALP).31 Recently, methods have been established for detecting ALP activity by carbon dots as fluorescent signal.32,33 Unfortunately, most of these methods are enzymatic assays based on fluorescence quenching mechanisms, while such turn-off enzymatic assays may provide false positive signals caused by interference from environmental stimulus such as other unexpected quenchers.34 Turn-off modes are not associated with high signal outputs and, because of high background signals, are usually less sensitive than turn-on methods. Therefore, it is of great interest and importance to develop novel metal NC based turn-on sensors for simple and sensitive detection of alkaline phosphatase. The inner filter effect (IFE) describes the absorption of excitation or emission from fluorophores within the detection system.32 The IFE can be caused by the overlapping between the absorption spectra of an absorbing species and the fluorescence excitation or emission spectra of the fluorophores. A series of fluorescent sensors based on IFE have been developed with enhanced sensitivity and selectivity compared with the pure absorbance-based methods,35−38 because changes in the absorbance of the absorbing species can translate into exponential changes in the measured fluorescence of the fluorescent substance. IFE based methods also offer considerable flexibility and are simpler to implement because there is no need for complicated modification of the metal NCs or for covalent linking between a fluorophore and a receptor. In the present work, we developed a new turn-on fluorescent sensor for simple and sensitive detection of ALP activity through the IFE of p-nitrophenylphosphate (PNPP) on the fluorescence of AuNCs. The AuNCs were synthesized using 11mercaptoundecanoic acid (MUA) as stabilized agent through a one-step synthesis method, on the basis of previous reports.39 The maximum excitation and emission wavelengths of the AuNCs were at 330 and 600 nm, respectively. PNPP was used as the ALP substrate in the detection system. The measured fluorescence of the AuNCs was decreased by competitive absorption, because the absorption spectra of PNPP has good overlap with the fluorescence excitation spectra of the prepared AuNCs. Thus, the fluorescence of AuNCs was significantly decreased by PNPP. However, in the presence of ALP, PNPP was catalytically hydrolyzed into p-nitrophenol (PNP), which has different absorption from that of PNPP and reduced overlap with the excitation spectra of the AuNCs. Thus, the IFE-decreased emission of AuNCs was recovered, allowing ALP to be detected by a simple yet sensitive way. Our proposed sensing approach provided a low detection limit of 0.002 U/L with a signal-to-noise ratio of 3. The proposed fluorescence method was also applied to investigate ALP inhibitors. We further applied this approach to assay ALP in human serum samples and obtained satisfactory results. As far as we know, this is the first report of using an IFE-based fluorescent sensing strategy with fluorescent AuNCs for sensing ALP. The developed method is simple to implement and highly sensitive and selective with potential to aid in clinical diagnosis applications. Furthermore, our approach may be extended to the development of other metal NC-based fluorescent turn-on sensors for a wide range of applications in biosensing.

2. EXPERIMENT 2.1. Instrumentation. Fluorescence spectra were recorded by a F-7000 fluorescence spectrophotometer (Hitachi Ltd., 121

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126

Research Article

ACS Applied Materials & Interfaces

signal. The morphology of the AuNCs is shown in Figure 2. Transmission electron microscopy (TEM) images confirmed

obtained and modulated to pH 7.0 utilizing NaOH solution. The treated serum samples were diluted 50 times using deionized water. The spiked samples were prepared by adding different activities of ALP solutions to the diluted serum samples. The spiked serum samples were then analyzed in accordance with the ALP activity assay procedure described in section 2.4.

3. RESULTS AND DISCUSSION 3.1. Characterization of AuNCs. The prepared AuNCs were colorless solutions under ambient light conditions and emitted strong orange fluorescence under irradiation with 312 nm UV light (inset of Figure 1B). Figure 1A displays UV−vis

Figure 2. (A) TEM image (scale bar: 10 nm) and (B) the size distribution histogram of the prepared AuNCs.

the presence of AuNCs with a uniform size distribution (Figure 2). The mean diameter of the AuNCs was estimated as 1.93 nm from TEM image analysis of 113 individual particles. The quantum yield (QY) of AuNCs in the aqueous solution was determined to be 12% employing rhodamine B (QY = 0.97 in methanol) as a reference41 (details shown in the Supporting Information). This value is comparable with those previously reported for fluorescent AuNCs stabilized with MUA.42,43 The photostability of the as-prepared AuNCs is an important factor for applications as a fluorescent probe. As shown in Figure S1, when excited at 330 nm, the fluorescence intensity of the AuNCs exhibited no significant change over 2 h, which indicates excellent photostability of the AuNC solution. 3.2. Principle of ALP Sensing Based on IFE. We proposed an IFE-based fluorescent sensor for simple and sensitive detection of ALP. In this sensor system, AuNCs function as fluorometric reporter, and PNPP serves as fluorescence absorber. As shown in Figure 3, the absorption spectra of PNPP have good overlaps with the excitation spectra of the AuNCs. Thus, the fluorescence intensity of the AuNCs is

Figure 1. (A) UV−vis absorption of the prepared AuNCs. (B) Fluorescence excitation (black line) and emission (blue line) spectra of the prepared AuNC solutions. The inset shows the photographs of AuNC solution (a) in ambient light and (b) under irradiation by 312 nm UV light.

absorption spectrum of the AuNCs. A strong and featureless absorption peak at 281 nm was observed, which was attributed to MUA units on the surface of the AuNCs. A characteristic surface plasmon resonance peak near 520 nm was not observed in the spectrum, indicating the absence of larger gold nanoparticles. The maximum excitation of the AuNCs was at 330 nm, and at this excitation wavelength, a strong emission wavelength was observed at 600 nm (Figure 1B). The potential interference from excitation is decreased because of the distance of 270 nm between the excitation and emission wavelengths, thereby increasing the intensity of the emission

Figure 3. Absorption spectra of PNPP (blue line) and fluorescence excitation spectra of the prepared AuNCs (black line). 122

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126

Research Article

ACS Applied Materials & Interfaces effectively quenched or decreased when the two substances are present. PNPP is often used as a substrate of ALP as it is low in cost and excellent in water-solubility and enzymatic utilization activity. Furthermore, the ALP hydrolysis product (PNP) has an absorbance maxima at 405 nm, which should not interfere with the absorbance/excitation of the AuNCs. The fluorescence of AuNCs is decreased in the presence of PNPP because of the intense absorption of PNPP. In the presence of ALP, PNPP is catalytically hydrolyzed to PNP, which then results in recovery of the AuNC fluorescence. According to this principle, ALP can be detected by a simple and sensitive way. To prove this design, different concentrations of PNPP were added to 1 mL AuNC solution, and the fluorescence was recorded with excitation at 330 nm. As shown in Figure S2, the fluorescence emission of the AuNCs diminished gradually as the concentration of PNPP increased (0−1 mM). The fluorescence intensity of the AuNCs could be modulated using PNPP. To ensure a low background signal in the sensing system, 1 mM of PNPP was used in further experiments. The UV absorption of the ALP sensing systems with different ALP concentrations was also recorded. As the ALP concentration increased, the absorbance at 310 nm decreased, and meanwhile, the absorbance at 405 nm increased (Figure 4). The

Scheme 1. (A) Preparation of AuNCs, Using 11Mercaptoundecanoic Acid (MUA) as a Stabilizing Agent, through One-Step Synthesis. (B) Principle of ALP Detection Based On Inner Filter Effect

concentration and AuNC fluorescence intensity (F − F0), where F0 is the fluorescence intensity of the AuNCs in the presence of 1 mM PNPP and 0.1 μM MgSO4, and F is the fluorescence intensity of the AuNCs after addition of various concentrations of ALP. In the concentration range 0.02−50 U/ L, the linear fitting equation could be expressed as follows: F − F0 = 26.14[ALP] + 12.14, U/L

(1)

The corresponding linear correlation coefficient was 0.98, and the ALP detection limit was as low as 0.002 U/L (S/N = 3). Compared with results of previously reported fluorescence assays for ALP, our method using AuNCs as fluorescent probe based on IFE can offer a lower detection limit (Table S1). The developed method is suitable for clinical diagnosis with easy implementation and high sensitivity. Furthermore, the developed method does not require complex synthesis or modification of the sensing probes. 3.4. Selectivity of the Sensing Method. To evaluate the specificity of the proposed method, we studied the effect of several enzymes, including galactosidase (Gal), glucose oxidase (GOX), thrombin, pepsin, and trypsin on the analytical system. The fluorescence of the AuNCs quenched by PNPP showed minimal changes after addition of 100 U/L of the previously listed enzymes (Figure 6). Furthermore, we investigated the interference of metal ions (Mg2+, Zn2+, Fe3+, K+, Na+, and Ca2+, 100 μM) on the fluorescence of AuNCs. The fluorescence of the sensing system was almost unchanged after adding the metal ions. However, the addition of 10 U/L ALP to these solutions again resulted in a dramatic fluorescence recovery (Figure 6). The interference experiments demonstrated that the earlier mentioned substances have negligible effect on the fluorescent intensity of the AuNCs and indicate that this sensing approach has a high selectivity toward ALP. 3.5. Investigation of ALP Inhibitors. The proposed fluorescent sensing assay was applied to evaluate enzyme inhibitor efficiency. As a common ALP inhibitor, Na3VO4 is widely used for inhibition assays. When Na3VO4 is added to the sensing system, the hydrolysis of PNPP is restricted because of inhibition of ALP, which results in depression of the fluorescent

Figure 4. Absorption spectra of the enzymatic reaction solution in the presence of various concentrations of alkaline phosphatase from 0 to 150 U/L with 1 mM PNPP and 0.1 μM MgSO4 incubated at 37 °C for 50 min.

corresponding color change of PNPP caused by ALP has been used for colorimetric detection of ALP. However, the colorimetric methods exhibit lower sensitivity than the fluorescence assays.44 The fluorescence lifetime, which changes for efficient fluorescence resonance energy transfer process, is expected to be unaffected by IFE.45 As shown in Figure S3, the fluorescence lifetime of AuNCs was unaffected by the presence of PNPP. This result further indicated that the quenched AuNC fluorescence can be attributed to an IFE between AuNCs and PNPP. 3.3. Fluorescence Assay for ALP Activity. As shown in Scheme 1, the fluorescence emission of AuNCs is effectively quenched by the IFE of PNPP. Nevertheless, the IFE-decreased fluorescence of AuNCs was recovered gradually as the ALP concentration increased from 0 to 300 U/L (Figure 5A). This results from the hydrolysis of PNPP by ALP. Figure 5B (inset) displays a good linear relationship between the ALP 123

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126

Research Article

ACS Applied Materials & Interfaces

Figure 6. Selectivity of ALP assays in the presence of galactosidase (Gal), thrombin (Thr), glucose oxidase (GOX), pepsin (Pep), trypsin (Try), and different metal ions. The concentration of ALP was 10 U/ L, the level of other enzymes was 100 U/L, and the concentrations of metal ions were 0.1 mM. F0 and F are the fluorescence intensities of AuNCs in the absence and presence of the tested substances, respectively.

Figure 5. (A) Fluorescence emission spectra of AuNCs in the presence of increasing concentrations of ALP (from bottom to top 0, 0.02, 0.05, 0.1, 0.5, 1, 2, 4, 5, 10, 20, 30, 40, 50, 100, 200, and 300 U/L) with 1 mM PNPP and 0.1 μM MgSO4 incubated at 37 °C for 50 min and (B) plots of F − F0 versus ALP concentration from 0 to 300 U/L (inset: its corresponding linear relationship from 0.02 to 50 U/L). F0 and F are the fluorescence intensities of AuNCs in the absence and presence of ALP, respectively. The fluorescence spectra were measured with excitation at 330 nm.

recovery. As shown in Figure 7, when the inhibitors were added to the ALP reaction system, the level of fluorescence recovery was limited in proportion to the amount of inhibitor. The inhibition rate I (%) was employed to express the enzyme inhibiting efficiency. The relevant equation is as follows: I(%) =

FI − F0 × 100 FB − F0

(2)

where FB is the initial fluorescence intensity of the AuNCs and PNPP mixture without ALP, F0 is the fluorescence intensity after recovery with ALP in the absence of the inhibitor, and FI is the fluorescence intensity in the presence of ALP and the inhibitor. The regression equation is I(%) = 1.22[Na3VO4 ] + 3.16

Figure 7. (A) Fluorescence intensity of AuNCs in the presence of various concentrations of Na3VO4 (from top to bottom 0, 5.6, 11.3, 22.5, 45, 90, 120, 240, 480, and 960 μM) and their calibration plots (B). The inhibitors were incubated with 20 U/L enzymes, 1 mM PNPP, and 0.1 μM MgSO4. The fluorescence spectra were measured with excitation at 330 nm.

(3)

The detection limit was determined to be 0.06 μM for Na3VO4 (S/N = 3). These results suggest that our approach could be used to screen for ALP inhibitors in drug discovery. 124

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126

ACS Applied Materials & Interfaces

■

3.6. Detection of ALP in Serum Samples. To assess the reliability of the developed approach in serum samples, the developed fluorescence sensor was used for ALP activity assays of human serum samples. The results detected by our standard addition approach are displayed in Table 1. The accuracy of the

added ALP (U/L)

found ALP (U/L)

recovery (%)

RSD (n = 3, %)

1 2 3 4

30 70 120 160

28.49 69.6 124.3 169.5

95 99 104 106

2.6 5 5 4.4

*E-mail: [email protected]. *Tel: 86-10-62733407. Fax: +86-10-62731016. E-mail: [email protected]. ORCID

Xueqin Ren: 0000-0002-7706-776X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Chinese National Scientific Foundation (21375146) and the Ministry of Science and Technology of China (2016YFC0501205).

approach was assessed by analyzing the recoveries of ALP in serum samples. The recoveries varied from 95% to 106%, and the relative standard deviation (RSD) was from 2.6% to 5%. To assess the reliability of the strategy in real sample detection, the proposed method was applied to detect ALP in human serum samples. The serum samples were collected from three adult volunteers by Peking University Third Hospital. These samples were analyzed by our method and the clinic method. In clinical method, ALP serum activity was detected with a commercial reagent kit by molecular absorbance spectrophotometry. The analytical results are shown in Figure S4. As shown in Figure S4, the results achieved by our method are in good agreement with those obtained by the clinical method. These results indicate that our proposed fluorescence sensing technology has potential applications for ALP determination in real samples.

■

REFERENCES

(1) Lorente, J. A.; Valenzuela, H.; Morote, J.; Gelabert, A. Serum Bone Alkaline Phosphatase Levels Enhance the Clinical Utility of Prostate Specific Antigen in the Staging of Newly Diagnosed Prostate Cancer Patients. Eur. J. Nucl. Med. Mol. Imaging 1999, 26, 625−632. (2) Kaliannan, K.; Hamarneh, S. R.; Economopoulos, K. P.; Alam, S. N.; Moaven, O.; Patel, P.; Malo, N. S.; Ray, M.; Abtahi, S. M.; Muhammad, N.; Raychowdhury, A.; Teshager, A.; Mohamed, M.; Moss, A. K.; Ahmed, R.; Hakimian, S.; Narisawa, S.; Millan, J. L.; Hohmann, E.; Warren, H. S.; Bhan, A. K.; Malo, M. S.; Hodin, R. A. Intestinal Alkaline Phosphatase Prevents Metabolic Syndrome in Mice. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7003−7008. (3) Ooi, K.; Shiraki, K.; Morishita, Y.; Nobori, T. High-Molecular Intestinal Alkaline Phosphatase in Chronic Liver Diseases. J. Clin. Lab. Anal. 2007, 21, 133−139. (4) Colombatto, P.; Randone, A.; Civitico, G.; Gorin, J. M.; Dolci, L.; Medaina, N.; Oliveri, F.; Verme, G.; Marchiaro, G.; Pagni, R.; Karayiannis, P.; Thomas, H. C.; Hess, G.; Bonino, F.; Brunetto, M. R. Hepatitis G Virus RNA in the Serum of Patients with Elevated Gamma Glutamyl Transpeptidase and Alkaline Phosphatase: A Specific Liver Disease. J. Viral Hepatitis 1996, 3, 301−306. (5) Fernley, H. N.; Bisaz, S. Studies on Alkaline Phosphatase Phosphorylation of Calf Intestinal Alkaline Phosphatase by 32PLabelled Pyrophosphate. Biochem. J. 1968, 107, 279−283. (6) Hasegawa, T.; Sugita, M.; Takatani, K.; Matsuura, H.; Umemura, T.; Haraguchi, H. Assay of Alkaline Phosphatase in Salmon Egg Cell Cytoplasm with Fluorescence Detection of Enzymatic Activity and Zinc Detection by ICP-MS in Relation to Metallomics Research. Bull. Chem. Soc. Jpn. 2006, 79, 1211−1214. (7) Choi, Y.; Ho, N. H.; Tung, C. H. Sensing Phosphatase Activity by Using Gold Nanoparticles. Angew. Chem., Int. Ed. 2007, 46, 707−709. (8) Wei, H.; Chen, C. G.; Han, B. Y.; Wang, E. K. Enzyme Colorimetric Assay Using Unmodified Silver Nanoparticles. Anal. Chem. 2008, 80, 7051−7055. (9) Diaz, A. N.; Sanchez, F. G.; Ramos, M. C.; Torijas, M. C. Horseradish Peroxidase Sol-Gel Immobilized for Chemiluminescence Measurements of Alkaline Phosphatase Activity. Sens. Actuators, B 2002, 82, 176−179. (10) Miao, P.; Ning, L. M.; Li, X. X.; Shu, Y. Q.; Li, G. X. An Electrochemical Alkaline Phosphatase Biosensor Fabricated with Two DNA Probes Coupled with Lambda Exonuclease. Biosens. Bioelectron. 2011, 27, 178−182. (11) Murata, T.; Yasukawa, T.; Shiku, H.; Matsue, T. Electrochemical Single-Cell Gene-Expression Assay Combining Dielectrophoretic Manipulation with Secreted Alkaline Phosphatase Reporter System. Biosens. Bioelectron. 2009, 25, 913−919. (12) Ino, K.; Kanno, Y.; Arai, T.; Inoue, K. Y.; Takahashi, Y.; Shiku, H.; Matsue, T. Novel Electrochemical Methodology for Activity Estimation of Alkaline Phosphatase Based on Solubility Difference. Anal. Chem. 2012, 84, 7593−7598.

4. CONCLUSION In conclusion, a novel turn-on fluorescent assay technology for ALP, on the basis of the inner filter effect, was developed using AuNC. The fluorescence intensity of the AuNCs was markedly quenched by PNPP via the inner filter effect. When ALP was incubated with PNPP, before the AuNCs were added, ALP catalyzed hydrolysis of PNPP and decreased its absorption. This then resulted in a recovery of IFE-decreased fluorescence of the AuNCs. This proposed IFE-based sensor provides a simple fluorescent turn-on platform for the detection of ALP with a detection limit of 0.002 U/L. Furthermore, the proposed method shows potential to aid in ALP inhibitor screening for drug discovery. The method was also successfully applied to detect ALP in human serum samples. This work not only provides a new avenue for ALP sensing in clinical diagnosis but also expands potential applications of metal nanoclusters.

■

AUTHOR INFORMATION

Corresponding Authors

Table 1. Determination of ALP in Human Serum Samples plasma samples

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b11920. Quantum yield measurement of AuNCs, estimation of the concentration of the as-prepared AuNCs, the photostability of the AuNCs measured by fluorescence spectrophotometer every 5 min, fluorescence intensity of AuNCs upon addition of various concentrations of PNPP from 0 to 1000 μM, effect of PNPP on the fluorescence lifetime of AuNCs, detection of ALP in three adult volunteer serums by the clinic method and by our method, comparison of analytical performance of some assays for ALP detection (PDF) 125

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126

Research Article

ACS Applied Materials & Interfaces (13) Ingram, A.; Moore, B. D.; Graham, D. Simultaneous Detection of Alkaline Phosphatase and Beta-Galactosidase Activity Using SERRS. Bioorg. Med. Chem. Lett. 2009, 19, 1569−1571. (14) Chen, J.; Jiao, H. P.; Li, W. Y.; Liao, D. L.; Zhou, H. P.; Yu, C. Real-Time Fluorescence Turn-On Detection of Alkaline Phosphatase Activity with a Novel Perylene Probe. Chem. - Asian J. 2013, 8, 276− 281. (15) Nutiu, R.; Yu, J.; Li, Y. F. Signaling Aptamers for Monitoring Enzymatic Activity and for Inhibitor Screening. ChemBioChem 2004, 5, 1139−1144. (16) Chen, Q.; Bian, N.; Cao, C.; Qiu, X. L.; Qi, A. D.; Han, B. H. Glucosamine Hydrochloride Functionalized Tetraphenylethylene: A Novel Fluorescent Probe for Alkaline Phosphatase Based on the Aggregation-Induced Emission. Chem. Commun. 2010, 46, 4067− 4069. (17) Zhang, H. M.; Xu, C. L.; Liu, J.; Li, X. H.; Guo, L.; Li, X. M. An Enzyme-Activatable Probe with a Self-Immolative Linker for Rapid and Sensitive Alkaline Phosphatase Detection and Cell Imaging Through a Cascade Reaction. Chem. Commun. 2015, 51, 7031−7034. (18) Zhao, X. Y.; Liu, Y.; Schanze, K. S. A Conjugated Polyelectrolyte-based Fluorescence Sensor for Pyrophosphate. Chem. Commun. 2007, 2914−2916. (19) An, L. L.; Tang, Y. L.; Feng, F. D.; He, F.; Wang, S. WaterSoluble Conjugated Polymers for Continuous and Sensitive Fluorescence Assays for Phosphatase and Peptidase. J. Mater. Chem. 2007, 17, 4147−4152. (20) Liu, Y.; Schanze, K. S. Conjugated Polyelectrolyte-Based RealTime Fluorescence Assay for Alkaline Phosphatase with Pyrophosphate as Substrate. Anal. Chem. 2008, 80, 8605−8612. (21) Liu, H.; Lv, Z. L.; Ding, K. G.; Liu, X. L.; Yuan, L.; Chen, H.; Li, X. M. Incorporation of Tyrosine Phosphate into Tetraphenylethylene Affords an Amphiphilic Molecule for Alkaline Phosphatase Detection, Hydrogelation and Calcium Mineralization. J. Mater. Chem. B 2013, 1, 5550−5556. (22) Liu, S. Y.; Pang, S.; Na, W. D.; Su, X. G. Near-infrared Fluorescence Probe for the Determination of Alkaline Phosphatase. Biosens. Bioelectron. 2014, 55, 249−254. (23) Jia, L.; Xu, J. P.; Li, D.; Pang, S. P.; Fang, Y. A.; Song, Z. G.; Ji, J. A. Fluorescence Detection of Alkaline Phosphatase Activity with BetaCyclodextrin-Modified Quantum Dots. Chem. Commun. 2010, 46, 7166−7168. (24) Freeman, R.; Finder, T.; Gill, R.; Willner, I. Probing Protein Kinase (CK2) and Alkaline Phosphatase with CdSe/ZnS Quantum Dots. Nano Lett. 2010, 10, 2192−2196. (25) Choi, S.; Dickson, R. M.; Yu, J. H. Developing Luminescent Silver Nanodots for Biological Applications. Chem. Soc. Rev. 2012, 41, 1867−1891. (26) Chen, L. Y.; Wang, C. W.; Yuan, Z. Q.; Chang, H. T. Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging. Anal. Chem. 2015, 87, 216−229. (27) Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Synthesis of Highly Fluorescent Gold Nanoparticles for Sensing Mercury(II). Angew. Chem., Int. Ed. 2007, 46, 6824−6828. (28) Lu, Y. Z.; Chen, W. Sub-Nanometre Sized Metal Clusters: from Synthetic Challenges to the Unique Property Discoveries. Chem. Soc. Rev. 2012, 41, 3594−3623. (29) Jin, L. H.; Shang, L.; Guo, S. J.; Fang, Y. X.; Wen, D.; Wang, L.; Yin, J. Y.; Dong, S. J. Biomolecule-Stabilized Au Nanoclusters as a Fluorescence Probe for Sensitive Detection of Glucose. Biosens. Bioelectron. 2011, 26, 1965−1969. (30) Sun, J.; Yang, F.; Zhao, D.; Yang, X. R. Highly Sensitive RealTime Assay of Inorganic Pyrophosphatase Activity Based on the Fluorescent Gold Nanoclusters. Anal. Chem. 2014, 86, 7883−7889. (31) Chen, Y.; Li, W. Y.; Wang, Y.; Yang, X. D.; Chen, J.; Jiang, Y. N.; Yu, C.; Lin, Q. Cysteine-Directed Fluorescent Gold Nanoclusters for the Sensing of Pyrophosphate and Alkaline Phosphatase. J. Mater. Chem. C 2014, 2, 4080−4085. (32) Li, G. L.; Fu, H. L.; Chen, X. J.; Gong, P. W.; Chen, G.; Xia, L.; Wang, H.; You, J. M.; Wu, Y. N. Facile and Sensitive Fluorescence

Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots Based on Inner Filter Effect. Anal. Chem. 2016, 88, 2720−2726. (33) Qian, Z. S.; Chai, L. J.; Huang, Y. Y.; Tang, C.; Shen, J. J.; Chen, J. R.; Feng, H. A Real-Time Fluorescent Assay for the Detection of Alkaline Phosphatase Activity Based on Carbon Quantum Dots. Biosens. Bioelectron. 2015, 68, 675−680. (34) Zhou, Z. X.; Du, Y.; Dong, S. J. DNA-Ag Nanoclusters as Fluorescence Probe for Turn-On Aptamer Sensor of Small Molecules. Biosens. Bioelectron. 2011, 28, 33−37. (35) Yang, S.; Wang, C. Y.; Liu, C. H.; Wang, Y. J.; Xiao, Y.; Li, J. S.; Li, Y. H.; Yang, R. H. Fluorescence Modulation by Absorbent on Solid Surface: An Improved Approach for Designing Fluorescent Sensor. Anal. Chem. 2014, 86, 7931−7938. (36) Chang, H. C.; Ho, J. Gold Nanocluster-Assisted Fluorescent Detection for Hydrogen Peroxide and Cholesterol Based on the Inner Filter Effect of Gold Nanoparticles. Anal. Chem. 2015, 87, 10362− 10367. (37) Yan, X.; Li, H. X.; Han, X. S.; Su, X. G. A Ratiometric Fluorescent Quantum Dots Based Biosensor for Organophosphorus Pesticides Detection by Inner Filter Effect. Biosens. Bioelectron. 2015, 74, 277−283. (38) Li, J.; Li, X.; Shi, X.; He, X.; Wei, W.; Ma, N.; Chen, H. Highly Sensitive Detection of Caspase-3 Activities via a Nonconjugated Gold Nanoparticle-Quantum Dot Pair Mediated by an Inner-Filter Effect. ACS Appl. Mater. Interfaces 2013, 5, 9798−9802. (39) Chang, H. C.; Chang, Y. F.; Fan, N. C.; Ho, J. Facile Preparation of High-Quantum-Yield Gold Nanoclusters: Application to Probing Mercuric Ions and Biothiols. ACS Appl. Mater. Interfaces 2014, 6, 18824−18831. (40) Shamsipur, M.; Shanehasz, M.; Khajeh, K.; Mollania, N.; Kazemi, S. H. A Novel Quantum Dot-Laccase Hybrid Nanobiosensor for Low Level Determination of Dopamine. Analyst 2012, 137, 5553− 5559. (41) Velapoldi, R. A.; Tonnesen, H. H. Corrected Emission Spectra and Quantum Yields for a Series of Fluorescent Compounds in the Visible Spectral Region. J. Fluoresc. 2004, 14, 465−472. (42) Sun, J.; Zhang, J.; Jin, Y. D. 11-Mercaptoundecanoic Acid Directed One-Pot Synthesis of Water-Soluble Fluorescent Gold Nanoclusters and Their Use as Probes for Sensitive and Selective Detection of Cr3+ and Cr6+. J. Mater. Chem. C 2013, 1, 138−143. (43) Sun, J.; Yue, Y.; Wang, P.; He, H. L.; Jin, Y. D. Facile and Rapid Synthesis of Water-Soluble Fluorescent Gold Nanoclusters for Sensitive and Selective Detection of Ag+. J. Mater. Chem. C 2013, 1, 908−913. (44) Zheng, F. Y.; Guo, S. H.; Zeng, F.; Li, J.; Wu, S. Z. Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on BetaineModified Polyethylenimine via Excimer/Monomer Conversion. Anal. Chem. 2014, 86, 9873−9879. (45) Zhang, M. W.; Cao, X. Y.; Li, H. K.; Guan, F. R.; Guo, J. J.; Shen, F.; Luo, Y. L.; Sun, C. Y.; Zhang, L. G. Sensitive Fluorescent Detection of Melamine in Raw Milk Based on the Inner Filter Effect of Au Nanoparticles on the Fluorescence of CdTe Quantum Dots. Food Chem. 2012, 135, 1894−1900.

126

DOI: 10.1021/acsami.6b11920 ACS Appl. Mater. Interfaces 2017, 9, 120−126