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Cite this: Chem. Commun., 2014, 50, 8677 Received 5th December 2013, Accepted 9th June 2014
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A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening† Yongqian Xu,*a Benhao Li,b Liangliang Xiao,a Jia Ouyang,d Shiguo Sun*a and Yi Pangc
DOI: 10.1039/c3cc49254k www.rsc.org/chemcomm
A dual-channel including a colorimetric and fluorescent probe based on the aggregation-caused quenching (ACQ) and enzymolysis approach has been presented to screen acid phosphatase (ACP) and its inhibitor. Moreover, the ACP activity was determined by real time assay.
Acid phosphatase (ACP) is a type of enzyme used to hydrolyze biomolecules to release phosphate groups during digestion. It is an essential phosphomonoesterase widely present in nature1 and found in mammalian tissues including prostate, liver and hematological system.2 Although the concentrations of ACP are found to be normally low in mammalian cells, it is involved in many important physiological processes, especially the movement of mammals. An abnormal level of ACP has been associated with a number of diseases, such as prostate cancer, Gaucher disease and enzyme disorders in kidneys, veins and bones.3 Due to its biological and clinical importance, the precise concentration measurement of ACP in physiological media has been regarded as an essential factor in the pathologic diagnosis of these relative diseases. In recent years, a few common strategies have been developed to detect ACP, including spectrophotometry,2a,4a–g surface acoustic wave (SAW) sensors4h and electrochemical methods.4i Although these methods are fairly sensitive, most of them require complicated instrumental setup6a and alkaline pH conditions. Among the more appealing spectrophotometry, either colorimetric or fluorometric substrates based on p-nitrophenyl phosphate ( pNPP) and 4-methyl-7-hydroxycoumarinyl phosphate (MUP) are utilized, respectively.5 However, they suffer from low selectivity, single channel, complex substrates and having a
College of Science, Northwest A&F University, Yangling, 712100, P. R. China. E-mail: [email protected], [email protected] b School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, P. R. China c Department of Chemistry & Maurice Morton Institute of Polymer Science, The University of Akron, Akron, OH, 44325, USA d School of Materials Science and Engineering, Northwestern Polytechnical University, Xi’an, 710062, P. R. China † Electronic supplementary information (ESI) available: Details of titration procedures, additional spectra and photograph. See DOI: 10.1039/c3cc49254k
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absorption and emission wavelengths in the UV region.6b Therefore, the development of facile methods with high sensitivity, dual channel screening, clinical diagnostic application, and more importantly, exhibiting its turn-on response in the near infrared (NIR) region to avoid short wavelength excitation-triggered photodamage, strong interference from biological medium and scattering light,6c is an important challenge. Squaraine (SQ) dyes are an interesting class of dyes showing sharp and intense fluorescence, typically in the red to NIR region (absorption maxima are found between 630 and 670 nm and emission maxima are between 650 and 700 nm).7 Because of NIR optical properties, they have been used as sensors for ions,8a and exploited in biomedical imaging.8b,c In solution, cationic squaraine (SQ) dyes favor to form an aggregate assembly with oppositely charged species via strong attractive electrostatic interactions.8b Accordingly, the fluorescence of SQ would dramatically decrease because of their aggregation-caused quenching (ACQ) property.8c,9 Reversely, as the aggregation equilibrium based on the electrostatic interactions is pertubed by introducing a competitive binding target in solution, SQ would deaggregate and their quenched fluorescence would recover, leading to a sensitive fluorescence turn-on assembly for target detection in the NIR region. In comparison with monomers, aggregates of SQ usually give different absorption bands which are enough to discriminate each other by color change.8d In other words, an effective dual-channel (both absorption and emission) method for detection of targets could be realized by controlling the state of SQ aggregation. Herein, we present a dual-channel method including colorimetric and fluorescent output for the detection of acid phosphatase based on the self-assembly of squaraines and sodium hexametaphosphate (NaPO3)6. The design rationale for the self-assembly detection is illustrated in Scheme 1. Commercially available sodium hexametaphosphate (NaPO3)6, with six negative charges in the six-membered ring, behaves as a building block to induce aggregate formation of positively charged SQ through intermolecular electrostatic interaction in phosphate buffer solution (pH 7.4), leading to an absorption shift and fluorescence quenching of the SQ solution. Then, ACP is added to the detection system, (NaPO3)6
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Scheme 1 (NaPO3)6.
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Illustration of a possible assembled assay between SQ and
is hydrolyzed to the phosphate segment by ACP. The process can be traced by recording both the color and fluorescent changes, which originate from variation of the squaraine aggregation states. Thus, a facile and label-free ACP fluorescent detection system has been established. It can be used for ACP detection in the neutral or weak alkali solution rather than under acidic conditions. To test the feasibility of this new method, different amounts of (NaPO3)6 were added to the SQ solution in phosphate buffer (5 mM, pH = 7.4). Fig. S1 and S2 (ESI†) reveal the absorption and fluorescence changes of the solution. The absorption spectra of SQ revealed three bands (lmax = 625, 567 and 540 nm), which were assigned to the monomer, hypsochromic dimer, and oligomer absorption, respectively (Fig. S1, ESI†).8b The monomer and dimer absorption gradually decreased with increasing concentrations of (NaPO3)6. Observation of a new band at about 520 nm indicated that the SQ molecules were transformed to H-aggregates.8b Upon addition of (NaPO3)6 (0–10 mM), the fluorescence of SQ was gradually quenched (Fig. S2, ESI†), leading to nearly 7.5 times of decrease, presumably due to the formation of SQ-(NaPO3)6 aggregates. The spectral response clearly indicated strong electrostatic interactions between SQ and (NaPO3)6. Moreover, addition of ACP into the SQ–(NaPO3)6 solution caused a gradual increase of the SQ monomer (at 625 nm), indicating the transformation of SQ from aggregates to monomer (Fig. S3, ESI†). In the presence of ACP, (NaPO3)6 was hydrolyzed to inorganic phosphoric acid, whose weak interaction with SQ could cause the dissociation of the SQ aggregate. In the spectra obtained from the control experiment, the new band at about 505 nm was attributed to SQ-ACP (Fig. S4, ESI†). Interestingly, the fluorescence intensity of the solution was remarkably increased (Fig. 1), while the emission peak was red-shifted by about 7 nm to 644 nm, coming from the possible interaction between ACP and the SQ monomer8b and avoiding the interference of the original wavelength. As the concentration of ACP added was up to 0.533 mM, the recovery of the fluorescence intensity levelled off, at which the pre-quenched emission intensity was recovered up to 94.5% of the original intensity at 644 nm (Fig. S2, ESI†). Titration of SQ–(NaPO3)6 with various concentrations of ACP in phosphate buffer solution was performed to investigate the viability of fluorescence detection of ACP (inset in Fig. 1). The fluorescence intensity at 644 nm was plotted as a function of the ACP concentration. Their fluorescence ratios (I644/I0) were linearly proportional to the ACP concentrations (0–0.533 mM).
8678 | Chem. Commun., 2014, 50, 8677--8680
Fig. 1 Fluorescence response of SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) upon addition of (NaPO3)6 (10 mM), then followed by ACP (0–0.533 mM). The arrow indicates the change in the fluorescence intensity with the ACP concentrations (lex = 600 nm). Inset: plot of the relative fluorescence intensity (I/I0) of the solution to ACP concentrations, where I and I0 stand for the fluorescence intensity at 644 nm in the absence and presence of ACP.
A concentration as low as 4.9 nM of ACP was detected with a signal-to-noise ratio of 3, indicating that this approach was applicable to NIR fluorescent detection of ACP. Furthermore, in diluted human serum samples, SQ–(NaPO3)6 shows turn-on fluorescence response to ACP, and a good linear relationship between the fluorescence intensity at 650 nm and ACP concentration was observed (Fig. S8, ESI†). These results suggest that SQ–(NaPO3)6 can be used for ACP detection in serum samples. Bilirubin (found in icteric samples) is a frequent interference for ACP detection.10 In the presence of 120 mmol L 1 bilirubin, the fluorescence response of SQ–(NaPO3)6 to ACP is similar to that in the absence of bilirubin, and the fluorescence ratios (I650/I0) were linearly proportional to the ACP concentrations, revealing that SQ–(NaPO3)6 can overcome the limitation of bilirubin to some extent (Fig. S10 and S11, ESI†). To demonstrate the feasibility of the method for monitoring the ACP activity in real time, the fluorescence spectral changes as a function of time upon incubation of (NaPO3)6 (10 mM) and ACP (0.53 mM) were investigated. As shown in Fig. S5 (ESI†), the quenched fluorescence intensity (line b) gradually increased with time duration as indicated, and reached maximum after 3 h. Kinetic parameters of ACP were further evaluated by assays using different initial (NaPO3)6 concentrations. The concentration changes of the hydrolyzed substrate (NaPO3)6 and unhydrolyzed (NaPO3)6 calculated from the fluorescence intensity as a function of time are shown in Fig. 2. The Michaelis constant Km value related to the substrate’s affinity to the enzyme was obtained from the Lineweaver–Burk plot using double reciprocal data of initial rate V vs. substrate (NaPO3)6 concentration [S]0 (Fig. 2c). The Km and Kcat/ Km value were determined to be 265.6 mM and 9.58 104 M 1 S 1, respectivily, consistent with the data reported in the literature.11 These results indicated that the SQ-(NaPO3)6 based ACP detection can be applied for real time ACP activity measurement. The method can also be used for the screening of potential ACP inhibitors, K2MoO4 was chosen as the inhibitor for ACP.12 As shown in Fig. 3, with increasing concentrations of K2MoO4, the emission intensity constantly decreases. This result suggests
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Fig. 4 Plots of relative fluorescence intensity changes (I/I0) of SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) in the presence of (NaPO3)6 (10 mM) to ACP and other enzymes with different concentrations, where I and I0 stand for the fluorescence intensity at 644 nm in the absence and presence of different enzymes. Fig. 2 Derived concentration of the hydrolyzed (NaPO3)6 (a) and unhydrolyzed (NaPO3)6 (b) as a function of time for ACP assays at different initial (NaPO3)6 concentrations. (c) Lineweaver–Burk plot for determination of the values of Km. Assays were carried out with ACP (0.53 mM) and SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) with different (NaPO3)6 concentrations as indicated at 37 1C.
Fig. 5 Photographs of color (a) and fluorescent change (b) of SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) in the presence of (NaPO3)6 (10 mM) upon addition of BSA, lysozyme, trypsin, Rnase A, pepsinum and ACP, respectively, where protein or enzyme concentrations are all 0.53 mM and the solution was excited by a hand-held UV lamp.
Fig. 3 Fluorescence spectral change of SQ (5 mM) in the presence of (NaPO3)6 (10 mM) and ACP (0.53 mM) in phosphate buffer (5 mM, pH = 7.4) with increasing concentrations of K2MoO4.
that the recovery of fluorescence intensity could attenuate in the presence of an inhibitor. The IC50 value of K2MoO4 obtained by using the sigmoidal fit of the experimental data was 5.30 mM (inset in Fig. 3). To evaluate the selectivity of the method based on the aggregation-caused quenching (ACQ) and enzymolysis, the absorption and fluorescence spectra response of the solution containing 10 mM (NaPO3)6 to various proteins and enzymes including BSA, lysozyme, trypsin, Rnase A and pepsinum were investigated (Fig. 4 and 5 and Fig. S12, ESI†). Obvious color change and remarkable fluorescence enhancement were observed for ACP, whereas other proteins and enzymes did not induce any changes in absorption and fluorescence intensity under identical conditions. This result proves that the assembly system is suitable for selective detection of ACP. In summary, we have reported a novel colorimetric and near infrared fluorescent assembly system that is inexpensive, efficient, and highly selective for detection of ACP. This design is based on the assembly of probe SQ with a commercially available substrate (NaPO3)6, which utilizes the aggregation-caused quenching (ACQ) and enzymolysis to trigger the optical response. Although with a
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simple design, this method can selectively detect ACP at a quite low concentration of less than 4.9 nM. Detection of ACP activity and ACP inhibitor could have potential application in disease diagnosis and evaluation. This work was supported by the National Natural Science Foundation of China (Grant No. 21206137), the Fundamental Research Funds for the Central Universities (2014YB027), Shaanxi Province Science and Technology (No. 2014K11-01-02-06) and the Scientific Research Foundation of Northwest A&F University (Z111021103 and Z111021107).
Notes and references 1 H. Bull, P. G. Murray, D. Thomas, A. M. Fraser and P. N. Nelson, Mol. Pathol., 2002, 55, 65. 2 (a) Y. Xie, Y. Tan, R. Liu, R. Zhao, C. Tan and Y. Jiang, ACS Appl. Mater. Interfaces, 2012, 4, 3784; (b) J. W. Seol, H.-B. Lee, N. S. Kim and S. Y. Park, Int. J. Mol. Med., 2009, 24, 57. 3 (a) E. S. Leman and R. H. Getzenberg, J. Cell. Biochem., 2009, 108, 3; (b) E. Schiffer, World J. Urol., 2007, 25, 557; (c) D. V. Makarov, S. Loeb, R. H. Getzenberg and A. W. Partin, Annu. Rev. Med., 2009, 60, 139. 4 (a) G. Davidson and T. B. Jalowiecki, Sci. Justice, 2012, 52, 106; (b) S. Wu, X. Gao, Q. Cai and C. A. Grimes, Sens. Actuators, B, 2007, 123, 856; (c) D. Ogonczyk, S. Glab and R. Koncki, Anal. Biochem., 2008, 381, 169; (d) D. Yang, Z. Li, Y. A. Diwu, H. Fu, J. Liao, C. Wei and Z. Diwu, Curr. Chem. Genomics, 2008, 2, 48; (e) A. Vaughan, G. G. Guilbault and D. Hackney, Anal. Chem., 1971, 43, 721; ( f ) Y. Liu and K. S. Schanze, Anal. Chem., 2008, 80, 8605; ( g) F. Xue and C. T. Seto, Org. Lett., 2010, 12, 1936; (h) R. H. Wang, Q. Y. Cai, D. F. Tong, L. H. Nie and S. Z. Yao, ´ndez, ˜´ Enzyme Microb. Technol., 1998, 22, 36; (i) J. Castan on-Ferna ´ndez-Abedul and A. Costa-Garcı´a, Anal. Chim. Acta, 2000, M. T. Ferna 413, 103.
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Communication 5 (a) J. Chernoff and H. C. Li, Arch. Biochem. Biophys., 1983, 226, 517; (b) H. N. Fernley and P. G. Walker, Biochem. J., 1965, 97, 95. 6 (a) Y. Xu, B. Li, P. Han, S. Sun and Y. Pang, Analyst, 2013, 138, 1004; (b) A. K. Dwivedi and P. K. Iyer, Anal. Methods, 2013, 5, 2374; (c) D. Tian, Z. Qian, Y. Xia and C. Zhu, Langmuir, 2012, 28, 3945. 7 (a) S. Das, K. G. Thomas, R. Ramanathan, M. V. George and P. V. Kamat, J. Phys. Chem., 1993, 97, 13625; (b) K. Y. Law, J. Phys. Chem., 1987, 91, 5184. 8 (a) Y. Xu, M. J. Panzner, X. Li, W. J. Youngs and Y. Pang, Chem. Commun., 2010, 46, 4073; (b) Y. Xu, M. Andery and Y. Pang, Chem. Commun., 2011, 47, 6662; (c) Y. Xu, Z. Li, M. Andery, S. Sun and Y. Pang, J. Phys. Chem. B, 2010, 114, 8574; (d) Y. Xu and Y. Pang, Org. Biomol. Chem., 2011, 9, 2878. 9 (a) B. Wang and C. Yu, Angew. Chem., Int. Ed., 2010, 49, 1485; (b) B. K. Gorityala, Z. Lu, M. L. Leow, J. Ma and X.-W. Liu, J. Am.
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ChemComm Chem. Soc., 2012, 134, 15229; (c) J. Chen, H. Jiao, W. Li, D. Liao, H. Zhou and C. Yu, Chem. – Asian J., 2013, 8, 276; (d) S. Yan, R. Huang, Y. Zhou, M. Zhang, M. Deng, X. Wang, X. Weng and X. Zhou, Chem. Commun., 2011, 47, 1273; (e) W. Li, J. Chen, H. Jiao, Q. Zhang, H. Zhou and C. Yu, Chem. Commun., 2012, 48, 10123; ( f ) Y. Yang, W. Li, H. Qi, Q. Zhang, J. Chen, Y. Wang, B. Wang, S. Wang and C. Yu, Anal. Biochem., 2012, 430, 48; (g) H. Jiao, B. Wang, J. Chen, D. Liao, W. Li and C. Yu, Chem. Commun., 2012, 48, 7862; (h) B. Wang, H. Jiao, W. Li, D. Liao, F. Wang and C. Yu, Chem. Commun., 2011, 47, 10269. 10 K. Lorentz, Clin. Chim. Acta, 2002, 326, 69–80. 11 (a) T. Kusudo, T. Sakaki and K. Inouye, Biosci., Biotechnol., Biochem., 2003, 67, 1609; (b) J. Montalibet, K. I. Skorey and B. P. Kennedy, Methods, 2005, 35, 2. 12 P. Guo, S. Yan, Y. Zhou, C. Wang, X. Xu, X. Weng and X. Zhou, Analyst, 2013, 138, 3365.
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Cite this: Chem. Commun., 2014, 50, 8677 Received 5th December 2013, Accepted 9th June 2014
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A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening† Yongqian Xu,*a Benhao Li,b Liangliang Xiao,a Jia Ouyang,d Shiguo Sun*a and Yi Pangc
DOI: 10.1039/c3cc49254k www.rsc.org/chemcomm
A dual-channel including a colorimetric and fluorescent probe based on the aggregation-caused quenching (ACQ) and enzymolysis approach has been presented to screen acid phosphatase (ACP) and its inhibitor. Moreover, the ACP activity was determined by real time assay.
Acid phosphatase (ACP) is a type of enzyme used to hydrolyze biomolecules to release phosphate groups during digestion. It is an essential phosphomonoesterase widely present in nature1 and found in mammalian tissues including prostate, liver and hematological system.2 Although the concentrations of ACP are found to be normally low in mammalian cells, it is involved in many important physiological processes, especially the movement of mammals. An abnormal level of ACP has been associated with a number of diseases, such as prostate cancer, Gaucher disease and enzyme disorders in kidneys, veins and bones.3 Due to its biological and clinical importance, the precise concentration measurement of ACP in physiological media has been regarded as an essential factor in the pathologic diagnosis of these relative diseases. In recent years, a few common strategies have been developed to detect ACP, including spectrophotometry,2a,4a–g surface acoustic wave (SAW) sensors4h and electrochemical methods.4i Although these methods are fairly sensitive, most of them require complicated instrumental setup6a and alkaline pH conditions. Among the more appealing spectrophotometry, either colorimetric or fluorometric substrates based on p-nitrophenyl phosphate ( pNPP) and 4-methyl-7-hydroxycoumarinyl phosphate (MUP) are utilized, respectively.5 However, they suffer from low selectivity, single channel, complex substrates and having a
College of Science, Northwest A&F University, Yangling, 712100, P. R. China. E-mail: [email protected], [email protected] b School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, P. R. China c Department of Chemistry & Maurice Morton Institute of Polymer Science, The University of Akron, Akron, OH, 44325, USA d School of Materials Science and Engineering, Northwestern Polytechnical University, Xi’an, 710062, P. R. China † Electronic supplementary information (ESI) available: Details of titration procedures, additional spectra and photograph. See DOI: 10.1039/c3cc49254k
This journal is © The Royal Society of Chemistry 2014
absorption and emission wavelengths in the UV region.6b Therefore, the development of facile methods with high sensitivity, dual channel screening, clinical diagnostic application, and more importantly, exhibiting its turn-on response in the near infrared (NIR) region to avoid short wavelength excitation-triggered photodamage, strong interference from biological medium and scattering light,6c is an important challenge. Squaraine (SQ) dyes are an interesting class of dyes showing sharp and intense fluorescence, typically in the red to NIR region (absorption maxima are found between 630 and 670 nm and emission maxima are between 650 and 700 nm).7 Because of NIR optical properties, they have been used as sensors for ions,8a and exploited in biomedical imaging.8b,c In solution, cationic squaraine (SQ) dyes favor to form an aggregate assembly with oppositely charged species via strong attractive electrostatic interactions.8b Accordingly, the fluorescence of SQ would dramatically decrease because of their aggregation-caused quenching (ACQ) property.8c,9 Reversely, as the aggregation equilibrium based on the electrostatic interactions is pertubed by introducing a competitive binding target in solution, SQ would deaggregate and their quenched fluorescence would recover, leading to a sensitive fluorescence turn-on assembly for target detection in the NIR region. In comparison with monomers, aggregates of SQ usually give different absorption bands which are enough to discriminate each other by color change.8d In other words, an effective dual-channel (both absorption and emission) method for detection of targets could be realized by controlling the state of SQ aggregation. Herein, we present a dual-channel method including colorimetric and fluorescent output for the detection of acid phosphatase based on the self-assembly of squaraines and sodium hexametaphosphate (NaPO3)6. The design rationale for the self-assembly detection is illustrated in Scheme 1. Commercially available sodium hexametaphosphate (NaPO3)6, with six negative charges in the six-membered ring, behaves as a building block to induce aggregate formation of positively charged SQ through intermolecular electrostatic interaction in phosphate buffer solution (pH 7.4), leading to an absorption shift and fluorescence quenching of the SQ solution. Then, ACP is added to the detection system, (NaPO3)6
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Scheme 1 (NaPO3)6.
ChemComm
Illustration of a possible assembled assay between SQ and
is hydrolyzed to the phosphate segment by ACP. The process can be traced by recording both the color and fluorescent changes, which originate from variation of the squaraine aggregation states. Thus, a facile and label-free ACP fluorescent detection system has been established. It can be used for ACP detection in the neutral or weak alkali solution rather than under acidic conditions. To test the feasibility of this new method, different amounts of (NaPO3)6 were added to the SQ solution in phosphate buffer (5 mM, pH = 7.4). Fig. S1 and S2 (ESI†) reveal the absorption and fluorescence changes of the solution. The absorption spectra of SQ revealed three bands (lmax = 625, 567 and 540 nm), which were assigned to the monomer, hypsochromic dimer, and oligomer absorption, respectively (Fig. S1, ESI†).8b The monomer and dimer absorption gradually decreased with increasing concentrations of (NaPO3)6. Observation of a new band at about 520 nm indicated that the SQ molecules were transformed to H-aggregates.8b Upon addition of (NaPO3)6 (0–10 mM), the fluorescence of SQ was gradually quenched (Fig. S2, ESI†), leading to nearly 7.5 times of decrease, presumably due to the formation of SQ-(NaPO3)6 aggregates. The spectral response clearly indicated strong electrostatic interactions between SQ and (NaPO3)6. Moreover, addition of ACP into the SQ–(NaPO3)6 solution caused a gradual increase of the SQ monomer (at 625 nm), indicating the transformation of SQ from aggregates to monomer (Fig. S3, ESI†). In the presence of ACP, (NaPO3)6 was hydrolyzed to inorganic phosphoric acid, whose weak interaction with SQ could cause the dissociation of the SQ aggregate. In the spectra obtained from the control experiment, the new band at about 505 nm was attributed to SQ-ACP (Fig. S4, ESI†). Interestingly, the fluorescence intensity of the solution was remarkably increased (Fig. 1), while the emission peak was red-shifted by about 7 nm to 644 nm, coming from the possible interaction between ACP and the SQ monomer8b and avoiding the interference of the original wavelength. As the concentration of ACP added was up to 0.533 mM, the recovery of the fluorescence intensity levelled off, at which the pre-quenched emission intensity was recovered up to 94.5% of the original intensity at 644 nm (Fig. S2, ESI†). Titration of SQ–(NaPO3)6 with various concentrations of ACP in phosphate buffer solution was performed to investigate the viability of fluorescence detection of ACP (inset in Fig. 1). The fluorescence intensity at 644 nm was plotted as a function of the ACP concentration. Their fluorescence ratios (I644/I0) were linearly proportional to the ACP concentrations (0–0.533 mM).
8678 | Chem. Commun., 2014, 50, 8677--8680
Fig. 1 Fluorescence response of SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) upon addition of (NaPO3)6 (10 mM), then followed by ACP (0–0.533 mM). The arrow indicates the change in the fluorescence intensity with the ACP concentrations (lex = 600 nm). Inset: plot of the relative fluorescence intensity (I/I0) of the solution to ACP concentrations, where I and I0 stand for the fluorescence intensity at 644 nm in the absence and presence of ACP.
A concentration as low as 4.9 nM of ACP was detected with a signal-to-noise ratio of 3, indicating that this approach was applicable to NIR fluorescent detection of ACP. Furthermore, in diluted human serum samples, SQ–(NaPO3)6 shows turn-on fluorescence response to ACP, and a good linear relationship between the fluorescence intensity at 650 nm and ACP concentration was observed (Fig. S8, ESI†). These results suggest that SQ–(NaPO3)6 can be used for ACP detection in serum samples. Bilirubin (found in icteric samples) is a frequent interference for ACP detection.10 In the presence of 120 mmol L 1 bilirubin, the fluorescence response of SQ–(NaPO3)6 to ACP is similar to that in the absence of bilirubin, and the fluorescence ratios (I650/I0) were linearly proportional to the ACP concentrations, revealing that SQ–(NaPO3)6 can overcome the limitation of bilirubin to some extent (Fig. S10 and S11, ESI†). To demonstrate the feasibility of the method for monitoring the ACP activity in real time, the fluorescence spectral changes as a function of time upon incubation of (NaPO3)6 (10 mM) and ACP (0.53 mM) were investigated. As shown in Fig. S5 (ESI†), the quenched fluorescence intensity (line b) gradually increased with time duration as indicated, and reached maximum after 3 h. Kinetic parameters of ACP were further evaluated by assays using different initial (NaPO3)6 concentrations. The concentration changes of the hydrolyzed substrate (NaPO3)6 and unhydrolyzed (NaPO3)6 calculated from the fluorescence intensity as a function of time are shown in Fig. 2. The Michaelis constant Km value related to the substrate’s affinity to the enzyme was obtained from the Lineweaver–Burk plot using double reciprocal data of initial rate V vs. substrate (NaPO3)6 concentration [S]0 (Fig. 2c). The Km and Kcat/ Km value were determined to be 265.6 mM and 9.58 104 M 1 S 1, respectivily, consistent with the data reported in the literature.11 These results indicated that the SQ-(NaPO3)6 based ACP detection can be applied for real time ACP activity measurement. The method can also be used for the screening of potential ACP inhibitors, K2MoO4 was chosen as the inhibitor for ACP.12 As shown in Fig. 3, with increasing concentrations of K2MoO4, the emission intensity constantly decreases. This result suggests
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Fig. 4 Plots of relative fluorescence intensity changes (I/I0) of SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) in the presence of (NaPO3)6 (10 mM) to ACP and other enzymes with different concentrations, where I and I0 stand for the fluorescence intensity at 644 nm in the absence and presence of different enzymes. Fig. 2 Derived concentration of the hydrolyzed (NaPO3)6 (a) and unhydrolyzed (NaPO3)6 (b) as a function of time for ACP assays at different initial (NaPO3)6 concentrations. (c) Lineweaver–Burk plot for determination of the values of Km. Assays were carried out with ACP (0.53 mM) and SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) with different (NaPO3)6 concentrations as indicated at 37 1C.
Fig. 5 Photographs of color (a) and fluorescent change (b) of SQ (5 mM) in phosphate buffer (5 mM, pH = 7.4) in the presence of (NaPO3)6 (10 mM) upon addition of BSA, lysozyme, trypsin, Rnase A, pepsinum and ACP, respectively, where protein or enzyme concentrations are all 0.53 mM and the solution was excited by a hand-held UV lamp.
Fig. 3 Fluorescence spectral change of SQ (5 mM) in the presence of (NaPO3)6 (10 mM) and ACP (0.53 mM) in phosphate buffer (5 mM, pH = 7.4) with increasing concentrations of K2MoO4.
that the recovery of fluorescence intensity could attenuate in the presence of an inhibitor. The IC50 value of K2MoO4 obtained by using the sigmoidal fit of the experimental data was 5.30 mM (inset in Fig. 3). To evaluate the selectivity of the method based on the aggregation-caused quenching (ACQ) and enzymolysis, the absorption and fluorescence spectra response of the solution containing 10 mM (NaPO3)6 to various proteins and enzymes including BSA, lysozyme, trypsin, Rnase A and pepsinum were investigated (Fig. 4 and 5 and Fig. S12, ESI†). Obvious color change and remarkable fluorescence enhancement were observed for ACP, whereas other proteins and enzymes did not induce any changes in absorption and fluorescence intensity under identical conditions. This result proves that the assembly system is suitable for selective detection of ACP. In summary, we have reported a novel colorimetric and near infrared fluorescent assembly system that is inexpensive, efficient, and highly selective for detection of ACP. This design is based on the assembly of probe SQ with a commercially available substrate (NaPO3)6, which utilizes the aggregation-caused quenching (ACQ) and enzymolysis to trigger the optical response. Although with a
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simple design, this method can selectively detect ACP at a quite low concentration of less than 4.9 nM. Detection of ACP activity and ACP inhibitor could have potential application in disease diagnosis and evaluation. This work was supported by the National Natural Science Foundation of China (Grant No. 21206137), the Fundamental Research Funds for the Central Universities (2014YB027), Shaanxi Province Science and Technology (No. 2014K11-01-02-06) and the Scientific Research Foundation of Northwest A&F University (Z111021103 and Z111021107).
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