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Label-free fluorescent assay for high sensitivity and selectivity detection of acid phosphatase and inhibitor screening
Sensors and Actuators B 234 (2016) 470–477
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Label-free fluorescent assay for high sensitivity and selectivity detection of acid phosphatase and inhibitor screening Jing Wang a,1 , Yu Yan a,1 , Xu Yan a,1 , Tianyu Hu a , Xiaojian Tang b , Xingguang Su a,∗ a b
Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu University, Changchun 130118, China
a r t i c l e
i n f o
Article history: Received 11 December 2015 Received in revised form 29 April 2016 Accepted 3 May 2016 Available online 7 May 2016 Keywords: Quantum dots Fluorescence enhancement Acid phosphatase Pesticide
a b s t r a c t In this work, we developed a convenient and label-free fluorescence sensing platform for sensitive detection of acid phosphatase (ACP) and its inhibitor. The selectivity fluorescent strategy was based on fluorescence enhancement mode of cysteamine-capped CdTe quantum dots (QDs). Upon addition of adenosine triphosphate (ATP), the amino groups on the surface of CdTe QDs can form both electrostatic and hydrogen bonding with ATP, leading to obvious fluorescence enhancement of QDs. ACP can easily catalyze the hydrolysis of ATP into adenosine and phosphate fragments under an acidic environment, causing dramatically decrease of the fluorescence intensity of QDs. Quantitative detection of ACP in a broad range from 1.0 to 50 U mL−1 with the detection limit of 0.45 U mL−1 can be achieved. The developed sensing platform has been successfully applied to the accurately analysis of ACP activity in human serum samples with good results. Furthermore, the proposed strategy also could be used for the detection of parathion-methyl which served as a model of ACP inhibitor. These results significantly demonstrated the established sensing platform can be used not only for ACP activity determination, but also for its inhibitor detection and screening. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acid phosphatase (ACP), which worked as digestive enzyme in mammalian body fluids and tissues, can efficiently catalyze the hydrolysis of phosphate esters under an acidic environment [1]. ACP in human serum is normally found in low concentrations but it plays a critical role in many mammalian physiological processes, especially the movement of humans [2]. An imbalance level of ACP may also cause a number of diseases, such as prostate cancer, multiple myeloma and Gaucher’s disease [3,4]. Clinically, the measurement of ACP activity has been used for monitoring cell viability [5]. In fact, it has already been recognized as an important biomarker of metastatic prostate cancer [6–8]. Therefore, the precise detection ACP activity is great significance in pathologic diagnosis, postsurgical evaluation and drug screening. Currently, a number of methods for ACP detection have been established, including high performance liquid chromatography [9], electrochemical methods [10,11] and immunoassay [12]. Although the above methods have good performance in sen-
∗ Corresponding author. E-mail address: [email protected] (X. Su). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.snb.2016.05.024 0925-4005/© 2016 Elsevier B.V. All rights reserved.
sitivity, the applications of these methods are limited due to tedious purification of samples, sophisticated instrumentation, costly bio-molecular reagents and time-costing immobilizing processes. Therefore, the development of a convenient, inexpensive and high sensitivity method for ACP detection is an important challenge to overcome. In order to circumvent these problems, fluorescence assay is regarded as a more desirable approach because of its convenience and high sensitivity. Up to date, few numbers of fluorometric assay using organic dyes [13,14] and fluorescent polymers [15] have been developed for ACP activity monitoring. Xu et al. established a fluorescent system for the determination of ACP based on the aggregation-caused quenching between cationic squaraine dyes and sodium hexametaphosphate [14]. Xie et al. established a sensitive fluorescent assay for ACP activity detection composed of a cationic conjugated polyelectrolyte and p-nitrophenyl phosphate [15]. As a result, most of established assays utilizing organic dyes are susceptible to poor stability, complex synthesis and tedious purification [16]. Compared with those organic dyes-based probes, quantum dots (QDs) offered several key merits, including better stability, easier preparation and size-tunable emission spectrum [17–19]. Recently, QDs have attracted increasing attention in chem/bio sensing, imaging and delivering drugs applications [20–22]. QDs-based fluorescent probe have already been regarded as a convenient system for sensitively analyzing for enzymes activ-
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Scheme 1. The schematic illustration of the novel nanosensor for ACP and inhibitor detection.
ity. For instance, Yan et al. developed ratiometric fluorescence sensor by hybridizing two differently sized QDs for selective detection of tyrosinase [16]. Li et al. established a QD-based fluorescence biosensor for the detection of matrix metalloproteinase-2 in vitro and in vivo [23]. Therefore, the promising advantages of QDs make them serve as ideal sensor and imaging platforms. With these insights, herein we designed a convenient assay with high sensitivity for ACP activity detection by using cysteaminecapped CdTe QDs as the fluorescent probe and adenosine triphosphate (ATP) as the substrate. As illustrated in Scheme 1, combining with ATP-triggered fluorescence enhancement and the ACP-caused catalytic hydrolysis, a novel sensing platform for ACP activity was proposed. The CdTe QDs capped with cysteamine possess strong green fluorescence at 539 nm. Upon addition of ATP into the system, the amino groups on the surface of QDs can interaction with the adenine base and phosphate group of ATP, resulting in the obvious fluorescence enhancement of the QDs. The presence of active ACP specifically catalyzes the hydrolysis of ATP into adenosine and phosphate fragments, and then the enhanced fluorescence of QDs can be dramatically quenched. On the basis of the fluorescence enhancement of QDs caused by ATP and following quenching with assistance of ACP, this label-free fluorescent assay possesses enough high sensitivity for ACP detection. Furthermore, the established fluorescence sensing system also could be used for the detection of the ACP inhibitor. In the presence of parathionmethyl (PM), the activity of ACP is inhibited [24], which prevents the hydrolyzation of ATP. This, in turn, will result in fluorescence recovery compared to that of QDs/ATP/ACP system. The present nanosensor based on ATP-triggered fluorescence enhancement of QDs for ACP activity detection has not been reported before.
purification. The water which used throughout the experimental process had a resistivity greater than 18 M cm−1 . Fourier transform infrared spectra (FTIR) were collected on a Bruker IFS66 V spectrometer equipped with a DGTS detector. UV–vis absorption spectra were obtained on a Shimadzu UV-1700 spectrophotometer (Shimadzu Co., Kyoto, Japan). The fluorescence spectra were carried out with RF-5301 PC spectrofluorophotometer (Shimadzu, Japan), where a xenon lamp worked as the excitation source. All pH measurements were made with a PHS-3C pH meter (Tuopu Co., Hangzhou, China). 2.2. Synthesis of CdTe QDs On the basis of our previous work [25], cysteamine capped CdTe QDs were synthesized by using a modified refluxing route. Briefly, sodium borohydride was firstly reacted with tellurium powder to produce sodium hydrogen telluride (NaHTe). NaHTe (0.25 mmol L−1 ), CdCl2 (1.25 mmol L−1 ) and cysteamine (1.87 mmol L−1 ) were mixed together at pH 5.7 in the presence of N2 protection. Then, the solution was subjected to a reflux at 250 ◦ C under condenser. Water-compatible cysteamine-capped CdTe QDs with fluorescence emission wavelength at 539 nm were obtained and used in the following experiments. 2.3. Fluorescence enhancement experiments induced by ATP
2. Experiment
Cysteamine capped CdTe QDs (100 L) and various concentrations of ATP (from 0 to 6.25 mol L−1 ) were introduced into 2.0 mL calibrated test tubes. And then, diluted to 2.0 mL with acetate buffer solution (10.0 mmol L−1 , pH = 5.0), followed by collecting the fluorescence spectrum with spectrofluorophotometer. The fluorescence emission spectra were measured with excitation wavelength at 360 nm.
2.1. Reagents and instruments
2.4. Acid phosphatase (ACP) detection
CdCl2 (99%), tellurium powder (99.8%), and NaBH4 (99%), Cysteamine (95%), acid phosphatase (ACP), parathion-methyl (PM), and adenosine-5-triphosphate (ATP) were purchased from SigmaAldrich Corporation. Acetic acid and sodium acetate trihydrate were purchased from Beijing Chemical Corp. Other solvents and reagents were of at least analytical grade and used without further
The ACP standard solution were prepared by dissolved in acetate buffer solution (10.0 mmol L−1 , pH = 5.0). Various concentrations of ACP standard solution were mixed with 5 mol L−1 ATP in a 2.0 mL calibrated test tube and kept in 37 ◦ C for 40 min. After reaction, 100 L CdTe QDs were introduced into the tube and diluted to 2.0 mL with acetate buffer solution (10.0 mmol L−1 , pH = 5.0). The
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mixed solution was shaken thoroughly for 1 min. Then, the fluorescence spectra were collected for ACP detection. 2.5. ACP detection in human serum samples For human serum samples analysis, healthy volunteer offered human blood samples through venipuncture at the China-Japan Union Hospital of Jilin University. The experiments were carried out in compliance with the relevant laws and institutional guidelines. Due to high protein in the blood plasma, some pretreatments were taken to eliminate the coexisting substances interferences [26,27]. Firstly, the blood samples were stand for 2 h at room temperature until the blood coagulation, and then samples were centrifuged at 10,000 rpm for 10 min at 4 ◦ C. Acetonitrile was introduced into serum samples with the fixed volume ratio 1.5:1 (acetonitrile/serum). After vigorously shaking for 15 min, the mixture solution was centrifuged at 10,000 rpm for 10 min. The supernatant serum samples were diluted 1000-fold with acetate buffer solution before analysis. 2.6. The inhibition effect of parathion-methyl (PM) The PM standard substance was dissolved in water solution containing 5% ethanol for preparing the standard solution. Different concentrations of PM were mixed with 50 U mL−1 of ACP for 30 min at 37 ◦ C. Then, 5 mol L−1 of ATP was introduced into the system for incubating 15 min. CdTe QDs (100 L) was introduced into the mixture solution for shaking 1 min and followed by recording the fluorescence emission spectrum of solution. 3. Results and discussion 3.1. Detection strategy of ACP In this paper, cysteamine capped QDs were employed as a fluorescent probe to monitoring ACP activity. Optical characteristics of the prepared CdTe QDs were studied by UV–vis absorption and fluorescence spectrophotometry. The CdTe QDs capped with cysteamine had a maximum absorption peak around 482 nm and had strong fluorescence emission intensity at 539 nm, respectively (Supplementary Fig. S1). As shown in Fig. 1A, the original fluorescence intensity of CdTe QDs (Black line) could be effectively enhanced by adding ATP (Red line). However, the fluorescence intensity of system was significantly quenched in the presence of ACP (Blue line). The changes in the intensity of QDs resulted in a visual fluorescence color change (Fig. 1B). This observation supported the assumption of quantitative determination of ACP activity using cysteamine capped CdTe QDs. To evaluate feasibility of this assay for ACP activity detection, we investigated the effect of ACP on the fluorescent of QDs. As shown in Supplementary Fig. S2, the fluorescence intensity of CdTe QDs would not be influenced by ACP (from 0 to 50 U mL−1 ), suggesting that the interaction between ACP and QDs could be ignored. The above results demonstrated that the proposed method could detect ACP based on the fluorescence enhancement of QDs. 3.2. The fluorescence enhancement effect of ATP on cysteamine-capped QDs When ATP was added into cysteamine capped QDs solution, the fluorescence intensity of CdTe QDs was enhanced accompanying a slightly red shift from 539 nm to 543 nm. The fluorescence properties of QDs had a close connection to surface capped layers [28], and the interaction between capping reagents and analyte can cause red-shifts of QDs emission [29]. According to previous study [30], the amine group of QDs, as an electron acceptor,
Fig. 1. (A) Fluorescence spectra of QDs, QDs/ATP and QDs/ATP/ACP. The final concentrations of ATP and ACP are 5 mol L−1 and 50 U mL−1 . (B) The corresponding color changes of QDs (a), QDs/ATP (b) and QDs/ATP/ACP (c) under UV lamp. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
can make the electron-hole recombination process lower which results in the weak fluorescence intensity. In the presence of ATP, the amine groups interact with phosphates through electrostatic bonding and form hydrogen bonding with the adenosine base, impeding the electron acceptor property of amine group. To better understand the interaction between ATP and QDs, a series of experiments were performed. As shown in Fig. 2A, the cysteaminecapped CdTe QDs were positively charged ( = +39.74 mV) while the ATP were negatively charged ( = −7.15 mV). Thus, there are strong electrostatic interactions between ATP and CdTe QDs. After mixed of CdTe QDs and ATP, the zeta potentials of system were +21.71 mV. The UV–vis absorption spectra of QDs, ATP and QDs/ATP were shown in Fig. 2B. There is no absorption band for ATP at 300–700 nm. Addition of quantitative ATP into QDs solution, the absorption band around 482 nm was considerably increased. This also confirmed the interaction between QDs and ATP. The FTIR spectrum of ATP, CdTe QDs and ATP/CdTe
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Fig. 2. (A) The zeta potentials of QDs, ATP and QDs/ATP mixture in the acetate buffer solution (10.0 mmol L−1 , pH = 5.0); (B) The UV–vis absorption spectra QDs, ATP and QDs/ATP mixture; (C) The FT-IR spectra of the QDs, ATP and QDs/ATP mixture. (D) Fluorescence spectra of QDs in the presence of different concentrations of ATP. The concentrations of ATP were 0, 0.5, 1.25, 2.5, 3.75, 5 and 6.25 mol L−1 , respectively. The inset was the linear plot of the fluorescence intensity ratio FE /FE0 versus the concentration of ATP. FE0 and FE were the fluorescence intensity of the QDs in the absence and presence of ATP, respectively.
QDs mixture were recorded in Fig. 2C. It could be obviously observed that the strong peak at 1711 cm−1 of ATP, which could be attributed to the C N stretching of the adenosine base, blue shifted to 1634 cm−1 due to the interaction with CdTe QDs. This indicated that the base moiety of ATP interacted with amine on the surface of CdTe QDs probably through hydrogen bonding effect. These results demonstrated that both hydrogen bonding and electrostatic interactions played significant role in the fluorescence enhancement of CdTe QDs induced by ATP. We also investigated the influence of ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine on the enhancement of CdTe QDs. As shown in Supplementary Fig. S3A, after adding 5 mol L−1 ATP, the fluorescence intensity of CdTe QDs is remarkably increased 143% of the original fluorescence intensity. In the presence of 5 mol L−1 ADP, AMP or adenosine, the lower enhancement (31% for ADP, 18% for AMP and 10% for adenosine) of the fluorescence intensities was obtained. Supplementary Fig. S3B illustrated that the fluorescence enhancement of CdTe QDs stystem response to analyte followed the order ATP > ADP > AMP > adenosine. The results clearly proved that the electrostatic bonding showed the main role in the fluorescence enhancement of this assay.
3.3. Optimization for ACP activity detection We systematically investigated the fluorescence enhancement of CdTe QDs caused by ATP. Fluorescence emission spectra of CdTe QDs measured as a function of ATP are illustrated in Fig. 2D. The fluorescence intensity at 539 nm of sensing platform gradually enhanced with the increasing ATP concentration and 5 mol L−1 of ATP was chosen for further study. To estiblish a fluorescent platform with excellent sensing performance for ACP detection, some related factors, such as pH of solution, reaction temperature, and reaction time are optimized. From Fig. 3A, we can see that pH had remarkable influence on the fluorescence intensity of CdTe QDs/ATP (Black Curve) and CdTe QDs/ATP/ACP (Red Curve) system. When the pH value changed from 5.0 to 6.0, the fluorescence intensity of QDs/ATP and QDs/ATP/ACP system gradually decreased. At pH 5.0, the system reached maximum quenching efficiency in the presence of 50 U mL−1 ACP. Therefore, pH 5.0 acetate buffer (10 mmol L−1 ) was selected as the working buffer for ACP activity detection. Reaction temperature was also a critical factor which could obviously affect the ACP activity; therefore, the temperature optimization was investigated in the pH 5.0 acetate buffer for 60 min. As shown in Fig. 3B, the results shown that the quenching effect
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reach the maximum at 37 ◦ C, which was consistent with the optimum temperature of the catalytic behavior of ACP. Thus, 37 ◦ C was chosen as the optimal incubation temperature for ACP detection. The incubation time of QDs/ATP system fluorescence intensity in the presence of different ACP concentrations (2.5, 25, 50 U mL−1 ) was investigated in Fig. 3C. The results demonstrated that the fluorescence quenching of QDs/ATP system occurred immediately and completed within 40 min. So we recorded the fluorescence intensity of QDs/ATP/ACP system after incubating 40 min. 3.4. Determination of ACP activity
Fig. 3. (A) The effect of pH on the fluorescence intensity of QDs/ATP and QDs/ATP/ACP system; (B) The effect of reaction temperature on the fluorescence intensity of QDs/ATP system in the absence or presence of 50 U mL−1 ACP; and (C) The effect of reaction time on the fluorescence intensity of QDs/ATP system in the presence of 2.5, 25, and 50 U mL−1 ACP. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
Under the optimal conditions, the QDs were mixed with 5.0 mol L−1 of ATP and different concentrations of ACP (0, 1.0, 2.5, 5.0, 10, 25 and 50 U mL−1 ) for 40 min at 37 ◦ C. As shown in Fig. 4A, the fluorescence intensity of QDs/ATP system was continuously quenched with the increasing of ACP concentration. The fluorescence intensity ratio (FQ /FQ0 ) showed good linear relationship with the ACP concentration in the range from 1.0 to 50 U mL−1 (inset in Fig. 4A). The regression equation was: FQ /FQ0 = 0.975 (±0.0039) − 0.00849 × 10−5 (±0.32 × 10−4 ) [ACP], U mL−1 . FQ0 and FQ were the fluorescence intensity of the QDs/ATP system in the absence and presence of ACP, respectively. The corresponding regression coefficient (R2 ) is 0.993, and the limit of detection (LOD) for ACP is 0.45 U mL−1 . The detection limit was obtained by the equation: LOD = (3/s), where is the standard deviation of blank signals (n = 9) and s is the slope of the calibration curve [31]. It has been reported that normal ACP level in blood serum is 35–123 mU mL−1 [32], thus our developed sensing platform could satisfy the detection requirements for ACP level detection in blood serum. Selective recognition capability is a critical parameter to evaluate the performance of the fluorescence sensing platform. Therefore, to test the specificity of the sensing platform for ACP detection, we studied the influences of various coexistence substances including inorganic salt (Na+ , K+ , Ca2+ , Cl− , SO4 2− and PO3 3− ), ascorbic acid, amino acid (glutamic acid, histidine, arginine, tryptophan, lysine, threonine, glycine and aspartic acid) and protein (papain, bovine serum albumin (BSA), trypsin, glucose oxidase (GO), human serum albumin (HSA), horseradish peroxidase (HRP) and urease). As shown in Fig. 4B, after the addition of above substance (0.5 g mL−1 ), the fluorescence intensity of QDs/ATP system remained nearly constant (fluorescence intensity change less than 5%), and only in the presence of ACP, the fluorescence intensity of system showed an obvious decrease (Blank, Fig. 4B). Those results demonstrated that the sensing platform showed excellent performance to ACP. We then studied the signal response of QDs/ATP sensing system toward 50 U mL−1 ACP (∼2 × 10−2 g mL−1 ) in the presence of 0.5 g mL−1 foreign substance. As shown in Fig. 4B, the QDs/ATP/ACP system still work the same in the presence of 0.5 g mL−1 interference, which indicates that established ACP sensing system could offer acceptable ability of resisting common coexisting substances. Thus, the proposed nanosensor exhibited an excellent sensitivity for the detection of ACP activity. Moreover, Supplementary Table S1 shows the comparison between our proposed sensing platform and existing strategies for ACP activity detection, proving that this sensing fluorescent method was comparable to most of the reported methods. So far, most previous fluorescent probes based on QDs for ACP activity detection utilized metal ion to switch off the fluorescence of QDs, then sodium pyrophosphate (PPi) or ATP was introduced to turn on the fluorescence intensity. In the presence of ACP, PPi or ATP was hydrolyzed, then metal ion was free to turn off the fluorescence again [5,33]. Different from these detection strategies for ACP, the present method was more convenient due to avoiding the use of metal ion. And the fluorescent probe for ACP detection based on fluorescence
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Table 1 Results of ACP determination in human serum samples. Sample
Original found (U mL−1 )
Added (U mL−1 )
Total found (U mL−1 )
Recovery
1
1.25
5.0 10.0
6.12 11.56
97 103
1.45 3.57
2
1.92
5.0 10.0
6.99 11.43
101 95
2.92 2.34
3
1.68
5.0 10.0
6.88 11.35
104 97
1.66 3.05
RSD (n = 3, %)
Fig. 5. (A) Fluorescence spectra of QDs/ATP/ACP system with different concentrations of PM. The concentrations of PM were 0, 0.001, 0.01, 0.1, 1.0, and 5.0 g mL−1 , respectively. The inset was the change trend of fluorescence intensity with different PM concentrations. (B) Relationship between the IE and the logarithm of the PM concentration.
Fig. 4. (A) The fluorescence spectra of QDs/ATP system in the presence of different concentration of ACP (0, 1.0, 2.5, 5.0, 10, 25 and 50 U mL−1 ). Inset: the linear plot of the fluorescence intensity ratio FQ /FQ0 versus the concentration of ACP. FQ0 and FQ were the fluorescence intensity of the QDs in the absence and presence of ATP, respectively. (B) The fluorescence intensity of the QDs/ATP system and QDs/ATP/ACP system in the presence of the interfering substances (0.5 g mL−1 ).
enhancement mode could significantly improve optical properties of fluorophores and detection sensitivity. Furthermore, the reaction time was short in compared with the previous ACP strategies (Supplementary Table S1). 3.5. Determination of ACP in human serum samples In order to investigate the accuracy, repeatability and applicability of sensing platform in real samples detection, the established fluorescence sensor was employed for the sensing of ACP activity in human serum samples. Different concentrations of standard
ACP solution (5.0 and 10.0 U mL−1 ) were added for the recovery test. From Table 1, the average recoveries of ACP in human serum samples ranged from 95% to 104% and the relative standard deviations (RSD) were lower than 3.57%. The results demonstrated the potential practical application of the QDs/ATP-based fluorescence platform for the ACP activity detection in real samples.
3.6. ACP inhibitor study Considering the inhibitors would reduce the ACP activity, our method could also be utilized for the determination of ACP inhibitors. According to previous report, organphosphorus pesticides (OPs) are common ACP inhibitors [24,34], which could efficiently inhibit the catalytic capacity of ACP. In this work, as a model of OPs, parathion-methyl (PM) is chosen as the inhibitor of ACP. When the ACP activity was inhibited by PM, both its efficiency to catalyze the hydrolysis of ATP and the degree of fluorescence quenching of QDs were obviously weakened. We use the inhibition
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efficiency (IE%) to ACP as a signal for the detection of PM. IE% was expressed by the following equation [16,35]: Inhibition Efficiency(IE) =
Finhibitor − Fno inhibitor × 100% F0 − Fnoinhibitor
where Finhibitor and Fnoinhibitor are the fluorescence intensity of QDs/ATP/ACP system in the presence and absence of PM, respectively. F0 refers to the fluorescence intensity of the proposed system in the absence of ACP and PM. As shown in Fig. 5A, the gradually increased fluorescence intensity was observed with the increasing concentrations of PM in the range of 0.001–10 g mL−1 . The inset illustrated the relationship between the fluorescence intensity and PM concentrations. The plot of IE versus the logarithm of the PM concentration (from 0.001 to 5.0 g mL−1 ) was observed in Fig. 5B. The regression equation is: IE (%) = 54.90 + 15.68 Log [PM], g mL−1 . The detection limit for PM is 0.5 ng mL−1 , which was much lower than the value (20 ng mg−1 ) of the maximum residue limit of PM set by the Chinese National food safety standard (GB-27632014). Moreover, this detection limit is comparable to or even lower than those of previously reported methods (detailed comparison is shown in Supplementary Table S2). Thus, the proposed sensing platform can satisfy most requirements for real-life uses. These results significantly demonstrated the established sensing probes can be used not only for ACP activity determination but also for its inhibitor detection and screening. 4. Conclusion In summary, a label-free fluorescence sensing platform was developed for sensitive detection of acid phosphatase (ACP) and its inhibitor using cysteamine-capped CdTe quantum dots. The facile, sensitive, and selective sensing system was based on ATPtriggered fluorescence enhancement and ACP-caused fluorescence quenching. Compared with previous fluorescence quenching strategy for ACP assay developed by our group, ATP-triggered emission enhancement system was employed as promising platform with advantages in terms of low background interference and superior resistance to photobleaching. The practical application is demonstrated by the assay of the human serum samples and obtains satisfactory results. More importantly, the system also provides a prospective platform for highly sensitive and selective detection PM based on enzyme inhibition. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grants 21075050, 21275063), the Science and Technology Development Project of Jilin Province, China (Grant 20110334), and the Graduate Innovation Fund of Jilin University (Grant 2015022). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.05.024. References [1] H. Bull, P.G. Murray, D. Thomas, A.M. Fraser, P.N. Nelson, Acid phosphatases, Mol. Pathol. 55 (2002) 65–72. [2] Z.P. Liu, Z.H. Lin, L.L. Liu, X.G. Su, A convenient and label-free fluorescence “turn off–on” nanosensor with high sensitivity and selectivity for acid phosphatase, Anal. Chim. Acta 876 (2015) 83–90. [3] E.S. Leman, R.H. Getzenberg, Biomarkers for prostate cancer, J. Cell. Biochem. 108 (2009) 3–9. [4] J. Sun, F. Yang, X.R. Yang, Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing, Nanoscale 7 (2015) 16372–16380.
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Biographies Jing Wang is admitted to Jilin University to study for a bachelor’s degree at the College of Chemistry in 2012. Her major research areas focus on the synthesis and functionalization of quantum dots and their application on sensors for proteins, pesticide and so on. Yu Yan is admitted to Jilin University to study for a bachelor’s degree at the College of Chemistry in 2012. His major research areas focus on the synthesis and functionalization of quantum dots and their application on sensors for proteins, pesticide and so on.
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Xu Yan is currently carrying out his graduate work for her Doctor’s degree under the guidance of Prof. Xingguang Su in Jilin University. His major research areas focus on the synthesis and functionalization of quantum dots and their application on sensors for proteins, pesticide and so on. Xiaojian Tang is a lecturer at Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu University. Her research focuses on the environmental pollutant degradation and detection. Xingguang Su is a professor at the Department of Analytical Chemistry at the College of Chemistry, Jilin University. She received her MS degree from Jilin University (China) in 1992 and her PhD degree from Jilin University (China) in 1999. Her research focuses on the synthesis, characterization, functionalization and application of quantum dots and quantum dots-tagged microspheres in biomedicine.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Label-free fluorescent assay for high sensitivity and selectivity detection of acid phosphatase and inhibitor screening Jing Wang a,1 , Yu Yan a,1 , Xu Yan a,1 , Tianyu Hu a , Xiaojian Tang b , Xingguang Su a,∗ a b
Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu University, Changchun 130118, China
a r t i c l e
i n f o
Article history: Received 11 December 2015 Received in revised form 29 April 2016 Accepted 3 May 2016 Available online 7 May 2016 Keywords: Quantum dots Fluorescence enhancement Acid phosphatase Pesticide
a b s t r a c t In this work, we developed a convenient and label-free fluorescence sensing platform for sensitive detection of acid phosphatase (ACP) and its inhibitor. The selectivity fluorescent strategy was based on fluorescence enhancement mode of cysteamine-capped CdTe quantum dots (QDs). Upon addition of adenosine triphosphate (ATP), the amino groups on the surface of CdTe QDs can form both electrostatic and hydrogen bonding with ATP, leading to obvious fluorescence enhancement of QDs. ACP can easily catalyze the hydrolysis of ATP into adenosine and phosphate fragments under an acidic environment, causing dramatically decrease of the fluorescence intensity of QDs. Quantitative detection of ACP in a broad range from 1.0 to 50 U mL−1 with the detection limit of 0.45 U mL−1 can be achieved. The developed sensing platform has been successfully applied to the accurately analysis of ACP activity in human serum samples with good results. Furthermore, the proposed strategy also could be used for the detection of parathion-methyl which served as a model of ACP inhibitor. These results significantly demonstrated the established sensing platform can be used not only for ACP activity determination, but also for its inhibitor detection and screening. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acid phosphatase (ACP), which worked as digestive enzyme in mammalian body fluids and tissues, can efficiently catalyze the hydrolysis of phosphate esters under an acidic environment [1]. ACP in human serum is normally found in low concentrations but it plays a critical role in many mammalian physiological processes, especially the movement of humans [2]. An imbalance level of ACP may also cause a number of diseases, such as prostate cancer, multiple myeloma and Gaucher’s disease [3,4]. Clinically, the measurement of ACP activity has been used for monitoring cell viability [5]. In fact, it has already been recognized as an important biomarker of metastatic prostate cancer [6–8]. Therefore, the precise detection ACP activity is great significance in pathologic diagnosis, postsurgical evaluation and drug screening. Currently, a number of methods for ACP detection have been established, including high performance liquid chromatography [9], electrochemical methods [10,11] and immunoassay [12]. Although the above methods have good performance in sen-
∗ Corresponding author. E-mail address: [email protected] (X. Su). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.snb.2016.05.024 0925-4005/© 2016 Elsevier B.V. All rights reserved.
sitivity, the applications of these methods are limited due to tedious purification of samples, sophisticated instrumentation, costly bio-molecular reagents and time-costing immobilizing processes. Therefore, the development of a convenient, inexpensive and high sensitivity method for ACP detection is an important challenge to overcome. In order to circumvent these problems, fluorescence assay is regarded as a more desirable approach because of its convenience and high sensitivity. Up to date, few numbers of fluorometric assay using organic dyes [13,14] and fluorescent polymers [15] have been developed for ACP activity monitoring. Xu et al. established a fluorescent system for the determination of ACP based on the aggregation-caused quenching between cationic squaraine dyes and sodium hexametaphosphate [14]. Xie et al. established a sensitive fluorescent assay for ACP activity detection composed of a cationic conjugated polyelectrolyte and p-nitrophenyl phosphate [15]. As a result, most of established assays utilizing organic dyes are susceptible to poor stability, complex synthesis and tedious purification [16]. Compared with those organic dyes-based probes, quantum dots (QDs) offered several key merits, including better stability, easier preparation and size-tunable emission spectrum [17–19]. Recently, QDs have attracted increasing attention in chem/bio sensing, imaging and delivering drugs applications [20–22]. QDs-based fluorescent probe have already been regarded as a convenient system for sensitively analyzing for enzymes activ-
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Scheme 1. The schematic illustration of the novel nanosensor for ACP and inhibitor detection.
ity. For instance, Yan et al. developed ratiometric fluorescence sensor by hybridizing two differently sized QDs for selective detection of tyrosinase [16]. Li et al. established a QD-based fluorescence biosensor for the detection of matrix metalloproteinase-2 in vitro and in vivo [23]. Therefore, the promising advantages of QDs make them serve as ideal sensor and imaging platforms. With these insights, herein we designed a convenient assay with high sensitivity for ACP activity detection by using cysteaminecapped CdTe QDs as the fluorescent probe and adenosine triphosphate (ATP) as the substrate. As illustrated in Scheme 1, combining with ATP-triggered fluorescence enhancement and the ACP-caused catalytic hydrolysis, a novel sensing platform for ACP activity was proposed. The CdTe QDs capped with cysteamine possess strong green fluorescence at 539 nm. Upon addition of ATP into the system, the amino groups on the surface of QDs can interaction with the adenine base and phosphate group of ATP, resulting in the obvious fluorescence enhancement of the QDs. The presence of active ACP specifically catalyzes the hydrolysis of ATP into adenosine and phosphate fragments, and then the enhanced fluorescence of QDs can be dramatically quenched. On the basis of the fluorescence enhancement of QDs caused by ATP and following quenching with assistance of ACP, this label-free fluorescent assay possesses enough high sensitivity for ACP detection. Furthermore, the established fluorescence sensing system also could be used for the detection of the ACP inhibitor. In the presence of parathionmethyl (PM), the activity of ACP is inhibited [24], which prevents the hydrolyzation of ATP. This, in turn, will result in fluorescence recovery compared to that of QDs/ATP/ACP system. The present nanosensor based on ATP-triggered fluorescence enhancement of QDs for ACP activity detection has not been reported before.
purification. The water which used throughout the experimental process had a resistivity greater than 18 M cm−1 . Fourier transform infrared spectra (FTIR) were collected on a Bruker IFS66 V spectrometer equipped with a DGTS detector. UV–vis absorption spectra were obtained on a Shimadzu UV-1700 spectrophotometer (Shimadzu Co., Kyoto, Japan). The fluorescence spectra were carried out with RF-5301 PC spectrofluorophotometer (Shimadzu, Japan), where a xenon lamp worked as the excitation source. All pH measurements were made with a PHS-3C pH meter (Tuopu Co., Hangzhou, China). 2.2. Synthesis of CdTe QDs On the basis of our previous work [25], cysteamine capped CdTe QDs were synthesized by using a modified refluxing route. Briefly, sodium borohydride was firstly reacted with tellurium powder to produce sodium hydrogen telluride (NaHTe). NaHTe (0.25 mmol L−1 ), CdCl2 (1.25 mmol L−1 ) and cysteamine (1.87 mmol L−1 ) were mixed together at pH 5.7 in the presence of N2 protection. Then, the solution was subjected to a reflux at 250 ◦ C under condenser. Water-compatible cysteamine-capped CdTe QDs with fluorescence emission wavelength at 539 nm were obtained and used in the following experiments. 2.3. Fluorescence enhancement experiments induced by ATP
2. Experiment
Cysteamine capped CdTe QDs (100 L) and various concentrations of ATP (from 0 to 6.25 mol L−1 ) were introduced into 2.0 mL calibrated test tubes. And then, diluted to 2.0 mL with acetate buffer solution (10.0 mmol L−1 , pH = 5.0), followed by collecting the fluorescence spectrum with spectrofluorophotometer. The fluorescence emission spectra were measured with excitation wavelength at 360 nm.
2.1. Reagents and instruments
2.4. Acid phosphatase (ACP) detection
CdCl2 (99%), tellurium powder (99.8%), and NaBH4 (99%), Cysteamine (95%), acid phosphatase (ACP), parathion-methyl (PM), and adenosine-5-triphosphate (ATP) were purchased from SigmaAldrich Corporation. Acetic acid and sodium acetate trihydrate were purchased from Beijing Chemical Corp. Other solvents and reagents were of at least analytical grade and used without further
The ACP standard solution were prepared by dissolved in acetate buffer solution (10.0 mmol L−1 , pH = 5.0). Various concentrations of ACP standard solution were mixed with 5 mol L−1 ATP in a 2.0 mL calibrated test tube and kept in 37 ◦ C for 40 min. After reaction, 100 L CdTe QDs were introduced into the tube and diluted to 2.0 mL with acetate buffer solution (10.0 mmol L−1 , pH = 5.0). The
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mixed solution was shaken thoroughly for 1 min. Then, the fluorescence spectra were collected for ACP detection. 2.5. ACP detection in human serum samples For human serum samples analysis, healthy volunteer offered human blood samples through venipuncture at the China-Japan Union Hospital of Jilin University. The experiments were carried out in compliance with the relevant laws and institutional guidelines. Due to high protein in the blood plasma, some pretreatments were taken to eliminate the coexisting substances interferences [26,27]. Firstly, the blood samples were stand for 2 h at room temperature until the blood coagulation, and then samples were centrifuged at 10,000 rpm for 10 min at 4 ◦ C. Acetonitrile was introduced into serum samples with the fixed volume ratio 1.5:1 (acetonitrile/serum). After vigorously shaking for 15 min, the mixture solution was centrifuged at 10,000 rpm for 10 min. The supernatant serum samples were diluted 1000-fold with acetate buffer solution before analysis. 2.6. The inhibition effect of parathion-methyl (PM) The PM standard substance was dissolved in water solution containing 5% ethanol for preparing the standard solution. Different concentrations of PM were mixed with 50 U mL−1 of ACP for 30 min at 37 ◦ C. Then, 5 mol L−1 of ATP was introduced into the system for incubating 15 min. CdTe QDs (100 L) was introduced into the mixture solution for shaking 1 min and followed by recording the fluorescence emission spectrum of solution. 3. Results and discussion 3.1. Detection strategy of ACP In this paper, cysteamine capped QDs were employed as a fluorescent probe to monitoring ACP activity. Optical characteristics of the prepared CdTe QDs were studied by UV–vis absorption and fluorescence spectrophotometry. The CdTe QDs capped with cysteamine had a maximum absorption peak around 482 nm and had strong fluorescence emission intensity at 539 nm, respectively (Supplementary Fig. S1). As shown in Fig. 1A, the original fluorescence intensity of CdTe QDs (Black line) could be effectively enhanced by adding ATP (Red line). However, the fluorescence intensity of system was significantly quenched in the presence of ACP (Blue line). The changes in the intensity of QDs resulted in a visual fluorescence color change (Fig. 1B). This observation supported the assumption of quantitative determination of ACP activity using cysteamine capped CdTe QDs. To evaluate feasibility of this assay for ACP activity detection, we investigated the effect of ACP on the fluorescent of QDs. As shown in Supplementary Fig. S2, the fluorescence intensity of CdTe QDs would not be influenced by ACP (from 0 to 50 U mL−1 ), suggesting that the interaction between ACP and QDs could be ignored. The above results demonstrated that the proposed method could detect ACP based on the fluorescence enhancement of QDs. 3.2. The fluorescence enhancement effect of ATP on cysteamine-capped QDs When ATP was added into cysteamine capped QDs solution, the fluorescence intensity of CdTe QDs was enhanced accompanying a slightly red shift from 539 nm to 543 nm. The fluorescence properties of QDs had a close connection to surface capped layers [28], and the interaction between capping reagents and analyte can cause red-shifts of QDs emission [29]. According to previous study [30], the amine group of QDs, as an electron acceptor,
Fig. 1. (A) Fluorescence spectra of QDs, QDs/ATP and QDs/ATP/ACP. The final concentrations of ATP and ACP are 5 mol L−1 and 50 U mL−1 . (B) The corresponding color changes of QDs (a), QDs/ATP (b) and QDs/ATP/ACP (c) under UV lamp. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
can make the electron-hole recombination process lower which results in the weak fluorescence intensity. In the presence of ATP, the amine groups interact with phosphates through electrostatic bonding and form hydrogen bonding with the adenosine base, impeding the electron acceptor property of amine group. To better understand the interaction between ATP and QDs, a series of experiments were performed. As shown in Fig. 2A, the cysteaminecapped CdTe QDs were positively charged ( = +39.74 mV) while the ATP were negatively charged ( = −7.15 mV). Thus, there are strong electrostatic interactions between ATP and CdTe QDs. After mixed of CdTe QDs and ATP, the zeta potentials of system were +21.71 mV. The UV–vis absorption spectra of QDs, ATP and QDs/ATP were shown in Fig. 2B. There is no absorption band for ATP at 300–700 nm. Addition of quantitative ATP into QDs solution, the absorption band around 482 nm was considerably increased. This also confirmed the interaction between QDs and ATP. The FTIR spectrum of ATP, CdTe QDs and ATP/CdTe
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Fig. 2. (A) The zeta potentials of QDs, ATP and QDs/ATP mixture in the acetate buffer solution (10.0 mmol L−1 , pH = 5.0); (B) The UV–vis absorption spectra QDs, ATP and QDs/ATP mixture; (C) The FT-IR spectra of the QDs, ATP and QDs/ATP mixture. (D) Fluorescence spectra of QDs in the presence of different concentrations of ATP. The concentrations of ATP were 0, 0.5, 1.25, 2.5, 3.75, 5 and 6.25 mol L−1 , respectively. The inset was the linear plot of the fluorescence intensity ratio FE /FE0 versus the concentration of ATP. FE0 and FE were the fluorescence intensity of the QDs in the absence and presence of ATP, respectively.
QDs mixture were recorded in Fig. 2C. It could be obviously observed that the strong peak at 1711 cm−1 of ATP, which could be attributed to the C N stretching of the adenosine base, blue shifted to 1634 cm−1 due to the interaction with CdTe QDs. This indicated that the base moiety of ATP interacted with amine on the surface of CdTe QDs probably through hydrogen bonding effect. These results demonstrated that both hydrogen bonding and electrostatic interactions played significant role in the fluorescence enhancement of CdTe QDs induced by ATP. We also investigated the influence of ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine on the enhancement of CdTe QDs. As shown in Supplementary Fig. S3A, after adding 5 mol L−1 ATP, the fluorescence intensity of CdTe QDs is remarkably increased 143% of the original fluorescence intensity. In the presence of 5 mol L−1 ADP, AMP or adenosine, the lower enhancement (31% for ADP, 18% for AMP and 10% for adenosine) of the fluorescence intensities was obtained. Supplementary Fig. S3B illustrated that the fluorescence enhancement of CdTe QDs stystem response to analyte followed the order ATP > ADP > AMP > adenosine. The results clearly proved that the electrostatic bonding showed the main role in the fluorescence enhancement of this assay.
3.3. Optimization for ACP activity detection We systematically investigated the fluorescence enhancement of CdTe QDs caused by ATP. Fluorescence emission spectra of CdTe QDs measured as a function of ATP are illustrated in Fig. 2D. The fluorescence intensity at 539 nm of sensing platform gradually enhanced with the increasing ATP concentration and 5 mol L−1 of ATP was chosen for further study. To estiblish a fluorescent platform with excellent sensing performance for ACP detection, some related factors, such as pH of solution, reaction temperature, and reaction time are optimized. From Fig. 3A, we can see that pH had remarkable influence on the fluorescence intensity of CdTe QDs/ATP (Black Curve) and CdTe QDs/ATP/ACP (Red Curve) system. When the pH value changed from 5.0 to 6.0, the fluorescence intensity of QDs/ATP and QDs/ATP/ACP system gradually decreased. At pH 5.0, the system reached maximum quenching efficiency in the presence of 50 U mL−1 ACP. Therefore, pH 5.0 acetate buffer (10 mmol L−1 ) was selected as the working buffer for ACP activity detection. Reaction temperature was also a critical factor which could obviously affect the ACP activity; therefore, the temperature optimization was investigated in the pH 5.0 acetate buffer for 60 min. As shown in Fig. 3B, the results shown that the quenching effect
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reach the maximum at 37 ◦ C, which was consistent with the optimum temperature of the catalytic behavior of ACP. Thus, 37 ◦ C was chosen as the optimal incubation temperature for ACP detection. The incubation time of QDs/ATP system fluorescence intensity in the presence of different ACP concentrations (2.5, 25, 50 U mL−1 ) was investigated in Fig. 3C. The results demonstrated that the fluorescence quenching of QDs/ATP system occurred immediately and completed within 40 min. So we recorded the fluorescence intensity of QDs/ATP/ACP system after incubating 40 min. 3.4. Determination of ACP activity
Fig. 3. (A) The effect of pH on the fluorescence intensity of QDs/ATP and QDs/ATP/ACP system; (B) The effect of reaction temperature on the fluorescence intensity of QDs/ATP system in the absence or presence of 50 U mL−1 ACP; and (C) The effect of reaction time on the fluorescence intensity of QDs/ATP system in the presence of 2.5, 25, and 50 U mL−1 ACP. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
Under the optimal conditions, the QDs were mixed with 5.0 mol L−1 of ATP and different concentrations of ACP (0, 1.0, 2.5, 5.0, 10, 25 and 50 U mL−1 ) for 40 min at 37 ◦ C. As shown in Fig. 4A, the fluorescence intensity of QDs/ATP system was continuously quenched with the increasing of ACP concentration. The fluorescence intensity ratio (FQ /FQ0 ) showed good linear relationship with the ACP concentration in the range from 1.0 to 50 U mL−1 (inset in Fig. 4A). The regression equation was: FQ /FQ0 = 0.975 (±0.0039) − 0.00849 × 10−5 (±0.32 × 10−4 ) [ACP], U mL−1 . FQ0 and FQ were the fluorescence intensity of the QDs/ATP system in the absence and presence of ACP, respectively. The corresponding regression coefficient (R2 ) is 0.993, and the limit of detection (LOD) for ACP is 0.45 U mL−1 . The detection limit was obtained by the equation: LOD = (3/s), where is the standard deviation of blank signals (n = 9) and s is the slope of the calibration curve [31]. It has been reported that normal ACP level in blood serum is 35–123 mU mL−1 [32], thus our developed sensing platform could satisfy the detection requirements for ACP level detection in blood serum. Selective recognition capability is a critical parameter to evaluate the performance of the fluorescence sensing platform. Therefore, to test the specificity of the sensing platform for ACP detection, we studied the influences of various coexistence substances including inorganic salt (Na+ , K+ , Ca2+ , Cl− , SO4 2− and PO3 3− ), ascorbic acid, amino acid (glutamic acid, histidine, arginine, tryptophan, lysine, threonine, glycine and aspartic acid) and protein (papain, bovine serum albumin (BSA), trypsin, glucose oxidase (GO), human serum albumin (HSA), horseradish peroxidase (HRP) and urease). As shown in Fig. 4B, after the addition of above substance (0.5 g mL−1 ), the fluorescence intensity of QDs/ATP system remained nearly constant (fluorescence intensity change less than 5%), and only in the presence of ACP, the fluorescence intensity of system showed an obvious decrease (Blank, Fig. 4B). Those results demonstrated that the sensing platform showed excellent performance to ACP. We then studied the signal response of QDs/ATP sensing system toward 50 U mL−1 ACP (∼2 × 10−2 g mL−1 ) in the presence of 0.5 g mL−1 foreign substance. As shown in Fig. 4B, the QDs/ATP/ACP system still work the same in the presence of 0.5 g mL−1 interference, which indicates that established ACP sensing system could offer acceptable ability of resisting common coexisting substances. Thus, the proposed nanosensor exhibited an excellent sensitivity for the detection of ACP activity. Moreover, Supplementary Table S1 shows the comparison between our proposed sensing platform and existing strategies for ACP activity detection, proving that this sensing fluorescent method was comparable to most of the reported methods. So far, most previous fluorescent probes based on QDs for ACP activity detection utilized metal ion to switch off the fluorescence of QDs, then sodium pyrophosphate (PPi) or ATP was introduced to turn on the fluorescence intensity. In the presence of ACP, PPi or ATP was hydrolyzed, then metal ion was free to turn off the fluorescence again [5,33]. Different from these detection strategies for ACP, the present method was more convenient due to avoiding the use of metal ion. And the fluorescent probe for ACP detection based on fluorescence
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Table 1 Results of ACP determination in human serum samples. Sample
Original found (U mL−1 )
Added (U mL−1 )
Total found (U mL−1 )
Recovery
1
1.25
5.0 10.0
6.12 11.56
97 103
1.45 3.57
2
1.92
5.0 10.0
6.99 11.43
101 95
2.92 2.34
3
1.68
5.0 10.0
6.88 11.35
104 97
1.66 3.05
RSD (n = 3, %)
Fig. 5. (A) Fluorescence spectra of QDs/ATP/ACP system with different concentrations of PM. The concentrations of PM were 0, 0.001, 0.01, 0.1, 1.0, and 5.0 g mL−1 , respectively. The inset was the change trend of fluorescence intensity with different PM concentrations. (B) Relationship between the IE and the logarithm of the PM concentration.
Fig. 4. (A) The fluorescence spectra of QDs/ATP system in the presence of different concentration of ACP (0, 1.0, 2.5, 5.0, 10, 25 and 50 U mL−1 ). Inset: the linear plot of the fluorescence intensity ratio FQ /FQ0 versus the concentration of ACP. FQ0 and FQ were the fluorescence intensity of the QDs in the absence and presence of ATP, respectively. (B) The fluorescence intensity of the QDs/ATP system and QDs/ATP/ACP system in the presence of the interfering substances (0.5 g mL−1 ).
enhancement mode could significantly improve optical properties of fluorophores and detection sensitivity. Furthermore, the reaction time was short in compared with the previous ACP strategies (Supplementary Table S1). 3.5. Determination of ACP in human serum samples In order to investigate the accuracy, repeatability and applicability of sensing platform in real samples detection, the established fluorescence sensor was employed for the sensing of ACP activity in human serum samples. Different concentrations of standard
ACP solution (5.0 and 10.0 U mL−1 ) were added for the recovery test. From Table 1, the average recoveries of ACP in human serum samples ranged from 95% to 104% and the relative standard deviations (RSD) were lower than 3.57%. The results demonstrated the potential practical application of the QDs/ATP-based fluorescence platform for the ACP activity detection in real samples.
3.6. ACP inhibitor study Considering the inhibitors would reduce the ACP activity, our method could also be utilized for the determination of ACP inhibitors. According to previous report, organphosphorus pesticides (OPs) are common ACP inhibitors [24,34], which could efficiently inhibit the catalytic capacity of ACP. In this work, as a model of OPs, parathion-methyl (PM) is chosen as the inhibitor of ACP. When the ACP activity was inhibited by PM, both its efficiency to catalyze the hydrolysis of ATP and the degree of fluorescence quenching of QDs were obviously weakened. We use the inhibition
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efficiency (IE%) to ACP as a signal for the detection of PM. IE% was expressed by the following equation [16,35]: Inhibition Efficiency(IE) =
Finhibitor − Fno inhibitor × 100% F0 − Fnoinhibitor
where Finhibitor and Fnoinhibitor are the fluorescence intensity of QDs/ATP/ACP system in the presence and absence of PM, respectively. F0 refers to the fluorescence intensity of the proposed system in the absence of ACP and PM. As shown in Fig. 5A, the gradually increased fluorescence intensity was observed with the increasing concentrations of PM in the range of 0.001–10 g mL−1 . The inset illustrated the relationship between the fluorescence intensity and PM concentrations. The plot of IE versus the logarithm of the PM concentration (from 0.001 to 5.0 g mL−1 ) was observed in Fig. 5B. The regression equation is: IE (%) = 54.90 + 15.68 Log [PM], g mL−1 . The detection limit for PM is 0.5 ng mL−1 , which was much lower than the value (20 ng mg−1 ) of the maximum residue limit of PM set by the Chinese National food safety standard (GB-27632014). Moreover, this detection limit is comparable to or even lower than those of previously reported methods (detailed comparison is shown in Supplementary Table S2). Thus, the proposed sensing platform can satisfy most requirements for real-life uses. These results significantly demonstrated the established sensing probes can be used not only for ACP activity determination but also for its inhibitor detection and screening. 4. Conclusion In summary, a label-free fluorescence sensing platform was developed for sensitive detection of acid phosphatase (ACP) and its inhibitor using cysteamine-capped CdTe quantum dots. The facile, sensitive, and selective sensing system was based on ATPtriggered fluorescence enhancement and ACP-caused fluorescence quenching. Compared with previous fluorescence quenching strategy for ACP assay developed by our group, ATP-triggered emission enhancement system was employed as promising platform with advantages in terms of low background interference and superior resistance to photobleaching. The practical application is demonstrated by the assay of the human serum samples and obtains satisfactory results. More importantly, the system also provides a prospective platform for highly sensitive and selective detection PM based on enzyme inhibition. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grants 21075050, 21275063), the Science and Technology Development Project of Jilin Province, China (Grant 20110334), and the Graduate Innovation Fund of Jilin University (Grant 2015022). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.05.024. References [1] H. Bull, P.G. Murray, D. Thomas, A.M. Fraser, P.N. Nelson, Acid phosphatases, Mol. Pathol. 55 (2002) 65–72. [2] Z.P. Liu, Z.H. Lin, L.L. Liu, X.G. Su, A convenient and label-free fluorescence “turn off–on” nanosensor with high sensitivity and selectivity for acid phosphatase, Anal. Chim. Acta 876 (2015) 83–90. [3] E.S. Leman, R.H. Getzenberg, Biomarkers for prostate cancer, J. Cell. Biochem. 108 (2009) 3–9. [4] J. Sun, F. Yang, X.R. Yang, Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing, Nanoscale 7 (2015) 16372–16380.
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Biographies Jing Wang is admitted to Jilin University to study for a bachelor’s degree at the College of Chemistry in 2012. Her major research areas focus on the synthesis and functionalization of quantum dots and their application on sensors for proteins, pesticide and so on. Yu Yan is admitted to Jilin University to study for a bachelor’s degree at the College of Chemistry in 2012. His major research areas focus on the synthesis and functionalization of quantum dots and their application on sensors for proteins, pesticide and so on.
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Xu Yan is currently carrying out his graduate work for her Doctor’s degree under the guidance of Prof. Xingguang Su in Jilin University. His major research areas focus on the synthesis and functionalization of quantum dots and their application on sensors for proteins, pesticide and so on. Xiaojian Tang is a lecturer at Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu University. Her research focuses on the environmental pollutant degradation and detection. Xingguang Su is a professor at the Department of Analytical Chemistry at the College of Chemistry, Jilin University. She received her MS degree from Jilin University (China) in 1992 and her PhD degree from Jilin University (China) in 1999. Her research focuses on the synthesis, characterization, functionalization and application of quantum dots and quantum dots-tagged microspheres in biomedicine.