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Enzyme-triggered inner filter effect on the fluorescence of gold nanoclusters for ratiometric detection of mercury(II) ions via a dual-signal responsive logic
Sensors and Actuators A 302 (2020) 111794
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Sensors and Actuators A: Physical journal homepage: www.elsevier.com/locate/sna
Enzyme-triggered inner filter effect on the fluorescence of gold nanoclusters for ratiometric detection of mercury(II) ions via a dual-signal responsive logic Wenjia Li, Dong Liu ∗ , Xiaoya Bi, Tianyan You ∗ Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education, School of Agricultural Equipment Engineering, Jiangsu University, Zhenjiang 212013, China
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Article history: Received 7 October 2019 Received in revised form 4 December 2019 Accepted 9 December 2019 Available online 23 December 2019 Keywords: Ratiometric fluorescence Gold nanocluster Enzyme inhibition Inner filter effect Mercury(II) ions analysis
a b s t r a c t Herein a novel ratiometric fluorescence strategy based on enzyme-triggered inner filter effect (IFE) was described to sensitively detect mercury ions (Hg2+ ) for the first time by using l-proline-protected gold nanoclusters (AuNCs) and 2,3-diaminophenazine (DAP) as IFE fluorophore and absorber. The IFE was derived from the overlap between the emission band of AuNCs and absorption band of DAP and confirmed by fluorescence lifetime decay tests. Based on IFE principle and ratiometric strategy, laccase (LACC)catalyzed o-phenylenediamine (OPD) oxidation was utilized to produce DAP, whereas the activity of LACC can be monitored by Hg2+ . In this way, the existence of Hg2+ could depress the emission of DAP while restore that of AuNCs, achieving the dual signal response of Hg2+ , and their emission intensity ratio was dependent on the concentration of Hg2+ . Under the optimized detection conditions, the linear range for Hg2+ determination was from 0.8 to 35 M with a detection limit of 0.27 M. Besides, it was successfully applied to the analysis of tap water and Yangtze river water. Our strategy can be used to assess the activity of LACC, and it also provided a novel way to construct other enzyme-based biosensors. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Mercury (Hg) has been classified as the most harmful heavy metal for its toxicity, and accumulated Hg2+ in human could seriously damage the central nervous system [1]. The sensitive analysis of Hg2+ has become essential to avoid Hg2+ poisoning and guarantee human healthy. Therebefore, many analytical techniques for Hg2+ have been developed, e.g. atomic absorption spectrometry [2], electrochemical [3], inductively coupled plasma-mass spectrometry [4] and fluorometry sensors [5]. Among these methods, fluorometry has the advantages of high sensitivity, rapid responsiveness and easy to use, allowing it highly useful for on-site testing [6]. Multiple fluorescent nanomaterials have been applied for Hg2+ detection, such as quantum dot (QDs), carbon dot (CDs) and noble metal clusters [7–9]. Recently, the fluorescent gold nanoclusters (AuNCs) have attracted special interest due to their molecule-like properties, high stability and facile synthesis. In terms of monitoring Hg2+ , its metallophilic interaction with Au+ could alter the electronic structure of AuNCs and induce the fluorescence quenching [10].
∗ Corresponding authors. E-mail addresses: [email protected] (D. Liu), [email protected] (T. You). https://doi.org/10.1016/j.sna.2019.111794 0924-4247/© 2019 Elsevier B.V. All rights reserved.
Shang et al. proposed a novel fluorescence assay for Hg2+ based on dihydrolipoic acid-capped AuNCs with a detection limit down to 0.5 nM, showing the excellent availability of AuNCs for analysis [11]. Accordingly, various AuNCs with different capping ligands have been explored for the precise examination of Hg2+ . Nevertheless, available AuNCs-based sensing systems mainly depend on the fluorescence quenching directly triggered by the target analyte, and such a single-signal responsive logic has some deficiencies in the further enhancement of analysis reliability and accuracy [12]. In this regard, particular interest has been devoted to ratiometric fluorescence assay in recent years for the attracting analytical properties relative to that of single emission fluorimetry [13,14]. Ratiometric fluorescence sensors collect the changes of two separated wavelengths and eliminate environmental interferences via self-calibration to improve the sensitivity and accuracy [15]. For fluorescent AuNCs, common strategies to achieve ratiometric sensing include synthesizing dual-emitting AuNCs and hybridizing AuNCs with other specie (QDs, CDs). Yang et al. synthesized dual-emitting AuNCs by using glutathione as reductant, and the two emissions served as reporter and reference signal for ratiometric sensing of Hg2+ [16]. Unfortunately, relatively few types of AuNCs satisfied both the dual emission and high feasibility for Hg2+ detection. On the contrary, AuNCs-based hybrids are extremely attracting
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Scheme 1. Schematic illustration of (A) the LACC catalyzed oxidation of OPD and (B) AuNCs/LACC/OPD sensing system based on IFE for Hg2+ determination.
in the construction of ratiometric sensors owing to the simple fabrication and easy regulation. Yan et al. proposed an ultrasensitive ratiometric fluorescence assay for Hg2+ analysis based on nanohybrid of CDs and AuNCs with a detection limit of 28 nM [17]. Currently, most of these ratiometric fluorescence assays are based on Hg2+ -dependent single signal logic by using the other emission as reference signal, yet such detection mode generally suffers from analyte-independent interferences [18]. Despite dual-signal detection mode can conquer the above issue, achieving dual signal responses to Hg2+ remains a challenge in sensing system based on fluorescent nanomaterials. Herein, we designed a novel AuNCs-based ratiometric fluorescent assay for the detection of Hg2+ via dual-signal response logic. Inspired by inner-filter effect (IFE), a sensing system consisting of l-proline protected AuNCs and 2,3-diaminophenazine (DAP) was proposed. Although there have been many reports on the detection of targets based on enzymatic reactions [19,20], but building a ratio sensor based on laccase inhibition to detect mercury has not been reported. To achieve the dual-signal response, laccase (LACC)catalyzed oxidation of o-phenylenediamine (OPD) was applied to generate DAP, whereas Hg2+ could efficiently inhibit the activity of LACC and in turn depress the IFE-induced fluorescence quenching of AuNCs. In this way, the AuNCs/OPD/LACC system could be utilized as a novel enzyme-triggered ratiometric fluorescence sensor for Hg2+ analysis. Based on this principle, the concept-of-proof experiment was performed as illustrated in Scheme 1. Briefly, the addition of OPD presented no influence on AuNCs. After the addition of LACC, two separated emissions could be observed from AuNCs and DAP. However, the presence of Hg2+ inhibited the activity of LACC and further depressed IFE; as a result, the emission
intensity of AuNCs restored while that of DAP showed a decrease. By recording relative changes of the two emissions, their intensity ratio was used as the yardstick for Hg2+ concentration. Consequently, our proposed assay for the determination of Hg2+ showed a linear range from 0.8 to 35 M with a detection limit of 0.27 M, and its feasibility in practical analysis was also confirmed.
2. Experimental 2.1. Materials and reagents Tetrachloroauric (III) acid trihydrate (HAuCl4 ·3H2 O, 99.9 %, ACROS Organics), l-proline (≥99 %, Sigma-Aldrich), laccase from Trametes versicolor (LACC, ≥0.5 U/mg, Sigma-Aldrich), ophenylenediamine (OPD, 99.5 %, Aladdin), 2,3-diaminophenazine (DAP, ≥90 %, Aladdin), and mercury (II) nitrate monohydrate (Hg(NO3 )2 ·H2 O, AR, SCRC) were utilized for the experiment. Both of Yangtze river water and tape water sample were collected locally in Zhenjiang. Millipore-Q water was used to prepare solutions, and all the reagents were used as received. 2.2. Apparatus Morphological characterization was performed using a transmission electron microscope (FEI TECNAI F20, USA). Fluorescence spectra were obtained with a spectrophotometer (Edinburgh FS5, UK). UV–vis absorption spectra were measured on a Shimadzu UV-2450 spectrophotometer (Japan). Malvern Nano-ZS Zeta Sizer (Malvern, UK) was employed to measure the Zeta potentials.
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Fig. 1. (A) UV/vis absorbance spectra of AuNCs, HAuCl4 and l-proline. (B) Fluorescence spectra, (C) TEM image and (D) fluorescence stability of AuNCs. Excitation wavelength: 340 nm. Inset of B: photograph of AuNCs under ambient and UV light (365 nm); inset of C: size distribution of Au NCs.
2.3. Preparation of AuNCs The fluorescent AuNCs were synthesized via a “heated method” [21]. Briefly, 8.0 mL of 10 M l-proline solution was rapidly dropped into a boiling solution of HAuCl4 (32.0 mL, 3.0 mM). The mixture was stirred for an additional 10 min and then it was removed from heating and cooled down to room temperature. Then the centrifugation (10,000 rpm, 15 min) was followed and the supernatant containing fluorescent AuNCs was collected. Finally, the product was stored at 4 ◦ C before use. The concentration of AuNCs was calculated to be 0.34 mM based on the molar concentration of HAuCl4 . 2.4. Inner filter effect of DAP on the fluorescent AuNCs To investigate the interaction between DAP and AuNCs, 450 L of their mixture consisting of 50 L AuNCs stock solution and DAP with different concentrations (0, 2, 5, 8, 10, 15, 20, 25, 30, 35, 40 and 50 M) were prepared with PBS solution (10 mM, pH 7.0). 2.5. Ratiometric fluorescent sensing of Hg2+ ions To estimate the analytical properties of proposed system toward Hg2+ , the mixture containing LACC (18 L, 2.25 U/mL) and Hg2+ with certain concentration was incubated at 37 ◦ C for 60 min. Subsequently, 15 L of OPD (2.4 mM) was added to react for another 70 min. After the addition of AuNCs (50 L), the solution was diluted to 450 L with PBS (10 mM, pH 7.0). All the fluorescence spectra in this work were tested with an excitation wavelength of 340 nm. 2.6. Detection of Hg2+ ions in water samples Tap water and Yangtze River water samples were obtained locally (Zhenjiang, China). Before testing, the samples were care-
fully filtered with a 0.45 m membrane, and then spiked with the standard Hg2+ stock solution.
3. Results and discussion 3.1. Characterization of AuNCs The UV/Vis spectra of AuNCs exhibits significant absorption in the range of 247–415 nm, whereas no absorption is observed for l-proline, indicating the presence of gold nanoparticle with dimension below 2 nm (Fig. 1A) [22]. AuNCs has strong fluorescence emission intensity at 340 nm excitation wavelength, but HAuCl4 and l-proline have no fluorescence emission (Fig. S1A). The results suggested that the AuNCs is successfully synthesized. Fig. S1B shows that the emission wavelength of AuNCs changes at different excitation wavelengths. The excitation and emission fluorescence maximum of AuNCs locates at 340 and 419 nm, respectively (Fig. 1B). The AuNCs solution produces the light blue fluorescence under 365 nm UV irradiation (inset of Fig. 1B). As evidenced by TEM images, AuNCs shows the uniform and well-dispersed morphology with narrow size distribution, and the average diameter is 1.3 nm (Fig. 1C). ESI mass spectrometry was applied to characterize the defined chemical composition of the generated Au NCs. Fig. S2A is the enlarged mass spectrum in the range of m/z = 800–1000. Fig. S2B and C show the peaks at m/z = 817.4 and 875.0 were assigned to [Au7 P2 +Na+H]2+ and [Au7 P3 +Na+H]2+ , respectively. The results suggested that the synthesized clusters consisted of seven gold atoms. Its room-temperature fluorescence QY was estimated to be 1.4 %. After storage for 30 days, the fluorescence intensity of AuNCs displays no obvious change, revealing the satisfactory long-term stability (Fig. 1D).
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Fig. 2. (A) Absorbance spectra of DAP and emission spectrum of AuNCs. (B) Emission spectra of AuNCs with various amounts of DAP (0, 2, 5, 8, 10, 15, 20, 25, 30, 35, 40, 50 M). (C) The fluorescence lifetimes and (D) absorbance spectra of AuNCs with DAP and without DAP.
3.2. Inner filter effect of DAP on the fluorescent AuNCs Typically, the absorption of absorber overlaps with the emission of fluorophore can induce the IFE [23]. Based on this principle, we designed a ratiometric fluorescent assay based on the enzymetriggered IFE for the detection of Hg2+ . As shown in Fig. 2A, the absorbance spectrum of DAP significantly overlaps with the emission of AuNCs, allowing the efficient IFE between AuNCs and DAP. Along with higher concentration of DAP, the emission intensity of AuNCs at 419 nm (I419 ) decreases while that at 576 nm (I576 ) assigned to DAP gradually increases (Fig. 2B). Moreover, Fig. S3 reveals that the intensity ratio of I576 /I419 increases linearly with the concentration of DAP raising. The linear function is I576 /I419 = 0.0078 + 0.026 C(DAP) with a correlation coefficient of 0.98, and the detection limit is 0.67 M (S/N = 3). It further confirms that DAP can quench the fluorescence of AuNCs. The fluorescence lifetime decay curve of AuNCs before and after the addition of DAP is shown in Fig. 2C. The calculated average lifetime of AuNCs was 6.771 ns and 6.772 ns, respectively, suggesting no complex formed [24]. On the other hand, the absorption spectrum of AuNCs have no obvious change in the presence and absence of DAP (Fig. 2D), which can conclude the complex have not formed between AuNCs and DAP. The zeta potential measurements revealed that DAP could not be adsorbed by AuNCs via electrostatic interaction because both of them are negatively charged (Fig. S4). Thus, the Förster resonance energy transfer between AuNCs and DAP could hardly occur, and it was IFE inducing the fluorescence quenching of AuNCs. 3.3. Principle of the Hg2+ determination based on IFE Based on the above observation, a ratiometric fluorescence strategy to detect Hg2+ was established, and the principle was schematically represented in Fig. 3A. Briefly, AuNCs and DAP generated from OPD oxidation could provide two fluorescence emissions
at 419 nm and 576 nm, respectively. However, the addition of Hg2+ inhibits the activity of LACC and leads the relative changes of their emissions. Its feasibility was evaluated in detail. The fluorescent intensity of AuNCs shows no obvious change after incubated with OPD, LACC, Hg2+ , OPD + Hg2+ or LACC + Hg2+ (Fig. 3B). After the addition of OPD and LACC, LACC oxidizes OPD to produce DAP, so the emission of AuNCs is strongly quenched along with a new emission assigned to DAP observed at 576 nm. When Hg2+ ions were added into the reaction system, the activity of LACC was inhibited and the amount of DAP was decreased, thus the fluorescence of AuNCs was recovered. Therefore, the proposed ratiometric fluorescence system can be used for Hg2+ monitoring. 3.4. Optimization of detection conditions In order to achieve satisfactory analysis properties toward Hg2+ , the optimization of critical experimental conditions was carried out via variable-controlling approach. The influences of following issues on the analytical performance were assessed in detail, including OPD concentration, LACC concentration, incubation temperature, pH value of solution, incubation time of OPD with LACC, incubation time of AuNCs with mixture. Fig. S5A shows that the fluorescence intensity ratio of I576 /I419 raised substantially with concentration of OPD (COPD ) increasing. To avoid excessive difference between I576 and I419 , COPD was selected to be 80 M with the I576 /I419 value of 1.93. Meanwhile, increasing the concentration of LACC (CLACC ) can also enlarge I576 /I419 (Fig. S5B), and 90 mU mL−1 of CLACC was chosen to regulate the value of I576 /I419 around 1.90. As shown in Fig. S5C, the reaction between OPD and LACC reaches equilibrium at 37 ◦ with the maximum reaction rate. 37 ◦ was chosen as the optimal incubation temperature. Fig. S5D shows that the fluorescence intensity ratios of I576 /I419 firstly increased with the increasing pH values and then decreased gradually with the largest ratios at the pH 7.0. LACC activity was inhibited under acidic or alkaline conditions. The stronger the acid-
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Fig. 3. (A) Principle of the Hg2+ determination; (B) Fluorescence emission spectra of AuNCs (1), AuNCs + OPD (2), AuNCs + LACC (3), AuNCs + Hg2+ (4), AuNCs + OPD + Hg2+ (5), AuNCs + LACC + Hg2+ (6), OPD + LACC (7), AuNCs + OPD + LACC (8), AuNCs + OPD + LACC + Hg2+ (9) ; [OPD] = 80 M, [LACC] = 90 mU mL−1 , [Hg2+ ] = 20 M, pH = 7.0.
Fig. 4. (A) Fluorescence spectra of AuNCs/OPD/LACC system in the presence of Hg2+ with different concentrations (from bottom to top: 0, 0.8, 1, 1.5, 2, 3.5, 5, 6.5, 8, 10, 15, 20, 35 M); (B) linear relationship between I576 /I419 and the logarithm of Hg2+ concentration.
ity or alkalinity, the stronger the inhibition. Therefore, the buffer solution with pH value of 7.0 was selected. The value of I576 /I419 reached a maximum at 70 min for the incubation of OPD and LACC, and 70 min was the optimal time for the incubation (Fig. S5E). As shown in Fig. S5F, the value of I576 /I419 remains unchanged in 10 min for the incubation of AuNCs and generated DAP. As a result, 3 min was chosen for the following experiments. 3.5. Analytical performance of IFE based fluorescent assay for Hg2+ ions sensing Under optimal conditions, Hg2+ ions were detected with the as-developed AuNCs-DAP system. The fluorescent spectra of AuNCs/OPD/LACC system upon the addition of Hg2+ with different concentrations were shown in Fig. 4A. The value of I419 increased while that of I576 decreased continuously with the growth of CHg2+ . A linear response between I576 /I419 and the logarithmic CHg2+ was obtained in the range of 0.8–35 M, and the linear regression equation was I576 /I419 = −2.9671 − 0.6981 Log CHg2+ (R2 = 0.9973) (Fig. 4B). The detection limit for Hg2+ was 0.27 M (S/N = 3). Table S1 lists some recent reports focused on the detection of Hg2+ . Compared with these sensors, the proposed assay is not among the best sensing ones in terms of linear range and detection limit, which was ascribed to the limited inhibition ability of Hg2+ toward LACC [25]. Nevertheless, such an assay can be employed to monitor the CHg2+ in sewage discharge; moreover, it also provides a new way to assess the enzyme activity of LACC with high accuracy and convenience [26,27].
Table 1 Analytical results for Hg2+ ions in water samples (n = 3). Sample
Tap water
Yangtze river water
Added (mol/L)
Found (mol/L)
Recovery (%, n = 3)
RSD (%, n = 3)
0 1.00 5.00 10.00 0 1.00 5.00 10.00
– 0.98 5.59 10.80 – 0.98 5.07 9.8
– 98 112 108 – 98 101 98
– 1.97 2.08 2.42 – 4.01 4.07 3.15
Subsequently, the selectivity was examined by measuring the affection of Hg2+ and foreign metal ions (K+ , Ca2+ , Fe2+ , Fe3+ , Pb2+ , Cu2+ , Ni2+ , Mn2+ , Cd2+ ) with the same concentration of 5 M on the ratiometric signal (I576 /I419 ). As shown in Fig. S6, the addition of Hg2+ induces the largest change of I576 /I419 , whereas other metal ions except Cu2+ and Cd2+ shows negligible effect on Hg2+ detection. The slight change of the ratiometric signal induced by Cu2+ and Cd2+ were due to their inhibition toward LACC. 3.6. Detection of Hg2+ ions in water samples The application feasibility of the IFE-based ratiometric fluorescence strategy was evaluated by standard addition method to determine Hg2+ in tap water and Yangtze River water. As shown in Table 1, the resultant recoveries in tap water samples ranges from
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98 % to 112 % with the RSD below 2.42 %, while that values for Yangtze river water samples ranges from 98 % to 101 % with the RSD less than 4.07 %, revealing the satisfactory reliability and accuracy. The proposed method possessed the potential in the practical applications to determine Hg2+ . 4. Conclusions A novel AuNCs/OPD/LACC-based ratiometric fluorescent assay was proposed for sensitive detection of Hg2+ . In such a sensing system, dual-signal response mode, namely the simultaneous fluorescent switch-off of DAP and switch-on of AuNCs, was evidently achieved by taking advantage of self-designed enzyme-triggered IFE. Consequently, the developed assay displayed a linear range from 0.8 to 35 M with a detection limit down to 0.27 M for Hg2+ detection. Satisfactory recoveries were obtained in practical analysis of tap water and Yangtze river water samples, demonstrating its feasibility and reliability for analytical applications. Despite its insufficient sensitivity relative to other state-of-the-art fluorescent sensors, our proposed assay provided a new enzyme-triggered IFE strategy to settle the dual-signal detection mode of ratiometric fluorescent sensors. We believed that such a strategy and sensing assay described here could inspire more investigations and boost the development of fluorescent sensors for high-accuracy monitoring of Hg2+ .
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
CRediT authorship contribution statement [17]
Wenjia Li: Investigation, Data curation, Writing - original draft. Dong Liu: Methodology, Conceptualization, Formal analysis, Writing - review & editing. Xiaoya Bi: Resources, Software. Tianyan You: Supervision, Conceptualization, Writing - review & editing.
[18]
[19]
Acknowledgments [20]
We would like to thank the supports from the National Natural Science Foundation of China (No. 21675065, 61901193), the Innovation/Entrepreneurship Program of Jiangsu Province, Natural Science Foundation of Jiangsu Province (No. BK20160490), and Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Appendix A. Supplementary data
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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.sna.2019. 111794.
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Biographies Wenjia Li is currently studying for doctor degree under the supervision of Prof. Tianyan You in school of Agricultural Equipment Engineering, Jiangsu University, China. Her research interest is the fabrication of biosensors for agriculture analysis. Dong Liu obtained his Ph.D. degree from Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences (CAS) in 2013. Currently, he is an associate professor at School of Agricultural Equipment Engineering, Jiangsu University, China.
W. Li, D. Liu, X. Bi et al. / Sensors and Actuators A 302 (2020) 111794 His current research interests are focused on the synthesis of functional nanomaterials and their applications in analysis and catalysis. Xiaoya Bi is studying for M.S. degree under the supervision of Prof. Tianyan You in school of Agricultural Equipment Engineering, Jiangsu University, China. Her research interest is agricultural biosensing.
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Prof. Tianyan You, received her Ph.D. degree in analytical chemistry from CIAC, CAS in 1999. Since Dec. 2003, she has been working in CIAC as a professor. Now, she is working in School of Agricultural Equipment Engineering, Jiangsu University as a professor. Her research interests are the functional nanomaterials and their applications in biosensing.
Contents lists available at ScienceDirect
Sensors and Actuators A: Physical journal homepage: www.elsevier.com/locate/sna
Enzyme-triggered inner filter effect on the fluorescence of gold nanoclusters for ratiometric detection of mercury(II) ions via a dual-signal responsive logic Wenjia Li, Dong Liu ∗ , Xiaoya Bi, Tianyan You ∗ Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education, School of Agricultural Equipment Engineering, Jiangsu University, Zhenjiang 212013, China
a r t i c l e
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Article history: Received 7 October 2019 Received in revised form 4 December 2019 Accepted 9 December 2019 Available online 23 December 2019 Keywords: Ratiometric fluorescence Gold nanocluster Enzyme inhibition Inner filter effect Mercury(II) ions analysis
a b s t r a c t Herein a novel ratiometric fluorescence strategy based on enzyme-triggered inner filter effect (IFE) was described to sensitively detect mercury ions (Hg2+ ) for the first time by using l-proline-protected gold nanoclusters (AuNCs) and 2,3-diaminophenazine (DAP) as IFE fluorophore and absorber. The IFE was derived from the overlap between the emission band of AuNCs and absorption band of DAP and confirmed by fluorescence lifetime decay tests. Based on IFE principle and ratiometric strategy, laccase (LACC)catalyzed o-phenylenediamine (OPD) oxidation was utilized to produce DAP, whereas the activity of LACC can be monitored by Hg2+ . In this way, the existence of Hg2+ could depress the emission of DAP while restore that of AuNCs, achieving the dual signal response of Hg2+ , and their emission intensity ratio was dependent on the concentration of Hg2+ . Under the optimized detection conditions, the linear range for Hg2+ determination was from 0.8 to 35 M with a detection limit of 0.27 M. Besides, it was successfully applied to the analysis of tap water and Yangtze river water. Our strategy can be used to assess the activity of LACC, and it also provided a novel way to construct other enzyme-based biosensors. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Mercury (Hg) has been classified as the most harmful heavy metal for its toxicity, and accumulated Hg2+ in human could seriously damage the central nervous system [1]. The sensitive analysis of Hg2+ has become essential to avoid Hg2+ poisoning and guarantee human healthy. Therebefore, many analytical techniques for Hg2+ have been developed, e.g. atomic absorption spectrometry [2], electrochemical [3], inductively coupled plasma-mass spectrometry [4] and fluorometry sensors [5]. Among these methods, fluorometry has the advantages of high sensitivity, rapid responsiveness and easy to use, allowing it highly useful for on-site testing [6]. Multiple fluorescent nanomaterials have been applied for Hg2+ detection, such as quantum dot (QDs), carbon dot (CDs) and noble metal clusters [7–9]. Recently, the fluorescent gold nanoclusters (AuNCs) have attracted special interest due to their molecule-like properties, high stability and facile synthesis. In terms of monitoring Hg2+ , its metallophilic interaction with Au+ could alter the electronic structure of AuNCs and induce the fluorescence quenching [10].
∗ Corresponding authors. E-mail addresses: [email protected] (D. Liu), [email protected] (T. You). https://doi.org/10.1016/j.sna.2019.111794 0924-4247/© 2019 Elsevier B.V. All rights reserved.
Shang et al. proposed a novel fluorescence assay for Hg2+ based on dihydrolipoic acid-capped AuNCs with a detection limit down to 0.5 nM, showing the excellent availability of AuNCs for analysis [11]. Accordingly, various AuNCs with different capping ligands have been explored for the precise examination of Hg2+ . Nevertheless, available AuNCs-based sensing systems mainly depend on the fluorescence quenching directly triggered by the target analyte, and such a single-signal responsive logic has some deficiencies in the further enhancement of analysis reliability and accuracy [12]. In this regard, particular interest has been devoted to ratiometric fluorescence assay in recent years for the attracting analytical properties relative to that of single emission fluorimetry [13,14]. Ratiometric fluorescence sensors collect the changes of two separated wavelengths and eliminate environmental interferences via self-calibration to improve the sensitivity and accuracy [15]. For fluorescent AuNCs, common strategies to achieve ratiometric sensing include synthesizing dual-emitting AuNCs and hybridizing AuNCs with other specie (QDs, CDs). Yang et al. synthesized dual-emitting AuNCs by using glutathione as reductant, and the two emissions served as reporter and reference signal for ratiometric sensing of Hg2+ [16]. Unfortunately, relatively few types of AuNCs satisfied both the dual emission and high feasibility for Hg2+ detection. On the contrary, AuNCs-based hybrids are extremely attracting
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Scheme 1. Schematic illustration of (A) the LACC catalyzed oxidation of OPD and (B) AuNCs/LACC/OPD sensing system based on IFE for Hg2+ determination.
in the construction of ratiometric sensors owing to the simple fabrication and easy regulation. Yan et al. proposed an ultrasensitive ratiometric fluorescence assay for Hg2+ analysis based on nanohybrid of CDs and AuNCs with a detection limit of 28 nM [17]. Currently, most of these ratiometric fluorescence assays are based on Hg2+ -dependent single signal logic by using the other emission as reference signal, yet such detection mode generally suffers from analyte-independent interferences [18]. Despite dual-signal detection mode can conquer the above issue, achieving dual signal responses to Hg2+ remains a challenge in sensing system based on fluorescent nanomaterials. Herein, we designed a novel AuNCs-based ratiometric fluorescent assay for the detection of Hg2+ via dual-signal response logic. Inspired by inner-filter effect (IFE), a sensing system consisting of l-proline protected AuNCs and 2,3-diaminophenazine (DAP) was proposed. Although there have been many reports on the detection of targets based on enzymatic reactions [19,20], but building a ratio sensor based on laccase inhibition to detect mercury has not been reported. To achieve the dual-signal response, laccase (LACC)catalyzed oxidation of o-phenylenediamine (OPD) was applied to generate DAP, whereas Hg2+ could efficiently inhibit the activity of LACC and in turn depress the IFE-induced fluorescence quenching of AuNCs. In this way, the AuNCs/OPD/LACC system could be utilized as a novel enzyme-triggered ratiometric fluorescence sensor for Hg2+ analysis. Based on this principle, the concept-of-proof experiment was performed as illustrated in Scheme 1. Briefly, the addition of OPD presented no influence on AuNCs. After the addition of LACC, two separated emissions could be observed from AuNCs and DAP. However, the presence of Hg2+ inhibited the activity of LACC and further depressed IFE; as a result, the emission
intensity of AuNCs restored while that of DAP showed a decrease. By recording relative changes of the two emissions, their intensity ratio was used as the yardstick for Hg2+ concentration. Consequently, our proposed assay for the determination of Hg2+ showed a linear range from 0.8 to 35 M with a detection limit of 0.27 M, and its feasibility in practical analysis was also confirmed.
2. Experimental 2.1. Materials and reagents Tetrachloroauric (III) acid trihydrate (HAuCl4 ·3H2 O, 99.9 %, ACROS Organics), l-proline (≥99 %, Sigma-Aldrich), laccase from Trametes versicolor (LACC, ≥0.5 U/mg, Sigma-Aldrich), ophenylenediamine (OPD, 99.5 %, Aladdin), 2,3-diaminophenazine (DAP, ≥90 %, Aladdin), and mercury (II) nitrate monohydrate (Hg(NO3 )2 ·H2 O, AR, SCRC) were utilized for the experiment. Both of Yangtze river water and tape water sample were collected locally in Zhenjiang. Millipore-Q water was used to prepare solutions, and all the reagents were used as received. 2.2. Apparatus Morphological characterization was performed using a transmission electron microscope (FEI TECNAI F20, USA). Fluorescence spectra were obtained with a spectrophotometer (Edinburgh FS5, UK). UV–vis absorption spectra were measured on a Shimadzu UV-2450 spectrophotometer (Japan). Malvern Nano-ZS Zeta Sizer (Malvern, UK) was employed to measure the Zeta potentials.
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Fig. 1. (A) UV/vis absorbance spectra of AuNCs, HAuCl4 and l-proline. (B) Fluorescence spectra, (C) TEM image and (D) fluorescence stability of AuNCs. Excitation wavelength: 340 nm. Inset of B: photograph of AuNCs under ambient and UV light (365 nm); inset of C: size distribution of Au NCs.
2.3. Preparation of AuNCs The fluorescent AuNCs were synthesized via a “heated method” [21]. Briefly, 8.0 mL of 10 M l-proline solution was rapidly dropped into a boiling solution of HAuCl4 (32.0 mL, 3.0 mM). The mixture was stirred for an additional 10 min and then it was removed from heating and cooled down to room temperature. Then the centrifugation (10,000 rpm, 15 min) was followed and the supernatant containing fluorescent AuNCs was collected. Finally, the product was stored at 4 ◦ C before use. The concentration of AuNCs was calculated to be 0.34 mM based on the molar concentration of HAuCl4 . 2.4. Inner filter effect of DAP on the fluorescent AuNCs To investigate the interaction between DAP and AuNCs, 450 L of their mixture consisting of 50 L AuNCs stock solution and DAP with different concentrations (0, 2, 5, 8, 10, 15, 20, 25, 30, 35, 40 and 50 M) were prepared with PBS solution (10 mM, pH 7.0). 2.5. Ratiometric fluorescent sensing of Hg2+ ions To estimate the analytical properties of proposed system toward Hg2+ , the mixture containing LACC (18 L, 2.25 U/mL) and Hg2+ with certain concentration was incubated at 37 ◦ C for 60 min. Subsequently, 15 L of OPD (2.4 mM) was added to react for another 70 min. After the addition of AuNCs (50 L), the solution was diluted to 450 L with PBS (10 mM, pH 7.0). All the fluorescence spectra in this work were tested with an excitation wavelength of 340 nm. 2.6. Detection of Hg2+ ions in water samples Tap water and Yangtze River water samples were obtained locally (Zhenjiang, China). Before testing, the samples were care-
fully filtered with a 0.45 m membrane, and then spiked with the standard Hg2+ stock solution.
3. Results and discussion 3.1. Characterization of AuNCs The UV/Vis spectra of AuNCs exhibits significant absorption in the range of 247–415 nm, whereas no absorption is observed for l-proline, indicating the presence of gold nanoparticle with dimension below 2 nm (Fig. 1A) [22]. AuNCs has strong fluorescence emission intensity at 340 nm excitation wavelength, but HAuCl4 and l-proline have no fluorescence emission (Fig. S1A). The results suggested that the AuNCs is successfully synthesized. Fig. S1B shows that the emission wavelength of AuNCs changes at different excitation wavelengths. The excitation and emission fluorescence maximum of AuNCs locates at 340 and 419 nm, respectively (Fig. 1B). The AuNCs solution produces the light blue fluorescence under 365 nm UV irradiation (inset of Fig. 1B). As evidenced by TEM images, AuNCs shows the uniform and well-dispersed morphology with narrow size distribution, and the average diameter is 1.3 nm (Fig. 1C). ESI mass spectrometry was applied to characterize the defined chemical composition of the generated Au NCs. Fig. S2A is the enlarged mass spectrum in the range of m/z = 800–1000. Fig. S2B and C show the peaks at m/z = 817.4 and 875.0 were assigned to [Au7 P2 +Na+H]2+ and [Au7 P3 +Na+H]2+ , respectively. The results suggested that the synthesized clusters consisted of seven gold atoms. Its room-temperature fluorescence QY was estimated to be 1.4 %. After storage for 30 days, the fluorescence intensity of AuNCs displays no obvious change, revealing the satisfactory long-term stability (Fig. 1D).
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Fig. 2. (A) Absorbance spectra of DAP and emission spectrum of AuNCs. (B) Emission spectra of AuNCs with various amounts of DAP (0, 2, 5, 8, 10, 15, 20, 25, 30, 35, 40, 50 M). (C) The fluorescence lifetimes and (D) absorbance spectra of AuNCs with DAP and without DAP.
3.2. Inner filter effect of DAP on the fluorescent AuNCs Typically, the absorption of absorber overlaps with the emission of fluorophore can induce the IFE [23]. Based on this principle, we designed a ratiometric fluorescent assay based on the enzymetriggered IFE for the detection of Hg2+ . As shown in Fig. 2A, the absorbance spectrum of DAP significantly overlaps with the emission of AuNCs, allowing the efficient IFE between AuNCs and DAP. Along with higher concentration of DAP, the emission intensity of AuNCs at 419 nm (I419 ) decreases while that at 576 nm (I576 ) assigned to DAP gradually increases (Fig. 2B). Moreover, Fig. S3 reveals that the intensity ratio of I576 /I419 increases linearly with the concentration of DAP raising. The linear function is I576 /I419 = 0.0078 + 0.026 C(DAP) with a correlation coefficient of 0.98, and the detection limit is 0.67 M (S/N = 3). It further confirms that DAP can quench the fluorescence of AuNCs. The fluorescence lifetime decay curve of AuNCs before and after the addition of DAP is shown in Fig. 2C. The calculated average lifetime of AuNCs was 6.771 ns and 6.772 ns, respectively, suggesting no complex formed [24]. On the other hand, the absorption spectrum of AuNCs have no obvious change in the presence and absence of DAP (Fig. 2D), which can conclude the complex have not formed between AuNCs and DAP. The zeta potential measurements revealed that DAP could not be adsorbed by AuNCs via electrostatic interaction because both of them are negatively charged (Fig. S4). Thus, the Förster resonance energy transfer between AuNCs and DAP could hardly occur, and it was IFE inducing the fluorescence quenching of AuNCs. 3.3. Principle of the Hg2+ determination based on IFE Based on the above observation, a ratiometric fluorescence strategy to detect Hg2+ was established, and the principle was schematically represented in Fig. 3A. Briefly, AuNCs and DAP generated from OPD oxidation could provide two fluorescence emissions
at 419 nm and 576 nm, respectively. However, the addition of Hg2+ inhibits the activity of LACC and leads the relative changes of their emissions. Its feasibility was evaluated in detail. The fluorescent intensity of AuNCs shows no obvious change after incubated with OPD, LACC, Hg2+ , OPD + Hg2+ or LACC + Hg2+ (Fig. 3B). After the addition of OPD and LACC, LACC oxidizes OPD to produce DAP, so the emission of AuNCs is strongly quenched along with a new emission assigned to DAP observed at 576 nm. When Hg2+ ions were added into the reaction system, the activity of LACC was inhibited and the amount of DAP was decreased, thus the fluorescence of AuNCs was recovered. Therefore, the proposed ratiometric fluorescence system can be used for Hg2+ monitoring. 3.4. Optimization of detection conditions In order to achieve satisfactory analysis properties toward Hg2+ , the optimization of critical experimental conditions was carried out via variable-controlling approach. The influences of following issues on the analytical performance were assessed in detail, including OPD concentration, LACC concentration, incubation temperature, pH value of solution, incubation time of OPD with LACC, incubation time of AuNCs with mixture. Fig. S5A shows that the fluorescence intensity ratio of I576 /I419 raised substantially with concentration of OPD (COPD ) increasing. To avoid excessive difference between I576 and I419 , COPD was selected to be 80 M with the I576 /I419 value of 1.93. Meanwhile, increasing the concentration of LACC (CLACC ) can also enlarge I576 /I419 (Fig. S5B), and 90 mU mL−1 of CLACC was chosen to regulate the value of I576 /I419 around 1.90. As shown in Fig. S5C, the reaction between OPD and LACC reaches equilibrium at 37 ◦ with the maximum reaction rate. 37 ◦ was chosen as the optimal incubation temperature. Fig. S5D shows that the fluorescence intensity ratios of I576 /I419 firstly increased with the increasing pH values and then decreased gradually with the largest ratios at the pH 7.0. LACC activity was inhibited under acidic or alkaline conditions. The stronger the acid-
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Fig. 3. (A) Principle of the Hg2+ determination; (B) Fluorescence emission spectra of AuNCs (1), AuNCs + OPD (2), AuNCs + LACC (3), AuNCs + Hg2+ (4), AuNCs + OPD + Hg2+ (5), AuNCs + LACC + Hg2+ (6), OPD + LACC (7), AuNCs + OPD + LACC (8), AuNCs + OPD + LACC + Hg2+ (9) ; [OPD] = 80 M, [LACC] = 90 mU mL−1 , [Hg2+ ] = 20 M, pH = 7.0.
Fig. 4. (A) Fluorescence spectra of AuNCs/OPD/LACC system in the presence of Hg2+ with different concentrations (from bottom to top: 0, 0.8, 1, 1.5, 2, 3.5, 5, 6.5, 8, 10, 15, 20, 35 M); (B) linear relationship between I576 /I419 and the logarithm of Hg2+ concentration.
ity or alkalinity, the stronger the inhibition. Therefore, the buffer solution with pH value of 7.0 was selected. The value of I576 /I419 reached a maximum at 70 min for the incubation of OPD and LACC, and 70 min was the optimal time for the incubation (Fig. S5E). As shown in Fig. S5F, the value of I576 /I419 remains unchanged in 10 min for the incubation of AuNCs and generated DAP. As a result, 3 min was chosen for the following experiments. 3.5. Analytical performance of IFE based fluorescent assay for Hg2+ ions sensing Under optimal conditions, Hg2+ ions were detected with the as-developed AuNCs-DAP system. The fluorescent spectra of AuNCs/OPD/LACC system upon the addition of Hg2+ with different concentrations were shown in Fig. 4A. The value of I419 increased while that of I576 decreased continuously with the growth of CHg2+ . A linear response between I576 /I419 and the logarithmic CHg2+ was obtained in the range of 0.8–35 M, and the linear regression equation was I576 /I419 = −2.9671 − 0.6981 Log CHg2+ (R2 = 0.9973) (Fig. 4B). The detection limit for Hg2+ was 0.27 M (S/N = 3). Table S1 lists some recent reports focused on the detection of Hg2+ . Compared with these sensors, the proposed assay is not among the best sensing ones in terms of linear range and detection limit, which was ascribed to the limited inhibition ability of Hg2+ toward LACC [25]. Nevertheless, such an assay can be employed to monitor the CHg2+ in sewage discharge; moreover, it also provides a new way to assess the enzyme activity of LACC with high accuracy and convenience [26,27].
Table 1 Analytical results for Hg2+ ions in water samples (n = 3). Sample
Tap water
Yangtze river water
Added (mol/L)
Found (mol/L)
Recovery (%, n = 3)
RSD (%, n = 3)
0 1.00 5.00 10.00 0 1.00 5.00 10.00
– 0.98 5.59 10.80 – 0.98 5.07 9.8
– 98 112 108 – 98 101 98
– 1.97 2.08 2.42 – 4.01 4.07 3.15
Subsequently, the selectivity was examined by measuring the affection of Hg2+ and foreign metal ions (K+ , Ca2+ , Fe2+ , Fe3+ , Pb2+ , Cu2+ , Ni2+ , Mn2+ , Cd2+ ) with the same concentration of 5 M on the ratiometric signal (I576 /I419 ). As shown in Fig. S6, the addition of Hg2+ induces the largest change of I576 /I419 , whereas other metal ions except Cu2+ and Cd2+ shows negligible effect on Hg2+ detection. The slight change of the ratiometric signal induced by Cu2+ and Cd2+ were due to their inhibition toward LACC. 3.6. Detection of Hg2+ ions in water samples The application feasibility of the IFE-based ratiometric fluorescence strategy was evaluated by standard addition method to determine Hg2+ in tap water and Yangtze River water. As shown in Table 1, the resultant recoveries in tap water samples ranges from
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98 % to 112 % with the RSD below 2.42 %, while that values for Yangtze river water samples ranges from 98 % to 101 % with the RSD less than 4.07 %, revealing the satisfactory reliability and accuracy. The proposed method possessed the potential in the practical applications to determine Hg2+ . 4. Conclusions A novel AuNCs/OPD/LACC-based ratiometric fluorescent assay was proposed for sensitive detection of Hg2+ . In such a sensing system, dual-signal response mode, namely the simultaneous fluorescent switch-off of DAP and switch-on of AuNCs, was evidently achieved by taking advantage of self-designed enzyme-triggered IFE. Consequently, the developed assay displayed a linear range from 0.8 to 35 M with a detection limit down to 0.27 M for Hg2+ detection. Satisfactory recoveries were obtained in practical analysis of tap water and Yangtze river water samples, demonstrating its feasibility and reliability for analytical applications. Despite its insufficient sensitivity relative to other state-of-the-art fluorescent sensors, our proposed assay provided a new enzyme-triggered IFE strategy to settle the dual-signal detection mode of ratiometric fluorescent sensors. We believed that such a strategy and sensing assay described here could inspire more investigations and boost the development of fluorescent sensors for high-accuracy monitoring of Hg2+ .
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CRediT authorship contribution statement [17]
Wenjia Li: Investigation, Data curation, Writing - original draft. Dong Liu: Methodology, Conceptualization, Formal analysis, Writing - review & editing. Xiaoya Bi: Resources, Software. Tianyan You: Supervision, Conceptualization, Writing - review & editing.
[18]
[19]
Acknowledgments [20]
We would like to thank the supports from the National Natural Science Foundation of China (No. 21675065, 61901193), the Innovation/Entrepreneurship Program of Jiangsu Province, Natural Science Foundation of Jiangsu Province (No. BK20160490), and Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Appendix A. Supplementary data
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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.sna.2019. 111794.
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Biographies Wenjia Li is currently studying for doctor degree under the supervision of Prof. Tianyan You in school of Agricultural Equipment Engineering, Jiangsu University, China. Her research interest is the fabrication of biosensors for agriculture analysis. Dong Liu obtained his Ph.D. degree from Changchun Institute of Applied Chemistry (CIAC), Chinese Academy of Sciences (CAS) in 2013. Currently, he is an associate professor at School of Agricultural Equipment Engineering, Jiangsu University, China.
W. Li, D. Liu, X. Bi et al. / Sensors and Actuators A 302 (2020) 111794 His current research interests are focused on the synthesis of functional nanomaterials and their applications in analysis and catalysis. Xiaoya Bi is studying for M.S. degree under the supervision of Prof. Tianyan You in school of Agricultural Equipment Engineering, Jiangsu University, China. Her research interest is agricultural biosensing.
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Prof. Tianyan You, received her Ph.D. degree in analytical chemistry from CIAC, CAS in 1999. Since Dec. 2003, she has been working in CIAC as a professor. Now, she is working in School of Agricultural Equipment Engineering, Jiangsu University as a professor. Her research interests are the functional nanomaterials and their applications in biosensing.