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Label-free and ultrasensitive fluorescence assay for Fe3+ detection using DNA-Templated Ag nanoclusters
Colloids and Surfaces A 579 (2019) 123656
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Label-free and ultrasensitive fluorescence assay for Fe3+ detection using DNA-Templated Ag nanoclusters ⁎
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Zhiqing Zhang , Tingting Liu, Changle Yue, Shanshan Wang, Jie Ma, Ting Zhou , Fang Wang, Xiufeng Wang, Guodong Zhang Department of Chemistry, College of Science, China University of Petroleum (East China), Qingdao, 266580, PR China
G R A P H I C A L A B S T R A C T
A non-labeling and ultrasensitive fluorescent probe for the detection of Fe3+ was constructed using DNA-templated Ag nanoclusters.
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanocluster DNA-templated Label-free fluorescent sensor Fe3+
We report for the first time the selective and sensitive detection of Fe3+ by a label-free fluorescent sensor base on AgNCs@C30. AgNCs@C30 was synthesized, and the averaged size is about 3.0 nm, and the maximal excitation and emission wavelengths were 335 nm and 480 nm, respectively. Fe3+ caused a significantly fluorescence quenching of AgNCs@C30, and a novel and label-free nanosensor for detecting Fe3+ was developed with optimum detection of 303.15 K and 2 min. The linear range for Fe3+ detection was 0.1˜12 μM with the corresponding limit of detection (LOD) of 86 nM. Furthermore, the preliminary detection mechanism was discussed in detail, and the fluorescence quenching was deemed to result from AgNCs@C30 aggregation induced with a relatively weak mutual collision between AgNCs@C30 and Fe3+. This strategy detection of Fe3+ will provide a valuable reference for the detection of metal ions by using nanocluster.
1. Introduction Metal ions detection in aqueous media is important for life systems and environmental fields. Among all metal ions, Fe3+ is an important factor for physiology, which is the most significant transition metal ion in organism, such as oxygen transportation, enzyme catalysis and the
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promotion of electron transition in cytochromes [1]. The contents of Fe3+ should be maintained within normal range, and deficiency or excess of Fe3+ ion may have a bad effect on human health and immunity, such as anemia, renal failure, cancer and Alzheimer’s disease [2,3]. Therefore, the assay of Fe3+ level is crucial to early discovery and diagnosis of these disease. Moreover, Fe3+ ion concentration is also an
Corresponding authors. E-mail addresses: [email protected] (Z. Zhang), [email protected] (T. Zhou).
https://doi.org/10.1016/j.colsurfa.2019.123656 Received 30 May 2019; Received in revised form 1 July 2019; Accepted 6 July 2019 Available online 13 July 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.
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concentration in the range from 0.1 to 12 μM (R2 = 0.9881), and the detection limit of Fe3+ was 86 nM. Moreover, the detection mechanism was discussed in detail, which might be dynamic quenching.
important factor to evaluate water quality or environment [4]. Various detective methods are used to measure the content of Fe3+, including voltammetry, absorption spectrometry and inductively coupled plasma mass spectrometry (ICP-MS) [5–7]. However, these methods still faced a number of problems on detection of Fe3+, such as sophisticated instruments, complicated synthesis, time-consuming detection procedures and a high detection limit. The fluorescence detection method is widely used due to highly selectivity, sensitivity, rapid and real-time detection of Fe3+ [8–10]. Typically, the fluorescence assay of Fe3+ are performed based on organic dyes such as rhodamine [1]. However, the sensitive detection of Fe3+ is prevented because of these organic dyes with the intrinsic low solubility and poor photo-stability. Nanoclusters (NCs) have received much attention due to its advantages such as distinctive optical properties, easy preparation, good stability and high resistance [11–19], which can almost be used as an ideal substitute for nanodots [20,21]. Many studies have investigated the detection of metal ions based on nanoclusters using various ligands such as amino acid, peptide, protein and polymer [22–26]. Huang et al. used highly fluorescent glutathione (GSH)-stabilized cooper nanocluster for Fe3+ sensing [22]. Chen et al. prepared GSH capped silver nanoclusters by facile sonochemical method in aqueous solution, and designed a new AgNCs@GSH fluorescent probe for the detection of Fe3+ [23]. Zheng et al. synthesized L-tryptophan-stabilized dual-emission fluorescent gold nanoclusters (GNCs), and Fe3+ ions were detected by the fabricated GNCs [24]. George et al. reported the surfactant-free utilization of DMF (dimethylformamide)-protected platinum nanocluster as a fluorescent sensor for the selective detection of Fe3+ ions in aqueous medium [25]. Xiao et al. introduced a fluorescent sensing platform based on L-glutathione templated Ag nanoclusters, which has been established for independent detection of Hg2+, Cu2+ and Fe3+ ions in a solution [26]. Obviously, the choice of the stabilizing agents can significantly affect the fluorescence properties of NCs. DNA-templated Ag nanoclusters (AgNCs@DNA) have been the subject of significant interest due to their brightness, stability, biocompatibility and simple synthesis [27–30]. There have been some reports that, AgNCs@DNA have been successfully used as a novel fluorescence probe to detect various targets such as metal ions [31–34], thioproteins [35,36] and small molecules [37,38]. Li et al. have constructed a label-free fluorescent probe using ssDNA-templated silver nanoclusters for the detection of Hg2+ [31]. Xu et al. designed a labelfree hairpin DNA-scaffolded AgNCs for fluorescent detection of Hg2+ using exonuclease III-assisted target recycling amplification [32]. Lee et al. reported fluorescence switch for silver ion detection based on Cyt12/AgNCs [33]. Guo et al. proposed a fluorescence assay of Fe3+ based on pH dependent 12 polycytosine oligonucleotide templated AgNCs [34]. According to literature [39–42], the properties of AgNCs prominently depend on the oligonucleotides with optimized strand length and base sequences, which provides the opportunity to choose a versatile oligonucleotides template for preparing AgNCs for a specific analyses. Moreover, we have also confirmed that oligonucleotides In this work, inspired by the template-dependent properties of AgNCs@DNA and their special response to metal ions, we herein describe a simple, label-free and ultra-sensitive strategy for the detection of Fe3+ based on DNA oligonucleotides C30 template-AgNCs (AgNCs@C30). The synthesis process and the basic detection principle is schematically illustrated in Scheme 1. In principle, C30 were used as a template for silver atom deposition, and the synthesis of AgNCs was achieved at the presence of NaBH4. AgNCs@C30 emitted blue fluorescence under UV light. The maximal excitation and emission wavelengths were 335 nm and 480 nm, respectively. Various metal ions showed little effect on fluorescence quenching of AgNCs@C30, but the fluorescence quenching phenomenon was evident after adding Fe3+, indicating the feasibility of Fe3+ selectivity detection. Several factors including temperature and response time were optimized to get the ideal quenching efficiency. Under the optimum detection condition, the change of fluorescent intensity has a good linear relationship with Fe3+
2. Materials and methods 2.1. Materials All DNA oligonucleotides (C5, C10, C20 and C30) used in this study were purchased and purified by Takara Co., Ltd (China). Silver nitrate and sodium borohydride were purchased from Alfa Aesar. All chemicals and reagents were used without further purification. The ultrapure water was used throughout all the experiments. 2.2. Preparation of AgNCs@C30 20 μL 100 μM C30 solution was mixed with 28 μL 1 mM AgNO3 and a certain volume of ultrapure water, then incubated for 20 min at 4℃ in the dark. 12 μL 1 mM ice-cold NaBH4 solution was added to the above mixture to reduce Ag ions. Finally, the mixture was kept in the dark for 12 h at 25 °C, and the color change of solution from colourless to pale yellow indicates the formation of AgNCs@C30. The concentrations of AgNCs@C30 were measured by Optima 8000 ICP-MS (PerkinElmer, USA). AgNO3 standard solution of 0.02 mg/L, 0.2 mg/L, and 2 mg/L were prepared and calibrated, respectively. 75 μL AgNCs@C30 and 25 mL ultrapure water were added into 50 mL tubes, and then ICP-MS was performed. 2.3. Fluorimetric detections of Fe3+ by AgNCs@C30 Fluorescence measurements were recorded on F-2700 spectrofluorometer (Hitachi, Japan). 2 μL prepared AgNCs@C30, a certain amount of ultrapure water and various contents of Fe3+ solutions were respectively added to centrifuge tubes. After incubation for 2 min at 25 °C, the fluorescence emission spectra of mixtures were measured at an excitation wavelength of 335 nm. The excitation and emission slits were both set at 10.0 nm. Fluorescent photographs were taken with a commercial digital camera under the ultraviolet light. 2.4. UV–vis spectra UV-vis measurement were recorded on a Lambda 950 UV–vis spectrometer (PerkinElmer, USA). UV-vis absorption spectra of AgNCs@C30 and AgNCs@C30/Fe3+ were obtained with wavelengths of 200˜800 nm. 2.5. Dynamic light scattering (DLS) The sizes of AgNCs@C30, AgNCs@C30/Fe3+, and AgNCs@C30/ Fe /EDTA were determined by DLS using a Zetasizer Nano-ZS instrument (Malvern, UK). All particle size data were referred to scattering intensity distribution. 3+
2.6. Zeta potential Zeta potential measurements were performed by laser Doppler electrophoresis using a Zetasizer Nano ZS spectrometer (Malvern, U.K.) in a standard DTS1060 C zeta cell. 3. Results and discussion 3.1. Optimization synthesis and characterization of AgNCs@C30 Strongly fluorescent Ag nanoclusters can be achieved in the presence of poly-cytosine oligonucleotides [28,29,39,41]. We investigated the effect of cytosine numbers on the fluorescence intensity of Ag 2
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Scheme 1. Schematic illustration of the assay strategy for the detection of Fe3+.
Fig. 1. The effect of cytosine numbers (a) and silver nitrate concentration (b) on the fluorescence intensity of AgNCs@DNA.
nanoclusters, as shown in Fig. 1a. The fluorescence of AgNCs@DNA was dramatically enhanced with the cytosine numbers increasing. Therefore, C30 was selected as the template to synthesis of AgNCs@C30. To further optimize the synthesis of AgNCs@C30, we fixed the C30 concentration (20 μM) and varied the amount of AgNO3 added (Fig. 1b). With increasing the concentration of AgNO3, the fluorescence of AgNCs@C30 increased firstly. Then it can be clearly seen from Fig. 1b that the fluorescence intensity passed through a maximum value at the concentration of 280˜320 μM and decreased with further increasing the concentration of AgNO3. In the above work, C30:Ag+ optimum ratio 20μM was about 300μM = 1: 15, meaning that coordination complex formed between cytosine and Ag+, the optimal mix proportion of cytosine and 20μM × 30 Ag+ was 300μM = 2: 1. This is in accordance with the result in literature [29]. As AgNO3 concentration increased to 300 μM, the fluorescence reached a maximum value, and this is due to three possible reasons: (1) The optimum ratio of cytosine and Ag+ was 2:1 in the AgNCs@C30 complex. (2)Ag+ dominated the reaction when increasing the concentration of AgNO3. The binding sites of Ag+ at C30 were much more than free Ag+ numbers, so increased the AgNO3 concentration was equal to increase the concentration of adsorbed ions (Ag+) in C30, which caused the enhancement of fluorescent intensity of AgNCs. (3) When the amount of Ag+ exceeded the binding sites of Ag+ at C30, residual Ag+ was reduced to a larger scale of nanoparticles, and the fluorescence was decreased. UV-vis absorption spectrum, fluorescence spectra and DLS were used to systematically characterize AgNCs@C30, as shown in Fig. 2. The fluorescence excitation spectrum showed two peaks at 270 nm and 335 nm, and the identical emission spectral shape of AgNCs@C30 was achieved by either exciting wavelength (Fig. 2a). The absorption maximum of C30 was approximately 270 nm, and 270 nm excitation wavelength indicated that AgNCs were associated with C30. So the directly excitation wavelength of AgNCs should be 335 nm, this excitation yielded the 480 nm blue fluorescent emission, which was
consistent with the report in the literature [28]. The inset in Fig. 2a were the photographs of AgNCs@C30 under daylight (left) or ultraviolet lamp (right), and AgNCs@C30 emitted the strongly blue fluorescence under UV light excitation. DLS was performed to determine the diameter of AgNCs@C30, and the results were shown in Fig. 2b. The synthesized AgNCs@C30 with diameter of about 3.0 nm can emit fluorescence upon photo excitation in the UV-vis range, because that the size of nanoclusters approaches the Fermi wavelength of electrons. The concentration of AgNCs@C30 was 0.019 mg/L by ICP-MS measurement. 3.2. Selectivity detection for Fe3+ using AgNCs@C30 In order to investigate the selectivity detection of metal ions, the fluorescence quenching of AgNCs@C30 after adding various metal ions (K+, Zn2+, Pb2+, Mn2+, Mg2+, Cu2+, Cd2+, Ba2+, Al3+, Cr3+and Fe3+, and the concentrations of ions were 10 μM) were shown in Fig. 3a and b. Other ions showed little effect, but the fluorescence quenching phenomenon was evident after adding Fe3+, indicating the specific selectivity of Fe3+ detection. 3.3. Optimization of detection conditions To confirm the feasibility of proposed detection strategy, we monitored the fluorescence change of AgNCs@C30 in the presence of Fe3+. As shown in Fig. 4a, the fluorescence signal of AgNCs@C30 dramatically decreased when 15 μM Fe3+ was added to the sample. Considering that the response of AgNCs@C30 to Fe3+ may be disturbed by various factors, therefore the detection conditions were optimized in the following. The effect of response time of AgNCs@C30 to Fe3+ were investigated firstly. In the presence of Fe3+, the rapid fluorescence signal observably decreased in less than 2 min, and the fluorescence intensity was maintained a constant value for longer period of time, meaning the reaction entirely finished. Thus, the optimal incubation time was 3
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Fig. 2. UV-vis and fluorescence spectra (a) and dynamic light scattering (b) results of AgNCs@C30.
480 nm gradually decreased with increasing of Fe3+ concentration, and the fluorescence was completely quenched at 12 μM Fe3+. In order to establish the quantitative relationship between the concentrations of Fe3+ and the fluorescence of AgNCs@C30, the fluorescence intensities of AgNCs@C30 at 480 nm were calculated (Fig. 5b). A good linear relationship between the quenching fluorescence efficiency (F0-F)/F0 and Fe3+ concentration in the range of 0˜12 μM was achieved, and the linear equation was (F0-F)/F0 = 0.07483x+0.00678 (R2 = 0.9881). Under the optimal conditions, according to the 3σ/s the limit of detection (LOD) of Fe3+ was 86 nM, where σ corresponds to the standard deviation of the blank sample and s is the slope in the plot of the fluorescence versus the quencher concentration [31]. By comparing Fe3+ detection limit of previously literature reported (Table 1), our strategy showed the high sensitivity for Fe3+ detection. Moreover, a limit of detection of 86 nM for the determination of Fe3+ is much lower than the maximum level of Fe3+ permitted in drinking water by the U. S. Environmental Protection Agency (0.3 mg⋅L−1, approximately equivalent to 5.4 μM) [8,9].
selected for 2 min in the following experiments. Generally, temperature often decreases the fluorescence signal of AgNCs@DNA by affecting the number and enrichment way of silver atoms on DNA, and by the enhancement of nonradiative decay [9,13]. The influence of temperature on the fluorescence intensity was shown in Fig. 4b. Values of F0/F indicate the degree of quenching, whereas, F0 and F are the fluorescent intensity of AgNCs@C30 without and with Fe3+, respectively, the higher F0/F value the stronger fluorescence quenching. The F0/F increased as the temperature increasing from 283.15 K to 303.15 K (Fig. 4b), but the degree of quenching reduced with further increasing temperature. Within a certain range of temperatures, the quenching degree increased with the rising of temperature under the existence of Fe3+, there are two possible reasons: On the one hand, the viscosity of sample reduced with the increase of temperature, and molecular diffusion speed was intensified. The greater opportunity of collision between AgNCs@C30 and Fe3+ increased, leading to the fluorescence quenching. On the other hand, the increase of temperature will promote the structure change of C30 template, which tends to be unstable providing less protection for AgNCs, and fluorescence intensity reduced. One of the main reasons why the quenching degree decreased with further rising of temperature (above 305.15 K) may be the internal energy conversion of the molecules. The higher temperature will promote the energy conversion within the molecules, and the excitation energy is firstly converted to the ground-state vibration energy, and then the vibration energy is lost through the vibration relaxation. As a result, the fluorescence quenching degree will decrease with further increase of temperature. As mentioned above, Fe3+ caused the maximal fluorescence signal change of AgNCs@C30 at 303.15 K. Therefore, 303.15 K was chosen as detection temperature in the further studies.
3.5. Preliminary mechanism of fluorescence quenching The change of fluorescence signal of AgNCs@C30 in the presence of Fe3+ might be caused by excited state reaction, collisional interaction (dynamic), static quenching or both or even through monomolecular mechanisms where fluorophore itself or other absorbing species attenuates the fluorescence intensity [10,11,14,17,22,25,26,31,43–46]. The fluorescence quenching was analyzed using Stern-Volmer equation [47]. F0/F = 1+Ksv [Q] where F0 and F are the fluorescent intensity of AgNCs@C30 without and with Fe3+, respectively. Ksv is the Stern-Volmer constant and [Q] is the concentration of the quencher. The corresponding Stern-Volmer plot of F0/F versus [Fe3+] showed a linear behavior with a Ksv value of 1.71 × 105 M−1 at 305.15 K. From the UV-vis results of AgNCs@C30,
3.4. Sensitively detection for Fe3+ We investigated the fluorescence intensity of AgNCs@C30 in the presence of different concentrations of Fe3+ under the optimal conditions. As shown in Fig. 5a, the fluorescence signal of AgNCs@C30 at
Fig. 3. Analysis of the selective detection of Fe3+. 4
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Fig. 4. (a) The fluorescence intensity changes of AgNCs@C30 before and after adding 15 μM Fe3+. (b) The effect of temperature on fluorescence signals of AgNCs@C30.
Fig. 5. (a) The fluorescence spectra of AgNCs@C30 in the presence of different content of Fe3+ (0–20 μM). (b) The fluorescence intensity of AgNCs@C30 versus the concentrations of Fe3+. A good linear relationship between (F0-F)/F0 and Fe3+ concentration in the range of 0–12 μM.
the larger Ag nanoparticles, resulting the aggregate of AgNCs. The aggregation of AgNCs@C30 might be the directly and primary cause of the fluorescence quenching. Based on the above results, we further investigated Zeta potential of AgNCs@C30 and AgNCs@C30/Fe3+ for gaining insights into the interaction mechanisms. The zeta potential value of AgNCs@C30 was -32.5 mV, indicating the excellent stability. Zeta potential values gradually decreased from-32.5 mV to -20.6 mV, -18.3 mV, -15.9 mV and -4.85 mV, with increasing of Fe3+ concentrations from 5 μM to 20 μM. The reduced zeta potentials indicated that decreased stability of AgNCs@C30 by introduced Fe3+ gradually. Because of the electrostatic attraction in opposite charges, the positive charges of Fe3+ was anxious to combined with the negative charges of AgNCs@C30, and aggregates formed between Fe3+ and AgNCs@C30, resulting the fluorescence quenching. Generally, ground-state complex formation (static quenching) frequently result in dramatically perturbation of the absorption spectra, and the location of absorption peak is markedly drifted [31]. On the contrary, dynamic quenching have effect on the excited states of the fluorophores, and slightly changes occur in the UV-vis absorption spectra [47], and the results of Fig. 6a are
Table 1 Comparison of the linear range and limit of detect for Fe3+ using different methods. Methods
Linear range
Limit of Detection
Reference
AgNCs@C30 AgNCs@GSH AgNCs@GSH GNCs@Try CuNCs@GSH Au/Ag@lipoic acid CQDs PtNCs@DMF
0–12 μM 0.1–18 μM 0.5–20 μM 1–500 μM 1–100 μM 1–80 μM 0–300 μM 0–50 μM
86 nM 50 nM 0.12 μM 0.16 μM 0.3 μM 0.5 μM 13.68 μM 4 μM
our work [26] [23] [24] [22] [9] [8] [25]
AgNCs@C30/Fe3+ and Fe3+ (Fig. 6a), the absorption wavelength of Fe3+ was 304 nm, and the peak of AgNCs@C30 was 402 nm. After adding 20 μM Fe3+, the UV-vis absorption peak of AgNCs@C30 was red shifted (from 402 nm to 410 nm), whereas the absorption intensity reduced. This result indicated that AgNCs became unstable or formatted
Fig. 6. (a) UV-vis spectra of AgNCs@C30, AgNCs@C30 + 20 μM Fe3+ and 20 μM Fe3+. (b) Zeta potentials of AuNCs@C30 in the presence of different Fe3+concentrations, respectively. 5
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Fig. 7. Fluorescence intensity of AgNCs@C30 (a) 12 μM Fe3+ was added firstly and then added 24 μM EDTA, (b) 24 μM EDTA was added firstly and then added12 μM Fe3+.
to 12 μM (R2 = 0.9881), and the detection limit of Fe3+ was 86 nM. Moreover, the preliminary mechanism of fluorescence quenching of AgNCs@C30 by Fe3+ was proposed, which was deemed to result from AgNCs@C30 aggregation induced with a relatively weak mutual collision between AgNCs@C30 and Fe3+.
consistent with this phenomenon. Moreover, aggregate formation may result in the decrease of stability of nanoclusters, which is agree with the zeta potential measurement (Fig. 6b). Therefore, we hypothesized that the mechanism of fluorescence detection for Fe3+ based on AgNCs@C30 was collision quenching. Moreover, in order to obtain more details about the fluorescence quenching mechanism, we attempted to add EDTA to AgNCs@C30 and Fe3+. As well known, EDTA owns strong binding ability with numerous metal ions [26,31], and the interactional type between Fe3+ and AgNCs@C30 can be indirectly proved by this principle. If there are weakly interaction between Fe3+ and AgNCs@C30, when EDTA was introduced to mixture of Fe3+ and AgNCs@C30, the fluorescence intensity was restored because of stronger combine affordability of EDTA and Fe3+. On the contrary, if the addition of EDTA cannot restore the fluorescence of AgNCs@C30/Fe3+, which would prove the strong force exist between AgNCs@C30 and Fe3+. The reaction type of Fe3+ and AgNCs@C30 was confirmed by analysis and comparison between the fluorescence spectra of AgNCs@C30, AgNCs@C30/Fe3+ and AgNCs@C30/Fe3+/EDTA. The original fluorescence intensity of AgNCs@C30 was 224. As shown in Fig. 7a, when 12 μM Fe3+ was added to AgNCs@C30, the fluorescence reduced to 67, and the quenching efficiency was approximately 70%. 24 μM EDTA was introduced to AgNCs@C30 and Fe3+ mixed solution, a nearly 64% of original fluorescence intensity (144) was recovered. Changing the adding order, that is adding EDTA firstly and then Fe3+ to AgNCs@C30 (Fig. 7b). The fluorescence of AgNCs@C30 was not influenced even in the presence of 24 μM EDTA, indicating non-complexation reaction between EDTA and AgNCs@C30. Further added Fe3+ to AgNCs@C30 and EDTA mixture, the fluorescence intensity quenched, and the value of fluorescent signal was approximately 148. Based on the above results, EDTA enabled the quenched fluorescence of AgNCs@C30 to recover, and the degree of fluorescence quench was the same for two added orders. This phenomenon indicated that the combination of AgNCs@C30 and Fe3+ was a relatively weak mutual collision, and the strong chelating agent EDTA can make Fe3+ depart from AgNCs@C30, leading to the fluorescence be restored. Taken together, our results revealed that the fluorescence intensity quenching of AgNCs@C30 by Fe3+ was deemed to AgNCs@C30 aggregation induced with dynamic quenching.
Declaration of Competing Interest None. Acknowledgements This work is supported by the National Natural Science Foundation of China (21703286, 21603276), the Natural Science Foundation of Shandong Province, China (ZR2019MB063, ZR2017MB045, ZR2016BL14), the Fundamental Research Funds for the Central Universities (19CX02049A, 19CX02057A, 19CX02060A) and the scholarship of China Scholarship Council, Qingdao Applied Basic Research Project (17-1-1-74-jch). References [1] D.T. Quang, J.S. Kim, Fluoro- and chromogenic chemodosimeters for heavy metal ion detection in solution and biospecimens, Chem. Rev. 110 (2010) 6280–6301. [2] J.D. Haas, T. Brownlie, Iron deficiency and reduced work capacity: a critical review of the research to determine a causal relationship, IV. J Nutr. 131 (2001) 676–688. [3] L. Zecca, M.B. Youdim, P. Riederer, J.R. Connor, R.R. Crichton, Iron, brain ageing and neurodegenerative disorders, Nat. Rev. Neurosci. 5 (2004) 863–873. [4] U. Hase, K. Yoshimura, Determination of trace amounts of iron in highly purified water by ion-exchanger phase absorptiometry combined with flow analysis, Analyst 117 (1992) 1501–1506. [5] S. Wang, E.S. Forzani, N. Tao, Detection of heavy metal ions in water by highresolution surface plasmon resonance spectroscopy combined with anodic stripping voltammetry, Anal. Chem. 79 (2007) 4427–4432. [6] S. Ghosh, S. Maji, A. Mondal, Study of selective sensing of Hg2+ ions by green synthesized silver nanoparticles suppressing the effect of Fe3+ ions, Colloid Surf. A 555 (2018) 324–331. [7] G.L. Arnold, S. Weyer, A.D. Anbar, Fe isotope variations in natural materials measured using high mass resolution multiple collector ICPMS, Anal. Chem. 76 (2004) 322–327. [8] Z.C. Xie, X.F. Sun, J.M. Jiao, X. Xin, Ionic liquid-functionalized carbon quantum dots as fluorescent probes for sensitive and selective detection of iron ion and ascorbic acid, Colloid Surf. A 529 (2017) 38–44. [9] H. Huang, H. Li, J.J. Feng, A.J. Wang, One-step green synthesis of fluorescent bimetallic Au/Ag nanoclusters for temperature sensing and in vitro detection of Fe3+, Sens. Actuators B Chem. 223 (2016) 550–556. [10] L. Zhao, X. Xin, P. Ding, A.X. Song, Z. Xie, J.L. Shen, G.Y. Xu, Fluorescent oligomer as a chemosensor for the label-free detection of Fe3+ and dopamine with selectivity and sensitivity, Anal. Chim. Acta 926 (2016) 99–106. [11] L. Shang, Sh.J. Dong, G.U. Nienhaus, Ultra-small fluorescent metal nanoclusters: synthesis and biological applications, Nano Today 6 (2016) 401–418. [12] T.X. Zhang, H.J. Liu, Y. Chen, Ultrabright gold-silver bimetallic nanoclusters: synthesis and their potential application in cysteine sensing, Colloid Surf. A 555 (2018) 572–579. [13] P. Yu, X. Wen, Y.R. Toh, J. Tang, Temperature-dependent fluorescence in Au10 nanoclusters, J. Phys. Chem. C 116 (2012) 6567–6571. [14] S. Ghosh, U. Anand, S. Mukherjee, Luminescent silver nanoclusters acting as a labelfree photoswitch in metal ion sensing, Anal. Chem. 86 (2014) 3188–3194.
4. Conclusions In this paper, the synthesis of AgNCs was achieved in the presence of oligonucleotides C30, and the maximal excitation and emission wavelengths of AgNCs@C30 were 335 nm and 480 nm, respectively, which emitted blue fluorescence under UV light. AgNCs@C30 was used as a label-free fluorescent sensor to detect Fe3+ with high selectivity and sensitivity. Under the optimum detection condition, (F0-F)/F0 has a good linear relationship with Fe3+ concentration in the range from 0.1 6
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Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Label-free and ultrasensitive fluorescence assay for Fe3+ detection using DNA-Templated Ag nanoclusters ⁎
T
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Zhiqing Zhang , Tingting Liu, Changle Yue, Shanshan Wang, Jie Ma, Ting Zhou , Fang Wang, Xiufeng Wang, Guodong Zhang Department of Chemistry, College of Science, China University of Petroleum (East China), Qingdao, 266580, PR China
G R A P H I C A L A B S T R A C T
A non-labeling and ultrasensitive fluorescent probe for the detection of Fe3+ was constructed using DNA-templated Ag nanoclusters.
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanocluster DNA-templated Label-free fluorescent sensor Fe3+
We report for the first time the selective and sensitive detection of Fe3+ by a label-free fluorescent sensor base on AgNCs@C30. AgNCs@C30 was synthesized, and the averaged size is about 3.0 nm, and the maximal excitation and emission wavelengths were 335 nm and 480 nm, respectively. Fe3+ caused a significantly fluorescence quenching of AgNCs@C30, and a novel and label-free nanosensor for detecting Fe3+ was developed with optimum detection of 303.15 K and 2 min. The linear range for Fe3+ detection was 0.1˜12 μM with the corresponding limit of detection (LOD) of 86 nM. Furthermore, the preliminary detection mechanism was discussed in detail, and the fluorescence quenching was deemed to result from AgNCs@C30 aggregation induced with a relatively weak mutual collision between AgNCs@C30 and Fe3+. This strategy detection of Fe3+ will provide a valuable reference for the detection of metal ions by using nanocluster.
1. Introduction Metal ions detection in aqueous media is important for life systems and environmental fields. Among all metal ions, Fe3+ is an important factor for physiology, which is the most significant transition metal ion in organism, such as oxygen transportation, enzyme catalysis and the
⁎
promotion of electron transition in cytochromes [1]. The contents of Fe3+ should be maintained within normal range, and deficiency or excess of Fe3+ ion may have a bad effect on human health and immunity, such as anemia, renal failure, cancer and Alzheimer’s disease [2,3]. Therefore, the assay of Fe3+ level is crucial to early discovery and diagnosis of these disease. Moreover, Fe3+ ion concentration is also an
Corresponding authors. E-mail addresses: [email protected] (Z. Zhang), [email protected] (T. Zhou).
https://doi.org/10.1016/j.colsurfa.2019.123656 Received 30 May 2019; Received in revised form 1 July 2019; Accepted 6 July 2019 Available online 13 July 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.
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concentration in the range from 0.1 to 12 μM (R2 = 0.9881), and the detection limit of Fe3+ was 86 nM. Moreover, the detection mechanism was discussed in detail, which might be dynamic quenching.
important factor to evaluate water quality or environment [4]. Various detective methods are used to measure the content of Fe3+, including voltammetry, absorption spectrometry and inductively coupled plasma mass spectrometry (ICP-MS) [5–7]. However, these methods still faced a number of problems on detection of Fe3+, such as sophisticated instruments, complicated synthesis, time-consuming detection procedures and a high detection limit. The fluorescence detection method is widely used due to highly selectivity, sensitivity, rapid and real-time detection of Fe3+ [8–10]. Typically, the fluorescence assay of Fe3+ are performed based on organic dyes such as rhodamine [1]. However, the sensitive detection of Fe3+ is prevented because of these organic dyes with the intrinsic low solubility and poor photo-stability. Nanoclusters (NCs) have received much attention due to its advantages such as distinctive optical properties, easy preparation, good stability and high resistance [11–19], which can almost be used as an ideal substitute for nanodots [20,21]. Many studies have investigated the detection of metal ions based on nanoclusters using various ligands such as amino acid, peptide, protein and polymer [22–26]. Huang et al. used highly fluorescent glutathione (GSH)-stabilized cooper nanocluster for Fe3+ sensing [22]. Chen et al. prepared GSH capped silver nanoclusters by facile sonochemical method in aqueous solution, and designed a new AgNCs@GSH fluorescent probe for the detection of Fe3+ [23]. Zheng et al. synthesized L-tryptophan-stabilized dual-emission fluorescent gold nanoclusters (GNCs), and Fe3+ ions were detected by the fabricated GNCs [24]. George et al. reported the surfactant-free utilization of DMF (dimethylformamide)-protected platinum nanocluster as a fluorescent sensor for the selective detection of Fe3+ ions in aqueous medium [25]. Xiao et al. introduced a fluorescent sensing platform based on L-glutathione templated Ag nanoclusters, which has been established for independent detection of Hg2+, Cu2+ and Fe3+ ions in a solution [26]. Obviously, the choice of the stabilizing agents can significantly affect the fluorescence properties of NCs. DNA-templated Ag nanoclusters (AgNCs@DNA) have been the subject of significant interest due to their brightness, stability, biocompatibility and simple synthesis [27–30]. There have been some reports that, AgNCs@DNA have been successfully used as a novel fluorescence probe to detect various targets such as metal ions [31–34], thioproteins [35,36] and small molecules [37,38]. Li et al. have constructed a label-free fluorescent probe using ssDNA-templated silver nanoclusters for the detection of Hg2+ [31]. Xu et al. designed a labelfree hairpin DNA-scaffolded AgNCs for fluorescent detection of Hg2+ using exonuclease III-assisted target recycling amplification [32]. Lee et al. reported fluorescence switch for silver ion detection based on Cyt12/AgNCs [33]. Guo et al. proposed a fluorescence assay of Fe3+ based on pH dependent 12 polycytosine oligonucleotide templated AgNCs [34]. According to literature [39–42], the properties of AgNCs prominently depend on the oligonucleotides with optimized strand length and base sequences, which provides the opportunity to choose a versatile oligonucleotides template for preparing AgNCs for a specific analyses. Moreover, we have also confirmed that oligonucleotides In this work, inspired by the template-dependent properties of AgNCs@DNA and their special response to metal ions, we herein describe a simple, label-free and ultra-sensitive strategy for the detection of Fe3+ based on DNA oligonucleotides C30 template-AgNCs (AgNCs@C30). The synthesis process and the basic detection principle is schematically illustrated in Scheme 1. In principle, C30 were used as a template for silver atom deposition, and the synthesis of AgNCs was achieved at the presence of NaBH4. AgNCs@C30 emitted blue fluorescence under UV light. The maximal excitation and emission wavelengths were 335 nm and 480 nm, respectively. Various metal ions showed little effect on fluorescence quenching of AgNCs@C30, but the fluorescence quenching phenomenon was evident after adding Fe3+, indicating the feasibility of Fe3+ selectivity detection. Several factors including temperature and response time were optimized to get the ideal quenching efficiency. Under the optimum detection condition, the change of fluorescent intensity has a good linear relationship with Fe3+
2. Materials and methods 2.1. Materials All DNA oligonucleotides (C5, C10, C20 and C30) used in this study were purchased and purified by Takara Co., Ltd (China). Silver nitrate and sodium borohydride were purchased from Alfa Aesar. All chemicals and reagents were used without further purification. The ultrapure water was used throughout all the experiments. 2.2. Preparation of AgNCs@C30 20 μL 100 μM C30 solution was mixed with 28 μL 1 mM AgNO3 and a certain volume of ultrapure water, then incubated for 20 min at 4℃ in the dark. 12 μL 1 mM ice-cold NaBH4 solution was added to the above mixture to reduce Ag ions. Finally, the mixture was kept in the dark for 12 h at 25 °C, and the color change of solution from colourless to pale yellow indicates the formation of AgNCs@C30. The concentrations of AgNCs@C30 were measured by Optima 8000 ICP-MS (PerkinElmer, USA). AgNO3 standard solution of 0.02 mg/L, 0.2 mg/L, and 2 mg/L were prepared and calibrated, respectively. 75 μL AgNCs@C30 and 25 mL ultrapure water were added into 50 mL tubes, and then ICP-MS was performed. 2.3. Fluorimetric detections of Fe3+ by AgNCs@C30 Fluorescence measurements were recorded on F-2700 spectrofluorometer (Hitachi, Japan). 2 μL prepared AgNCs@C30, a certain amount of ultrapure water and various contents of Fe3+ solutions were respectively added to centrifuge tubes. After incubation for 2 min at 25 °C, the fluorescence emission spectra of mixtures were measured at an excitation wavelength of 335 nm. The excitation and emission slits were both set at 10.0 nm. Fluorescent photographs were taken with a commercial digital camera under the ultraviolet light. 2.4. UV–vis spectra UV-vis measurement were recorded on a Lambda 950 UV–vis spectrometer (PerkinElmer, USA). UV-vis absorption spectra of AgNCs@C30 and AgNCs@C30/Fe3+ were obtained with wavelengths of 200˜800 nm. 2.5. Dynamic light scattering (DLS) The sizes of AgNCs@C30, AgNCs@C30/Fe3+, and AgNCs@C30/ Fe /EDTA were determined by DLS using a Zetasizer Nano-ZS instrument (Malvern, UK). All particle size data were referred to scattering intensity distribution. 3+
2.6. Zeta potential Zeta potential measurements were performed by laser Doppler electrophoresis using a Zetasizer Nano ZS spectrometer (Malvern, U.K.) in a standard DTS1060 C zeta cell. 3. Results and discussion 3.1. Optimization synthesis and characterization of AgNCs@C30 Strongly fluorescent Ag nanoclusters can be achieved in the presence of poly-cytosine oligonucleotides [28,29,39,41]. We investigated the effect of cytosine numbers on the fluorescence intensity of Ag 2
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Scheme 1. Schematic illustration of the assay strategy for the detection of Fe3+.
Fig. 1. The effect of cytosine numbers (a) and silver nitrate concentration (b) on the fluorescence intensity of AgNCs@DNA.
nanoclusters, as shown in Fig. 1a. The fluorescence of AgNCs@DNA was dramatically enhanced with the cytosine numbers increasing. Therefore, C30 was selected as the template to synthesis of AgNCs@C30. To further optimize the synthesis of AgNCs@C30, we fixed the C30 concentration (20 μM) and varied the amount of AgNO3 added (Fig. 1b). With increasing the concentration of AgNO3, the fluorescence of AgNCs@C30 increased firstly. Then it can be clearly seen from Fig. 1b that the fluorescence intensity passed through a maximum value at the concentration of 280˜320 μM and decreased with further increasing the concentration of AgNO3. In the above work, C30:Ag+ optimum ratio 20μM was about 300μM = 1: 15, meaning that coordination complex formed between cytosine and Ag+, the optimal mix proportion of cytosine and 20μM × 30 Ag+ was 300μM = 2: 1. This is in accordance with the result in literature [29]. As AgNO3 concentration increased to 300 μM, the fluorescence reached a maximum value, and this is due to three possible reasons: (1) The optimum ratio of cytosine and Ag+ was 2:1 in the AgNCs@C30 complex. (2)Ag+ dominated the reaction when increasing the concentration of AgNO3. The binding sites of Ag+ at C30 were much more than free Ag+ numbers, so increased the AgNO3 concentration was equal to increase the concentration of adsorbed ions (Ag+) in C30, which caused the enhancement of fluorescent intensity of AgNCs. (3) When the amount of Ag+ exceeded the binding sites of Ag+ at C30, residual Ag+ was reduced to a larger scale of nanoparticles, and the fluorescence was decreased. UV-vis absorption spectrum, fluorescence spectra and DLS were used to systematically characterize AgNCs@C30, as shown in Fig. 2. The fluorescence excitation spectrum showed two peaks at 270 nm and 335 nm, and the identical emission spectral shape of AgNCs@C30 was achieved by either exciting wavelength (Fig. 2a). The absorption maximum of C30 was approximately 270 nm, and 270 nm excitation wavelength indicated that AgNCs were associated with C30. So the directly excitation wavelength of AgNCs should be 335 nm, this excitation yielded the 480 nm blue fluorescent emission, which was
consistent with the report in the literature [28]. The inset in Fig. 2a were the photographs of AgNCs@C30 under daylight (left) or ultraviolet lamp (right), and AgNCs@C30 emitted the strongly blue fluorescence under UV light excitation. DLS was performed to determine the diameter of AgNCs@C30, and the results were shown in Fig. 2b. The synthesized AgNCs@C30 with diameter of about 3.0 nm can emit fluorescence upon photo excitation in the UV-vis range, because that the size of nanoclusters approaches the Fermi wavelength of electrons. The concentration of AgNCs@C30 was 0.019 mg/L by ICP-MS measurement. 3.2. Selectivity detection for Fe3+ using AgNCs@C30 In order to investigate the selectivity detection of metal ions, the fluorescence quenching of AgNCs@C30 after adding various metal ions (K+, Zn2+, Pb2+, Mn2+, Mg2+, Cu2+, Cd2+, Ba2+, Al3+, Cr3+and Fe3+, and the concentrations of ions were 10 μM) were shown in Fig. 3a and b. Other ions showed little effect, but the fluorescence quenching phenomenon was evident after adding Fe3+, indicating the specific selectivity of Fe3+ detection. 3.3. Optimization of detection conditions To confirm the feasibility of proposed detection strategy, we monitored the fluorescence change of AgNCs@C30 in the presence of Fe3+. As shown in Fig. 4a, the fluorescence signal of AgNCs@C30 dramatically decreased when 15 μM Fe3+ was added to the sample. Considering that the response of AgNCs@C30 to Fe3+ may be disturbed by various factors, therefore the detection conditions were optimized in the following. The effect of response time of AgNCs@C30 to Fe3+ were investigated firstly. In the presence of Fe3+, the rapid fluorescence signal observably decreased in less than 2 min, and the fluorescence intensity was maintained a constant value for longer period of time, meaning the reaction entirely finished. Thus, the optimal incubation time was 3
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Fig. 2. UV-vis and fluorescence spectra (a) and dynamic light scattering (b) results of AgNCs@C30.
480 nm gradually decreased with increasing of Fe3+ concentration, and the fluorescence was completely quenched at 12 μM Fe3+. In order to establish the quantitative relationship between the concentrations of Fe3+ and the fluorescence of AgNCs@C30, the fluorescence intensities of AgNCs@C30 at 480 nm were calculated (Fig. 5b). A good linear relationship between the quenching fluorescence efficiency (F0-F)/F0 and Fe3+ concentration in the range of 0˜12 μM was achieved, and the linear equation was (F0-F)/F0 = 0.07483x+0.00678 (R2 = 0.9881). Under the optimal conditions, according to the 3σ/s the limit of detection (LOD) of Fe3+ was 86 nM, where σ corresponds to the standard deviation of the blank sample and s is the slope in the plot of the fluorescence versus the quencher concentration [31]. By comparing Fe3+ detection limit of previously literature reported (Table 1), our strategy showed the high sensitivity for Fe3+ detection. Moreover, a limit of detection of 86 nM for the determination of Fe3+ is much lower than the maximum level of Fe3+ permitted in drinking water by the U. S. Environmental Protection Agency (0.3 mg⋅L−1, approximately equivalent to 5.4 μM) [8,9].
selected for 2 min in the following experiments. Generally, temperature often decreases the fluorescence signal of AgNCs@DNA by affecting the number and enrichment way of silver atoms on DNA, and by the enhancement of nonradiative decay [9,13]. The influence of temperature on the fluorescence intensity was shown in Fig. 4b. Values of F0/F indicate the degree of quenching, whereas, F0 and F are the fluorescent intensity of AgNCs@C30 without and with Fe3+, respectively, the higher F0/F value the stronger fluorescence quenching. The F0/F increased as the temperature increasing from 283.15 K to 303.15 K (Fig. 4b), but the degree of quenching reduced with further increasing temperature. Within a certain range of temperatures, the quenching degree increased with the rising of temperature under the existence of Fe3+, there are two possible reasons: On the one hand, the viscosity of sample reduced with the increase of temperature, and molecular diffusion speed was intensified. The greater opportunity of collision between AgNCs@C30 and Fe3+ increased, leading to the fluorescence quenching. On the other hand, the increase of temperature will promote the structure change of C30 template, which tends to be unstable providing less protection for AgNCs, and fluorescence intensity reduced. One of the main reasons why the quenching degree decreased with further rising of temperature (above 305.15 K) may be the internal energy conversion of the molecules. The higher temperature will promote the energy conversion within the molecules, and the excitation energy is firstly converted to the ground-state vibration energy, and then the vibration energy is lost through the vibration relaxation. As a result, the fluorescence quenching degree will decrease with further increase of temperature. As mentioned above, Fe3+ caused the maximal fluorescence signal change of AgNCs@C30 at 303.15 K. Therefore, 303.15 K was chosen as detection temperature in the further studies.
3.5. Preliminary mechanism of fluorescence quenching The change of fluorescence signal of AgNCs@C30 in the presence of Fe3+ might be caused by excited state reaction, collisional interaction (dynamic), static quenching or both or even through monomolecular mechanisms where fluorophore itself or other absorbing species attenuates the fluorescence intensity [10,11,14,17,22,25,26,31,43–46]. The fluorescence quenching was analyzed using Stern-Volmer equation [47]. F0/F = 1+Ksv [Q] where F0 and F are the fluorescent intensity of AgNCs@C30 without and with Fe3+, respectively. Ksv is the Stern-Volmer constant and [Q] is the concentration of the quencher. The corresponding Stern-Volmer plot of F0/F versus [Fe3+] showed a linear behavior with a Ksv value of 1.71 × 105 M−1 at 305.15 K. From the UV-vis results of AgNCs@C30,
3.4. Sensitively detection for Fe3+ We investigated the fluorescence intensity of AgNCs@C30 in the presence of different concentrations of Fe3+ under the optimal conditions. As shown in Fig. 5a, the fluorescence signal of AgNCs@C30 at
Fig. 3. Analysis of the selective detection of Fe3+. 4
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Fig. 4. (a) The fluorescence intensity changes of AgNCs@C30 before and after adding 15 μM Fe3+. (b) The effect of temperature on fluorescence signals of AgNCs@C30.
Fig. 5. (a) The fluorescence spectra of AgNCs@C30 in the presence of different content of Fe3+ (0–20 μM). (b) The fluorescence intensity of AgNCs@C30 versus the concentrations of Fe3+. A good linear relationship between (F0-F)/F0 and Fe3+ concentration in the range of 0–12 μM.
the larger Ag nanoparticles, resulting the aggregate of AgNCs. The aggregation of AgNCs@C30 might be the directly and primary cause of the fluorescence quenching. Based on the above results, we further investigated Zeta potential of AgNCs@C30 and AgNCs@C30/Fe3+ for gaining insights into the interaction mechanisms. The zeta potential value of AgNCs@C30 was -32.5 mV, indicating the excellent stability. Zeta potential values gradually decreased from-32.5 mV to -20.6 mV, -18.3 mV, -15.9 mV and -4.85 mV, with increasing of Fe3+ concentrations from 5 μM to 20 μM. The reduced zeta potentials indicated that decreased stability of AgNCs@C30 by introduced Fe3+ gradually. Because of the electrostatic attraction in opposite charges, the positive charges of Fe3+ was anxious to combined with the negative charges of AgNCs@C30, and aggregates formed between Fe3+ and AgNCs@C30, resulting the fluorescence quenching. Generally, ground-state complex formation (static quenching) frequently result in dramatically perturbation of the absorption spectra, and the location of absorption peak is markedly drifted [31]. On the contrary, dynamic quenching have effect on the excited states of the fluorophores, and slightly changes occur in the UV-vis absorption spectra [47], and the results of Fig. 6a are
Table 1 Comparison of the linear range and limit of detect for Fe3+ using different methods. Methods
Linear range
Limit of Detection
Reference
AgNCs@C30 AgNCs@GSH AgNCs@GSH GNCs@Try CuNCs@GSH Au/Ag@lipoic acid CQDs PtNCs@DMF
0–12 μM 0.1–18 μM 0.5–20 μM 1–500 μM 1–100 μM 1–80 μM 0–300 μM 0–50 μM
86 nM 50 nM 0.12 μM 0.16 μM 0.3 μM 0.5 μM 13.68 μM 4 μM
our work [26] [23] [24] [22] [9] [8] [25]
AgNCs@C30/Fe3+ and Fe3+ (Fig. 6a), the absorption wavelength of Fe3+ was 304 nm, and the peak of AgNCs@C30 was 402 nm. After adding 20 μM Fe3+, the UV-vis absorption peak of AgNCs@C30 was red shifted (from 402 nm to 410 nm), whereas the absorption intensity reduced. This result indicated that AgNCs became unstable or formatted
Fig. 6. (a) UV-vis spectra of AgNCs@C30, AgNCs@C30 + 20 μM Fe3+ and 20 μM Fe3+. (b) Zeta potentials of AuNCs@C30 in the presence of different Fe3+concentrations, respectively. 5
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Fig. 7. Fluorescence intensity of AgNCs@C30 (a) 12 μM Fe3+ was added firstly and then added 24 μM EDTA, (b) 24 μM EDTA was added firstly and then added12 μM Fe3+.
to 12 μM (R2 = 0.9881), and the detection limit of Fe3+ was 86 nM. Moreover, the preliminary mechanism of fluorescence quenching of AgNCs@C30 by Fe3+ was proposed, which was deemed to result from AgNCs@C30 aggregation induced with a relatively weak mutual collision between AgNCs@C30 and Fe3+.
consistent with this phenomenon. Moreover, aggregate formation may result in the decrease of stability of nanoclusters, which is agree with the zeta potential measurement (Fig. 6b). Therefore, we hypothesized that the mechanism of fluorescence detection for Fe3+ based on AgNCs@C30 was collision quenching. Moreover, in order to obtain more details about the fluorescence quenching mechanism, we attempted to add EDTA to AgNCs@C30 and Fe3+. As well known, EDTA owns strong binding ability with numerous metal ions [26,31], and the interactional type between Fe3+ and AgNCs@C30 can be indirectly proved by this principle. If there are weakly interaction between Fe3+ and AgNCs@C30, when EDTA was introduced to mixture of Fe3+ and AgNCs@C30, the fluorescence intensity was restored because of stronger combine affordability of EDTA and Fe3+. On the contrary, if the addition of EDTA cannot restore the fluorescence of AgNCs@C30/Fe3+, which would prove the strong force exist between AgNCs@C30 and Fe3+. The reaction type of Fe3+ and AgNCs@C30 was confirmed by analysis and comparison between the fluorescence spectra of AgNCs@C30, AgNCs@C30/Fe3+ and AgNCs@C30/Fe3+/EDTA. The original fluorescence intensity of AgNCs@C30 was 224. As shown in Fig. 7a, when 12 μM Fe3+ was added to AgNCs@C30, the fluorescence reduced to 67, and the quenching efficiency was approximately 70%. 24 μM EDTA was introduced to AgNCs@C30 and Fe3+ mixed solution, a nearly 64% of original fluorescence intensity (144) was recovered. Changing the adding order, that is adding EDTA firstly and then Fe3+ to AgNCs@C30 (Fig. 7b). The fluorescence of AgNCs@C30 was not influenced even in the presence of 24 μM EDTA, indicating non-complexation reaction between EDTA and AgNCs@C30. Further added Fe3+ to AgNCs@C30 and EDTA mixture, the fluorescence intensity quenched, and the value of fluorescent signal was approximately 148. Based on the above results, EDTA enabled the quenched fluorescence of AgNCs@C30 to recover, and the degree of fluorescence quench was the same for two added orders. This phenomenon indicated that the combination of AgNCs@C30 and Fe3+ was a relatively weak mutual collision, and the strong chelating agent EDTA can make Fe3+ depart from AgNCs@C30, leading to the fluorescence be restored. Taken together, our results revealed that the fluorescence intensity quenching of AgNCs@C30 by Fe3+ was deemed to AgNCs@C30 aggregation induced with dynamic quenching.
Declaration of Competing Interest None. Acknowledgements This work is supported by the National Natural Science Foundation of China (21703286, 21603276), the Natural Science Foundation of Shandong Province, China (ZR2019MB063, ZR2017MB045, ZR2016BL14), the Fundamental Research Funds for the Central Universities (19CX02049A, 19CX02057A, 19CX02060A) and the scholarship of China Scholarship Council, Qingdao Applied Basic Research Project (17-1-1-74-jch). References [1] D.T. Quang, J.S. Kim, Fluoro- and chromogenic chemodosimeters for heavy metal ion detection in solution and biospecimens, Chem. Rev. 110 (2010) 6280–6301. [2] J.D. Haas, T. Brownlie, Iron deficiency and reduced work capacity: a critical review of the research to determine a causal relationship, IV. J Nutr. 131 (2001) 676–688. [3] L. Zecca, M.B. Youdim, P. Riederer, J.R. Connor, R.R. Crichton, Iron, brain ageing and neurodegenerative disorders, Nat. Rev. Neurosci. 5 (2004) 863–873. [4] U. Hase, K. Yoshimura, Determination of trace amounts of iron in highly purified water by ion-exchanger phase absorptiometry combined with flow analysis, Analyst 117 (1992) 1501–1506. [5] S. Wang, E.S. Forzani, N. Tao, Detection of heavy metal ions in water by highresolution surface plasmon resonance spectroscopy combined with anodic stripping voltammetry, Anal. Chem. 79 (2007) 4427–4432. [6] S. Ghosh, S. Maji, A. Mondal, Study of selective sensing of Hg2+ ions by green synthesized silver nanoparticles suppressing the effect of Fe3+ ions, Colloid Surf. A 555 (2018) 324–331. [7] G.L. Arnold, S. Weyer, A.D. Anbar, Fe isotope variations in natural materials measured using high mass resolution multiple collector ICPMS, Anal. Chem. 76 (2004) 322–327. [8] Z.C. Xie, X.F. Sun, J.M. Jiao, X. Xin, Ionic liquid-functionalized carbon quantum dots as fluorescent probes for sensitive and selective detection of iron ion and ascorbic acid, Colloid Surf. A 529 (2017) 38–44. [9] H. Huang, H. Li, J.J. Feng, A.J. Wang, One-step green synthesis of fluorescent bimetallic Au/Ag nanoclusters for temperature sensing and in vitro detection of Fe3+, Sens. Actuators B Chem. 223 (2016) 550–556. [10] L. Zhao, X. Xin, P. Ding, A.X. Song, Z. Xie, J.L. Shen, G.Y. Xu, Fluorescent oligomer as a chemosensor for the label-free detection of Fe3+ and dopamine with selectivity and sensitivity, Anal. Chim. Acta 926 (2016) 99–106. [11] L. Shang, Sh.J. Dong, G.U. Nienhaus, Ultra-small fluorescent metal nanoclusters: synthesis and biological applications, Nano Today 6 (2016) 401–418. [12] T.X. Zhang, H.J. Liu, Y. Chen, Ultrabright gold-silver bimetallic nanoclusters: synthesis and their potential application in cysteine sensing, Colloid Surf. A 555 (2018) 572–579. [13] P. Yu, X. Wen, Y.R. Toh, J. Tang, Temperature-dependent fluorescence in Au10 nanoclusters, J. Phys. Chem. C 116 (2012) 6567–6571. [14] S. Ghosh, U. Anand, S. Mukherjee, Luminescent silver nanoclusters acting as a labelfree photoswitch in metal ion sensing, Anal. Chem. 86 (2014) 3188–3194.
4. Conclusions In this paper, the synthesis of AgNCs was achieved in the presence of oligonucleotides C30, and the maximal excitation and emission wavelengths of AgNCs@C30 were 335 nm and 480 nm, respectively, which emitted blue fluorescence under UV light. AgNCs@C30 was used as a label-free fluorescent sensor to detect Fe3+ with high selectivity and sensitivity. Under the optimum detection condition, (F0-F)/F0 has a good linear relationship with Fe3+ concentration in the range from 0.1 6
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