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gold nanoclusters nanohybrid for “on-off-on” bifunctional detection and cellular imaging of mercury (II) ions and cysteine
Journal Pre-proof Efficient Ratiometric Fluorescence Probe Utilizing Silicon Particles/Gold Nanoclusters Nanohybrid for “on-off-on” Bifunctional Detection and Cellular Imaging of Mercury (II) Ions and Cysteine Fan Ru, Peiyao Du, Xiaoquan Lu PII:
S0003-2670(20)30038-6
DOI:
https://doi.org/10.1016/j.aca.2020.01.020
Reference:
ACA 237379
To appear in:
Analytica Chimica Acta
Received Date: 4 November 2019 Revised Date:
21 December 2019
Accepted Date: 8 January 2020
Please cite this article as: F. Ru, P. Du, X. Lu, Efficient Ratiometric Fluorescence Probe Utilizing Silicon Particles/Gold Nanoclusters Nanohybrid for “on-off-on” Bifunctional Detection and Cellular Imaging of Mercury (II) Ions and Cysteine, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier B.V. All rights reserved.
Biographies Fan Ru is master's graduate students in the College of Chemistry & Chemical Engineering of Northwest Normal University, China. His main research interests are fluorescence sensor and nanomaterials. Peiyao Du graduated from Nankai University in 2018 and received her PhD degree. She joined Prof. Xiaoquan Lu’s group at Tianjin University after completing her PhD degree. Her main research interests are the development and application of fluorescence sensors. Xiaoquan Lu is the director of Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu, China. He received his Ph. D. from Zhongshan University in 1997. He was awarded “Yangtze Scholars Program” of ministry of education, distinguished professor in 2012. His research interest focuses on bioelectrochemistry, fluorescent detection, etc.
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Herein, we reported an effective dual-emission ratiometric probe for “on-off-on” detection of Hg2+ and cysteine.
Efficient
Ratiometric
Particles/Gold
Fluorescence
Nanoclusters
Probe
Nanohybrid
Utilizing for
Silicon
“on-off-on”
Bifunctional Detection and Cellular Imaging of Mercury (II) Ions and Cysteine
Fan Ru, ‡ Peiyao Du, *,† and Xiaoquan Lu †, ‡
†
Tianjin Key Laboratory of Molecular Optoelectronic, Department of Chemistry, School of
Science, Tianjin University, Tianjin, 300072, P. R. China ‡
Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College
of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou, 730070, P. R. China
Corresponding Authors *E-mail: [email protected] (P. Du).
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ABSTRACT
An effective ratiometric fluorescent probe based on silicon particles/gold nanoclusters (SiNPs/AuNCs) nanohybrid has been fabricated and applied to be a “on-off-on” switch sensing platform for detection of Hg2+ and cysteine. In this elaborated sensing platform, the SiNPs just acted as internal reference signal, providing a build-in correction for background interferences and environmental effects, to which the AuNCs as a signal report unit for Hg2+ response was covalently grafted by amidation reaction. The fluorescence intensity of SiNPs/AuNCs could be effectively quenched upon adding Hg2+, accompanied with an easily distinguishable fluorescent color change. The ratiometric fluorescence signal (F649/F511) of the established nanoprobe was linearly proportional to the concentration of Hg2+ ranging from 0.02-24 µM with a low detection limit of 5.6 nM, which is below the guideline value of Hg2+ in drinking water set by the World Health Organization. Interestingly, upon addition of cysteine, the Hg2+-quenched fluorescence intensity was recovered gradually. Furthermore, the approach developed has also been utilized for Hg2+ detection in real complex biological samples with satisfactory results. More importantly, benefiting from the good water-solubility and excellent biocompatibility, this nanoprobe can monitor the intracellular Hg2+ and cysteine in living cells，indicating its potential applications in advanced biosensing and bioimaging.
Keyword: Ratiometric fluorescence probe; Cellular imaging; “ON-OFF-ON” switch; Mercury (Ⅱ)
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1. Introduction Mercury (Ⅱ) ion, as a ubiquitous and hypertoxicity heavy metal pollutants, can cause a series of environmental and health problems. By binding the -SH group in protein strongly, Hg2+ can accumulate continuously in the living organism through the food chain, which may induce seriously disorders and permanent damages to the nervous system, endocrine system and immune system even with low concentration [1,2]. On the other hand, L-Cysteine (Cys) is an important thiol-containing natural amino acid that plays essential biological roles associated with the synthesis of protein，detoxification and metabolism [3,4]. However, Cys at abnormal levels in organism is usually associated with several human sicknesses, such as liver injuries, Alzheimer’s disease, skin lesions, and so forth [5-6]. Thus, the monitoring the Hg2+ and Cys is very important and attracting large attentions, especially in real complex biological fluids and living cells. As we all know, numerous analytical methods for Hg2+ or Cys have been explored up now, including the electrochemical method, chemiluminescence, as well as surface-enhanced Raman scattering [7-9]. However, most of these detection technologies have been suffering expensive instrumentation, time-consuming and complicated procedures, which still restrained their further applications. It is still imminently and greatly desirable to develop a facile approach for Hg2+ and Cys sensing with characteristics of ultra-high sensitivity and selectivity, as well as convenient and excellent biocompatibility. To address the above problems, fluorescence sensing method could be considered as an alternative way. Fluorescence nanomaterials have been widely accepted as the promising efficient sensing 3
materials and attracted widespread attention, owing to their intrinsic superiorities such as simple synthesis, ultrahigh photostability in terms of monitoring the trace amounts of analytes in environment and in vivo [10-16]. Among the reported fluorescent nanomaterial, especially, the gold nanocluster (AuNCs) has received more attention because of their ultrafine sizes, high quantum yield, and low toxicity [17-18]. Up till now, the functionalized AuNCs have been applied to detect the metal ions and bioanalytes as the fluorescent probes [19-20]. However, the majority of the reported fluorescent probes using the sole “turn-off” sensing signals are unsuitable for practical use because their fluorescence color has no or a minor change during the sensing procedure causing a failure in the naked-eye sensing mode, and their reproducibility is not outstanding with the interference from the instrument background and external environmental conditions [21]. In contrast, ratiometric fluorescent nanoprobes employing two isolated emission peaks under one excitation wavelength and operating through “on-off-on” mechanism is particular attractive, which can effectively avoid the aforementioned drawbacks by means of self-calibration, exhibiting a distinctly improve the accuracy and reliability of the detection results. Nevertheless, most of ratiometric AuNCs-based probes mainly consist of organic dyes, which are susceptible to irreversible photobleaching, have narrow excitation bandwidths and broad emission spectra to result in the poor efficient fluorescence sensing [22]. Based on the above considerations, it is important to choose one material serves as a reference signal during the sensing process, while AuNCs displays an intensity response toward analytes. As a novel fantastic fluorescent material, silicon nanoparticles (SiNPs) possess many inherent advantages, including rich raw material, simply green synthesis, and antiphotobleaching capability [23]. Inspired these, we attempted to integrate of AuNCs with SiNPs to construct nanohybrid as a ratiometric fluorescent probe.
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Herein, as shown in Scheme 1, a novel dual-emission ratiometric nanohybrid probe was designed and synthesized based on the red-emission from AuNCs and green-emission from SiNPs as the reference signal. By adding Hg2+ to the probe, the red fluorescence emission of AuNCs (I649) is quenched, whereas the green fluorescence emission of SiNPs remains nearly constant. Meanwhile, under the irradiation of a UV lamp, a noticeable fluorescence change during the sensing procedure from orange to lime and to green can be easily seized by the naked eyes without any complicated instruments. It’s worth mentioning that the constructed ratiometric luminescent sensor with strong visible color change is more sensitive and reliable than the single fluorescent signal response, which only AuNCs was used as probe. Under the optimized conditions, the LOD is much lower than the guideline value for Hg2+ in drinking water recommended the WHO. The proposed sensor has also been applied for visual identification of Hg2+ in complicated body fluids. Interestingly, the quenched fluorescence can be recovered gradually after adding L-cysteine (Cys). Therefore, a facile "on-off-on" style sensor was accomplished by the proposed nanoprobe for effective Hg2+ and Cys monitoring. Based on the low cytotoxicity and good PL properties of SiNPs/AuNCs, it can be efficaciously applied in the ratiometric intracellular imaging.
2. Experimental section 2.1 Fabrication of Dual-Emission Ratiometric Fluorescent Probe SiNPs/AuNCs To obtain the dual-emissive fluorescent nanosensor, the red emissive AuNCs with carboxylic groups are chemically connected to the green emissive amino-modified SiNPs by a condensation reaction. Briefly, excess red emissive AuNCs were dissolved in a 19 mL PBS buffers (10 mM, pH 7.4). 120 mg EEDQ was added into the solution by stirring in a 50 mL flask for 24 h. 4 mL amino-modified SiNPs was added into the activated AuNCs solution, and the mixture was
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vigorously stirred for 12 h in the dark [24]. The resulting SiNPs/AuNCs were centrifuged at 8000 r/min, then dissolved in a 10 mL PBS buffers. 2.2 Fluorescence Detection of Hg2+ Ions 600 µL of prepared probe solution was injected into 1.9 mL of PBS buffer solution (10 mM, pH 7.4) in a spectrophotometer quartz cuvette as the working solution, followed by adding the calculated amount of Hg2+. The final concentrations of the Hg2+ were 0.02, 0.04, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 4.0, 4.8, 6.0, 7.2, 8, 10, 16, 20, 24 and 30 µM, respectively. After shaking thoroughly for 30 s, the fluorescence emission spectra were recorded when excited at 390 nm. For the selectivity of the assay, other metal ions or amino acids instead of Hg2+ were tested under the same experiment conditions. For the anti-interference testing, 0.6 mM of other common metal ions were mixed with 30 µM of Hg2+ ions, and the mixtures were added to 2.5 mL as-prepared probe, respectively. 2.3 Detection of Hg2+ in Human Urine and Serum Samples Biological fluid samples used in this study were provided by the first affiliated hospital of Lanzhou University and were diluted 50 and 100 times before analysis, respectively. And then the diluted samples were spiked with different known concentrations of Hg2+. 2.4 Fluorescence “Turn-on” detection of Cys Different concentrations of Cys were mixed with 30 µM Hg2+ thoroughly, and the solution was injected to 2.5 mL ratiometric probe solution. The selectivity and interference of the fluorescence probe toward Cys was evaluated by using the same methods as mentioned above. 2.5 Cytotoxicity Assays The cytotoxicity effect of the SiNPs/AuNCs on the Hela cells and mouse embryo fibroblasts
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NIH3T3 was evaluated with Cell Counting Kit-8 (CCK-8). Prior to be tested, 5×105 cells were seeded in triplicate in 96-well cell culture plates at 37 ºC for 24 h. The HeLa cells and the NIH3T3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS) and F12 medium containing fetal bovine serum (FBS) for 24 h, respectively. The SiNPs/AuNCs ratiometric probe at a range of concentrations (0, 100, 200, 300, 400, 500 and 600 µg/mL) were then introduced to the medium and incubated for a further 24 h. Then, cells were washed twice with fresh PBS (10 mM, pH 7.4) before adding 100 µL fresh medium and 10 µL the CCK8 reagent (Dojindo, Japan) to each well. The cells were incubated for additional 2 h at 37 Ⅱ with 5% CO2. The optical density at 450 nm (OD450) of the mixture was measured using a microplate reader. 2.6 Vivo Fluorescence Imaging of Hg2+ and Cys in living cells First, NIH3T3 cells and HeLa cells were cultured for 24 h. Then, the cells were treated by 370 µg/mL SiNPs/AuNCs (prepared in a PBS buffer, pH 7.4) at 37 Ⅱ for 2 h, and stained by Hg2+ (30 µM/mL) and the mixture of Hg2+ (30 µM/mL) and Cys (120 µM/mL) for another 2 h, respectively. The cells were washed with PBS three times followed by fixing with methanol for 30 min. The cells were then stained by DAPI (final concentration of 5 µg/mL) [25]. All confocal images were observed by an Olympus, FV1000 confocal microscope from Japan.
3. Results and discussion 3.1 Characterization of SiNPs/AuNCs SiNPs/AuNCs nanohybrid was prepared by SiNPs and AuNCs through a simple carbodiimide-activated coupling reaction, as described in the ESI†. First, the morphology of the as-prepared SiNPs/AuNCs nanohybrid was characterized by transmission electron microscope (TEM) images, under which the SiNPs and AuNCs exhibited spherical particles with good 7
monodispersibility. The detailed structural and the atomic lattice fringes of as-synthesized SiNPs/AuNCs were further confirmed by high-resolution TEM. The lattice spacing of 0.31 nm and 0.24 nm are attributed to the SiNPs (111) layers and Au (111) facet (Fig. 1A inset). Meanwhile, the EDX mapping of SiNPs/AuNCs nanohybrid shows the presence and distribution of Si and Au (Fig. S1). The powder X-ray diffraction (XRD) analysis shows that the prepared SiNPs and AuNCs are both in the amorphous phase (Fig. S2). In addition, FT-IR was employed to identify the characteristic functional groups on the surface of SiNPs, AuNCs and SiNPs/AuNCs nanohybrid. In the FT-IR spectrum of the pure AuNCs, the broad peak centered at about 3350 cm-1 can be assigned to the O-H groups stretching vibration mode. The high intensity peaks at 1655 cm-1 ascribed to the C=O stretching vibrations (Fig. 1B, black curve). The presence of the hydrophilic groups including -OH, -COOH impart good water solubility of AuNCs [26]. In the spectrum of SiNPs (Fig. 1B, red curve), the absorption peaks at 2930 cm−1 is attributed to C-H bending vibration, and the sharp peak signal at 1128 cm−1 is assigned to the vibrational stretch of Si-O bonding. Two distinct bands observed at 1600 and 3355 cm-1 are characteristics of N-H bending and stretching vibration, respectively, revealing that abundant amino groups are functionalized in the SiNPs surface. As for the SiNPs/AuNCs nanohybrid (Fig. 1B, blue curve), some similar peaks of SiNPs and AuNCs were exhibited，which initially confirms that the SiNPs/AuNCs is successfully assembled. Compared with SiNPs, a shift of the characteristic peak associated with the C-N stretching vibration from 1030 to 1034 cm-1 was observed for SiNPs/AuNCs nanohybrid. To shed more light on the surface composition and chemical states of the prepared SiNPs/AuNCs nanohybrid, X-ray photoelectron spectroscope (XPS) measurements were also
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performed. All the expected elements (Au, Si, C, N and O) were detected at their specific peak positions (Fig. S3A). The contents of the Au 4f, Si 2p, Si 2s, C1s, N 1s and O 1s are 67.90%, 4.70%, 20.52%, 4.86% and 2.02%, respectively. The high resolution XPS spectrum of C 1s (Fig. S3B) can be deconvoluted into five main peaks corresponding to the C-Si (284.1 eV), C-N (284.6 eV), C-O (285.2 eV), C-O (286.1 eV) and C=O (287.4 eV), which demonstrates that amido-bond is existed in the SiNPs/AuNCs nanohybrid. Besides, the N1s (Fig. S3C) spectrum reveals three relative nitrogen species of N-H (398.4 eV), C-N-C (399.2 eV) and N-(C3) (400.3 eV), indicating the SiNPs of the nanohybrid are rich in amino groups on the surface. The peaks shown in Fig. S3D located at 531.0, 532.5 and 533.7 eV in the XPS spectrum of O 1s are attributed to the C=O, C-O-C and Si-O groups, respectively. The XPS spectrum of Si 2p (Fig. S3E) can be divided into three main peaks corresponded to Si-C (100.1 eV), Si-N (100.7 eV) and Si-O (101.5 eV) respectively, which is similar to the Si 2p in the individual SiNPs. Furthermore, the Au7/2 spectrum can be divided into two distinct components centering at the 84.0 and 85.2 eV binding energy (Fig. S3F), suggesting that Au (0) and Au (I) species coexist in the nanohybrid. According to the above results, the nanohybrid still preserve the same structure and composition as individual SiNPs and AuNCs. 3.2 Optical Properties A set of optical characterizations of the as-synthesized SiNPs/AuNCs nanohybrid were studied. In UV-vis absorption spectrum, SiNPs shows an absorption peak at 235 nm, which is attributed to the π-π* transition of the C=C bond (Fig. 2A, blue curve). For AuNCs, the maximum peak at 275 nm is ascribed to n-π* transition of the carbonyl group in BSA (Fig. 2A, red curve). There are no obvious absorption peaks in the visible region of both SiNPs and AuNCs, suggesting
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that there are no large particles exists. After forming the SiNPs/AuNCs nanohybrid, an additional broad band appears around 300-400 nm. Under the irradiation of UV lamp at 365 nm, the aqueous solutions of the prepared individual SiNPs and AuNCs exhibit green and red fluorescence, respectively, which are strong enough to be seen by the naked eyes (Inset of Fig. S4). For SiNPs, the optimal fluorescence excitation and emission peaks are 420 nm and 510 nm, respectively (Fig. S4A). The optimal fluorescence excitation and emission peaks of the AuNCs located at 380 nm and 653 nm (Fig. S4B). The emission wavelengths of SiNPs and AuNCs have a great spectra shift, which can avoid the serve spectrum overlap for the emission bands of the SiNPs/AuNCs hybrid. The effect of SiNPs on the fluorescence of AuNCs was also investigated. The experimental result shows that the fluorescence intensity of AuNCs undergoes a slight decrease with the addition of SiNPs (Fig. S5). After forming the SiNPs/AuNCs ratiometric probe, it is found that the optimal excitation wavelength located at 390 nm which ensures that SiNPs and AuNCs can be excited simultaneously. At the optimal excitation wavelength, the SiNPs/AuNCs nanohybrid displays two well-resolved dual emission peaks at 649 nm and 511 nm (Fig. 2B, blue curve), and emits strong orange fluorescence. It’s worth noting that the red fluorescence emission peak of AuNCs in nanohybrid probe around 649 nm is very beneficial for bioimaging. Before the SiNPs/AuNCs nanohybrid is prepared for further practical sensing applications, it is essential to investigate the stability in many kinds of environments. The fluorescence intensities of the obtained materials under continuous irradiation and in different pH conditions were then measured. The slight decrease in the fluorescence intensity of AuNCs and SiNPs were observed after the UV irradiation for 90 min (Fig. S6). For SiNPs/AuNCs nanohybrid, no significant change
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of fluorescence intensity ratio (F649/F511) were detected under the same conditions, which indicates that the nanohybrid is stable toward photobleaching (Fig. S7). Both of the individual SiNPs and the AuNCs exhibit great photostability even under extreme pH conditions (Fig. S8). After forming the SiNPs/AuNCs nanohybrid, the fluorescence intensity of the solution is nearly constant at the pH 4-11, which is attributed to the protonation−deprotonation variation of the surface charge in SiNPs/AuNCs. The outstanding stability of the as-synthesized ratiometric probe toward the ambient environment makes it suitable for further sensing applications. 3.3 Fluorescence “Turn-off” response to Hg2+ Ions The fluorescence responses of the SiNPs/AuNCs nanohybrid by adding different amounts of Hg2+ were measured to assess the sensing sensitivity. By adding Hg2+, the emission intensity of red-emission AuNCs gradually decreases until quenches, while that of green-emission SiNPs remains constant, resulting in fluorescence ratiometric change. A distinguishable fluorescence color change (Inset of Fig. 3A) has been taken with a slight variation of the intensity ratio (F649/F511), which is available for the visual detection of Hg2+. In order to further improve the sensitivity of the SiNPs/AuNCs nanohybrid towards Hg2+, the fluorescence quenching response under various pH conditions was measured. As shown in Fig. S9A, considering biological environment and fluorescence quenching effect, pH 7.4 was chosen as the detection condition. The ratio of the fluorescence intensity (F649/F511) were found to follow a good linear relationship (F649/F511= 1.586-0.0572×[Hg2+]) with Hg2+ concentration ranging from 0.02 to 24 µM (Fig. 3). Furthermore, the limit of detection (LOD) of Hg2+ in this assay was 5.6 nM. As can be seen from Table S1, the sensing performance of this nanoprobe for Hg2+ detection is at least comparable to, or even better than most of the previously reported. It should also be mentioned, the fluorescence
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quenching reaction is completed within 1 min (Fig. S9B), which suggests that the prepared ratiometric probe possesses favorable potentiality for real-time tracking of Hg2+ in the biological and environmental system. To verify the advantage of the proposed ratiometric SiNPs/AuNCs probe toward Hg2+, for comparison, the fluorescence response of the red-emission AuNCs for the visual Hg2+ detection was also examined. Under the same detection conditions, the emission intensity of the AuNCs can also be quenched by gradually adding Hg2+ from 0 to 30 µM (Fig. S10). Clearly, the color changes of the individual AuNCs fluorescence quenching is hard to distinguish from the original background by the naked eye. These results clearly reveal that the ratiometric SiNPs/AuNCs probe possesses a higher sensitivity and reliability in contrast to the individual AuNCs fluorescence probe. 3.4 Selectivity for detection of Hg2+ As high specificity is another key factor in evaluating the detection performance of the proposed nanoprobe, competitive experiments were studied in the presence of other co-existing metal ions and amine acids. No obvious fluorescence quenching was observed in the fluorescence intensity ratio or accompanied remarkable fluorescent color changes for the majority interferences, and the obtained results were shown in Fig. 4. Although Cu2+ and Ag+ have a slight quenching effect on the red fluorescence of the sensor, fortunately, such interferences from Cu2+ and Ag+ can be effectively circumvented by adding 2,6-Pyridinedicarboxylic acid (PDCA) and NaCl, respectively, owing to the formation of the water-insoluble ion-associate. Subsequently, the anti-interference performance of the SiNPs/AuNCs probe was also evaluated considering the cross reactivity by adding a mixture consisting of 30 µM Hg2+ and other metal ions which are 20-fold
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higher than that of Hg2+ to the SiNPs/AuNCs solution. Little influence on the fluorescent intensity was observed, and is compared to that of the system which only exist Hg2+ (Figure S11). All these results suggest that the ratiometric SiNPs/AuNCs probe exhibits satisfying selectivity for Hg2+, which provides a basic for the detection of Hg2+ in complex biological systems. 3.5 Fluorescence “Turn-on” Detecting of Cys It has been reported that the stability constants for the Cys-hydrosulfuryl complexes are much higher than that of the Hg-carboxylic acid [27]. Therefore, by adding Cys, Hg2+ can form new polymeric species with Cys instead of AuNCs. Then, the fluorescence response of the nanohybrid/Hg2+ sensing system to Cys was investigated. Fortunately, the fluorescence of the quenching sensor can recover or even enhance after adding Cys, which is an attractive proposed assay because fluorescence “turn-on” type sensor preferred to enhance visible distinguish ability and avoid false signal response. Specifically, the fluorescence intensity of the SiNPs/AuNCs probe containing Hg2+ (30 µM) is gradually recovered by increasing the concentration of the Cys, accompany with the fluorescence colors change continuously as demonstrated in the inset of Fig. 5A. A good linear relationship between the value of fluorescence intensity change of the SiNPs/AuNCs nanohybrid (log (F649/F511)) and the concentration of the Cys was obtained over the range of 2-60 µM with a correlation coefficient (R2) of 0.9956 (Fig. 5B, inset). The limit of detection (LOD) for Cys was calculated to be 320 nM. Next, the selectivity of the quench sensor of SiNPs/AuNCs probe containing Hg2+ (30 µM) toward Cys was investigated by adding other amino acids. Under the same conditions, the investigated amino acids had minor or no interference on the fluorescence recover (Fig. S12). Meanwhile, when the mixture consisting of equal concentrations of Cys and other foreign substances which are 20-fold higher than that of Cys were
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added into the SiNPs/AuNCs probe solution containing Hg2+, negligible changes were observed (Fig. S13). Above observations suggest that the “off” state of SiNPs/AuNCs probe containing Hg2+ presents an excellent selectivity towards Cys. 3.6 Proposed Mechanism Involved in Determination of Hg2+ and Cys The above results demonstrate that SiNPs/AuNCs probe presents great sensitivity and selectivity for Hg2+ sensing. The possible mechanism involved in fluorescence sensing process was explored. The UV-vis spectral changes can be explained as the dispersed Hg2+ can be wrapped around the AuNCs because of the strong affinity between Hg2+ and AuNCs (Fig. S14) [28] However, it is irrational that the Hg2+ on the surface of the AuNCs can form the non-fluorescence gold amalgamation because the redox cannot be taken place in the system [29]. The structure of AuNCs is gold atoms coated by the Au (I). The mechanism of the Hg2+ quenching of the ratiometric probe is probably coursed by the strong and specific d10-d10 interaction between Hg2+ (4f145d10) and the coated Au+ (4f145d10), which is called the metallophilic effect [30]. As previously reported in the literature, the Hg-Au metallophilic bond exhibits high affinity and specificity which permitted establishing a sensing platform for Hg2+ [31-32]. The quenching process might be governed by a static or dynamic mechanism, and which can be recognized by measuring the temperature dependence of the slope in the plot of F649/F511 versus [Hg2+]. In Fig. S15, the plots are linear and slope decreased with the increasing temperature [33]. It can be concluded that the quenching is not initiated by static quenching, which is consistent with the above conclusions. Furthermore, the subsequent cysteine-induced fluorescent recovery may be attributable to the formation of Hg–S bonds and/or the reformation of agglomerated structures [34].
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3.7 Determination of Hg2+ in Biological Matrix To evaluate the feasibility of the prepared SiNPs/AuNCs for practical applications in determination of Hg2+, spiking experiments were conducted. As shown in Table 1, the recovery values ranged from 95.5 to 105.2% and the relative standard deviation (RSD) value was below 5%. The satisfactory results also indicated the highly accurate and reliable of our proposed method for Hg2+ detection in the human biological samples. 3.8 Cytotoxicity Assays and Cellular Imaging Encouraged by the desirable sensing performance of SiNPs/AuNCs, we attempted to explore its fluorescence imaging ability. Prior to the application of SiNPs/AuNCs in cell imaging, the in vivo cytotoxicity of the ratiometric probe was examined by CCK8-based cell viability assay. The cell viability was determined by incubating SiNPs/AuNCs (0-600 mg ml-1) in HeLa cells and NIH3T3 cells, respectively. As depicted in Fig. 6, only a slight damage was observed at the higher concentration, and the cell viability was above 85% upon incubating with 500 µg/mL SiNPs/AuNCs. Such low toxicity and excellent biocompatibility may be owing to the two components of the nanohybrid, silicon and gold, which do little harm to the cells. The extremely low cell toxicity and superior optical properties of SiNPs/AuNCs provide a significant possibility and suitability for vivo cellular imaging. As expected, confocal microscopy showed that the SiNPs/AuNCs-treated cells (including HeLa cells and NIH3T3 cells) have a significant intracellular red emission and green emission (Fig. 7, a-d for HeLa cells, m-p for NIH3T3 cells). When the cells were supplemented with the Hg2+ prior to the treatment with the SiNPs/AuNCs, a strong decrease in the red emission was observed in the intracellular region (Fig. 7, e-h for HeLa cells, q-t for NIH3T3 cells), while the green fluorescent
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signal remained (Fig. 7g and s). However, after adding Cys to the Hela cells and NIH3T3 cells incubated by SiNPs/AuNCs and 30 µM of Hg2+, the quenched emission in the red channel was almost recovery thoroughly and the green emission remained almost unchanged. These results confirm the potential of SiNPs/AuNCs for monitoring intracellular Hg2+ and Cys in living cells via luminescence imaging based on on-off-on strategy.
4. Conclusions In summary, a “on-off-on” ultrasensitive fluorescence sensing method based on SiNPs/AuNCs nanoprobe was successfully developed for the detection Hg2+ and Cys. This nanoprobe takes advantages of the excellent stability of SiNPs and the high sensitivity of AuNCs towards Hg2+ to provide an effective sensing platform. Upon the addition of Hg2+, the fluorescence color changes of SiNPs/AuNCs nanoprobe can be easily distinguished with the naked eye under UV irradiation, which exhibits the enhanced visual detection selectivity and reliability compared with individual AuNCs. Meanwhile, in the present of Cys, the quenched fluorescence of the SiNPs/AuNCs system was recovered. More importantly, this nonoprobe can be used for fluorescence bioimaging of Hg2+ and Cys in living cells. Compared with previous reported methods, the proposed nanoprobe in this paper possesses several impressive advantages. First, the reference material and the response party are both covalently linked, which results in a stable nanoprobe and a reliable reference. Second, the significant distinction between the fluorescent colors enhances the distinguishability of the visual detection. Third, the superior water-soluble of the ratiometric probe endows the detection of Hg2+ and Cys with simplicity and immediateness. Finally, with the high biocompatibility, the fluorescence nanomaterial has the excellent potential applicability in vivo. 16
Acknowledgments The authors would like to thank the financial support given through Natural Science Foundation of China (21575115); the Program of Innovation and Entrepreneurial for Talent, Lan Zhou, Gansu Province, China (Grant No. 2014-RC-39); the Program for Chang Jiang Scholars and Innovative Research Team, Ministry of Education, China (Grant No. IRT-16R61). We also thank Dr. Qilin Yu (College of Life Science, Nankai University) for the cytotoxicity experiment and discussion. Appendix A. Supplementary data
The following is Supplementary data to this article:
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125 (2012) 1551-1562. [6] M. Yang, Y. Yan, H. Shi, C. Wang, E. Liu, X. Hu, J. Fan, Efficient inner filter effect sensors based on CdTeS quantum dots and Ag nanoparticles for sensitive detection of l-cysteine, J. Alloys Compd., 781 (2019) 1021-1027. [7] T. Liu, Z. Chu, W. Jin, Electrochemical mercury biosensors based on advanced nanomaterials, J. Mater. Chem. B, 7 (2019) 3620-3632. [8] Y. Tang, J. Li, Q. Guo, G. Nie, An ultrasensitive electrochemiluminescence assay for Hg2+ through graphene quantum dots and poly (5-formylindole) nanocomposite, Sens. Actuators, B 282 (2019) 824-830. [9] Y. Wang, M. Shang, Y. Wang, Z. Xu, Droplet-based microfluidic synthesis of (Au nanorod@ Ag)– polyaniline Janus nanoparticles and their application as a surface-enhanced Raman scattering nanosensor for mercury detection, Anal. Methods, 11 (2019) 3966-3973. [10] W. Zhong, Nanomaterials in fluorescence-based biosensing, Anal. Bioanal. Chem., 394 (2009) 47-59. [11] B. Song, Y. He, Fluorescent silicon nanomaterials: From synthesis to functionalization and application, Nano Today, (2019). [12] M. Holzinger, A. Le Goff, S. Cosnier, Nanomaterials for biosensing applications: a review, Front. Chem., 2 (2014) 63. [13] J. Chen, J. Liu, X. Chen, H. Qiu, Recent progress in nanomaterial-enhanced fluorescence polarization/anisotropy sensors, Chin. Chem. Lett., (2019). [14] X. Zhang, M.A. Ballem, Z.J. Hu, P. Bergman, K. Uvdal, Nanoscale LightⅡHarvesting Metal– Organic Frameworks, Angew. Chem. Int. Ed., 50 (2011) 5729-5733.
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[15] Y. Luo, C. Zhu, D. Du, Y. Lin, A review of optical probes based on nanomaterials for the detection of hydrogen sulfide in biosystems, Anal. Chim. Acta, (2019). [16] X. Ji, H. Wang, B. Song, B. Chu, Y. He, Silicon nanomaterials for biosensing and bioimaging analysis, Front. Chem., 6 (2018) 38. [17] M. Zhuang, C. Ding, A. Zhu, Y. Tian, Ratiometric fluorescence probe for monitoring hydroxyl radical in live cells based on gold nanoclusters, Anal. Chem., 86 (2014) 1829-1836. [18] X. Wang, C. Wang, N. Yang, J. Xia, L. Li, Preparation of fluorescent nanocomposites based on gold nanoclusters self-assembly, Colloids Surf., A 548 (2018) 27-31. [19] F. Sang, X. Zhang, F. Shen, Fluorescent methionine-capped gold nanoclusters for ultra-sensitive determination of copper (II) and cobalt (II), and their use in a test strip, Microchim. Acta 186 (2019) 373. [20] Z. He, T. Shu, L. Su, X. Zhang, Strategies of Luminescent Gold Nanoclusters for Chemo-/Bio-Sensing, Molecules, 24 (2019) 3045. [21] A.M. Desai, P.K. Singh, An Ultrafast Molecular-Rotor-Based Fluorescent Turn-On Sensor for the Perrhenate Anion in Aqueous Solution, Chem. -Eur. J. 25 (2019) 2035-2042. [22] H. Ma, T. Zhou, Z. Dai, J. Hu, J. Liu, In Situ SelfⅡAssembly of Ultrastable Crosslinked Luminescent Gold Nanoparticle and Organic Dye Nanohybrids toward Ultrasensitive and Reversible Ratiometric Thermal Imaging, Adv. Opt. Mater., (2019) 1900326. [23] Y. Zhong, B. Chu, X. Bo, Y. He, C. Zhao, Aqueous synthesis of three-dimensional fluorescent silicon-based nanoscale networks featuring unusual anti-photobleaching properties, Chem. Commun., 55 (2019) 652-655. [24] Y. Wang, C. Zhang, X. Chen, B. Yang, L. Yang, C. Jiang, Z. Zhang, Ratiometric fluorescent paper
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sensor utilizing hybrid carbon dots–quantum dots for the visual determination of copper ions, Nanoscale, 8 (2016) 5977-5984. [25] Q. Yu, B. Zhang, J. Li, M. Li, The design of peptide-grafted graphene oxide targeting the actin cytoskeleton for efficient cancer therapy, Chem. Commun., 53 (2017) 11433-11436. [26] C. Ding, A. Zhu, Y. Tian, Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging, Acc. Chem. Res., 47 (2013) 20-30. [27] M. Ravichandran, Interactions between mercury and dissolved organic matter-a review, Chemosphere, 55 (2004) 319-331. [28] S. Maiti, C. Pezzato, S. Garcia Martin, L.J. Prins, Multivalent interactions regulate signal transduction in a self-assembled Hg2+ sensor, J. Am. Chem. Soc., 136 (2014) 11288-11291. [29] S.F.L. Mertens, M. Gara, A.S. Sologubenko, J. Mayer, S. Szidat, K.W. Krämer, T. Jacob, D.J. Schiffrin, T. Wandlowski, Au@Hg Nanoalloy Formation Through Direct Amalgamation: Structural, Spectroscopic, and Computational Evidence for Slow Nanoscale Diffusion, Adv. Funct. Mater., 21 (2011) 3259-3267. [30] P. Pyykkö, Theoretical chemistry of gold, Angew. Chem. Int. Ed., 43 (2004) 4412-4456. [31] Z. Yan, M. Yan, J. Jiang, P. Gao, S. Shuang, Highly selective and sensitive nanoprobes for Hg(II) ions based on photoluminescent gold nanoclusters, Sens. Actuators, B 235 (2016) 386-393. [32] Y.C. Lin, J. Wu, Y.-W. Lin, Fluorescence sensing of mercury (II) and melamine in aqueous solutions through microwave-assisted synthesis of egg-white-protected gold nanoclusters, Anal. Methods, 10 (2018) 1624-1632. [33] U. Mote, S. Bhattar, S. Patil, G. Kolekar, Interaction between felodipine and bovine serum albumin: fluorescence quenching study, Luminescence, 25 (2010) 1-8.
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[34] Z. Li, Y. Wang, Y. Ni, S. Kokot, A rapid and label-free dual detection of Hg (II) and cysteine with the use of fluorescence switching of graphene quantum dots, Sens. Actuators, B, 207 (2015) 490-497.
Scheme 1. Design Strategy and the Proposed Sensing Mechanism of SiNPs/AuNCs.
Fig. 1. (A) TEM image of the SiNPs/AuNCs. (B) FT-IR spectra of SiNPs (red curve), AuNCs (black curve) and SiNPs/AuNCs nanohybrid (blue curve).
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Fig. 2. (A) UV-vis absorption spectra of SiNPs, AuNCs and ratiometric probe. (B) Fluorescence emission spectra (λex = 390 nm) of SiNPs (a), AuNCs (b) and ratiometric probe (c), respectively. The inset shows the corresponding photographs under UV light illumination at 365 nm.
Fig. 3. (A) The corresponding fluorescence spectra (λex = 390 nm) of the SiNPs/AuNCs probe upon the exposure to different Hg2+ concentrations. (From top to bottom, 0, 0.02, 0.04, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 4, 4.8, 6, 7.2, 8, 10, 16, 20, 24 and 30 µM). The inset shows the corresponding fluorescence photos taken under a UV lamp. (B) The linear calibration plot between F619/F511 and the concentrations of Hg2+ (0.02-24 µM).
22
Fig. 4. (A) The selectivity of the ratiometric probe to various metal ions in the PBS buffer (pH = 7.4, 10 mM). The various bars represent the addition of these metal ions at 30 µM. The inset images show the corresponding fluorescence colors under a UV lamp. (B) Fluorescence responses of the ratiometric probe towards 30 µM Hg2+, Ag+, 30 µM Hg2+ + 0.6 mM Ag+, and pretreating with NaCl; (C) Fluorescence responses of the ratiometric probe towards 30 µM Hg2+, Cu2+, 30 µM Hg2+ + 0.6 mM Cu2+, and pretreatment with PDCA.
Fig. 5. (A) Fluorescence emission spectra of SiNPs/AuNCs containing Hg2+ (30 µM) upon addition of different concentrations of Cys (0-120 µM). Inset is the corresponding fluorescence photos taken under a UV lamp. (B) The relationship between the fluorescence of SiNPs/AuNCs/Hg2+ and Cys from 0 to 60 µM. Inset: linear fitting curve of F649/F511 versus a 23
different concentration of Cys.
Fig 6. Cell viabilities of HeLa cells and NIH3T3 cells in the presence of different concentrations of the SiNPs/AuNCs.
Fig. 7. Fluorescence confocal images of HeLa cells (a-d) and NIH3T3 cells (m-p) incubated with SiNPs/AuNCs, HeLa cells (e-h), and NIH3T3 (q-t) cells treated with Hg2+ prior to the treatment with SiNPs/AuNCs, HeLa cells (i-l) and NIH3T3 (u-x) cells treated with Cys prior to the treatment with SiNPs/AuNCs/Hg2+. Left images: the fluorescence confocal images of Hela cells and NIH3T3 cells treated with DAPI of blue channel. Middle images: fluorescence confocal images of red channel and green channel, respectively. Right images: merge images of previously channels. Scale bar, 20 µm.
24
Table 1 Assay results for the Hg2+ detection in real samples. serum
spiked
human urine
concentration
found
recovery
RSC
found
recovery
RSC
(µM)
(ppb)
(%)
(%)
(ppb)
(%)
(%)
1
0.96
96.7
6.4
0.96
95.5
4.8
5
4.89
97.8
4.2
4.83
96.6
3.6
20
20.8
104.1
4.8
21
105.2
2.1
25
For TOC only:
26
Highlights 1.
A
novel
efficient
water-soluble
dual-emission
fluorescent
probe
for
simultaneously and visual detecting Hg2+ and L-Cys was constructed. 2. The probe could simultaneously detection of Hg2+ in human serum and urine samples. 3. Due to the ratiometric fluorescence model, the background interference was extincted. 4. The developed probe was successfully used for the fluorescence bioimaging of Hg2+ and L-Cys in live cells.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0003-2670(20)30038-6
DOI:
https://doi.org/10.1016/j.aca.2020.01.020
Reference:
ACA 237379
To appear in:
Analytica Chimica Acta
Received Date: 4 November 2019 Revised Date:
21 December 2019
Accepted Date: 8 January 2020
Please cite this article as: F. Ru, P. Du, X. Lu, Efficient Ratiometric Fluorescence Probe Utilizing Silicon Particles/Gold Nanoclusters Nanohybrid for “on-off-on” Bifunctional Detection and Cellular Imaging of Mercury (II) Ions and Cysteine, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier B.V. All rights reserved.
Biographies Fan Ru is master's graduate students in the College of Chemistry & Chemical Engineering of Northwest Normal University, China. His main research interests are fluorescence sensor and nanomaterials. Peiyao Du graduated from Nankai University in 2018 and received her PhD degree. She joined Prof. Xiaoquan Lu’s group at Tianjin University after completing her PhD degree. Her main research interests are the development and application of fluorescence sensors. Xiaoquan Lu is the director of Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu, China. He received his Ph. D. from Zhongshan University in 1997. He was awarded “Yangtze Scholars Program” of ministry of education, distinguished professor in 2012. His research interest focuses on bioelectrochemistry, fluorescent detection, etc.
1
Herein, we reported an effective dual-emission ratiometric probe for “on-off-on” detection of Hg2+ and cysteine.
Efficient
Ratiometric
Particles/Gold
Fluorescence
Nanoclusters
Probe
Nanohybrid
Utilizing for
Silicon
“on-off-on”
Bifunctional Detection and Cellular Imaging of Mercury (II) Ions and Cysteine
Fan Ru, ‡ Peiyao Du, *,† and Xiaoquan Lu †, ‡
†
Tianjin Key Laboratory of Molecular Optoelectronic, Department of Chemistry, School of
Science, Tianjin University, Tianjin, 300072, P. R. China ‡
Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College
of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou, 730070, P. R. China
Corresponding Authors *E-mail: [email protected] (P. Du).
1
ABSTRACT
An effective ratiometric fluorescent probe based on silicon particles/gold nanoclusters (SiNPs/AuNCs) nanohybrid has been fabricated and applied to be a “on-off-on” switch sensing platform for detection of Hg2+ and cysteine. In this elaborated sensing platform, the SiNPs just acted as internal reference signal, providing a build-in correction for background interferences and environmental effects, to which the AuNCs as a signal report unit for Hg2+ response was covalently grafted by amidation reaction. The fluorescence intensity of SiNPs/AuNCs could be effectively quenched upon adding Hg2+, accompanied with an easily distinguishable fluorescent color change. The ratiometric fluorescence signal (F649/F511) of the established nanoprobe was linearly proportional to the concentration of Hg2+ ranging from 0.02-24 µM with a low detection limit of 5.6 nM, which is below the guideline value of Hg2+ in drinking water set by the World Health Organization. Interestingly, upon addition of cysteine, the Hg2+-quenched fluorescence intensity was recovered gradually. Furthermore, the approach developed has also been utilized for Hg2+ detection in real complex biological samples with satisfactory results. More importantly, benefiting from the good water-solubility and excellent biocompatibility, this nanoprobe can monitor the intracellular Hg2+ and cysteine in living cells，indicating its potential applications in advanced biosensing and bioimaging.
Keyword: Ratiometric fluorescence probe; Cellular imaging; “ON-OFF-ON” switch; Mercury (Ⅱ)
2
1. Introduction Mercury (Ⅱ) ion, as a ubiquitous and hypertoxicity heavy metal pollutants, can cause a series of environmental and health problems. By binding the -SH group in protein strongly, Hg2+ can accumulate continuously in the living organism through the food chain, which may induce seriously disorders and permanent damages to the nervous system, endocrine system and immune system even with low concentration [1,2]. On the other hand, L-Cysteine (Cys) is an important thiol-containing natural amino acid that plays essential biological roles associated with the synthesis of protein，detoxification and metabolism [3,4]. However, Cys at abnormal levels in organism is usually associated with several human sicknesses, such as liver injuries, Alzheimer’s disease, skin lesions, and so forth [5-6]. Thus, the monitoring the Hg2+ and Cys is very important and attracting large attentions, especially in real complex biological fluids and living cells. As we all know, numerous analytical methods for Hg2+ or Cys have been explored up now, including the electrochemical method, chemiluminescence, as well as surface-enhanced Raman scattering [7-9]. However, most of these detection technologies have been suffering expensive instrumentation, time-consuming and complicated procedures, which still restrained their further applications. It is still imminently and greatly desirable to develop a facile approach for Hg2+ and Cys sensing with characteristics of ultra-high sensitivity and selectivity, as well as convenient and excellent biocompatibility. To address the above problems, fluorescence sensing method could be considered as an alternative way. Fluorescence nanomaterials have been widely accepted as the promising efficient sensing 3
materials and attracted widespread attention, owing to their intrinsic superiorities such as simple synthesis, ultrahigh photostability in terms of monitoring the trace amounts of analytes in environment and in vivo [10-16]. Among the reported fluorescent nanomaterial, especially, the gold nanocluster (AuNCs) has received more attention because of their ultrafine sizes, high quantum yield, and low toxicity [17-18]. Up till now, the functionalized AuNCs have been applied to detect the metal ions and bioanalytes as the fluorescent probes [19-20]. However, the majority of the reported fluorescent probes using the sole “turn-off” sensing signals are unsuitable for practical use because their fluorescence color has no or a minor change during the sensing procedure causing a failure in the naked-eye sensing mode, and their reproducibility is not outstanding with the interference from the instrument background and external environmental conditions [21]. In contrast, ratiometric fluorescent nanoprobes employing two isolated emission peaks under one excitation wavelength and operating through “on-off-on” mechanism is particular attractive, which can effectively avoid the aforementioned drawbacks by means of self-calibration, exhibiting a distinctly improve the accuracy and reliability of the detection results. Nevertheless, most of ratiometric AuNCs-based probes mainly consist of organic dyes, which are susceptible to irreversible photobleaching, have narrow excitation bandwidths and broad emission spectra to result in the poor efficient fluorescence sensing [22]. Based on the above considerations, it is important to choose one material serves as a reference signal during the sensing process, while AuNCs displays an intensity response toward analytes. As a novel fantastic fluorescent material, silicon nanoparticles (SiNPs) possess many inherent advantages, including rich raw material, simply green synthesis, and antiphotobleaching capability [23]. Inspired these, we attempted to integrate of AuNCs with SiNPs to construct nanohybrid as a ratiometric fluorescent probe.
4
Herein, as shown in Scheme 1, a novel dual-emission ratiometric nanohybrid probe was designed and synthesized based on the red-emission from AuNCs and green-emission from SiNPs as the reference signal. By adding Hg2+ to the probe, the red fluorescence emission of AuNCs (I649) is quenched, whereas the green fluorescence emission of SiNPs remains nearly constant. Meanwhile, under the irradiation of a UV lamp, a noticeable fluorescence change during the sensing procedure from orange to lime and to green can be easily seized by the naked eyes without any complicated instruments. It’s worth mentioning that the constructed ratiometric luminescent sensor with strong visible color change is more sensitive and reliable than the single fluorescent signal response, which only AuNCs was used as probe. Under the optimized conditions, the LOD is much lower than the guideline value for Hg2+ in drinking water recommended the WHO. The proposed sensor has also been applied for visual identification of Hg2+ in complicated body fluids. Interestingly, the quenched fluorescence can be recovered gradually after adding L-cysteine (Cys). Therefore, a facile "on-off-on" style sensor was accomplished by the proposed nanoprobe for effective Hg2+ and Cys monitoring. Based on the low cytotoxicity and good PL properties of SiNPs/AuNCs, it can be efficaciously applied in the ratiometric intracellular imaging.
2. Experimental section 2.1 Fabrication of Dual-Emission Ratiometric Fluorescent Probe SiNPs/AuNCs To obtain the dual-emissive fluorescent nanosensor, the red emissive AuNCs with carboxylic groups are chemically connected to the green emissive amino-modified SiNPs by a condensation reaction. Briefly, excess red emissive AuNCs were dissolved in a 19 mL PBS buffers (10 mM, pH 7.4). 120 mg EEDQ was added into the solution by stirring in a 50 mL flask for 24 h. 4 mL amino-modified SiNPs was added into the activated AuNCs solution, and the mixture was
5
vigorously stirred for 12 h in the dark [24]. The resulting SiNPs/AuNCs were centrifuged at 8000 r/min, then dissolved in a 10 mL PBS buffers. 2.2 Fluorescence Detection of Hg2+ Ions 600 µL of prepared probe solution was injected into 1.9 mL of PBS buffer solution (10 mM, pH 7.4) in a spectrophotometer quartz cuvette as the working solution, followed by adding the calculated amount of Hg2+. The final concentrations of the Hg2+ were 0.02, 0.04, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 4.0, 4.8, 6.0, 7.2, 8, 10, 16, 20, 24 and 30 µM, respectively. After shaking thoroughly for 30 s, the fluorescence emission spectra were recorded when excited at 390 nm. For the selectivity of the assay, other metal ions or amino acids instead of Hg2+ were tested under the same experiment conditions. For the anti-interference testing, 0.6 mM of other common metal ions were mixed with 30 µM of Hg2+ ions, and the mixtures were added to 2.5 mL as-prepared probe, respectively. 2.3 Detection of Hg2+ in Human Urine and Serum Samples Biological fluid samples used in this study were provided by the first affiliated hospital of Lanzhou University and were diluted 50 and 100 times before analysis, respectively. And then the diluted samples were spiked with different known concentrations of Hg2+. 2.4 Fluorescence “Turn-on” detection of Cys Different concentrations of Cys were mixed with 30 µM Hg2+ thoroughly, and the solution was injected to 2.5 mL ratiometric probe solution. The selectivity and interference of the fluorescence probe toward Cys was evaluated by using the same methods as mentioned above. 2.5 Cytotoxicity Assays The cytotoxicity effect of the SiNPs/AuNCs on the Hela cells and mouse embryo fibroblasts
6
NIH3T3 was evaluated with Cell Counting Kit-8 (CCK-8). Prior to be tested, 5×105 cells were seeded in triplicate in 96-well cell culture plates at 37 ºC for 24 h. The HeLa cells and the NIH3T3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS) and F12 medium containing fetal bovine serum (FBS) for 24 h, respectively. The SiNPs/AuNCs ratiometric probe at a range of concentrations (0, 100, 200, 300, 400, 500 and 600 µg/mL) were then introduced to the medium and incubated for a further 24 h. Then, cells were washed twice with fresh PBS (10 mM, pH 7.4) before adding 100 µL fresh medium and 10 µL the CCK8 reagent (Dojindo, Japan) to each well. The cells were incubated for additional 2 h at 37 Ⅱ with 5% CO2. The optical density at 450 nm (OD450) of the mixture was measured using a microplate reader. 2.6 Vivo Fluorescence Imaging of Hg2+ and Cys in living cells First, NIH3T3 cells and HeLa cells were cultured for 24 h. Then, the cells were treated by 370 µg/mL SiNPs/AuNCs (prepared in a PBS buffer, pH 7.4) at 37 Ⅱ for 2 h, and stained by Hg2+ (30 µM/mL) and the mixture of Hg2+ (30 µM/mL) and Cys (120 µM/mL) for another 2 h, respectively. The cells were washed with PBS three times followed by fixing with methanol for 30 min. The cells were then stained by DAPI (final concentration of 5 µg/mL) [25]. All confocal images were observed by an Olympus, FV1000 confocal microscope from Japan.
3. Results and discussion 3.1 Characterization of SiNPs/AuNCs SiNPs/AuNCs nanohybrid was prepared by SiNPs and AuNCs through a simple carbodiimide-activated coupling reaction, as described in the ESI†. First, the morphology of the as-prepared SiNPs/AuNCs nanohybrid was characterized by transmission electron microscope (TEM) images, under which the SiNPs and AuNCs exhibited spherical particles with good 7
monodispersibility. The detailed structural and the atomic lattice fringes of as-synthesized SiNPs/AuNCs were further confirmed by high-resolution TEM. The lattice spacing of 0.31 nm and 0.24 nm are attributed to the SiNPs (111) layers and Au (111) facet (Fig. 1A inset). Meanwhile, the EDX mapping of SiNPs/AuNCs nanohybrid shows the presence and distribution of Si and Au (Fig. S1). The powder X-ray diffraction (XRD) analysis shows that the prepared SiNPs and AuNCs are both in the amorphous phase (Fig. S2). In addition, FT-IR was employed to identify the characteristic functional groups on the surface of SiNPs, AuNCs and SiNPs/AuNCs nanohybrid. In the FT-IR spectrum of the pure AuNCs, the broad peak centered at about 3350 cm-1 can be assigned to the O-H groups stretching vibration mode. The high intensity peaks at 1655 cm-1 ascribed to the C=O stretching vibrations (Fig. 1B, black curve). The presence of the hydrophilic groups including -OH, -COOH impart good water solubility of AuNCs [26]. In the spectrum of SiNPs (Fig. 1B, red curve), the absorption peaks at 2930 cm−1 is attributed to C-H bending vibration, and the sharp peak signal at 1128 cm−1 is assigned to the vibrational stretch of Si-O bonding. Two distinct bands observed at 1600 and 3355 cm-1 are characteristics of N-H bending and stretching vibration, respectively, revealing that abundant amino groups are functionalized in the SiNPs surface. As for the SiNPs/AuNCs nanohybrid (Fig. 1B, blue curve), some similar peaks of SiNPs and AuNCs were exhibited，which initially confirms that the SiNPs/AuNCs is successfully assembled. Compared with SiNPs, a shift of the characteristic peak associated with the C-N stretching vibration from 1030 to 1034 cm-1 was observed for SiNPs/AuNCs nanohybrid. To shed more light on the surface composition and chemical states of the prepared SiNPs/AuNCs nanohybrid, X-ray photoelectron spectroscope (XPS) measurements were also
8
performed. All the expected elements (Au, Si, C, N and O) were detected at their specific peak positions (Fig. S3A). The contents of the Au 4f, Si 2p, Si 2s, C1s, N 1s and O 1s are 67.90%, 4.70%, 20.52%, 4.86% and 2.02%, respectively. The high resolution XPS spectrum of C 1s (Fig. S3B) can be deconvoluted into five main peaks corresponding to the C-Si (284.1 eV), C-N (284.6 eV), C-O (285.2 eV), C-O (286.1 eV) and C=O (287.4 eV), which demonstrates that amido-bond is existed in the SiNPs/AuNCs nanohybrid. Besides, the N1s (Fig. S3C) spectrum reveals three relative nitrogen species of N-H (398.4 eV), C-N-C (399.2 eV) and N-(C3) (400.3 eV), indicating the SiNPs of the nanohybrid are rich in amino groups on the surface. The peaks shown in Fig. S3D located at 531.0, 532.5 and 533.7 eV in the XPS spectrum of O 1s are attributed to the C=O, C-O-C and Si-O groups, respectively. The XPS spectrum of Si 2p (Fig. S3E) can be divided into three main peaks corresponded to Si-C (100.1 eV), Si-N (100.7 eV) and Si-O (101.5 eV) respectively, which is similar to the Si 2p in the individual SiNPs. Furthermore, the Au7/2 spectrum can be divided into two distinct components centering at the 84.0 and 85.2 eV binding energy (Fig. S3F), suggesting that Au (0) and Au (I) species coexist in the nanohybrid. According to the above results, the nanohybrid still preserve the same structure and composition as individual SiNPs and AuNCs. 3.2 Optical Properties A set of optical characterizations of the as-synthesized SiNPs/AuNCs nanohybrid were studied. In UV-vis absorption spectrum, SiNPs shows an absorption peak at 235 nm, which is attributed to the π-π* transition of the C=C bond (Fig. 2A, blue curve). For AuNCs, the maximum peak at 275 nm is ascribed to n-π* transition of the carbonyl group in BSA (Fig. 2A, red curve). There are no obvious absorption peaks in the visible region of both SiNPs and AuNCs, suggesting
9
that there are no large particles exists. After forming the SiNPs/AuNCs nanohybrid, an additional broad band appears around 300-400 nm. Under the irradiation of UV lamp at 365 nm, the aqueous solutions of the prepared individual SiNPs and AuNCs exhibit green and red fluorescence, respectively, which are strong enough to be seen by the naked eyes (Inset of Fig. S4). For SiNPs, the optimal fluorescence excitation and emission peaks are 420 nm and 510 nm, respectively (Fig. S4A). The optimal fluorescence excitation and emission peaks of the AuNCs located at 380 nm and 653 nm (Fig. S4B). The emission wavelengths of SiNPs and AuNCs have a great spectra shift, which can avoid the serve spectrum overlap for the emission bands of the SiNPs/AuNCs hybrid. The effect of SiNPs on the fluorescence of AuNCs was also investigated. The experimental result shows that the fluorescence intensity of AuNCs undergoes a slight decrease with the addition of SiNPs (Fig. S5). After forming the SiNPs/AuNCs ratiometric probe, it is found that the optimal excitation wavelength located at 390 nm which ensures that SiNPs and AuNCs can be excited simultaneously. At the optimal excitation wavelength, the SiNPs/AuNCs nanohybrid displays two well-resolved dual emission peaks at 649 nm and 511 nm (Fig. 2B, blue curve), and emits strong orange fluorescence. It’s worth noting that the red fluorescence emission peak of AuNCs in nanohybrid probe around 649 nm is very beneficial for bioimaging. Before the SiNPs/AuNCs nanohybrid is prepared for further practical sensing applications, it is essential to investigate the stability in many kinds of environments. The fluorescence intensities of the obtained materials under continuous irradiation and in different pH conditions were then measured. The slight decrease in the fluorescence intensity of AuNCs and SiNPs were observed after the UV irradiation for 90 min (Fig. S6). For SiNPs/AuNCs nanohybrid, no significant change
10
of fluorescence intensity ratio (F649/F511) were detected under the same conditions, which indicates that the nanohybrid is stable toward photobleaching (Fig. S7). Both of the individual SiNPs and the AuNCs exhibit great photostability even under extreme pH conditions (Fig. S8). After forming the SiNPs/AuNCs nanohybrid, the fluorescence intensity of the solution is nearly constant at the pH 4-11, which is attributed to the protonation−deprotonation variation of the surface charge in SiNPs/AuNCs. The outstanding stability of the as-synthesized ratiometric probe toward the ambient environment makes it suitable for further sensing applications. 3.3 Fluorescence “Turn-off” response to Hg2+ Ions The fluorescence responses of the SiNPs/AuNCs nanohybrid by adding different amounts of Hg2+ were measured to assess the sensing sensitivity. By adding Hg2+, the emission intensity of red-emission AuNCs gradually decreases until quenches, while that of green-emission SiNPs remains constant, resulting in fluorescence ratiometric change. A distinguishable fluorescence color change (Inset of Fig. 3A) has been taken with a slight variation of the intensity ratio (F649/F511), which is available for the visual detection of Hg2+. In order to further improve the sensitivity of the SiNPs/AuNCs nanohybrid towards Hg2+, the fluorescence quenching response under various pH conditions was measured. As shown in Fig. S9A, considering biological environment and fluorescence quenching effect, pH 7.4 was chosen as the detection condition. The ratio of the fluorescence intensity (F649/F511) were found to follow a good linear relationship (F649/F511= 1.586-0.0572×[Hg2+]) with Hg2+ concentration ranging from 0.02 to 24 µM (Fig. 3). Furthermore, the limit of detection (LOD) of Hg2+ in this assay was 5.6 nM. As can be seen from Table S1, the sensing performance of this nanoprobe for Hg2+ detection is at least comparable to, or even better than most of the previously reported. It should also be mentioned, the fluorescence
11
quenching reaction is completed within 1 min (Fig. S9B), which suggests that the prepared ratiometric probe possesses favorable potentiality for real-time tracking of Hg2+ in the biological and environmental system. To verify the advantage of the proposed ratiometric SiNPs/AuNCs probe toward Hg2+, for comparison, the fluorescence response of the red-emission AuNCs for the visual Hg2+ detection was also examined. Under the same detection conditions, the emission intensity of the AuNCs can also be quenched by gradually adding Hg2+ from 0 to 30 µM (Fig. S10). Clearly, the color changes of the individual AuNCs fluorescence quenching is hard to distinguish from the original background by the naked eye. These results clearly reveal that the ratiometric SiNPs/AuNCs probe possesses a higher sensitivity and reliability in contrast to the individual AuNCs fluorescence probe. 3.4 Selectivity for detection of Hg2+ As high specificity is another key factor in evaluating the detection performance of the proposed nanoprobe, competitive experiments were studied in the presence of other co-existing metal ions and amine acids. No obvious fluorescence quenching was observed in the fluorescence intensity ratio or accompanied remarkable fluorescent color changes for the majority interferences, and the obtained results were shown in Fig. 4. Although Cu2+ and Ag+ have a slight quenching effect on the red fluorescence of the sensor, fortunately, such interferences from Cu2+ and Ag+ can be effectively circumvented by adding 2,6-Pyridinedicarboxylic acid (PDCA) and NaCl, respectively, owing to the formation of the water-insoluble ion-associate. Subsequently, the anti-interference performance of the SiNPs/AuNCs probe was also evaluated considering the cross reactivity by adding a mixture consisting of 30 µM Hg2+ and other metal ions which are 20-fold
12
higher than that of Hg2+ to the SiNPs/AuNCs solution. Little influence on the fluorescent intensity was observed, and is compared to that of the system which only exist Hg2+ (Figure S11). All these results suggest that the ratiometric SiNPs/AuNCs probe exhibits satisfying selectivity for Hg2+, which provides a basic for the detection of Hg2+ in complex biological systems. 3.5 Fluorescence “Turn-on” Detecting of Cys It has been reported that the stability constants for the Cys-hydrosulfuryl complexes are much higher than that of the Hg-carboxylic acid [27]. Therefore, by adding Cys, Hg2+ can form new polymeric species with Cys instead of AuNCs. Then, the fluorescence response of the nanohybrid/Hg2+ sensing system to Cys was investigated. Fortunately, the fluorescence of the quenching sensor can recover or even enhance after adding Cys, which is an attractive proposed assay because fluorescence “turn-on” type sensor preferred to enhance visible distinguish ability and avoid false signal response. Specifically, the fluorescence intensity of the SiNPs/AuNCs probe containing Hg2+ (30 µM) is gradually recovered by increasing the concentration of the Cys, accompany with the fluorescence colors change continuously as demonstrated in the inset of Fig. 5A. A good linear relationship between the value of fluorescence intensity change of the SiNPs/AuNCs nanohybrid (log (F649/F511)) and the concentration of the Cys was obtained over the range of 2-60 µM with a correlation coefficient (R2) of 0.9956 (Fig. 5B, inset). The limit of detection (LOD) for Cys was calculated to be 320 nM. Next, the selectivity of the quench sensor of SiNPs/AuNCs probe containing Hg2+ (30 µM) toward Cys was investigated by adding other amino acids. Under the same conditions, the investigated amino acids had minor or no interference on the fluorescence recover (Fig. S12). Meanwhile, when the mixture consisting of equal concentrations of Cys and other foreign substances which are 20-fold higher than that of Cys were
13
added into the SiNPs/AuNCs probe solution containing Hg2+, negligible changes were observed (Fig. S13). Above observations suggest that the “off” state of SiNPs/AuNCs probe containing Hg2+ presents an excellent selectivity towards Cys. 3.6 Proposed Mechanism Involved in Determination of Hg2+ and Cys The above results demonstrate that SiNPs/AuNCs probe presents great sensitivity and selectivity for Hg2+ sensing. The possible mechanism involved in fluorescence sensing process was explored. The UV-vis spectral changes can be explained as the dispersed Hg2+ can be wrapped around the AuNCs because of the strong affinity between Hg2+ and AuNCs (Fig. S14) [28] However, it is irrational that the Hg2+ on the surface of the AuNCs can form the non-fluorescence gold amalgamation because the redox cannot be taken place in the system [29]. The structure of AuNCs is gold atoms coated by the Au (I). The mechanism of the Hg2+ quenching of the ratiometric probe is probably coursed by the strong and specific d10-d10 interaction between Hg2+ (4f145d10) and the coated Au+ (4f145d10), which is called the metallophilic effect [30]. As previously reported in the literature, the Hg-Au metallophilic bond exhibits high affinity and specificity which permitted establishing a sensing platform for Hg2+ [31-32]. The quenching process might be governed by a static or dynamic mechanism, and which can be recognized by measuring the temperature dependence of the slope in the plot of F649/F511 versus [Hg2+]. In Fig. S15, the plots are linear and slope decreased with the increasing temperature [33]. It can be concluded that the quenching is not initiated by static quenching, which is consistent with the above conclusions. Furthermore, the subsequent cysteine-induced fluorescent recovery may be attributable to the formation of Hg–S bonds and/or the reformation of agglomerated structures [34].
14
3.7 Determination of Hg2+ in Biological Matrix To evaluate the feasibility of the prepared SiNPs/AuNCs for practical applications in determination of Hg2+, spiking experiments were conducted. As shown in Table 1, the recovery values ranged from 95.5 to 105.2% and the relative standard deviation (RSD) value was below 5%. The satisfactory results also indicated the highly accurate and reliable of our proposed method for Hg2+ detection in the human biological samples. 3.8 Cytotoxicity Assays and Cellular Imaging Encouraged by the desirable sensing performance of SiNPs/AuNCs, we attempted to explore its fluorescence imaging ability. Prior to the application of SiNPs/AuNCs in cell imaging, the in vivo cytotoxicity of the ratiometric probe was examined by CCK8-based cell viability assay. The cell viability was determined by incubating SiNPs/AuNCs (0-600 mg ml-1) in HeLa cells and NIH3T3 cells, respectively. As depicted in Fig. 6, only a slight damage was observed at the higher concentration, and the cell viability was above 85% upon incubating with 500 µg/mL SiNPs/AuNCs. Such low toxicity and excellent biocompatibility may be owing to the two components of the nanohybrid, silicon and gold, which do little harm to the cells. The extremely low cell toxicity and superior optical properties of SiNPs/AuNCs provide a significant possibility and suitability for vivo cellular imaging. As expected, confocal microscopy showed that the SiNPs/AuNCs-treated cells (including HeLa cells and NIH3T3 cells) have a significant intracellular red emission and green emission (Fig. 7, a-d for HeLa cells, m-p for NIH3T3 cells). When the cells were supplemented with the Hg2+ prior to the treatment with the SiNPs/AuNCs, a strong decrease in the red emission was observed in the intracellular region (Fig. 7, e-h for HeLa cells, q-t for NIH3T3 cells), while the green fluorescent
15
signal remained (Fig. 7g and s). However, after adding Cys to the Hela cells and NIH3T3 cells incubated by SiNPs/AuNCs and 30 µM of Hg2+, the quenched emission in the red channel was almost recovery thoroughly and the green emission remained almost unchanged. These results confirm the potential of SiNPs/AuNCs for monitoring intracellular Hg2+ and Cys in living cells via luminescence imaging based on on-off-on strategy.
4. Conclusions In summary, a “on-off-on” ultrasensitive fluorescence sensing method based on SiNPs/AuNCs nanoprobe was successfully developed for the detection Hg2+ and Cys. This nanoprobe takes advantages of the excellent stability of SiNPs and the high sensitivity of AuNCs towards Hg2+ to provide an effective sensing platform. Upon the addition of Hg2+, the fluorescence color changes of SiNPs/AuNCs nanoprobe can be easily distinguished with the naked eye under UV irradiation, which exhibits the enhanced visual detection selectivity and reliability compared with individual AuNCs. Meanwhile, in the present of Cys, the quenched fluorescence of the SiNPs/AuNCs system was recovered. More importantly, this nonoprobe can be used for fluorescence bioimaging of Hg2+ and Cys in living cells. Compared with previous reported methods, the proposed nanoprobe in this paper possesses several impressive advantages. First, the reference material and the response party are both covalently linked, which results in a stable nanoprobe and a reliable reference. Second, the significant distinction between the fluorescent colors enhances the distinguishability of the visual detection. Third, the superior water-soluble of the ratiometric probe endows the detection of Hg2+ and Cys with simplicity and immediateness. Finally, with the high biocompatibility, the fluorescence nanomaterial has the excellent potential applicability in vivo. 16
Acknowledgments The authors would like to thank the financial support given through Natural Science Foundation of China (21575115); the Program of Innovation and Entrepreneurial for Talent, Lan Zhou, Gansu Province, China (Grant No. 2014-RC-39); the Program for Chang Jiang Scholars and Innovative Research Team, Ministry of Education, China (Grant No. IRT-16R61). We also thank Dr. Qilin Yu (College of Life Science, Nankai University) for the cytotoxicity experiment and discussion. Appendix A. Supplementary data
The following is Supplementary data to this article:
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Scheme 1. Design Strategy and the Proposed Sensing Mechanism of SiNPs/AuNCs.
Fig. 1. (A) TEM image of the SiNPs/AuNCs. (B) FT-IR spectra of SiNPs (red curve), AuNCs (black curve) and SiNPs/AuNCs nanohybrid (blue curve).
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Fig. 2. (A) UV-vis absorption spectra of SiNPs, AuNCs and ratiometric probe. (B) Fluorescence emission spectra (λex = 390 nm) of SiNPs (a), AuNCs (b) and ratiometric probe (c), respectively. The inset shows the corresponding photographs under UV light illumination at 365 nm.
Fig. 3. (A) The corresponding fluorescence spectra (λex = 390 nm) of the SiNPs/AuNCs probe upon the exposure to different Hg2+ concentrations. (From top to bottom, 0, 0.02, 0.04, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 4, 4.8, 6, 7.2, 8, 10, 16, 20, 24 and 30 µM). The inset shows the corresponding fluorescence photos taken under a UV lamp. (B) The linear calibration plot between F619/F511 and the concentrations of Hg2+ (0.02-24 µM).
22
Fig. 4. (A) The selectivity of the ratiometric probe to various metal ions in the PBS buffer (pH = 7.4, 10 mM). The various bars represent the addition of these metal ions at 30 µM. The inset images show the corresponding fluorescence colors under a UV lamp. (B) Fluorescence responses of the ratiometric probe towards 30 µM Hg2+, Ag+, 30 µM Hg2+ + 0.6 mM Ag+, and pretreating with NaCl; (C) Fluorescence responses of the ratiometric probe towards 30 µM Hg2+, Cu2+, 30 µM Hg2+ + 0.6 mM Cu2+, and pretreatment with PDCA.
Fig. 5. (A) Fluorescence emission spectra of SiNPs/AuNCs containing Hg2+ (30 µM) upon addition of different concentrations of Cys (0-120 µM). Inset is the corresponding fluorescence photos taken under a UV lamp. (B) The relationship between the fluorescence of SiNPs/AuNCs/Hg2+ and Cys from 0 to 60 µM. Inset: linear fitting curve of F649/F511 versus a 23
different concentration of Cys.
Fig 6. Cell viabilities of HeLa cells and NIH3T3 cells in the presence of different concentrations of the SiNPs/AuNCs.
Fig. 7. Fluorescence confocal images of HeLa cells (a-d) and NIH3T3 cells (m-p) incubated with SiNPs/AuNCs, HeLa cells (e-h), and NIH3T3 (q-t) cells treated with Hg2+ prior to the treatment with SiNPs/AuNCs, HeLa cells (i-l) and NIH3T3 (u-x) cells treated with Cys prior to the treatment with SiNPs/AuNCs/Hg2+. Left images: the fluorescence confocal images of Hela cells and NIH3T3 cells treated with DAPI of blue channel. Middle images: fluorescence confocal images of red channel and green channel, respectively. Right images: merge images of previously channels. Scale bar, 20 µm.
24
Table 1 Assay results for the Hg2+ detection in real samples. serum
spiked
human urine
concentration
found
recovery
RSC
found
recovery
RSC
(µM)
(ppb)
(%)
(%)
(ppb)
(%)
(%)
1
0.96
96.7
6.4
0.96
95.5
4.8
5
4.89
97.8
4.2
4.83
96.6
3.6
20
20.8
104.1
4.8
21
105.2
2.1
25
For TOC only:
26
Highlights 1.
A
novel
efficient
water-soluble
dual-emission
fluorescent
probe
for
simultaneously and visual detecting Hg2+ and L-Cys was constructed. 2. The probe could simultaneously detection of Hg2+ in human serum and urine samples. 3. Due to the ratiometric fluorescence model, the background interference was extincted. 4. The developed probe was successfully used for the fluorescence bioimaging of Hg2+ and L-Cys in live cells.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: