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Dithiothreitol-Facilitated Synthesis of Bovine Serum Albumin-Gold Nanoclusters for Pb(II) Ion Detection on Paper Substrates and in Live Cells Peuli Nath, Manosree Chatterjee, and Nripen Chanda ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01191 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 5, 2018
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Dithiothreitol-Facilitated Synthesis of Bovine Serum AlbuminGold Nanoclusters for Pb(II) Ion Detection on Paper Substrates and in Live Cells Peuli Nath,a,b Manosree Chatterjee,a and Nripen Chandaa,b* a
Micro System Technology Laboratory, CSIR-Central Mechanical Engineering Research Institute, MG Avenue, Durgapur 713209, India.
b
Academy of Scientific and Innovative Research (AcSIR), CSIR Campus, CSIR Road, Taramani, Chennai 600113, India KEYWORDS. Fluorescent gold nanosensor, bovine serum albumin, dithiothreitol, lead, detection, live cells.
ABSTRACT: In this work, a bright red fluorescent protein-capped gold nanocluster, AuNC@BSA is developed in a green synthesis approach and its application as a sensor for detection of PbII ion in water and in live cells is demonstrated. AuNC@BSA is prepared by dithiothreitol (DTT) mediated activation of bovine serum albumin (BSA) followed by the reduction of HAuCl4 in an aqueous medium. The incorporation of DTT assists in the reduction of disulfide bridges present in cysteine residues which in turn increases the reducing power of BSA forming a significant number of the Au25 clusters that enhances the bright red fluorescence of AuNC@BSA at 660 nm when excited at 520 nm with a highest quantum yield of ~20.0 % reported so far. AuNC@BSA as nanosensor selectively detect PbII ion in water with a detection limit of 1.0 ppb, lower than the WHO limit (10.0 ppb) and follow quenching based sensing mechanism through metallophilic interaction between AuI and PbII ion. In presence of a strong chelating agent, EDTA, AuNC@BSA shows regeneration ability after the treatment with PbII ion. On paper-based substrate AuNC@BSA also exhibits a distinct change in red fluorescent color with metal ion concentration. The cellular study with AuNC@BSA demonstrates excellent biocompatibility that makes them a suitable candidate for biosensing of PbII ion in biological medium. In HeLa cells, AuNC@BSA shows bright red fluorescence under the confocal microscope, which is quenched when incubated with PbII ion enriched cells up to a limit of 1.0 ppm proving their imaging ability for in vitro applications. For the first time, such efficient green synthesis of a gold nanosensor for a sensitive and selective detection of PbII ion on paper and in live cells is reported in this study. Clusters of gold owing to sub-nanometer dimensions possess unique photo-electronic properties like strong emission of light on excitation in UV-vis region and thus show immense potential in sensor technology.1-10 Specifically, gold nanoclusters (AuNCs) of 2-3 nm in size is very useful as it is highly fluorescent, photo-physically stable, less toxic, with a huge surface-to-volume ratio, which makes them sensitive to monitor the interactions of any biologically and environmentally relevant species including metal toxins. Mainly two different types of molecules i.e. large-sized protein, nucleic acid, dendrimer and small-sized peptide, amino acid, thiol are widely used for the synthesis of fluorescent AuNCs.11-13 Clusters obtained by proteins, mainly by bovine serum albumin (BSA) are well studied as sensors as they show strong red emission at ~640 nm on excitation at 520 nm with a maximum quantum yield (QY) ~10%.6-7, 9, 14-16 For example, Govindaraju et al. reported fluorescent AuNC using BSA with QY ~8 % for dopamine detection in cerebrospinal fluid.15 Zhang et al. have prepared BSA stabilized AuNC with QY ~9.4 % (rhodamine B as reference) and studied their use in bioimaging of cancer cells.16 There are also a good number of
examples in the literature of small molecules like glutathione (GSH), dithiothreitol (DTT), L-arginine, and cysteine-mediated synthesis of fluorescent AuNCs, which generally require either a long time or high temperature or both.8, 10, 17 Most of the AuNCs obtained by this procedure possess low to high QY (3.5-65%). For example, Bain et al recently developed a green fluorescent gold nanoclusters using GSH in an aqueous medium that exhibits QY ~3.15 % (quinine sulfate as reference).18 Ding et al. prepared dithiothreitol-capped fluorescent gold nanoclusters with a QY ~2.09% (rhodamine B as reference).19 Deng et al. developed a green fluorescent AuNCs with a photoluminescence quantum yield (QY) as high as 65% using 6-aza-2-thiothymine and L-arginine (Arg).20 Roy et al made a water-soluble green fluorescent gold nanoclusters using a dipeptide L-cysteinyl-L-cysteine with a QY ~41%.21 Thus, many reports have been published on small molecule stabilized gold nanoclusters for various applications, but there is still a requirement of an efficient system, specifically, protein stabilized gold nanoclusters with higher QY, so that it can be effectively used not only in an aqueous medium but also in the biological domain. In recent past, AuNCs containing BSA as stabilizer have been significantly used for the detection of toxic
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heavy metal ions.5, 12, 22-23 BSA containing multi-functional groups makes AuNCs suitable for detection of various metal ions like copper, mercury, iron, and lead based on fluorescence quenching technique. For example, Hsu et al. reported fluorescence based detection of HgII ion with a LOD of 2.98 nM (0.6 ppb) using BSA stabilized gold nanoclusters.6 In another study, Durgadas et al. prepared fluorescent gold nanoclusters for detection of CuII ion in live cells with a detection limit of 0.05 mM (3.17 ppm). They studied the quenching of AuNCs in the presence of CuII ion inside HeLa cells.5 In their work, the fluorescent gold nanocluster shows strong fluorescence under fluorescence microscope but in pre-treated cells with CuII ion, did not exhibit any fluorescence which proves that the internalized CuII ion in cytoplasm quenched the emission of the AuNCs. This study demonstrates that BSA stabilized AuNCs could be an excellent candidate in order to detect metal ion inside live cells, as BSA can act as transport protein in transferring exogenous material from outside to inside the cells thus working as an excellent carrier for nanoparticulate species to detect metal ions in live cells.13 Among various heavy metal ions, the analysis of lead (Pb) contamination is highly required, as it possesses a serious threat to human health. Lead when ingested causes continuous accumulation inside living cells of the nervous system and several neurotoxic effects such as behavioral abnormalities, learning impairment, decreased hearing, and impaired cognitive functions in human. This metal ion easily crosses blood-brain barrier and is accumulated in cellular matrix of brain.24-25 WHO has made a safe limit of 10 ppb for the lead, above which the water is unfit for drinking. Therefore, there is a need for efficient, cost effective sensor system to detect lead ions in water as well as in the biological system with tracer level sensitivity. Among various AuNC-based systems, gold nanoclusters with a QY of 2.1 % (quinine sulfate as reference) reported by Lee at al. exhibited the lead ion detection in water with a limit of 4.8 nM (~1 ppb).7 Zhu et al. developed a green fluorescent nanohybrid of gold nanoclusters and quantum dots using glutathione as stabilizing agent and mercaptopropionic acid as ligand that showed the sensitivity to lead ion with a detection limit of 3.5 nM (~0.72 ppb).3 Bain et al. recently demonstrated PbII detection in water using green fluorescent gold nanoclusters with a lowest detection limit of 10 nM (~2.1 ppb).18 Thus, there are several AuNCbased methods for the detection of PbII ion in an aqueous medium, but no reports were found where PbII ion detection is achieved in both aqueous medium and intracellular matrix using a single gold nanocluster with high quantum yield. In this study, we report the green synthesis of BSA stabilized fluorescent gold nanocluster (AuNC@BSA) in presence of a biocompatible thiol compound i.e. dithiothreitol (DTT) and demonstrate its use as a nanosensor for the detection of PbII ion in both aqueous solution and live cells. The introduction of DTT molecules in AuNC@BSA synthesis enhances the quantum yield significantly up to ~20.0 % as DTT helps in
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Fig. 1 Schematic illustration showing synthesis of red fluorescent gold nanocluster, AuNC@BSA with dithiothreitol (DTT) (A) and working principle of II AuNC@BSA in absence and presence of Pb ion when excited at 365 nm UV lamp and recovery of quenched fluorescence in presence of EDTA (B).
reducing the disulfide linkage26 present in cysteine residues of BSA, thus increasing the population of fluorescent Au25 clusters through gold-thiol interaction. The gold nanocluster was characterized using FTIR, UVvis spectroscopy, fluorescence spectroscopy and XPS analysis. The system shows maximum red fluorescence emission peak at ~660 nm at pH ~7.5 when excited at 520 nm. There is no surface resonance peak at 520 nm in UVvis spectroscopy owing to its smaller size than 3.0 nm. AuNC@BSA can detect PbII ion at pH ~7.5 with high sensitivity (~1.0 ppb) by quenching mechanism. The nanosensor has regeneration ability after treatment of the AuNC@BSA-PbII complex with a strong chelating agent, EDTA. Paper based detection of PbII using AuNC@BSA shows same efficiency as in solution with gradual change in red fluorescence color against different metal ion concentrations and involves low consumption of the chemicals and less waste production during the sensing process. In another study, the gold nanocluster exhibits excellent photostability property inside HeLa cells showing bright red fluorescence under the confocal microscope and its ability to detect PbII ion inside live cells, making them a suitable platform as bioimaging or biosensing agents. To the best of our knowledge, such DTT mediated protein-based gold nanocluster synthesis and its efficient application in PbII detection on the paper strip as well as in live cells through fluorescence imaging has not been reported earlier. A detailed study on the mechanism of sensing is also discussed in this work.
EXPERIMENTAL SECTION Materials. Bovine serum albumin (BSA-MW ~66 kDa), dithiothreitol (DTT), lead chloride (PbCl2), cysteine, was procured from Sigma aldrich. Hydrogen tetracholoroaurate (III) dihydrate (HAuCl4.2H2O) was purchased from Himedia, India. All other metal salts used were purchased from Merck,
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Germany. Cell lines were supplied by National Centre for Cell Science (NCCS), Pune, India. Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS), 10X phosphate buffer saline (PBS) and 1% penicillin-streptomycin (penstrip) were purchased from Gibco, Thermofisher Scientific Pvt. Ltd, India. Cell culture grade Milli Q water was used throughout the experiments and all other reagents used was of high analytical grade. Instrumentations. UV-vis absorption spectra were recorded on a Cary 60 Agilent technologies spectrophotometer at room temperature. Fluorescence spectra were taken on a Cary Eclipse fluorescence spectrometer. Size and charge were measured using NS500 (NanoSight) instrument. Transmission electron microscopy (TEM) was performed on a Technai G 20 (FEI) microscope operated at an accelerating voltage of 200 kV. Detailed surface characterizations of the nanocluster were performed using X-ray photoelectron spectroscopy (XPS, PHI 5000 Versa Probe II, FEI Inc.). The matrix assisted laser desorption ionization –time of flight was performed in Applied Biosystems Q10 4800 MALDI-TOF analyzer. The time correlated single photon counting (TCSPC) analysis was carried out in Horiba Jobin Yvon Fluorescence Lifetime System. Fourier Transform Infra Red (FTIR) spectroscopy was carried on Jasco 4700 FT/IR instrument. Circular dichroism (CD) spectra were measured in Chirascan spectropolarimeter (Applied Photophysics, UK). Slides containing cells were observed under DM 900 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Thermo scientific Multiscan FC microplate reader was used for MTT assay. Synthesis of AuNC@BSA. Fluorescent gold nanoclusters with BSA protein i.e, AuNC@BSA was prepared using 27 previous reports with slight modification. An aqueous solution of 10 mM HAuCl4.H2O was added to 1 ml of 2.5 mg DTT (dithiothreitol). The mixture solution is brown in color was then added to 3 ml bovine serum albumin (BSA) solution (50 mg/ml). The pH of the solution was adjusted to pH ~11 by addition of sodium hydroxide (1M NaOH; 500 µl) and the mixture was stirred for 1 hr at 50 °C. The color of the solution gradually changed from dark yellow to dark orange indicating the formation of fluorescent gold nanoclusters. The dark orange solution emitted strong red fluorescence under UV lamp of 365 nm wavelength. The synthesized nanocluster was purified by centrifugation technique using a centrifuge tube of molecular cut-off of 10 kDa to remove any kind of small impurities. The purified solution was dried and kept at 4 °C for further use. BSA stabilized fluorescent gold nanoclusters (AuNC) with same reaction condition but without DTT was also synthesized for comparison with AuNC@BSA. Characterization of AuNC@BSA. AuNC@BSA was characterized by UV-vis spectroscopy, particle size and charge analysis and transmission electron microscopy (TEM) techniques. Fluorescence spectroscopy was used to record the emission spectra of AuNC@BSA at an excitation wavelength of 520 nm. Since BSA was used in the synthesis, protein folding changes during the process were monitored using circular dichroism spectropolarimeter at 25 °C. The samples were scanned in far UV region from 190-270 nm in a cleaned quartz cuvette of 10 mm path length. The spectra were expressed in terms of molar ellipticity. XPS study was
performed to determine the composition of fluorescent gold nanocluster after synthesis. The raw XPS data were corrected using the binding energy of the C 1s peak at 284.8 eV. FTIR spectra were recorded to determine the functional groups present within BSA network. The FTIR spectrum was blank subtracted and baseline corrected using the software. The mass of the AuNC@BSA, BSA and AuNCs was calculated from MALDI-TOF mass spectrometry experiment. The TCSPC analysis was carried out to determine the fluorescence decay of AuNC@BSA excited at 455 nm wavelength. The charge and size of AuNC@BSA were evaluated to determine the stability of the nanosensor in solution. The average diameter was measured by NS500 in aqueous medium at 25 °C. The surface charge was determined by zeta potential measurement according to the manufacturer’s instructions for measurement in high ionic strength media at 25 °C. All measurements were performed in triplicate following dilution of nanosensor by dispersing in cell culture grade HPLC water (1.0 mg/mL). Next, the wellII characterized AuNC@BSA was tested for Pb binding affinity using UV-vis, fluorescence, TCSPC, XPS and FTIR techniques. The values of size and charge for AuNC@BSA at different pH solutions are shown in Table S1. The stability study was performed by mixing AuNC@BSA with 10% NaCl, 0.05% HSA, 0.05% cysteine and different pH solutions. The stability was measured with fluorescence spectrophotometer over a period of 2 hr to 24 hr. The similar study was also performed with only BSA and AuNCs made without DTT as controls. Slides containing cells treated with AuNC@BSA and II Pb ion was monitored under confocal microscope at 480 nm. Quantum Yield measurement of AuNC@BSA. The quantum yield (QY) of AuNC@BSA was calculated with respect to Rhodamine B as a reference using a comparative method. The below equation was followed to calculate QY of 18 AuNC@BSA: 2 QYsample = QYreference x (Gradsample/Gradreference) (η sample/ 2 η reference) where Gradsample and Gradreference is the gradient of the plot of integrated fluorescence intensity vs absorbance of the sample and reference respectively, and QYreference is the quantum yield of the reference Rhodamine B (31% in water) used to obtain QYsample i.e. AuNC@BSA. The refractive indexes (ηsample and ηreference) of the solvent were 1.33 for both sample and reference, as water was used for the experiment. Fluorescence spectrophotometer was used to measure the fluorescence intensity at 520 nm excitation wavelength. II
Pb detection using visual and spectroscopic techniques. A stock solution of AuNC@BSA (25.0 mg/ml) and lead ion (10.0, 1.0, 0.1, 0.01, 0.001 ppm) were prepared II with MilliQ water. Fluorometric detection of Pb ion was performed to determine the selectivity and lowest concentration detection ability of the nanosensor. During the fluorometric study, 0.1 ml of different concentrations of each lead ion (10.0, 1.0, 0.1, 0.01, 0.001 ppm) was added into AuNC@BSA solution (0.1 ml) to estimate the lowest limit of detection by the naked eye under UV lamp of 365 nm wavelength and also monitored by fluorescence II spectrophotometer. Selectivity of Pb ion for the synthesized AuNC@BSA was tested against various metal ions. 0.1 ml of
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nanosensor in aqueous phase was mixed separately with 0.1 I I ml (1.0 ppm) of various common metal ions- Na (NaCl), K II II II III (KCl), Cu (CuCl2), Cd (Cd(NO3)2), Mg (MgCl2), Cr (CrCl3), III II II II Fe (FeCl3), Hg (Hg(NO3)2), Ni (NiSO4), and Ca (CaCl2). Change in fluorescence of the solutions was observed visually by naked eye using UV lamp of 365 nm wavelength. The corresponding emission spectra were recorded in the fluorescence spectrophotometer. Following this experiment, the sensitivity test was also performed in straight paper strips. For this purpose, Whatman filter paper was cut using CO2 laser engraving system (VLS 2.30, Universal Laser Inc., USA) at 3 Watt. The engraved strip parameters were as follows: length (l) = 15.0 mm, width (w) = 5.0 mm and height (h) = 0.1 mm (100 μm). The filter paper strips were soaked in warm HPLC grade water for removing impurities, if any. AuNC@BSA was adsorbed and dried on the straight paper strips. The nanosensor modified paper strips were then kept II vertically on petridishes having the solution of Pb ion with different concentrations. In another experiment, a custom II made lead free tap water spiked with Pb ion solution was also prepared to monitor the performance analysis of AuNC@BSA on the paper strip. As the solution containing II Pb ion comes in contact with the nanosensor, differences in red fluorescence colors were observed under UV lamp of 365 nm wavelength and recorded using a digital camera. To II validate the concentration of the Pb ion spiked in the tap II water, a series of Pb ion concentration (0, 0.00025, 0.0025, 0.025, 0.25, 2.5 ppm) was prepared. A calibration curve was obtained from the ratio of fluorescence intensity I0/I vs II concentration of Pb ion, where I0 is the fluorescence intensity of the control and I is the fluorescence intensity of II AuNC@BSA after Pb ion addition. The I0/I was also calculated for the spiked sample and the lead concentration was obtained from the plotted calibration curve. Lifetime measurement study of AuNC@BSA and after Pb interaction. The time correlated single photon counting (TCSPC) analysis of AuNC@BSA nanosensor in absence and presence of Pb(II) ion was performed using Horiba Jobin Yvon Fluorescence lifetime system. The fluorescence decay curves were fitted by a double 15 exponential function using the following equation: II
where D indicates the fluorescence decay, τ is the lifetime, τi is the lifetime of various fluorescent components and i is corresponding pre-exponential factors. The fluorescence lifetime decay was estimated at 455 nm excitation wavelength using a colloidal silica particle solution as a reference. The average lifetime (τav) was calculated from below equation:
Reversibility of
[email protected] reversibility study of AuNC@BSA was performed by observing the fluorescent
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color of the nanosensor after addition of strong chelating agent EDTA to a mixture of AuNC@BSA and lead ion -4 solution (10 ppm). Upon addition of 1.0 x 10 M EDTA, the red fluorescence of the nanosensor recovered back to the original state under UV lamp of 365 nm wavelength. This reversibility of the nanosensor was also monitored through fluorescence spectroscopic analysis before and after EDTA treatment. Cell culture and toxicity evaluation of AuNC@BSA. Cervical cancer cell line HeLa purchased from NCCS, Pune II was used for intracellular Pb ion detection study. The cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) medium with 10% fetal bovine serum (FBS) and 1% penstrip (penicillin-streptomycin) at 5% CO2 incubator at 37 °C. In vitro cytotoxicity evaluation of AuNC@BSA was 28 4 performed as described in previous literature. Briefly, 1×10 Hela cells at their exponential growth phase were seeded in each well of a flat-bottomed 96-well polystyrene coated plate and were incubated at 37 °C for 24 hr in an incubator at 5% CO2 environment. A series of concentrations - 0, 0.25, 0.5, 0.75 and 1 mg/mL of the synthesized nanosensor were made in cell culture grade water. Each concentration was added to the plate in triplet manner. After 3 hr incubation, all the media was removed and 100 µL of fresh medium was added. 10 μL of MTT per well (stock solution 5 mg in 1ml PBS) was added and incubated for 4 hr at 37 °C, and the formazan crystals so formed were dissolved in 50 μL DMSO (dimethyl sulfoxide). The plates were kept for 10 min in dark at 37 °C to dissolve all crystals, and the intensity of developed color was measured in a micro plate reader (Thermo Scientific Multiskan FC) operating at 570 nm wavelength. Wells with complete medium, nanosensor, and MTT, but without cells, were used as blanks. Untreated cells were considered 100% viable. II
Detection of Pb ion in live cells using confocal II microscopic study. Intracellular sensing of Pb ion in live cells was performed with HeLa cell line. HeLa cells were grown in poly-L-lysine coated coverslip (washed and cleaned with piranha solution) up to 85% confluency and were incubated with AuNC@BSA for 3 hr and after that, the cells with the internalized nanosensor was treated with different II concentrations of Pb ion -10.0 ppm and 1.0 ppm. Control set II was done without the addition of Pb ion. Another set was II incubated with Pb ion of different concentration, kept in CO2 incubator at 37 °C for 3 hr for internalization, and then incubated with AuNC@BSA for another 3 hr. After incubation, the coverslips were removed from the medium and washed several times with ice-cold 1X PBS buffer. The coverslips were then fixed with 4% p-formaldehyde for 10 min at room temperature and the nuclei were stained with DAPI for fluorescence imaging and fixed on a microscopic slide. The slides were then observed under fluorescence confocal microscope at an excitation wavelength of 480 nm.
RESULTS AND DISCUSSION
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Design and synthesis of AuNC@BSA nanosensor. Synthesis of AuNC@BSA and its working principle as nanosensor for detecting PbII ion in aqueous solution are shown in schematic presentation Fig. 1. An aqueous solution of HAuCl4 was added to DTT solution at room temperature to form a brown color complex which represents the formation of Au(III)-DTT thiolate species. As soon as the thiolate complex was added to an aqueous BSA solution followed by the addition of 1.0 M NaOH, the color of the mixture immediately changed to pale yellow. This may happen due to the reduction of AuIII to Au0 through AuI state and stabilization of the nanocluster by AuI-thiolate bond formation at 50 °C.27 NaOH increased the pH value of the solution to pH ~11 which in turn increased the reducing power of BSA.29 The color of the solution finally turned into dark yellow to dark orange within 1.0 hr time and showed red emitting fluorescence under UV lamp indicating the formation of fluorescent AuNC@BSA nanocluster. The reaction time, temperature and pH values play an important role in the formation of AuNC@BSA with bright and stable fluorescence. It is known that the preparation of other AuNCs takes ~12 hrs at 37 °C and the reaction time decreases to 10 min if the reaction temperature is maintained at 80 °C.16, 27 In our study, optimal temperature of 50 °C was selected for the reaction as temperature above that can cause degradation of BSA leading to weaker interaction to surface gold atoms.30 At this temperature, the formation of AuNC@BSA happened within 1.0 hr time, which may be due to the activation of BSA in presence of DTT at pH ~ 11. DTT mediated synthesis of AuNC@BSA not only reduced the reaction time, but also enhanced the quantum yield significantly to ~20.0% when pH is adjusted to ~7.5. This enhancement is approximately 2-times higher in comparison to most of the BSA mediated fluorescent gold nanoclusters reported so far (Table S1).15-16, 27 It is also observed that the quantum yield is highly dependent on DTT concentration. If the concentration of DTT increases, the quantum yield of AuNC@BSA increases as shown in Fig. S1. Since maximum intensity with bright red fluorescence was observed at pH ~ 7.5, all the characterization studies of AuNC@BSA were performed at this pH medium. In the UV-vis spectrum, no significant surface plasmon resonance (SPR) peak was observed at ~520 nm suggesting the fact that there are no larger sized gold nanoparticles formed during synthesis.31-32 However, like native BSA, an absorption peak at ~276 nm was observed which is attributed to the presence of π-π* transition of aromatic amino acids in the protein chain of AuNC@BSA (Fig. S2).33 In Fig. 2A and Fig. S3A, AuNC@BSA exhibited an emission maximum at 660 nm and corresponding excitation profile at 375 nm and 520 nm that is similar to the reported values for the BSA stabilized gold nanocluster.34 Wen et al. reported a correlation between the structure and optical properties of the Au25 nanocluster stabilized by BSA.35 They observed two fitted peaks ─one at 640 nm due to the 6 x (–S-Au-S-Au-S-) staples and the other at 710 nm due to Au013 icosahedral metal core. Based on these observations, we deconvoluted
Fig. 2 (A) Emission spectra of AuNC@BSA at 660 nm in II absence (1) and presence (2) of Pb ion when excited at 520 nm, (B) Fluorescent color developed under visible and UV light (365 nm). (C) TEM image of AuNC@BSA (circled in red) and corresponding size distribution showing size
