Fluorescent Indicator Displacement Assay: Ultrasensitive Detection of

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Fluorescent Indicator Displacement Assay: Ultrasensitive Detection of Glutathione and Selective Cancer Cell Imaging Krishnendu Das, Saheli Sarkar, and Prasanta Kumar Das ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06353 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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ACS Applied Materials & Interfaces

Fluorescent Indicator Displacement Assay: Ultrasensitive Detection of Glutathione and Selective Cancer Cell Imaging Krishnendu Das, Saheli Sarkar and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata – 700 032, India

*To whom correspondence should be addressed. E-mail: [email protected]

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ABSTRACT The present article reports the development of nanohybrid comprising of anionic carbon dots (ACD) protected gold nanoparticle (GNP). ACD directly cap GNP through its anionic surface functionalization leading to the formation of stable aqueous GNP dispersion. This newly developed ACD-GNP nanohybrid has been thoroughly characterized by different spectroscopic and microscopic techniques. This nanohybrid is successfully employed towards the selective sensing of glutathione (GSH). The mechanism of GSH sensing by this nanosensor is based on the GSH triggered displacement of fluorescent indicator ACD from the GNP surface. Upon capping GNP, intrinsic fluorescence of ACD gets quenched. Addition of GSH displaces the fluorescent indicator ACD from GNP surface and restores the fluorescence signal of ACD. This nanosensor exhibits very high selectivity as well as sensitivity towards glutathione over the other biothiols and can detect as low as 6 nM of GSH. More importantly, selective imaging of the cancer cells over the non-cancerous cells was achieved by this ACD-GNP hybrid implying its potential applications in biosensing as well as in cancer diagnosis.

KEYWORDS: carbon dots, glutathione, gold nanoparticle, imaging, nanohybrid, sensing.

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1. INTRODUCTION Glutathione (GSH), a thiolated tripeptide, is the most abundant endogenous antioxidant which plays crucial role in defense against toxins and free radicals within mammalian cells.1,2 It protects different cellular constituents by scavenging reactive oxygen species (ROS) like free radicals, peroxides and lipid peroxides through reversible redox process. On the course of neutralizing the ROS, glutathione itself oxidised to GSSG, which further reduced by the glutathione reductase (GSR) to reform GSH. Thus, the ratio of GSSG and GSH (GSSG/GSH) is a key indicator of the cellular redox environment and also the oxidative stress inside the cells.3 Alteration of GSH level is often associated with several diseases like cancer, HIV, mitochondrial damage in brain, Parkinson’s disease etc.3,4 In particular, elevated level of GSH was found within the doxorubicin resistant erythroleukaemia K562 cells.5 Increased level of GSH was also found within squamous cell carcinoma of the lung cell lines, which are resistant to 4hydroxyifosfamide, cisplatin, and methotrexate upon long-term exposure to combination chemotherapy.6 In malignant tumour, GSH protects cancerous cells by rendering resistance to the chemotherapeutic drugs.7 Systematic experiment on HepG2 cells showed that alteration of intracellular GSH plays crucial role in cancer cell responsiveness to cisplatin.8 It is evident that GSH content within tumor cells is significantly higher than that of non-cancerous cells.9 Hence, sensing of intra cellular GSH level is an important task towards fighting against cancer. Different fluorometric techniques based on conventional organic fluorophores and also the combinations of nanomaterials have been exploited for GSH detection.4,9-20 Photobleaching and low sensitivity have restricted the use of organic fluorophores for long-term assay4,9. Nanomaterial based fluorescence “off-on” sensor suffers from low sensitivity and strong interference from other thiol containing functionalities.11,18,19 Recently Shi et al reported a GSH

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sensor based on cationic carbon dots induced aggregation of gold nanoparticle (GNP) and GSH mediated disaggregation of GNP towards the discriminative detection of GSH.11 Electrostatic interaction between amine surface functionalities of carbon dots and citrate capped GNP led to the aggregation of GNP and this aggregated carbon dot-GNP sensor was utilized for GSH sensing. Instead of this dual mode sensing, more simplistic sensing mechanism could improve the applicability of the nanosensor. Furthermore, the said nanosensor suffers interference from other biothiols like cysteine, homocysteine and could not distinguish between cancer and noncancer cells. In this regard Zhang et al had developed GSH detection and selective cancer cell imaging technique based on GSH mediated fluorescence signal amplification of gold nanoclusture comprised of HAuCl4 and histidine.20 Though the detection technique is quite simple but detection limit is quite high (0.2 µM) compared to the other reported techniques.20 Thus, development of fluorometric biosensors for selective detection of GSH in a relatively simple mechanism and simultaneously for selective imaging of cancer cells is a challenging task. Herein, we have developed a nanosensor based on fluorescent carbon dot protected gold nanoparticle (GNP) towards ultrasensitive and selective detection of GSH. Carbon dots have gained much attention in biotechnology because of its intrinsic fluorescence, high photostability, superior biocompatibility and cost effective easy synthesis.21-23 Carbon dots have been exploited in different fields like imaging, sensing, and drug delivery.24-29 Gold nanoparticle (GNP) is also a fascinating class of nanomaterial due to its unique physicochemical and optoelectronic properties. It has been extensively used in sensing, drug delivery and many other fields.30-35 In general, different chemical ligands are used as capping agent to stabilize GNPs of varying dimension in aqueous medium. However, example of nanomaterial protected GNP is really scarce. Herein for the first time we utilized carbon dots as the capping agent of GNPs. Carbon

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dots were chosen as capping agent of GNP because of its significant biocompatibility and intrinsic fluorescence property. The anionic carbon dot (ACD) protected GNP nanosensor can selectively detect as low as 6 nM GSH over the other biothiols. Moreover this nanosensor was used for selective imaging of cancerous cells. 2. EXPERIMENTAL SECTION 2.1. Materials. Citric acid was purchased from Spectrochem (India). L-glutathione, glycine, L-cysteine were purchased from SRL Chemicals (India). L-Glutathione oxidised, L-homocysteine, gold (III) chloride (HAuCl4) solution and MTT were procured from Sigma Aldrich. All experiments were carried out using Milli-Q water. The absorption spectra were taken in PerkinElmer spectrophotometer. Varian Cary Eclipse luminescence spectrometer was used to record fluorescence spectra. Centrifugation was done using Thermo Scientific Espresso centrifuge. Nano-ZS of Malvern Instruments Limited was used to measure zeta potential. Perkin Elmer Spectrum 100 was used to record FTIR spectra a. Heat inactivated fetal bovine serum (FBS), dulbecco’s Modified Eagles’ Medium (DMEM), and trypsin (from porcine pancreas) were purchased from Himedia. B16F10 and CHO cells were received from NCCS, Pune, India. 2.2. Synthesis of Anionic Carbon Dot (ACD). Anionic carbon dots (ACD) have been synthesized following the reported protocols.21 Towards the synthesis of ACDs, sodium salt of glycine (1.1 g, 14 mmol) was prepared upon mixing with equivalent quantity of NaOH (2 mL aqueous solution). Citric acid (3 g, 14 mmol) taken in water (2 mL) was added to this carboxylate salt to form a homogeneous mixture. This mixture of citric acid and carboxylate salt was subjected to lyophilization to get the solid mass, which was followed by grinded to prepare fine powder. This powder taken in a porcelain crucible was

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heated in a furnace at 200 °C for 2 h and then allowed to cool to room temperature. The brownish-black mass was extracted using water (25 mL). The suspension was centrifuged at 12000 rpm for 30 min and the residue was discarded. The supernatant was lyophilized to get the ACDs with the yield of ~60%. 2.3. Quantum Yield of ACD. Quantum yield of ACDs was measured following the previously reported methods.23 The ACD was taken up to a concentration so that the absorbance remains restricted below 0.01. Integrated emission signal of the solution was determined at excitation maxima (340 nm). The quantum yield (QY) of the ACDs was calculated by the equation depicted below: Q = Qst(Ism/Ist)(ODst/ODsm)(ηsm2 /ηst 2 )

(1)

where Q is the quantum yield, I designates the measured integrated emission intensity, η refers to refractive index, and OD represents optical density, which was restricted below 0.01. The subscript ‘sm’ and ‘st’ refers to sample and standard fluorophore of known fluorescence, respectively. Quinine sulfate (solution in 0.1 M H2SO4) was used as a standard, whose QY is 0.54. 2.4. Synthesis of ACD protected GNP. Stable aqueous dispersion of gold nanoparticle (GNP) was synthesized by following the conventional NaBH4 reduction protocol. Freshly prepared aqueous NaBH4 solution (1.0 mM) was maintained at ice cold condition and followed by added to aqueous HAuCl4 solution (0.5 mM) under stirring condition. Required volume of aqueous solution of anionic carbon dots (ACD) was added immediately to achieve final concentration of 25, 50, 100, 150, 200, 300 and 500 µgmL-1 in 2 mL GNP solution (0.5 mM). 2.5. Synthesis of Citrate capped GNP.

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Citrate capped GNP was synthesized by following the previously reported protocol.33 To a 2 mL aqueous solution of HAuCl4 (ice-cold), freshly prepared aqueous NaBH4 solution was added along with trisodium citrate. The final concentrations of HAuCl4, trisodium citrate and NaBH4 were 0.5 mM, 0.5 mM and 1.0 mM, respectively. The solution became wine red and it showed surface plasmon resonance (SPR) peak at 533 nm. 2.6. Characterization. Samples for transmission electron microscopy (TEM) were prepared by casting 4 µL of the synthesized ACDs solution on Cu-coated TEM grid (300-mesh) and subjected to drying under vacuum for 4 h prior to imaging. JEOL JEM microscope (2100F UHR) was used for taking TEM images. The ACD solution (10 µL) was placed on a rectangular Cu plate followed by drying under vacuum for 8 h before the X-ray photoelectron spectroscopy (XPS) experiment. XPS analysis was carried out in X-ray photoelectron spectrometer (Omicron, series 0571). X-ray diffraction (XRD) spectra of powdered ACD and ACD protected GNP were recorded on a Bruker D8 Advance diffractometer and the source was CuKα radiation (α= 0.15406 nm) with a voltage 40 kV and current 30 mA. 2.7. Fluorescence Decay Experiment. Time correlation single photon count (TCSPC) measurement was carried out in a picosecond diode laser IBH-405. Native ACD (10 µgmL-1) solution of 2 mL and a solution of 25 µM ACDGNP (2 mL) conjugate (prepared using 25 µM GNP and 10 µgmL-1 of ACD) were subjected to the analysis. Samples were excited at 340 nm. IBH DAS6 software was used for fluorescence decay analysis. Time resolved fluorescence decay p(t) was analyzed using equation (2)

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where n = number of discrete emissive species, b = baseline correction (“dc” offset), i

= pre-exponential factors related with the ith component

i

= excited state fluorescence lifetimes related with the ith component. The average lifetime for multi exponential decays was analyzed using equation (3).

Where

which refers to the contribution of a decay component.

2.8. Glutathione (GSH) Sensing Using ACD-GNP Nanohybrid. The fluorescence response of this ACD-GNP nanosensor towards GSH was investigated by adding varying concentration of GSH (0-50 µM) within 25 µM ACD-GNP nanohybrid. The fluorescence intensity was measured at 420 nm upon exciting the ACD-GNP nanohybrid at 340 nm. 2.9. Cell culture. B16F10 (melanoma cells) and CHO (Chinese Hamster Ovary) cells were received from National Centre for Cell Science, Pune and cultured in FBS (10%) supplemented DMEM media in presence of streptomycin (100 mg/L) and penicillin (100 IU/mL). The cells were grown in culture flask (25 mL) and maintained at 37 °C under humidified atmosphere of 5% CO2 to ~ 7080% confluence. Sub-culture of both cells was carried out in every 2-3 days. FBS supplemented media was replaced after 48-72 h. Trypsinization was done to detach the adherent cells from the culture flask surface. These cells were used for cytotoxicity and imaging experiments. 8


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2.10. Stability Experiments. The stability of the ACD-GNP nanohybrid in pure water, pH 7.4 PBS buffer and FBS supplemented media was measured by adding the ACD-GNP conjugate in the respective medium and kept for desired time span. In all cases, the final concentration of ACD-GNP was 100 µM. The absorbance of these solutions was recorded at 533 nm (corresponding to the SPR peak of the GNP) at different time interval to determine the suspension stability index. Suspension Stability Index (SSI) = (At/A0) x 100

(4)

Where At = Absorbance of the solutions at 533 nm at different time intervals. A0 = Initial absorbance of the solutions at 533 nm. 2.11. MTT Assay. Cell viability of ACD protected GNP (ACD-GNP) was investigated by the microculture MTT reduction method.36 This method involves the conversion of tetrazolium salt to a colored formazan (water insoluble) product by mitochondrial dehydrogenase of alive cells. The quantity of formazan was estimated by measuring the absorbance of the product upon dissolving in DMSO. The formation of formazan is directly related to the number of live cells. B16F10 and CHO cells were separately cultured with 15,000 cells per well in a 96-well microtiter plate 18-24 h before the assay. The amount of ACD-GNP was varied from 10-50 µM in the 96-well plate. The cells were incubated under 5% CO2 at 37 °C for 24 h followed by incubation with 15 µL MTT solution (5 mg/mL) for 4 h. Water insoluble formazan was dissolved in DMSO and absorbance was recorded at 570 nm using Elisa Reader. The number of alive cells were expressed as percent viability = (A570(treated cells)-background)/ A570(untreated cells)background) × 100. 9



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2.12. Bioimaging. B16F10 and CHO cells were cultured at a concentration of 104 cells per well within a chamber slide 18-24 h prior to the experiment. Cells were treated individually with 25 µM of ACD-GNP solutions for 6 h. In case of imaging of CHO cells pretreated with glutathione, 104 cells were preincubated with 5 mM GSH for 6 h and then excess GSH was taken away from the medium. After that 25 µM of ACD-GNP solutions was added within the cells and kept for 6 h. The treated cells were washed with PBS buffer (phosphate-buffered saline), followed by fixed with paraformaldehyde (4%) for 30 min. Thereafter, glycerol solution (50%) was used to mount the cells on slide, which was covered with a cover slip and set aside for 24 h. Imaging experiment was carried out in fluorescence microscope (having excitation filter BP330-385 nm and a band absorbance filter covering wavelength below 405 nm). 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Anionic Carbon Dots (ACD). Carbon dots are the interesting class of nanomaterials because of their unique physicochemical properties. Among many other distinct features of carbon dots, our objective was to exploit the intrinsic fluorescence character of carbon dots in biosensing. To this end, anionic carbon dot (ACD) has been synthesized following the reported protocols (detail is given in Experimental Section).21 Zeta potential of the aqueous solution of carbon dots was found to be -21 mV, which implies the anionic surface charge of the carbon dots and high aqueous stability. Dynamic light scattering (DLS) experiment showed that the hydrodynamic diameter of the ACDs are around 710 nm (Figure S1a, Supporting Information (SI)). The ACDs were found to be 3-5 nm in size from scanning transmission electron microscopic (STEM) images (Figure 1a). These ACDs showed blue emission under UV exposure (365 nm) and upon excitation at 340 nm, strong

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fluorescence signal was observed at 420 nm (Figure 2a). FTIR spectrum of ACD showed two distinct peaks at 1700 and 1638 cm-1 indicating the presence of amide linkage and carboxylate groups on ACD surface (Figure S2a, SI).21 This confirms the successful thermal coupling between Na-salt of glycine and citric acid towards the surface functionalization of ACD with Nasalt of glycine where citric acid forms the carbon core. The quantum yield of the intrinsically fluorescent ACD was found to be 3.7 % with respect to quinine sulfate as standard. These synthesized ACDs were used as the capping agent of GNP. 3.2. Synthesis and Characterization of ACD Protected Gold Nanoparticle (ACD-GNP). Gold nanoparticles (GNPs) are gaining attention

in different research arena, including

biosensing, nanobiotechnology, and others due to its unique optoelectronic and molecular recognition properties.37-46 Different chemical ligands including task specific unit have been exploited to form the stable aqueous dispersion of GNPs.43-46 Interestingly, nanomaterials have not been explored yet as capping agent to develop stable aqueous dispersion of GNPs. Herein, we utilized ACD towards the preparation of stable aqueous dispersion of GNPs. ACD protected gold nanoparticles were synthesized following the conventional NaBH4 reduction protocol. Concentration ratio of HAuCl4 (0.5 mM) and NaBH4 (1.0 mM) was maintained as 1:2 and the ACD concentration was varied from 25 to 500 µgmL-1 (detail synthetic procedure is given in experimental section, Table S1, SI). Stable aqueous GNP solution was formed at and above 200 µgmL-1 of ACDs (Figure 3). Below 200 µgmL-1 of ACDs, insufficient capping agent probably led to the instability of GNP solution. The carboxylate (COO-1) functionalities of ACDs probably anchors on the GNP surface in a similar way that of citrate capped GNP.47 ACD capped GNPs (ACD-GNP) showed characteristic SPR peak at 533 nm in absorption spectroscopy indicating the successful formation of distinct GNP (Figure 2b).48 The ACD-GNP hybrid was further

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characterized with FTIR spectroscopy. FTIR spectrum of ACD-GNP conjugate showed two distinct peaks at 1700 and 1638 cm-1 indicating the existence of amide linkage as well as free carboxylate groups (Figure S2b, SI). The zeta potential of the nanohybrid was found to be -27 mV due to the presence of free carboxylate moiety of ACD-GNP hybrid. The intrinsic fluorescence intensity of ACDs (10 µgmL-1) got drastically quenched upon capping on the surface of GNP (25 µM), which is a well known fluorescence quencher (Figure 2a).49,50 Close association of fluorescent molecules towards the GNP leads to the quenching of emission intensity of the fluorescent molecule through resonance energy transfer.49 Interestingly, separate mixing of citrate capped GNP (25 µM) with ACD (10 µgmL-1) could not induce any notable quenching in the fluorescence signal of native ACD (Figure S3a, SI) which indicates that ACD and citrated capped GNP are not in close association under the mentioned experimental condition. In contrast, upon formation of the ACD-GNP hybrid by capping of GNP with ACD, emission intensity of ACD notably quenched ACD (Figure 2). This discriminating behaviour of separately added ACD and citrate capped GNP with the ACD capped GNP clearly delineates the close association of ACD on GNP surface for ACD-GNP hybrid possibly because of the capping of GNP through carboxylate functionalities of ACD. At 300 µgmL-1 and above concentration of ACDs (15 and 25 µgmL-1, respectively after dilution), moderate fluorescence intensity was observed for ACD-GNP hybrid possibly due to the presence of excess free carbon dots in the medium (Figure S3b, SI). STEM images revealed that the size of GNP is around 20 nm and the presence of ACDs around it confirms the successful capping of GNP with ACDs (Figure 1b). The size of ACD-GNP conjugate was found to be around 25-30 nm from DLS study (Figure S1b, SI) which is in concurrence with the observed size of the nanohybrid in STEM image (Figure 1b).

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ACD-GNP was further characterized with X-ray diffraction (XRD) analysis. A broad peak around 24-30° is indicative of amorphous carbon dots and sharp peaks around 38° (GNP111), 45° (GNP200) and 65° (GNP220) indicate the presence of crystalline GNP (Figure S4, SI).51,52 Thus simultaneous presence of characteristic peaks of both ACDs and GNP confirms the coexistence of crystalline GNP and ACD. Moreover, XPS analysis was performed to investigate the ACD protected GNP. XPS analysis of ACD-GNP showed characteristic peaks of ACD at 285 eV (C 1s), 418 eV (N 1s), 532 eV (O 1s) (Figure 4a).23 Peaks at 87.71 eV (Au 4f5/2), 83.7 eV (Au 4f7/2), 337 eV (Au 4d5/2), 338.4 (Au 4d3/2) indicate the presence of elemental gold (Au0) (Figure 4b, 4c).47 At this point XPS analysis was carried out for analyzing of the surface chemistry of ACD-GNP nanohybrid. Coordination of functional groups is strongly related with binding energy of electrons of an atom as well as the electronic environment of adjacent atom. Deconvoluted C1s spectrum consisting of three distinct peaks at 284.8, 287.5 and 290.6 eV are characteristic peaks of carbon (C-C or CH), coordinated carboxylate (COO-Au) and free carboxyl moiety (COO-1), respectively (Figure 4d).47 Interestingly, The C1s spectra of native ACD showed a single peak at 284.8 eV (characteristic peak of carbon (C-C or C-H (Figure S5, SI). Deconvoluted C1s spectra of native ACD did not show any peak at 287.5 eV corresponding to the Au coordinated carboxylate (COO-Au) (Figure S5, SI). This COO-Au peak could only be generated if ACD caps GNP through its carboxylate surface functionalities. Thus, presence of a peak at 287.5 eV in the deconvoluted C1s spectra of ACD-GNP hybrid clearly suggests the successful formation of ACD capped GNP. We also carried out fluorescence decay experiment (TCSPC) to investigate the fluorescence quenching mechanism. The decay time of native ACD and ACD-GNP were

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measured by exciting the samples at 340 nm. The lifetime of ACD was found to be 6.38 ns whereas after capping with GNP, the life time of ACD-GNP hybrid decreased to 4.55 ns (Figure S6, SI). The shorter lifetime reveals the dynamic quenching of ACD upon anchoring on the GNP surface.18 Close association of ACD to the GNP leads to the quenching of fluorescence signal of ACD through resonance energy transfer.49 This result also supports the formation of ACD-GNP conjugate through capping of GNP with the carboxylate surface functionalities of ACD. 3.3 Standardization of Nanohybrid Concentration for Glutathione (GSH) Sensing. At this point we intend to utilize this ACD capped GNP towards selective sensing of GSH. GNP (0.5 mM) stabilized with ACD (200 µgmL-1) was taken as the standard solution. GNP stabilized with higher concentration of ACDs (300 and 500 µgmL-1) was avoided to minimize the background signal (Figure S3b, Figure S7, SI). To employ this ACD-GNP nanosensor for GSH sensing, the standard solution was diluted 20 times to have final concentration of GNP (25 µM) and ACD at 10 µgmL-1. Now, the fluorescence response of this diluted solution of ACD-GNP (25 µM with respect to GNP, 10 µgmL-1 with respect to ACD) against a fixed concentration of GSH (50 µM) was investigated with time. The fluorescence signal of ACD at 420 nm gradually improved with increase in time and did not change any further after 5 min (Figure S8, SI). Thus for GSH detection experiment, a 10 min incubation period was always maintained to get the maximum fluorescence intensity of released ACDs (Scheme 1). Now, to standardize the optimum concentration of ACD-GNP to be used for GSH sensing, we diluted the standard ACDGNP solution (0.5 mM GNP stabilized with 200 gmL-1 ACD) into three different concentrations of 10, 25 and 50 µM solution with respect to gold concentration where ACD concentration gets fixed at 4, 10, 20 µgmL-1, respectively (Figure S7, SI). To each of this diluted solution of ACDGNP, 50 µM of GSH was added and kept for 10 min. The maximum fluorescence response was 14


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found at 25 µM concentration of ACD-GNP (Figure 5a). Hence, ACD-GNP (25 µM with respect to gold) having 10 µgmL-1 of ACD was used for further studies. 3.4 Glutathione Sensing with ACD-GNP Nanohybrid. To investigate the fluorescence response of this ACD-GNP nanosensor towards GSH, varying concentration (0-50 µM) of GSH was incubated with ACD-GNP (25 µM). Upon increase in the GSH concentration, the fluorescence signal of ACD at 420 nm gradually increased (Figure 5b). At 50 µM GSH, almost 75% fluorescence intensity was restored with respect to the fluorescence intensity of native ACD (10 µgmL-1). This fluorescence enhancement of ACD in presence of GSH could be attributed to the capping of GNP by GSH and displacement of the fluorescent indicator (ACD) from the GNP (quencher) surface (Scheme 1). The multidentate anchoring ability of GSH enables it to bind with GNP surface in strong affinity by replacing the ACDs. The detection limit was as low as 6 nM. Importantly, the plot of fluorescence intensity vs GSH concentration resembles polynomial curve over a concentration range of GSH from 0 to 1 µM (Figure 5c). Moreover the plot of fluorescence response ((F-F0)/F0, where F0 is the emission intensity of ACD-GNP and F is the emission intensity of displaced ACD in presence of GSH) vs. concentration of GSH showed two linear relationship in two different concentration range (0 to 100 nM and 100 to 1 µM) (Figure 5d).20 Now it would be interesting to investigate whether there is any change in shape and size of the GNP after replacement of carbon dots with GSH as capping agent. The aqueous solution of ACD did not show any distinct peaks in the UV-vis spectrum (Figure 6).21 The native SPR peak of ACD capped GNP at 533 nm remain almost unaltered in presence of 6 nM as well as very high concentration of GSH (50 µM) (Figure 6). This implies no change in shape and size of GNP took place by the displacement of ACDs in presence of GSH. Similarly, according to the

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STEM image, absence of ACD around the GNP in presence of GSH (50 µM) demonstrates the GSH triggered displacement of ACD from GNP surface (Figure S7, SI). Also, STEM image clearly delineates that size of GNP remains unaltered upon displacement of ACD from GNP surface in presence of GSH (Figure S9, SI). The sensing mechanism is governed by the displacement of fluorescence indicator ACD from the quencher GNP surface. ACDs directly cap GNP through its carboxylate surface functionalities. As a consequence GNP, a well known fluorescence quencher drops the emission intensity of the fluorescent indicator ACD. GSH, having a unique steric structure and multidentate anchoring ability, scavenges GNP from the ACD-GNP conjugate and releases the ACD from the GNP surface which eventually leads to the restoration of fluorescence signal of ACD as it gets away from the surface of GNP. This capping agent exchange resulted in the fluorescence turn on of ACD (Scheme 1). 3.5 Selectivity towards Glutathione (GSH). The selectivity of this ACD-GNP nanosensor towards the GSH was also investigated using control experiments with competitive biothiols like cysteine (Cys), homocysteine (HCy) and glutathione oxidised (GSSG) under similar experimental conditions. In comparison to GSH, other competitive biothiols at 50 µM could not induce any notable change in the fluorescence signal of ACD (Figure 7). This clearly indicates the selective sensing of GSH over the other biothiols by the ACD-GNP hybrid. In this context, previous studies revealed that Cys and HCy are more capable of inducing aggregation of GNP than that of GSH.53 The difference in steric hindrance effect and coordination capability of GSH over the other biothiols possibly led to the discriminative detection of GSH by this ACD-GNP nanosensor. To evaluate the interference from other amino acids, this ACD-GNP nanosensor (25 µM) was screened through seven other

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naturally occurring amino acids at a concentration of 200 µM. Interestingly, they could not induce any notable change in the fluorescence signal of ACD-GNP nanosensor (Figure S10a, SI). More importantly, Cys, HCy and GSSG even at high concentration (200 µM) could not affect the fluorescence signal in a notable extent (Figure S10a, SI). Moreover, the nanosensor was screened through other biologically relevant molecules such as ascorbic acid, vitamin-E, DTT, BSA protein, glucose and ions such as Na+, K+ and S2- which also could not affect the fluorescence signal of ACD-GNP nanosensor (50 µM) in notable extent (Figure S10b, SI). Thus, the newly developed ACD-GNP nanosensor can selectively detect GSH over the other biothiols and other biologically relevant molecules. Also the GSH detection limit of this ACD-GNP hybrid is as low as 6 nM. Unlike the work of Shi et al,11 in the present investigation, the direct attachment of ACD on GNP surface possibly made this nanosensor more sensitive as well as more selective towards GSH.11 The simplistic and selective sensing mechanism of GSH by this ACD-GNP nanohybrid is found to be more advantageous in comparison to the previously reported nanomaterial based fluorometric detection techniques (Table S2, SI). 3.6 Selective Cancer Cell Imaging. Herein, this ACD-GNP nanosensor was used to sense GSH inside the mammalian cells. However, the stability and the cell viability of the ACD-GNP nanohybrid in biological milieu need to be examined prior to its application in biosensing. To this objective, we investigated the stability of this ACD-GNP nanosensor (100 µM) in pure water, pH 7.4 PBS buffer and FBS supplemented DMEM media. ACD-GNP nanosensor (100 µM) showed superior stability in the FBS supplemented DMEM media over 24 h incubation period and no coagulation or precipitation of ACD-GNP was observed even at high concentration of FBS which also reflected in the corresponding suspension stability index (Figure S11a, SI). This indicates the superior

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stability of the nanosensor in biological milieu. ACD-GNP nanosensor at a 100 µM concentration also showed superior stability in pure water and pH 7.4 PBS buffer over 5 days span (Figure S11b, S11c, SI). The cell viability of ACD-GNP hybrid was evaluated using MTT assay. B16F10 and CHO cells were incubated with 10-50 µM of ACD-GNP hybrid for 24 h. The nanosensor showed considerable (~70-95%) cell viability towards both cancerous melanoma cells and normal CHO cells even after 24 h incubation (Figure 8). Hence, this ACD-GNP hybrid met the essential prerequisites for utilization in cellular study. The glutathione concentration within cancer cells is reported to be higher than that of non-cancerous cells.3,13,54,55 Moreover, different studies including metastatic behaviour of melanoma cells, resistance of cancer cells towards anticancer drugs, tumor growth promoting mechanism reported very high glutathione content within melanoma cells B16F10.3,6-8 Carretero et al have shown that amount of GSH enhanced in B16 melanoma cells at the early stage of exponential growth in vitro, to attain maximum of ~37 nmol/106 cells after 12 h of plating, and then progressively reduced to base values (~10 nmol/106 cells) when cultures approached confluence.56 Interestingly, Saha et al has reported that the GSH concentration within CHO cells is around 2.8 fmol/ CHO cells.55 These two reports clearly indicate the notable difference in the GSH content within melanoma cell line B16F10 cells and noncancerous CHO cells. To this end, B16F10 and CHO cells were treated with ACD-GNP (25 µM) for 6 h. After the incubation period, B16F10 cells showed blue emission under fluorescence microscope while the CHO cells did not show any notable fluorescence inside the cells (Figure 9). The discriminating fluorescence response of ACD-GNP nanosensor towards cancerous and non-cancerous cells can be explained on the basis of GSH content in different cells. The difference between GSH content within B16F10 and CHO cells enables ACD-GNP nanosensor to detect cancer cell (B16F10)

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selectively through GSH mediated fluorescence turn on technique. High concentration of GSH in cancerous B16F10 cell readily displaces the ACD from the GNP (quencher) surface (Scheme 1) and thereby ACD showed its intrinsic blue emission inside the cells. In contrast, the low content of GSH in normal CHO cells could not induce the displacement of ACD from GNP surface at a given period of time and therefore did not show notable intracellular fluorescence. In this context, incubation of native ACD (10 mgmL-1) with both B16F10 and CHO cells showed blue fluorescence inside the cells under fluorescence microscope (Figure S12, SI). Moreover, CHO cells pre-incubated with 5 mM GSH, showed moderate blue fluorescence inside cells after incubation with 25 µM ACD-GNP nanosensor (Figure S13, SI). This is possibly because of the increase in GSH content inside the CHO cells upon pre-incubation with GSH that led to GSH mediated fluorescence turn on of ACD-GNP nanosensor. Thus, the newly developed ACD-GNP nanosensor can selectively image the cancerous cells through intracellular GSH mediated fluorescence ‘turn on’ technique. 4. CONCLUSION In conclusion, we developed a new class of nanohybrid consisting of anionic carbon dots protected gold nanoparticle. Carbon dots directly cap GNP to form stable aqueous dispersion of GNP. This nanohybrid acts as fluorescence ‘turn on’ sensor for selective detection of GSH over the other biothiols. Upon capping GNP, intrinsic fluorescence of ACD gets quenched. Introduction of glutathione gradually restores the fluorescence signal of ACD. This nanosensor can detect GSH as low as 6 nM. GSH triggered fluorescence ‘on’ technique of the nanohybrid has been successfully employed in selective labelling of cancer cells on the contrary to normal cells which could be beneficial for cancer diagnosis in future.

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ASSOCIATED CONTENT Supporting Information. Table for synthesis of GNP with different concentration of ACD, comparison between different fluorometric detection techniques of GSH, DLS plot of ACD and ACD-GNP, FTIR spectra of ACD and ACD-GNP hybrid, fluorescence spectra of ACD in presence of citrate capped GNP and ACD-GNP with varying concentration of ACD, XRD spectra of ACD-GNP, deconvoluted XPS spectra of C1s of native ACD, time-resolved fluorescence decay of ACD and ACD-GNP, standardization of ACD-GNP nanohybrid concentration for GSH sensing conjugate, time dependent fluorescence response of ACD-GNP in presence of 50 µM GSH, STEM image of ACD-GNP in presence of GSH (50 µM), fluorescence response of ACD-GNP in presence of different amino acids, GSSG, biothiols, ions, sugars, vitamins, optical images and suspension stability index of ACD-GNP hybrid in pure water, pH 7.4 buffer and FBS supplemented DMEM media, bright field and fluorescence microscopic images of B16F10 and CHO cells after 6 h incubation with native ACD, and CHO cells pre-incubated with GSH for 6 h followed by incubation with ACD-GNP hybrid.

ACKNOWLEDGEMENTS P.K.D. is thankful to Council of Scientific and Industrial Research (CSIR), India (ADD, CSC0302) for financial assistance. K. D. and S. S. acknowledge CSIR, India and Department of Science and Technology (DST), India, respectively for research fellowships. The authors are thankful to DST Unit of Nanoscience, IACS for the XPS and TEM facility.

5. References:

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Scheme 1. Glutathione triggered fluorescence ‘turn on’ of anionic carbon dots (ACD).

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Figure 1. STEM images of (a) anionic carbon dots (ACD) and (b) ACD protected gold nanoparticle (GNP).

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Figure 2. (a) Fluorescence signal of ACD and ACD protected GNP upon excitation at 340 nm. Inset images depict the fluorescence of aqueous solution of ACD and ACD-GNP upon UV exposure (365 nm). (b) UV-vis absorption spectra of ACD capped GNP.

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Figure 3. Optical images of ACD capped GNP solutions with varying ACD concentration (GNP concentration was fixed at 0.5 mM and ACD concentration was varied over 25, 50, 100, 150, 200, 300 and 500 µg mL-1).

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Figure 4. (a) XPS spectra of ACD-GNP, (b) characteristic peak of Au 4f, (c) characteristic peak of Au 4d, (d) deconvoluted XPS spectra of C1s.

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Figure 5. (a) Fluorescence response of ACD-GNP with varying concentration of GNP at GSH concentration of 50 µM. F0 is the fluorescence intensity of ACD-GNP and F is the fluorescence intensity of displaced ACD in presence of GSH. (b) Fluorescence response of ACD-GNP upon adding GSH over a concentration range 0-50 µM. (c) Correlation between fluorescence intensity against GSH over a concentration range of 0-1 µM. (d) Two linear range of fluorescence response of ACD-GNP with varying concentration of GSH.

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Figure 6. UV-vis spectra of ACD-GNP (25 µM) in presence and absence of GSH.

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Figure 7. Fluorescence response of ACD-GNP (25 µM) in presence of 50 µM of GSH and other biothiols.

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Figure 8. Percentage cell viability of (a) B16F10 and (b) CHO with varying concentrations of ACD-GNP (10-50 µM) after 24 h incubation. The experimental errors were in the range of 3-5 % in triplicate experiments.

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Figure 9. Bright field and fluorescence microscopic images of cells after 6 h incubation with ACD-GNP (25 µM), (a,b) B16F10 and (c,d) CHO cells.

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Fluorescent Indicator Displacement Assay: Ultrasensitive Detection of

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