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Carbon Dot Based, Naphthalimide Coupled FRET Pair for Highly Selective Ratiometric Detection of Thioredoxin Reductase and Cancer Screening Jagpreet Singh Sidhu, Ashutosh Singh, Neha Garg, and Narinder Singh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07046 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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ACS Applied Materials & Interfaces

Carbon Dot Based, Naphthalimide Coupled FRET Pair for Highly Selective Ratiometric Detection of Thioredoxin Reductase and Cancer Screening Jagpreet Singh Sidhu†, Ashutosh Singh††, Neha Garg††, Narinder Singh†* †

Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar, Punjab 140001, India ††

School of Basic Sciences, Indian Institute of Technology Mandi, Kamand, Mandi, Himachal Pradesh-175005, India

*Corresponding author (Narinder Singh) E-mail: [email protected]; Tel: +91-1881242176 Abstract The Fluorescence resonance energy transfer (FRET) mechanism has been established between the carbon dots (CDs) and naphthalimide to monitor the activity of thioredoxin reductase (TrxR), which is often overexpressed in many cancer cells. The naphthalimide moiety was covalently attached to the surface of CDs through a disulfide linkage. In the normal cell conditions (when devoid of high concentrations of TrxR), the CDs act as an energy donor and naphthalimide acts as an acceptor, which establishes the FRET pair as interpreted from the emission at λem =565 nm, when excited at λex =360 nm. However, contrary to this, the elevated levels of TrxR cause the breakage of disulfide bonds and consequently abolishes the FRET pair through the release of naphthalimide moiety from the surface of CDs. This process was studied by monitoring of fluorescence intensity at λem = 565 nm and λem = 440 nm, when excited at the same wavelength (λex =360 nm). The TrxR based ratiometric quenching and enhancement of fluorescence intensity offers an interesting opportunity to monitor the enzyme activities and has many advantages over the conventional monitoring of fluorescence intensity at a single wavelength to avoid interference of external factors. Fluorescence images of cancer cells in response to nanosensor were visualized under confocal microscope. Cytotoxicity study of nanosensor retards the growth of HeLa and MCF7 cell lines in the presence of visible light. Therefore, nanosensor also acts as theranostic agent to diagnose as well treatment of cancer cells.

Keywords: FRET pair, Carbon Dots, Naphthalimide, Sensor, Cancer Diagnostics 1

ACS Paragon Plus Environment


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Introduction In recent years, detection of cancer cells has fascinated significant attentions among the scientist to distinguish normal cell from affected ones.1 The exact recognition of cancer cells is mandatory to establish for specific removal and treatment of tumor cells from suffered body part without compromising the safety parameters.2,3 Although diverse range of fluorescent probes for sensing of biomolecules have been reported, still there is room for the improvements in term of their aqueous solubility, fast response, broad pH range, and independence from the interference of background signals.4-7 Thus, tremendous efforts are required to conquer these limitations. To adress such problems for sensing of tumor cells, carbon dots were employed as a more effective fluorescence probe, because of their facile synthesis, inert nature towards normal cells, high photostability, good water solubility and easily surface functionalization compared to other imaging techniques.8-11 To distinguish cancer cells from the normal ones, some of the distinct features of tumor cells can be used as a target for development of sensor probes. In this context, we focused the thioredoxin reductase (TrxR) enzyme which is often overexpressed in most types of cancers.12 In normal physiological condition, TrxR play a pivotal role in maintaining the redox homeostasis in the presence of NADP through thio and disulfide reaction mechanism. TrxR is targeted for its ablation to reverse the proliferation of tumor cells and differentiate the affected cells from well functional, normal ones.12-14 Most of the optical sensors for TxrR reported in literature require lysis of cells prior to assay, which is a sluggish and laborious process. Some of the reported fluorescence sensors offer conventional monitoring of fluorescence intensity at single wavelength to analyte.15 Zhang et al. synthesized the first naphthalimide based fluorescent probe for the mammalian TrxR working on the principle of fluorescence “ONOFF” phenomenon.16 Further, Ma et al. also developed a red emission probe for TrxR sensing that also gives change in fluorescence intensity at the single emission wavelength.17 However, a single emission point gets easily influenced in the presence of external factors such as solvent system, environment temperature, concentration, excitation source and nonuniform mixing of receptor solution.18,19 Consequently, quantitative analysis of fluorescence signal is quite difficult, which affects reproducibility of the results. Hence, apposite and consistent sensor probes are highly required to monitor the activity of TrxR Fluorescence resonance energy transfer (FRET) system reduces the fluctuation in detection and also offer freedom from the effect of external factor through considering the fluorescence intensity ratio of two interconnected donor and acceptor units.20,21 Hence, to eliminate such problems, we 2


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have developed a constructive ratiometric nanosensor ‘Biotin-CDs-Naphthalimide’ to target TrxR in cancer cells. This nanosensor is fabricated with disulfide linker that connects the CDs and naphthalimide unit together to transfer the energy from donor to acceptor unit. For this ratiometric nanosensor, 3-aminonaphthalimide moiety was chosen as an energy acceptor unit that emits at 565 nm in the presence of energy donor CDs having emission wavelength 450 nm when excited at 360 nm. CDs in comparison to organic dye as an energy donor unit hold better optical properties such as nonradiative deactivation, tunable emission profile, and high photostability, unlike the conventional dyes which render the CDs more attractive for sensing applications.22 An important feature of CDs for the biological system is their water solubility and well dispersity inside the cells, that enables their utilization for biosensing and bioimaging applications.23-25 Therefore, to utilize a sensor for cell imaging, we have chosen the CDs as an energy donor unit in nanosensor complex. For efficient energy transfer from CDs to naphthalimide, an appropriate distance between these two units was adjusted with cystamine that covalently joined the acceptor and donor units together. Higher level of TrxR in cancer environment reduces the disulfide bond which will liberate naphthalimide from the CDs surface and eliminates the transfer of energy between two chromophores, results in predominance of emission from CDs over the acceptor unit.26-28 Further the overexpression of biotin receptors such as avidin and streptavidin on surface of rapidly proliferating cancer cells, biotin-conjugates favorably get integrated at a cancer site in comparison to normal cells. Thus, conjugation of biotin unit will enhance the uptake of nanosensor more selective to cancer cells hrough endocytosis.29-33 Moreover, 3-aminonaphthalimide derivatives such as amonafide have been explored for their anticancer activity and in the presence of light, redox species are generated from CDs surface also inhibit the growth of tumor cells.34-36 Hence, this nanosensor has also been explored for anticancer activity in the presence as well as absence of visible light. Fluorescence images of cancer cells in response to nanosensor were collected using confocal laser scanning microscope after 45 min and 2 h of treatment. Clear change in color emission from orange yellow to blue was observed in cancer cell after 2 h of treatment. Thus, FRET based nanosensor would assist in the development of new fluorescent probe for sensing of TrxR activity in the living system and screening of cancer cells. Results and Discussions Synthesis and Characterization of Nanosensor: In order to synthesize designed nanosensor for sensing of a TrxR enzyme in cancer cells, the 3-aminonaphthalimide and CDs were synthesized separately. The luminescent CDs bearing the -COOH and -NH2 group were 3



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prepared through the pyrolysis of citric acid and ethylenediamine at 180 °C for 0.5 h.37 The TEM images of CDs reveals the spherical morphology having 5 nm size and monodispersity of dots in solution (Fig. S1). The Powder X-ray diffraction (XRD) pattern of CDs exhibit broad diffraction peaks positioned at 18° (d= 0.42) and 45° (d = 0.12), figure out the disorder of carbon atoms and this is consistent with literature reports (Fig. S2).38 In order to study the size range of particles in solution, the particle size was analyzed with dynamic light scattering and the histogram reveals the particle size (7 nm) slightly higher than the one estimated with TEM (Fig. S3A). The UV-Vis absorption study reveals the band at 245 nm and 350 nm correspondence to π-π* and n- π* transition respectively (Fig. 1A).39 CDs has exhibited characteristics tunable excitation features, i.e. excitation dependent fluorescence emission in aqueous solution (Fig. 1B). The excitation-dependent emission profile was recorded using different excitation wavelength varying from 360 nm to 500 nm. As the excitation wavelength move from 360 nm to 500 nm, the emission intensity was quenched along with a red shift in emission maxima. The exact mechanism of excitation dependent emission of CDs is still obscure. Different theories have been put forward to explain the observations such as quantum confinement, surface traps model, giant red-edge effect, edge states and heteroatom atom models.40-43 Recently, sharma et al. explained the presence of discrete multiple electronic states which depends upon the presence of different functional groups on the surface of CDs responsible for emission properties of CDs.44 Due to this behavior, emission profile of CDs having the desired energy (emission energy of CDs overlapped with the absorption energy of naphthalimide) can be obtained by changing the energy of excitation wavelength. Therefore, without choosing any specific energy donor units, energy from CDs to the acceptor unit in a FRET pair system can be easily adjusted through changing the excitation wavelength of CDs.

4


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A

B

CDs

0.6 0.5 0.4 0.3 0.2 0.1 0.0 200

250

300

350

400

450

λex 360 nm 380 nm 400 nm 420 nm 440 nm 460 nm 480 nm 500 nm

Fluorescence Intensity (a.u.)

0.7

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


500

400

450

Wavelength (nm)

500

550

600

650

Wavelength (nm)

Figure 1. (A) UV-Vis absorption spectrum of as synthesized parent CDs shows absorption band at 345 nm and 245 nm corresponding to π-π* and n- π* transition respectively. (B) Fluorescence emission spectra of CDs at different excitation wavelengths varying from 360 nm to 500 nm. Furthermore, the functional group prevailed over the surface of CDs were analyzed with FTIR spectroscopy (Fig. 2, black line). Broad vibration band from 3500-3200 cm-1 are ascribed to –OH and –NH2 stretching.45 Strong vibration band at 1720 cm-1 (-C=O stretches) and 1240 cm-1 confirms the presence of -COOH group on the surface of CDs. The band at 1420 cm-1 indicate the C-N stretches of CDs along with 1540 cm-1 attributes to N-H bendings.46 Band at 1349 cm-1 corresponds to the deformation of CH2 and –CH3 deformation and at 726 cm-1 for -CH2 alkyl rocking,47 proves the successful synthesis of CDs bearing COOH and -NH2 groups. Energy acceptor 3-aminonaphthalic anhydride was covalently attached to the -COOH of CDs through disulfide linker cystamine via an amide linkage. 3aminonaphthalic anhydride (S3) was synthesized in two steps as outlined in Scheme1. The first step involves the nitration of naphthalic anhydride followed by a reduction of –NO2 in the presence of SnCl2. The linker unit was framed by the protection of its one end amino group with t-Boc and another end of cystamine was easily condensed with acceptor unit in ethanol with 76% yield. To attach the synthesized assembly with parent CDs, t-boc amine was hydrolyzed with TFA to obtain compound S5. The whole synthesized organic assembly was characterized with NMR and Mass spectroscopy (Fig. S16 – S20). Further, compound S5 reacted with -COOH of CDs in the presence of EDC and DMAP (Scheme 1). Thus prepared designed Naph-CDs receptor was purified and characterized by FTIR spectroscopy and DLS to ascertain the formation of amide linkage between CDs and naphthalimide (Fig. 2 red line).

5



CDs Naph-CDs Biotin-Naph-CDs (nanosensor)

Transmitance (%)

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4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Figure 2. FTIR spectrum of parent CDs (black line), naphthalimide conjugated CDs (NaphCDs red line) and nanosensor (Biotin-CD-Naph blue line). As compared to IR spectra of parent CDs, Naph-CDs exhibit an additional vibrational band at 1612 cm-1 and 1496 cm-1 which corresponding to the aromatic ring skeleton of naphthalimde moiety. Intensified vibrational band at 2950 cm-1 along with a small band at 2853 cm-1 represents the –CH2 stretches. Reduction in –OH stretching and C-O bending’s of parent CDs in IR spectra of Naph-CDs clearly evident for the dehydration process and band and 3363 cm-1 stretches indicate –NH stretch of amide linkage confirmed the coupling of CDs with naphthalimide unit.47 Zeta potential of Naph-CDs was investigated to analyze to conjugation of naphthalimide with CDs surface. Decrease in negative charge of CDs from 21.5 mV to 15.7 mV suggests consumption of –COO- due to binding with –NH2 of naphthalimide unit (Fig. S9).48

6


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Scheme 1: Synthesis details of organic receptor and conjugating at the surface of CDs to generate the final Nanosensor (Biotin-CD-Naph). Moreover, coupling of naphthalimide and CDs was also recognized from the changes in the hydrodynamic diameter from 7 nm to 16 nm as analysed from histogram of DLS (Fig. S3B). Further, the biotin moiety was also covalently coupled to Naph-CDs through an amide bond and after purification the desired nanosensor complex was characterized with IR spectroscopy (Fig. 2 blue line), particle diameter (DLS) and zeta potential measurement. Zeta 7



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potential of Naph-CDs changed from -15.7 mV to -18.4 mV may suggest the reaction of – NH2 of CDs with –COOH of biotin. Consumption of –NH2 results into a less number of free amine groups on CDs surface leads to decrease in zeta potential of CDs.48 In IR spectra of nanosensor, new additional absorption band at 3380 cm-1 appeared and broad absorption peak correspondence to –NH2 stretches disappeared suggest the coupling of biotin unit. Additionally, the particle diameter increased from 16 nm to 26 nm highlights the construction of designed Biotin-Naph-CDs nanosensor (Fig. S3C). The UV-Vis absorption spectra of conjugate nanosensor showed a new absorption band at 433 nm (as compared to the absorption profile of parent CDs) correspondence to n-π* transition of naphthalimide unit; along with a slight blue shift in the band at 347 confirms the synthesis of nanosensor (Fig. S4). Emission profile of parent CDs displays significant overlapping with the absorption spectrum of S5 (Fig. S5A, S5B) and nanosensor complex exhibit fluorescence maxima at 565 nm when excited at 360 nm ensure the presence of FRET pair between CDs and naphthalimide moiety. Further, for confirmation of FRET between CDs and naphthalimide in nanosensor, decay time measurements were performed using time-resolved fluorescence spectroscopy. Fluorescence lifetime denotes the average time of the excited state of any molecule when excited from the ground state to excited state.49 Therefore, here, we measured the lifetime of parent CDs and nanosensor because the attachment of energy acceptor to CDs may cause a change in the life time of excited state using 360 nm as excitation wavelength (Fig. S6). Tail fitting decay time curve of parent CDs reveals that CDs manifest average decay time 6 ns while nanosensor exhibit average decay time is 2.9 ns. Decrease in decay time of CDs indicates the efficient transfer of energy from CDs to naphthalimide moiety (Table 1). Shorter decay time (τ /ns) in parent CDs and nanosensor is relates to various 2

trapped states during recombination’s.49 Table. 1 Fluorescence decay and quantum yield measurement of CDs, nanosensor and nanosensor + TrxR Materials

τ /ns

τ /ns

χ

CDs

6.8

5.1

0.996

25

Nanosensor

3.8

1.2

1.04

11

Nanosensor + TrxR

4.2

2.4

1.02

19

1

2

2

Quantum yield (%)

τ: life time, χ : goodness of fit 2

8


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Evaluation of FRET Pair of Nanosensor and TrxR Activities: For efficient transfer of energy from CDs to naphthalimide, the specific excitation wavelength was selected on the basis of absorption maxima of naphthalimide and λem of CDs. As it was observed from the UV-Vis and fluorescence spectroscopic data, absorption maxima of naphthalimide and emission maxima of CDs are noteworthy complementary to each other, when CDs were excited at 360 nm wavelength (Fig. S5A). Therefore, λex 360 nm was selected as most appropriate excitation wavelength for FRET system. Stability of FRET pair system was analyzed by measuring fluorescence intensity ratio I565/I450 at different pH and salt strength (with varying concentrations of NaCl). The pH stability studies of nanosensor demonstrate that a FRET pair system remains significantly intact at the pH range of 4-10. Nanosensor also withstands salt concentration up to 1 µg/mL as evident from insignificant modulation of emission spectra which is meaningful in cell imaging (Fig. S7A, S7B). Further, nanosensor was evaluated for TrxR sensing assay using 30 ug/mL of nanosensor. On addition of TrxR (50 nM), a remarkable change in fluorescence emission profile at 565 nm and 450 nm was observed in time-dependent manner. After 80 min of incubation, plateau phase in fluorescence intensity is reached which was noticed by plotting emission intensity ratio (I450/I565) vs. incubation time as shown in Fig. 3A – 3C. Thus, within time frame of 0 – 80 min fluorescence intensity of donor unit at 450 nm enhanced while the emission intensity of acceptor unit diminished, which is accompanied by a change in emission from orange-yellow to blue (Fig. 3D). Nanosensor exhibit faster response (80 min to reach plateau) to TrxR compared to reported molecule TRFS-red (120 min plateau) and TRFS-green (>180 min).16,17 The addition of TrxR in nanosensor solution causes breakage of disulfide bond of linker units which results into two fluorophore units go far apart from each other leads to the elimination of energy transfer process and FRET pair is ‘switched off’. Elimination of FRET between CDs and naphthalimide also confirmed by time resolved spectroscopy. Average decay time of nanosensor increased from 2.9 ns (nanosensor) to τav 5 ns which was close to the parent CDs proves that CDs are released from the covalent bonding and transfer of energy to acceptor moiety dimnished. Quantum yield of nanosensor after the treatment with TrxR was increased from 11% to 19% also indicate the breakage of disulphide bond and CDs are released from nanosensor complex. Further, nanosensor mediated enzyme activities were determined by calculating the Km/Kcat value. Km represents the amount of substrate which covers the half of the active site of the enzyme. Lower the Km value indicates the higher binding of substrate with enzyme and vice versa.50, 51 The change in emission intensity shows 9



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ratiometric changes with time scale at 565 nm and 450 nm with Km/Kcat were determined to be 7 µg/2.4 s-1 (Fig. S8A). To recognize the specificity of the prepared nanosensor, fluorescence response was recorded using 5 µM concentrations of other small molecules which are present in the mammalian system (cysteine, homocysteine, glutathione and NADH) (Fig. 3E and 3F). In the presence of same molecules, interference study was also performed to examine the effect of interference ions over senstivity of nanosensor (Fig. S10). It was observed that nanosensor is quite stable and senstive towards specific analyte ‘TrxR’ and fluorescence intensity ratio (I450/I565) was negligibly influenced in the presence of interfering ions. Impact of change in pH and salt concentration over the fluorescence intensity ratio in the presence of thioreductase is recorded by changing the pH from 4.5 to 8.5 and increasing the concentration of NaCl. Fluorescence intensity remains fairly stable in the pH range of 5-7.5 (Fig. S11A) designates that change in energy transfer from donor to acceptor unit was remained stable at a wide range of pH and even in the presence of salt from 0-1 µg/mL (Fig. S11B) The limit of detection was found to be 7.2 × 10-8 M (Fig. S8B) and determined as dividing the 3 times of standard deviation of blank signal with a slope of the calibration curve. Therefore, such a nanosensor could serve as a rational ratiometric fluorescence sensor for TrxR in different physiological condition’s condition. Application of Nanosensor in Cellular Environment to Monitor the Thioredoxin Reductase Activities: To quantify the real-time application of nanosensor, human breast cancer MCF-7 and cervical cancer HeLa cell lines were engaged in this study. For the cell imaging experiment, 1 × 105 numbers of cells were grown in 6 well plates at 37 oC for 24 h. After washing with PBS, both types of cells were incubated with nanosensor conjugate in PBS for 1h and 2 h at 37 oC. CLSM images of cells were recorded at 401 nm

10


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Figure 3. (A) Fluorescence spectra of nanosensor (30 µg/mL) in response to TrxR (50 nM) with time (B) Fluorescence intensity (I450/I565) ratio of sensor in response to TrxR (C) Linear fluorescence changes in nanosensor with TrxR at 450 nm and 565 nm (D) Schematic representation of sensing mechanism of TrxR (E) Fluorescence intensity ratio response of nanosensor (30 µg/mL) in the different disulfide reducing molecules present in mammalian system (50 nM) (F) Bar graph of Figure E. All spectra recorded in the presence of 125 µM of NADPH at pH 7.4. excitation wavelength. After 45 min of treatment of cells, the yellow emission intensity was more intense relative to blue emission, in both types of cells, demonstrate the presence of FRET pair between two units. Due to optimum quantum yield, higher contrast between the fluorescence of nanosensor and background signal in cells was observed. This contrast was higher in both types of cells after 2 h of treatment in comparison to 45 min of treatment indicates the the liberation of free CDs. Weak blue emission in 45 min of treatment cells 11



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signifies the reduction of disulfide bond of cystamine as the nanosensor was internalized in cells. After 2 h of treatment, owing to release of naphthalimide moiety from CDs surface (elimination of FRET pair), yellow colored intensities decreased significantly and the blue emission dominated (Fig. 4B, 4D). The change in color emission from yellow to blue proves that nanosensor conjugate is quite effective and sensitive to reductase enzyme in a cellular environment which helps in screening of cancer cells on the basis of color emission of cells. CLSM images of cells demonstrating that the prepared nanosensor remains in cytoplasm of cells and nucleus remains intact nanosensor remains in dispersed form in cytoplasm of MCF7 and HeLa cells even after 2 h of treatment (Fig. 4A-4D).

Fig. 4: Confocal images of MCF-7 and HeLa cells. Fluorescence images of HeLa cells (A) and MCF-7 (C) after 45 min of treatment with nanosensor shows intense yellow emission over blue, signifies the presence of FRET pair of nanosensor. (B, D) After 2 h of treatment in HeLa (B) and MCF-7 (D) blue emission predominent owing to cleavage of disulfide bond demonstrate the emission intensity of free CDs released from nanosensor complex in the cytoplasm. Cytotoxicity Assay: It is worth mentioning here that the parent CDs has not pronounced any significant ability to retard the growth of living cells, more than 90% of cells remains viable upon treatment with 100 ug/ml concentration of CDs. However, nanosensor reduces the cell viability of cancer cells up to 70% at a dose of 100 µg/mL (Fig. 12S). The cytotoxicity of nanosensor was interpreted as follows: the cytoplasm of cancer cells having elevated level of TrxR break the disulfide bond and releases free naphthalimide entity. The structure of 12


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naphthalimide derivative of nanosensor is very close to the structure of amonafide (a chemotherapeutic drug), which is extensively being studied for the treatment of cancer as a candidate of topoisomerase inhibitor and DNA intercalator.52 Therefore reduction of cell growth after treatment with nanosensor may be related to the release of free naphthalimide unit. Furthermore, as the CDs may involve in photocatalytic oxidation and reduction reactions through the redox system,53,54 we utilized this effect to halt the growth of cells in the light conditions. For this, MCF-7 and HeLa cells were incubated with nanosensor in the presence of visible light to evaluate the effect of light on the growth of treated cells. Growth of cells in the presence of light without any treatment was chosen as a blank sample and found that cell viability reduced to 40% in light treatment cell which may be attributing to the formation of reactive oxygen species (ROS) from CDs in response to light (Fig. S13A, S13B).55 Generation of ROS in response to light from CDs was analyzed by recording the fluorescence intensity of 2′,7′-dichlorofluorescein (DCF) in hypoxic PBS. An increase in fluorescence intensity of DCFH was observed with time, when DCFH solution in CDs exposed to visible light. Increase in fluorescence intensity is owing to oxidation of DCFH to DCF demonstrating the formation of free radicals that are responsible for the killing of cells in light conditions (Fig. S14).55 To predict whether biotin conjugated nanosensor could be endocytosis into the cells through highly up-regulated biotin receptor present on the cell wall or not, cells were treated with clathrin pathway inhibitor chlorpromazine before incubation with nanosensor for 3 h and then cell viability assay was performed in the presence of light. In bothtype of cell, ≥ 80% cell remains viable which proved our prediction that biotin conjugated nanosensor predominantly entered into cells by endocytosis (Fig. S15). Conclusion In conclusion, fluorescence sensor encompassing FRET based energy transfer from CDs to naphthalimide for the detection of cancer cells using TrxR sensitive probe has been developed. Before the addition of TrxR, energy got transferred from CDs to naphthalimide unit which emits at 565 nm. However, upon addition of TrxR, distortion of energy transfer happened within 80 minutes, that resulted in enhancement of fluorescence intensity of CDs and gave blue emission. This sensor exhibited high sensitivity towards TrxR over other thiols used in the present sudy. Confocal microscope study revealed that the nanosensor precisely entered into the cancer cells through biotin receptor-mediated endocytosis and it has been further confirmed by chlorpromazine treatment. Change in the emission intensity of cancer cells from orange-yellow to blue was clearly observed under CLSM. Cytotoxicity assay of 13



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nanosensor has been performed under both light and dark conditions. In the presence of visible light, growth of cells reduced to 40%, while in dark, 70% cells remained viable. The reduction in cell viability in the presence of light indicates the generation of some free radicals from the surface of the CDs, which disrupt the cellular redox potential leading to cell death. Generation of reactive oxygen species has been verified by measuring fluorescence intensity of 2′,7′-dichlorofluorescein in a hypoxic environment. Experimental Section Chemicals and Instruments: 1, 8-naphthalic anhydride, citric acid, glutathione, and thioreductase were purchased from Sigma-Aldrich. Cystaminedihydrochloride, DMAP (dimethylaminopyridine),

EDC.HCl

(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride), biotin, BOC anhydride (tert-butyloxycarbonyl), and trifluoracetic acid (TFA) were procured from Avra Synthesis. Fetal bovine serum (FBS), DMEM and DMSO supplied by Hi-Media. All the chemicals were used without further purification. Milli-Q water was used in all synthetic and analytical experiments. Compound S2 and S3 were synthesized by following the protocol reported in the literature.56,57 1

HNMR and

respectively.

13

CNMR were measured on Jeol instrument operated on 400 and 100 MHz Photophysical

properties

were

recorded

on

Shimadzu

UV-2400

spectrophotometer and Perkin Elmer LS 55 fluorescence spectrophotometer. PL quantum yield and decay time recorded on PicoQuant FluoTime 300 High Performance Fluorescence Lifetime Spectrometer using a Time-Correlated Single Photon Counting (TCSPC) technique. Single exponential function was used to measure fitted PL decay curve. The Response function of instrument acquired with LUDOX. Morphology of CDs was characterized by TEM (Transmission electron microscope) using Hitachi (H-7500) instrument. Particle diameter was measured by using DLS (Dynamic Light Scattering) with an external probe of Metrohm Microtrac Ultra Nanotrac particle size analyzer. IR spectrums of solid samples were recorded by using a solid cell technique on Bruker Tensor 27 spectrophotometer. Powder XRD pattern of CDs estimated from analytical X’PERT PRO instrument. Synthesis of S1: Synthesized by following the protocol reported in literature.58 Briefly, Cystamine dihydrochloride (1g, 4.4 mmol) and 16 mmol of triethylamine were dissolved in methanol and stirred the mixture at 0 °C. After 30 min, Boc anhydride (0.959 g, 4.4 mmol) was added over the 15 min and let the reaction mixture to stir for another 45 min. Solvent was evaporated under vacuum and washed with diethyl ether three times. Solid residues were 14


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dissolved in water and basified with 1 M NaOH (pH 9) and extracted with ethyl acetate. Organic layer was dried over sodium sulfate and evaporated to get pale yellowish oil. Synthesis of S4: 3-amino naphthalic anhydride (1g, 4.69 mmol) and S1 (1.26 g, 5 mmol) was dissolved in ethanol and refluxed for 6 h. Reaction mixture was cooled to 4 °C and filtered to get yellow colored precipitates. Precipitates were washed with ethanol and dried under vacuum to get pure yellow powder S4 (57% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.01 (dd, J = 17.6, 7.6 Hz, 2H), 7.93 (d, J = 2.0 Hz, 1H), 7.25 (s, 1H), 5.94 (s, 2H), 4.33 – 4.24 (m, 2H), 3.18 (q, J = 6.2 Hz, 2H), 3.00 – 2.92 (m, 2H), 2.75 (t, J = 6.9 Hz, 2H), 1.32 (s, 10H). 13C NMR (101 MHz, DMSO-D6) δ 164.25, 164.07, 156.00, 148.41, 134.97, 134.09, 131.35, 127.47, 126.03, 123.01, 122.32, 122.22, 121.16, 112.36, 78.30, 38.18, 35.81, 28.73. Synthesis of S5: S4 (1 mmol) was dissolved in TFA and stirred the reaction mixture for 1 h. Progress of the reaction was monitored by TLC. After completion of the reaction, TFA was evaporated under vacuum and 2 mL water containing triethylamine was added. A light yellow colored product was precipitated and collected by filtration. Purified product was sticky yellow in color. 1H NMR (400 MHz, DMSO-d6) δ 8.07 – 7.96 (m, 2H), 7.92 (s, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.23 (s, 1H) 5.92 (s, 2H), 4.26 (d, J = 7.3 Hz, 3H), 3.18 – 3.15 (m, 2H), 2.97 – 2.91 (m, 2H), 2.74 (t, J = 6.9 Hz, 2H); HRMS m/z (ESI) : calcd for C16H17N3O2S2 [M+H]+ 348.0840, found 348.1014 Synthesis of Nanosensor (Biotin-CD-Naph): CDs were prepared according to reported literature with some modification.59 In brief, 0.5 g of citric acid and 0.1 mL of ethylenediamine was heated at 180 °C for 1 h. A reddish brown colored powder was washed with water and ethyl alcohol. Finally, CDs were dialyzed against water and dried under vacuum. CDs were stored at 4 °C in dark for further use. The 10 mg of CDs were dissolved in deionized water and 7 mg EDC.HCl was added followed by DMAP (1 mg). Reaction mixture was stirred for 1 h under nitrogen atmosphere. The ligand S5 (5 mg) was dissolved in 1 mL of DMSO added to the reaction mixture and let the reaction mixture to stirred for another 36 h in dark. After, that solution was dialyzed against deionized water for 48 h and product was collected by centrifugation and dried under vacuum. Biotin (4 mg) and 2 mg of EDC.HCl were dissolved in 1 mL DMSO and 0.5 mg of DMAP was added into it and stirred for 1 h at room temperature (rt) under nitrogen. 4 mg of naphthalimide conjugated CDs in water was added drop-wise and let the reaction mixture to stirrer for 40 h at rt in dark. Thus prepared nanosensor was dialyzed against water for 72 h and product was freeze dried. 15



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Fluorescence Sensing of Thioredoxin Reductase: 30 µg/mL solution of nanosensor was prepared in HEPES buffer having pH 7.4. Stock solution (1 mM) TrxR was prepared and diluted further sensing studies to 50 nM. Concentration of nanosensor and TrxR was kept constant and fluorescence response was recorded with respect to time. Quantum Yield Measurement: Quantum yield was measured at room temperature employing the absolute method. 10 mm path length quartz cuvette was used for all the liquid samples. Solvent without any sample in the same cuvette is used as a blank sample. 2aminopyridine (QY: 84%) and rhodamine 101 (QY: 99%) were chosen as a reference standard to test the accuracy of apparatus. Sensing and Cell Imaging: MCF-7 and HeLa cell lines at a density 105 were cultured using 10% FBS in DMEM media at 37 °C in 5% CO2 in 6 well plates for 24 h containing cell culturing glass slides. Culture media was replaced with new DMEM media containing 30 µg/mL concentration of nanosensor and incubated for 45 min at 37 °C. After 45 min cell were washed three times with PBS and glass slide was taken out and images of cells were collected under confocal laser scanning microscope (CLSM). For invitro assay of TrxR, cells were incubated with 30 µg/mL nanosensor for 45 min. Cells were washed three times with PBS to remove excess of nanosensor and further cells were incubated for 2 h in DMEM media at 37 °C in 5% CO2. Cell images were recorded under CLSM and imaged using Nikon Eclipse Ti-U inverted microscope. Cell Viability Assay: 96 well plates were incubated with MCF-7 and HeLa cells in DMEM medium supplemented with 10% FBS at 37 °C in 5% CO2 for 24 h. After 24 h incubation media was removed and washed with PBS and cells were treated with different concentration containing nanosensor for 24 h in the dark. Afterward, cells were washed with PBS and treated with MTT for 2 h. The absorbance of DMSO lysed cells was measured. For the light treatment, cells were grown in the presence of day light using xenon lamp. Effect of light on the viability of cells was recorded from 0 to 80 min. Supporting Information: Uv-Vis, Fluorescence, Mass and NMR spectra of nanosensor, DLS histogram, TEM images, powder XRD of nanosensor, Cytotoxicity assay and decay time profile. Acknowledgement:

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TrxR

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