Multispectroscopic and bioimaging approach for the interaction of rhodamine 6G capped gold nanoparticles with bovine serum albumin

Accepted Manuscript Multispectroscopic and bioimaging approach for the interaction of rhodamine 6G capped gold nanoparticles with bovine serum albumin

N. Manjubaashini, Mookkandi Palsamy Jegathalaprathaban Rajesh, T. Daniel Thangadurai

Kesavan,

PII: DOI: Reference:

S1011-1344(18)30205-7 doi:10.1016/j.jphotobiol.2018.05.005 JPB 11239

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

22 February 2018 30 April 2018 4 May 2018

Please cite this article as: N. Manjubaashini, Mookkandi Palsamy Kesavan, Jegathalaprathaban Rajesh, T. Daniel Thangadurai , Multispectroscopic and bioimaging approach for the interaction of rhodamine 6G capped gold nanoparticles with bovine serum albumin. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jpb(2017), doi:10.1016/j.jphotobiol.2018.05.005

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ACCEPTED MANUSCRIPT Multispectroscopic and Bioimaging approach for the interaction of Rhodamine 6G capped Gold Nanoparticles with Bovine Serum Albumin N. Manjubaashinia, Mookkandi Palsamy Kesavanb, Jegathalaprathaban Rajeshb, T. Daniel Thangaduraia* a

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Department of Nanoscience and Technology, Sri Ramakrishana Engineering College, Coimbatore 641 022, Tamilnadu, India. b

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Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai 623 806, Tamilnadu, India

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Corresponding author: Tel.: +91 422 246 1588; fax: +91 422 246 1089.

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E-mail address: [email protected]

ACCEPTED MANUSCRIPT Abstract Binding interaction of Bovine Serum Albumin (BSA) with newly prepared rhodamine 6Gcapped gold nanoparticles (Rh6G-Au NPs) under physiological conditions (pH 7.2) was investigated by a wide range of photophysical techniques. Rh6G-Au NPs caused the static quenching of the intrinsic fluorescence of BSA that resulted from the formation of ground-state

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complex between BSA and Rh6G-Au NPs. The binding constant from fluorescence quenching

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method (Ka = 1.04×104 L/mol; LoD = 14.0 µM) is in accordance with apparent association

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constant (Kapp = 1.14×101 M−1), which is obtained from absorption spectral studies. Förster resonance energy transfer (FRET) efficiency between the tryptophan (Trp) residue of BSA and fluorophore of Rh6G-Au NPs during the interaction was calculated to be 90%. The free energy

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change (ΔG = –23.07 kJ/mol) of BSA–Rh6G-Au NPs complex was calculated based on modified Stern-Volmer Plot. The time-resolved fluorescence analysis confirmed that quenching of BSA

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follows static mechanism through the formation of ground state complex. Furthermore, synchronous and three-dimensional fluorescence measurement, Raman spectral analysis and

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Circular Dichroism spectrum results corroborate the strong binding between Rh6G-Au NPs and BSA, which causes the conformational changes on BSA molecule. In addition, fluorescence

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imaging experiments of BSA in living human breast cancer (HeLa) cells was successfully demonstrated, which articulated the value of Rh6G-Au NPs practical applications in biological

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systems.

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Key Words: Rhodamine 6G; Au NPs; BSA; Static quenching; FRET; Bioimaging

ACCEPTED MANUSCRIPT 1. Introduction Serum albumins, major soluble protein components, have many physiological functions such as distribution and transportation of various exogenous and endogenous ligands, namely fatty acids, amino acids, steroids, metal ions, and a variety of drugs, and maintain the osmotic pressure and pH of the blood [1]. In addition, albumins efficiently increase the solubility of hydrophobic

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drugs in plasma and alter their delivery to cells; however, the binding site and molar ratio might

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differ based on the molecular and physicochemical properties of the interacting ligands [2]. The interaction between proteins and drugs has been playing a very vital role in pharmacology [3].

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Therefore, developing a new model drug which appends more on protein with high binding affinity and highest energy acceptance efficiency is a big challenge for the researchers, an area

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which should be addressed soon for the progression of clinical diagnostics. Bovine serum albumin (BSA) and human serum albumin (HSA) are the two of the most

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extensively studied serum albumins having the similarities in confirmation, number of disulfide bridges, series of loops and assembly in subdomains [4]. Based on the analysis, the main

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difference between these two serum albumins is the tryptophan (Trp) numbers; BSA has two Trp residues (Trp-212 and Trp-134) whereas HSA has a single Trp residue (Trp-214) [5]. The

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investigation on interaction between serum albumin and drugs has been studied effectively over few decades to elucidate conformational changes, action mechanism and toxicity, distribution

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volume of drugs and elimination rate of drugs owing to the significance in the biomedical applications [6]. For the current work, BSA is selected as our protein replica due to its medicinal

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importance, ready availability, low cost, and unusual ligand-binding properties, which made the study an interesting one [7].

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In recent years, gold nanoparticles (AuNPs) are drawing much attention owing to their applications in diverse fields of medicine, catalysis and industry with respect to their novel optical, electrical, electronic and catalytic properties [8]. AuNPs play a vital role in clinical trials such as gene silencing, cell labeling, cancer therapy, drug delivery systems, and biosensing [9] based on their distinct size and shape-dependent optical properties and quantum confinement effects [10]. Owing to their biological compatibility with living cells and ability of fast removal from the organism, the gold nanoparticles are used as photosensitizers [11a]. The direct interaction between AuNPs and protein was investigated mainly by fluorescence quenching technique due to its advantage of high sensitivity even at low analyte concentration, rapidity and

ACCEPTED MANUSCRIPT ease to handle; it also provides the information on essential binding phenomenon [11]. Though there are few reports available for AuNPs-BSA interaction, the interaction of fluorescent-capped metal NPs with BSA has not been reported in the literature before to the best of our knowledge [12]. The fluorescent-capped AuNPs are not only useful in studying the binding nature of BSA with fluorescent moiety, but also useful for bioimaging the protein molecule in living cells.

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Based on the above insights, we attempted to develop a new drug model with organic-

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capping agent to prevent non-radiative recombination at surface sites with intent to interact with

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protein molecule through hydrophobic interaction or hydrogen bonding. In this report, we explore the interaction between Rh6G-Au NPs and BSA under physiological conditions through UV-Visible absorption, steady state emission, time-resolved fluorescence, synchronous emission,

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3D emission, Raman and Circular Dichroism (CD) spectral studies. Additionally, we applied Rh6G-Au NPs for BSA sensing in live cancer cell (HeLa) imaging application. These new

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findings are expected to afford imperative insight into the interaction mechanism of physiologically important protein, serum albumin, with fluorescent-capped metal NPs, which

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may be a useful guideline for pharmacology sector.

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2.1. Materials and Methods

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2. Experimental

All the reagents and solvents involved in synthesis were analytical grade and used without

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any further purification. Bovine serum albumin (BSA) was purchased from Sigma and used as such. Rhodamine 6G, gold (III) chloride, Tris HCl, Sodium chloride, Sodium hydroxide, and

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solvents were purchased from Sigma Aldrich, India, TCI Chemicals and Himedia. Rhodamine 6G-capped AuNPs were prepared by simple reduction method. X-ray diffraction measurements were carried out using x-ray source of Cu Kα radiation (λ = 1.542 Å) (Xpert Powder diffractometer, PANanlytical, The Netherlands). The surface morphology was analyzed by HRTEM (200 kV FE-TEM, model JEM-2100F, JEOL) at CUSAT, Kochi. UV-Visible absorption was carried out on Analytik Jena spectrophotometer (Model SZ-100, Germany). Fluorescence experiments were recorded on Agilent Technologies spectrofluorophotometer (G9800A) at 298. K. Raman spectra recorded on Bruker RFS 27 with 532 nm laser at SAIF IITMadras, Chennai. Time-dependent fluorescence spectra were recorded on Horiba Jobin Yvon

ACCEPTED MANUSCRIPT with 280 nm LED source at Anna University-Chennai. CD spectral analysis was conducted with JASCO J-1500 Circular Dichroism Spectrophotometer at CLRI, Chennai. Synchronus spectrum was recorded on JASCO Fluorescence Spectrophotometer (FP-6600) at Bharathiar University, Coimbatore. Thermal studies were carried out in Perkin Elmer Diamond thermal analysis instrument at CUSAT, Cochin, India. Fluorescence imaging of HeLa cells was carried out by

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using Wexwox FM3000 fluorescence microscope at VIT, Vellore, India.

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2.2. Synthesis of Rh6G-Au NPs by reduction method

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To a continuous stirring distilled water solution of gold (III) chloride (1.0 M) were added ~20 mL of Rhodamine 6G (0.005 M) and the calculated quantity of reducing agent (NaBH4, 1%) in

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room temperature. The resulting reaction mixture was continuously stirred for 3 h. Then the solution was refrigerated for 2 days to reduce the NPs agglomeration and better dispersion stability. Finally, the NPs were washed by distilled water (2×10 mL) and centrifuged to remove

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excess/unreactive chemicals and dried in hot air oven for 6 h at 100 ºC.

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2.3. Preparation of stock solutions

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The reagent stock solution was freshly prepared in double distilled water before every experiment. Buffer solution was prepared in 250 mL standard measuring flask by the addition of Tris HCl (5 mmol) and NaCl (50 mmol). The pH (≈7.2) of the resulting solution was adjusted by

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the drop-wise addition of NaOH (0.1 M). The as-prepared BSA stock solution (0.01 g of BSA in 10 mL of buffer solution) was refrigerated for 30 min to attain equilibrium. The Rh6G-Au NPs

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stock solution (12.5 mg in 5 mL of H2O) was prepared to carry out experiments.

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2.3. Absorption studies

The UV-Visible absorption spectral studies were carried out to BSA and Rh6G-Au NPs solutions separately in the range of 200-800 nm. BSA and Rh6G-Au NPs exhibit a strong absorption peak at 278 and 525 nm, respectively. The intensity changes of absorption peak of BSA at 278 nm upon increasing the concentration of Rh6G-Au NPs were monitored. The apparent association constant (Kapp) was calculated by using Benesi-Hildebrand plot.

ACCEPTED MANUSCRIPT 2.4. Fluorescence studies Fluorescence spectra were measured by using Quartz cells. The emission value for BSA (λem = 347 nm) was obtained upon excitation at 278 nm. We carried out fluorescence titration experiments by measuring the changes in fluorescence emission of BSA upon successive addition of 4 µL of aqueous solution of Rh6G-Au NPs. For all the measurements, excitation was

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at 278 nm and emission slit width was 5 nm. The initial volume of BSA was 3 mL. Titrations

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were manually done by using micro-pipette for the addition of Rh6G-Au NPs. The titration

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experiment was repeated at least thrice to check the consistency. Stern–Volmer and modified Stern–Volmer equations were used to calculate the quenching rate constant (Kq), binding constant (Ka) and free energy (ΔG) changes during the interaction. The association constant

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(Kass) and binding stoichiometry was calculated by Scatchard plot and the dissociation constant

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(Kd) was calculated by Hill plot analysis.

Excited state fluorescence lifetime, three dimensional and synchronous fluorescence

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measurement for BSA was performed in the absence and presence of Rh6G-Au NPs upon excitation at 278 nm. Exactly the same concentration of BSA and Rh6G-Au NPs was used and

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for the synchronous fluorescence emission, the spectra were measured at two different Δλ values

2.5. Circular Dichroism

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such as 15 and 60 nm.

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Circular dichroism (CD) spectra were recorded on JASCO J-1500 Circular Dichroism Spectrophotometer at room temperature. The Rh6G-Au NPs were added in small aliquots to

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protein solution (1 mg/mL). Each spectrum represents an average of 20 scans. The experiments were performed at 25 °C in a 2 mm path cuvette. Mean Residue Ellipticity (MRE) is expressed in millidegrees.

2.6. Bioimaging studies For bioimaging experiments, exponentially growing HeLa cells were seeded in 6-well plates; each well plates containing approximately 0.1 – 0.2×106 cells. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) for 24 h at 37 °C and 5 % CO2. For control group, HeLa cells were incubated with Rh6G-Au NPs

ACCEPTED MANUSCRIPT (1.0×10−5 M; 100 μL) for 30 min at 37 °C, and then washed by PBS buffer before imaging. For the experimental groups, the HeLa cells were incubated with Rh6G-Au NPs (1.0×10−5 M; 100 μL) for 30 min, followed by loading with BSA (1.25×10-6 M) and incubated for 30 min at 37 °C and again washed with PBS buffer before imaging. For labeling, the cells were mounted on glass slides with DAPI stain medium and imaged. The images were acquired using a fluorescence microscope through UV-region, blue and green channels. Excitation was fixed at 525 nm and

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emission was monitored from 550 to 600 nm. To find out the cell viability and the IC50 value of

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Rh6G-Au NPs, the cytotoxicity test was also carried out. 3. Results and Discussion

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The protein–nanoparticles binding in biosystem will bring significant benefits to medical field to enable earlier and more specific bimolecular monitoring. The process of retaining and

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releasing oxygen to hemoglobin at particular pressure is termed as colloidal osmotic pressure balancing for proper functioning of respiratory and circulatory system. BSA, bovine serum

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albumin, is a major protein content in blood plasma, which acts as a center for stabilizing extracellular fluid and maintaining osmotic pressure to transfer body fluid between blood vessels

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and body tissues with the help of oxygen carriers [13].

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3.1. Characterization of Rh6G-Au NPs

Multiphotophysical techniques were used to study and characterize various aspects of the

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BSA and Rh6G-Au NPs interaction. These analytical methods led us to determine the number of basic properties such as association constant of BSA with Rh6G-Au NPs, binding kinetics, and

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conformational changes in BSA upon binding. The Rh6G-Au NPs were prepared by the simple reduction method and characterized by the standard physiochemical techniques. 3.1.1. X-Ray Diffraction studies To obtain purity, crystalline nature and structural information for the Rh6G-Au NPs, the XRD diffraction analysis was performed for newly synthesized Rh6G-Au NPs (Figure 1). The noticeable sharp and intense diffraction peaks appeared at 2θ = 38.55°, 44.57°, 65.20°, and 77.82° are assigned to (111), (200), (220) and (311) orientation plane of Au, respectively. These intense peaks indicate that Rh6G-Au NPs are highly crystalline, well arranged in specific

ACCEPTED MANUSCRIPT orientation and in face-centered cubic structure (JCPDS No. 65-2870) [14]. No other peaks of impurity were observed, which revealed that the newly synthesized Rh6G-Au NPs are pure. Insert Figure 1

3.1.2. TEM analysis

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The surface morphology of Rh6G-Au NPs was characterized using transmission electron

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microscopy. TEM samples were prepared by introducing a drop of a dilute hexane dispersion of

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Rh6G-Au NPs on the surface of a 400-mesh copper grid backed with Formvar and were dried in a vacuum chamber for 30 min. The HRTEM image shows that Rh6G-capped AuNPs are in

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dendritic architecture with average particle size of ~6 nm [15] with Au metal in approximately spherical shape (Figure 2). The high magnification image yields the lattice images of single

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Rh6G-Au NPs (Figure 2b,c) and the selected area electron diffraction (SAED) pattern illustrates the crystalline nature of the Rh6G-Au NPs (Figure 2d), which is in good harmony with XRD

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pattern.

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3.1.3. Thermal stability studies

Thermal stability of the newly synthesized Rh6G-Au NPs was examined by Thermo-

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Gravimetric analyses (TGA) and Differential-Scanning Calorimetric (DSC) techniques in a heating range from 40 to 740 °C with a heating rate of 20 °C/min. TG curves exhibit the

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decomposition, which is undergone in a single step (Fig. SI6). The weight loss of 7.187 % around 450 °C and the weight retained signify Au present in the sample. This weight loss

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originated from the evaporation of Rhodamine 6G moieties. DSC analysis results imply a distinct exothermic peak accompanying a significant weight loss, which is observed around 405 °C indicating that there is one chemical reaction involved in the process (Fig. SI7). These thermal studies revealed that the Rh6G-Au NPs are highly stable and confirm the presence of Au in the sample.

3.2. Rh6G-Au NPs and BSA interaction studies

ACCEPTED MANUSCRIPT 3.2.1. Absorption spectrum analysis To elucidate the excited state reaction and to identify the type of interaction between Rh6GAuNPs (acceptor) and BSA (donor), we performed absorption titration studies. The UV-Visible spectrum of BSA (1.0×10−5 M) exhibits a strong absorption band at 278 nm in the absence of Rh6G-Au NPs at pH 7.2 (Figure 3a). Addition of Rh6G-AuNPs (0 – 100 µL) gradually increases

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the intensity of BSA absorption band, i.e., hyperchromism. This increase in absorption intensity

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clearly indicates the strong static interaction between Rh6G-Au NPs with BSA, which resulted in

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the formation of ground state complex [1,16]. Consequently, a structural change in BSA occurred and equilibrium existed between BSA–Rh6G-Au NPs [17].

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by using Benesi-Hildebrand method (eq. 1) [18]

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Based on the absorption titration results, the apparent association constant (Kapp) was calculated

)[

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(eq.1)

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(

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where ‘Ao’ and ‘Ac’ are the absorbance of BSA alone and the fully bound form of BSA with Rh6G-Au NPs at 278 nm, respectively. Aobs is the absorbance of the BSA upon the addition of

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different concentration of Rh6G-Au NPs at 278 nm. The apparent association constant value [

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and shows the binding affinity of

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(Kapp) was calculated by plotting

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Rh6G-Au NPs with BSA in physiological condition (Figure 3b).

3.2.2. Fluorescence titration studies The emission traits of tryptophan, tyrosine, and phenylalanine residues present in proteins can provide information on the binding nature and conformation changes upon association with NPs [19]. The current study led us to attain the detail that fluorescent-capped gold NPs efficiently quench the intrinsic fluorescence emission of BSA. To explore the binding interaction between Rh6G-Au NPs and BSA, we measured the fluorescence emission intensity changes of BSA that occurred upon successive addition of Rh6G-Au NPs. The fluorescence emission

ACCEPTED MANUSCRIPT intensity of BSA (1.0× 10−6 M) was steadily decreased upon the addition of Rh6G-Au NPs (1.0×10−5 M; 0 – 56 µL) at physiological condition (Figure 4). The appearance of isosbestic point at 482 nm indicates equilibrium is attained between BSA and Rh6G-Au NPs and revealed the formation of the ground state complex. The following Stern-Volmer equation (eq. 2) clearly

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]

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(eq.2)

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[

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explains the quenching of BSA fluorescence by the Rh6G-Au NPs.

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where ‘Fo’ and ‘F’ are the fluorescence intensities of free BSA and Rh6G-Au NPs-bound BSA, respectively. ‘KSV’ is Stern-Volmer constant, [Rh6G-Au NPs] which is the concentration of the Rh6G-Au NPs, ‘kq’ is the quenching rate constant, and ‘τ0’ is the average lifetime of the free

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BSA molecule, which is 10−8 s [20]. According to Stern-Volmer equation, the value of KSV was calculated by Fo/F plotted against [Rh6G-Au NPs] (Figure 5). The obtained linear Stern-Volmer

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quenching relationship might be due to the interaction of Trp residues of BSA with fluorophore

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with absorption spectral results.

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of Rh6G-Au NPs causing the formation of the ground state complex, which is in good concord

3.2.3. Affinity constant calculation

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The binding affinity constant (Ka) for Rh6G-Au NPs–BSA interaction was calculated by modified Stern–Volmer equation (eq. 3) based on measuring the steady state fluorescence data of BSA with the consecutive addition of Rh6G-Au NPs at physiological pH [21]

[

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(eq.3)

ACCEPTED MANUSCRIPT where ‘fa’ is the fraction of accessible fluorescence. The obtained Ka value (1.04×104 M-1) implies that Rh6G-Au NPs showed an excellent binding affinity with Trp residues of BSA through hydrophobic interaction or hydrogen bonding (Figure 6) [1, 22].

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3.2.4. Determination of free energy change

Generally, the signs of the thermodynamic parameters such as electrostatic forces,

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hydrophobic forces, van der Waal's interactions and hydrogen bonding furnish the idea about the nature of the forces involved in the drug-protein interaction. The following Gibbs–Helmholtz

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equation (eq. 4) was used to calculate the free energy change during the interaction between BSA

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and Rh6G-Au NPs ∆G = –RT ln Ka

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(eq.4)

where ‘Ka’ is the binding affinity constant at corresponding temperature ‘T’, and ‘R’ is the gas

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constant. In the current work, the experimentally attained negative free energy value (ΔG = −23.07 kJ/mol) indicates that the interaction between Rh6G-Au NPs and BSA is extremely

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favorable and also affords an additional support to the formation of stable ground state complex in physiological condition (scheme 1) [23].

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3.2.5.Determination of Binding Stoichiometry and Association Constant The binding molar ratio association constant (Kass) and number of site (n) for the interaction

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of Rh6G-Au NPs on a BSA surface was evaluated by the Scatchard plot analysis (eq. 5) [24]. The association constant (Kass) was calculated from the intercept and the binding site ‘n’ was determined as the slope of the plot

(

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intrinsic fluorescence intensity of BSA upon increasing the concentration of Rh6G-Au NPs.

(

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(eq.5)

ACCEPTED MANUSCRIPT The association constant (Kass) 4.77×109 M-1 with ‘n’ value of 2 obtained from linear Scatchard plot revealed that BSA strongly binds with Rh6G-Au NPs through hydrophobic interaction or hydrogen bonding in 1:2 stoichiometric ratio at pH 7.2 (Figure 7a, b).

3.2.6.Cooperative binding analysis

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To understand the binding ability of the acceptor molecule, we calculated the Hill coefficient

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using eq.6. The Hill coefficient is, more often than not, used to estimate the number of donor

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(

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molecules/particles required to bind to the acceptor to produce a functional effect [25]

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(eq.6)

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where ‘θ’ is fraction of active sites on BSA occupied by the Rh6G-Au NPs, ‘nH’ is Hill coefficient, [Rh6G-Au NPs] is concentration of Rh6G-Au NPs and Kd is dissociation constant.

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Based on the experiments, the obtained Hill coefficient value (nH = 2.1368) revealed that there is

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a positive cooperative binding between acceptor and the donor molecules during the interaction (Figure 8). This indicates that the first bounded Rh6G-Au NPs molecule persuades the affinity for the second Rh6G-Au NPs molecule, which resulted in 1:2 stoichiometry (donor:acceptor

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ratio) with high dissociation constant (Kd = 5.21×109 M-1).

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3.2.7. Determination of Energy Transfer Förster Resonance Energy Transfer (FRET) is used to investigate the protein interaction in which energy transferred from the donor to the acceptor, i.e., the emission band of donor overlapping with the absorption band of the acceptor. To determine the efficiency of energy transfer and to calculate the distance between Rh6G-Au NPs as acceptors and the Trp residue of BSA proteins as donors during the interaction, we utilized FRET (eq. 7)

(eq.7)

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where ‘r’ is the effective binding distance between the donor and acceptor, ‘R0’ is the critical energy transfer distance at which 50% of the donor excitation energy is transferred to the acceptor, which was calculated by the following equation (eq. 8) R0 = 8.79×10-25 k2 n-4 Jφ

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(eq.8)

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where ‘k2’ is the spatial orientation factor of BSA and Rh6G-Au NPs, ‘n’ is the average

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refractive index of the medium, ‘φ’ is the fluorescence quantum yield of the BSA in the absence of the Rh6G-Au NPs, and ‘J’ is the overlap integral of the fluorescence emission spectrum of the

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BSA and the absorption spectrum of the Rh6G-Au NPs. In the current experimental condition, k2= 2/3, n = 1.336 and φ of BSA = 0.11. The value of J was calculated by the following equation

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( ) ( )

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(eq.9)

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{

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(eq. 9)

where ‘F(λ)’ is the fluorescence intensity of BSA at wavelength 278 nm, ‘εA(λ)’ is the molar

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extinction coefficient of BSA at wavelength 278 nm, and ‘∆λ’ is (absorption wavelength of Rh6G-Au NPs – emission wavelength of BSA). ‘E’ and ‘J’ values were obtained experimentally.

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The lifetime of BSA molecule is very important for the rate of energy transfer (kET)

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(eq. 10)

where ‘τD’ is the lifetime of the BSA in the absence of the Rh6G-Au NPs. According to the equations (7)-(9), the values of the parameters were found to be J = 2.6175×1014 J, R0 = 1.65 nm, E = 0.90 and r = 1.15 nm, kET = 8.72×108 s−1. From the experimental results, it has been observed that the emission spectrum of donor (BSA) overlapped with the absorption spectrum of acceptor (Rh-6G-Au NPs) molecules (Figure 9). In

ACCEPTED MANUSCRIPT the current system, the calculated donor-to-acceptor distance ‘r’ of 1.15 nm is less than that of the maximum distance of 8 nm for the FRET interaction to occur, and it satisfies another condition of 0.5Ro < r < 1.5Ro. These observations clearly indicate that the non-radiative energy transfer from BSA to Rh6G-Au NPs occurred with highest possibility of 90% and undergoes a ground-state complex formation [26]. This is in harmony with fundamental requirement for the incidence of FRET theory and indicated again the static quenching between BSA and Rh6G-Au

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NPs (Scheme 2). The 90% of energy transfer was compatible with or better than those obtained

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using other nanomaterial-based probes for BSA interaction (References 1 to 6 in SI; Table SI1).

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The good sensitivity and selectivity with wide linearity for the BSA interaction suggest great

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3.2.8. Time resolved fluorescence measurements

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potential of Rh6G-Au NPs for the application in bioassays.

To examine the effect of Rh6G-Au NPs on fluoresence lifetime of BSA, we have carried out

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the fluorescence decay of BSA in the absence and presence of Rh6G-Au NPs (Figure 10). Intially, in the absence of Rh6G-Au NPs, the fluorescence lifetime profile of BSA is best fitted

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with triexponential function to yield an average lifetime of 6.40 ns. The first addition of low concenrtration of Rh6G-Au NPs (10 µL) changed the decay curve from triexponential to

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biexponential with average lifetime value of 6.32 ns (Table 7). The biexponential decay profiles may arise due to the presence of an interacting and non-interacting fraction of BSA molecule.

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The shorter lifetime component present in BSA disappeared after the energy transfer and therefore a biexponential decay profile exists. Further increase in the concentraion of Rh6G-Au

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NPs (50 and 100 µL), unaltered the fluorescence lifetime of BSA (6.30 ns and 6.31 ns, respectively), which indicates that the quenching of BSA follows static mechanism. Since there is no significant change in the decay time of BSA at the maximum concentration of Rh6G-Au NPs, the formation of a ground state complex between BSA and Rh6G-Au NPs is confirmed through static quenching mechanism of BSA [27]. The quenching of intrinsic fluorescence emission of donor (BSA) decreases the donor lifetime, which was confirmed by the fluorescence experiments.

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3.3. Evaluation of conformational changes 3.3.1. Characteristics of synchronous fluorescence spectra

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The effect of fluorescent-capped AuNPs on the conformational changes of BSA was

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assessed by synchronous fluorescence method by simultaneous scanning of excitation and emission monochromators. This method provides the information about the molecular

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microenvironment in the neighborhood of fluorophore functional groups, characteristic informations about tyrosine residues and tryptophan residues when difference between excitation

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and emission wavelength (Δλ) is stabilized at 15 nm and 60 nm, respectively [28]. The fluorescence intensity of both tryptophan and tyrosine were decreased, but the emission

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wavelength of tryptophan was red shifted (~2 nm) with increasing concentration of rhodamine 6G-capped Au NPs (Figure 11). Comparing the emission wavelength of tyrosine, no significant

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change was observed at 305 nm (Fig. SI8), which indicated that the interaction of rhodamine 6G -capped Au NPs with BSA, does not affect the conformation of tyrosine micro-region. Firstly,

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combined with tyrosine, rhodamine 6G-capped Au NPs gradually interact with tryptophan and bring changes to BSA and resulted in red-shift of fluorescence wavelength (345 → 347 nm). It is

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possibly due to the fact that hydrophobic amino acid structure surrounding tryptophan residues in BSA tends to collapse slightly and thus tryptophan residues are exposed more to the aqueous

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phase [29]. Similar types of observations have already been reported [30]. Furthermore, the fluorescence intensity decreased regularly with the addition of Rh6G-Au NPs, which further

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demonstrated the occurrence of fluorescence quenching in the binding process.

3.3.2. Three dimensional fluorescence spectra Rh6G-Au NPs induced confirmational changes on BSA, which was further investigated by 3D emission spectroscopy. The fluorescence emission spectrum and contour map of BSA was recorded in the absence and presence of Rh6G-Au NPs (Figure 12). In the absence of Rh6G-Au NPs, the 3D spectrum of BSA exhibits two peaks, namely peak A and peak 1. Among these two peaks, peak A is a first ordered Rayleigh scattering peak (λex = λem) and peak 1((λex = 280 nm,

ACCEPTED MANUSCRIPT λem = 348 nm) corresponds to the spectral behavior of Trp and Tyr residues. [31] Addition of 50 µL of Rh6G-Au NPs increases the 3D emission intensities of peak A and in contrast, decreases the intensity of peak 1 of BSA. The increasing intensities of peak A upon the addition of Rh6GAu NPs owing to an increase in the macromolecular diameter of BSA due to the binding of Rh6G-Au NPs. The decrease in emission intensity of peak 1 is attributed to changes in the microenvironment of Trp or Tyr residues and Rh6G-Au NPs-induced conformational changes in

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the secondary structure of BSA [32]. The observation on contour intensity of BSA upon the

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addition of Rh6G-Au NPs additionally confirms that the Rh6G-Au NPs molecules bind to BSA

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(Figure 12c,d).

3.3.3. Raman spectral analysis

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For the additional confirmation on the conformational changes of BSA due to interaction with Rh6G-Au NPs at physiological pH condition, we carried out Raman spectrum of BSA before and after the addition of Rh6G-Au NPs. A strong band was observed at ca. 1420 cm−1,

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which is characteristic of amide III (α–helix) peak of BSA owing to the coupling of C–N

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stretching and N–H bending mode (Figure 13). Addition of Rh6G-Au NPs (50 µL) not only shifts the amide III (α–helix) peak of BSA to lower frequency (1414 cm −1), but also decreases

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the intensities of skeletal (α–helix) peak of BSA (ca. 955 and 1012 cm-1), which confirms the structural changes taking place in BSA. The amide I band ca. 1650 cm−1 (C=O stretching)

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assinged to tryptophan amino acid residues [33] shifted to higher wave number (ca. 1676 cm-1) upon complexation with Rh6G-Au NPs. Therefore, the raman spectrum results further confirm

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the biochemical environmental changes of BSA during the interaction with Rh6G-Au NPs.

3.3.4. Circular dichroism (CD)

To monitor the conformational changes in BSA induced by the addition of Rh6G-Au NPs, we carried out far-UV circular dichroism (CD) spectroscopy analysis (Figure 14). The CD spectrum of BSA protein showed two negative bands in the far-UV region at ~209 nm and ~223 nm, which is characteristic of α-helical structure of protein [34]. The band at 209 nm attributed

ACCEPTED MANUSCRIPT to π→π* transition of the α–helix, whereas, the band at 223 nm is corresponds to n→π* transition for both the α–helix and random coils. [35] In the presence of Rh6G-Au NPs, there is a slight decrease in ellipticity values of both the negative bands at 209 and 223 nm with no disparity in shape indicating the conformational change. Mean Residue Ellipticity (MRE) (° cm2 dmol-1) was calculated according to the following equation )

T

(

)

(eq. 11)

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(

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where ‘Cp’ is the concentration of protein, ‘n’ is the number of amino acid residues, and ‘l’ is the path length. The α–helical content of free and bound protein was determined from the MRE

[

]

)

(eq. 12)

AN

(

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value at 209 nm using eq. 12

where ‘MRE209’ is the observed MRE value at 209 nm, 4000 is the MRE of β-form and random

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coil conformation at 209 nm, and 33000 is the MRE value of a pure α–helix at 209 nm.

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The quantitative analysis outcome of the α–helix in the secondary structure of BSA was obtained by applying the eq. 12. The percentage of α–helical structure of BSA decreased (77.53 → 73.76

PT

→ 47.79%) upon increasing the concentration of Rh6G-Au NPs (0 → 50 → 100 µL, respectively). A similar observation has been already reported for the interaction of small

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molecules with hemoglobin [36]. This percentage decrease indicates the loss of α–helical protein structure upon the interaction and Rh6G-Au NPs bound with the amino acid residues of protein

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obliterated the hydrogen bonding networks [37]. CD spectra results revealed that the binding of Rh6G-Au NPs to BSA induces small conformational changes in the secondary structure of BSA protein along with a decrease in α–helical stability. The obtained conformational changes through Raman spectra, synchronous and the three-dimensional fluorescence spectra techniques are compatible with the CD spectra results.

3.4. Living cell Bioimaging studies

ACCEPTED MANUSCRIPT To investigate the practical aptitude of Rh6G-Au NPs to sense BSA within living HeLa cancer cells, we carried out fluorescence bioimaging experiment in different cell lines at physiological conditions (Figure 15). When HeLa cells were incubated seperately with BSA (1.25×10-6 M) and Rh6G-Au NPs (1.0×10−5 M; 100 μL) at 37°C for 30 min, a very weak fluorescence response and no cell outline was observed, respectively (Figure 15b,c). However, when HeLa cells were pretreated with BSA (1.25×10-6 M) for 30 min, washed with PBS buffer

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and incubated with Rh6G-Au NPs (1.0×10−5 M; 100 μL) for an additional 30 min, a significant

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increase in the fluorescence from the intracellular area and no morphological change of cell was

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observed (Figure 15d,e). The cell viability (90%) and the IC50 value (1.689 μM) provides further significant support to the role of Rh6G-Au NPs in the biological studies (Fig. SI9). The addition

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of less concentrations of Rh6G-Au NPs (10 and 50 µL) were not adequate enough to produce a clear imaging through fluorescence microscope (Fig. SI11). These observations clearly confirms

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that the cells were feasible throughout the imaging experiments [38]. The interaction of BSA with Rh6G-Au NPs facilitates to spot BSA in the inner circle of the cell, which was influenced

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by the brightness of the Rh6G-Au NPs. These interesting results demonstrate that Rh6G-Au NPs was capable of permeating into HeLa cells and reacting with BSA to generate the blue as well as

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green channel fluorescence images [39]. Hence, Rh6G-Au NPs could be used as a reliable and

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4. Conclusions

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practical probe to detect the role of BSA in living cancer cells through fluorescence imaging.

In summary, we have competently analyzed the interaction between Rhodamine 6G-capped

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AuNPs with BSA under physiological condition. Fluorescence emission experiment results indicate that the intrinsic fluorescence of BSA was quenched through static quenching process through complex formation. From the relevant fluorescence data, quenching rate constant (4.0653×1012 Lmol-1 s-1), binding constant, and binding stoichiometry (BSA: Rh6G-Au NPs = 1:2), association constant (4.77×109 M-1) and dissociation constant (5.21×109 M-1) were calculated. Non-radiative energy transfer from BSA to Rh6G-Au NPs was enhanced by the very short effective binding distance (r 1.15 nm) and critical energy transfer distance (R0 1.65 nm) between BSA and Rh6G-Au NPs. To best of our knowledge, in comparison with already existing reports, this report proclaims the maximum FRET efficiency (90%) between the donor

ACCEPTED MANUSCRIPT (tryptophan (Trp) residue of BSA) and acceptor (fluorophore of Rh6G-Au NPs) during the interaction. The disappearance of very short lifetime component of BSA upon addition of Rh6GAu NPs confirmed the static quenching mechanism during the interaction. From the synchronous and three dimensional fluorescence spectra, Raman and CD spectra results, it is revealed that the confirmation and microenvironment of BSA molecule was changed especially in the tryptophan micro-region in the presence of Rh6G-Au NPs. Finally, we demonstrated that Rh6G-Au NPs

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were capable of permeating into human breast cancer living cells and react with BSA to generate

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the fluorescence images in different channels. The current findings enlighten that Au NPs capped

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with fluorescent molecule can act as a good oxygen carrier and useful tool in detecting blood

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species.

Acknowledgements

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We gratefully acknowledge the financial support by The Council of Scientific and Industrial Research (01(2818)/14/EMR-II), New Delhi. The authors acknowledge IIT - Madras for Raman

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spectra; CECRI - Karaikudi for CD spectral analysis; Anna University - Chennai for Fluorescence decay measurements; CUSAT-Kochi for HR-TEM and Thermal analysis;

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Bharathiar University – Coimbatore for synchronous fluorescence spectra; Vellore Institute of Technology University - Vellore for Bioimaging studies and SNR Sons and Charitable Trust -

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Coimbatore, for providing XRD, Fluorescence, and UV-Visible characterization facilities. The authors also thank DST-FIST Laboratory facilities at Sri Ramakrishna Engineering College,

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constant support.

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Coimbatore, and Dr. G. Rajagopal, Chikkanna Government Arts College, Tiruppur, India, for his

Supplementary data Spectroscopic characterization of Rh6G-Au NPs, synchronous fluorescence studies, thermal analysis, LoD calculation and cytotoxicity test. To reveal the comparative importance of Rh6GAu NPs, the control experiment, BSA interaction with AuNPs under similar experimental condition were carried out and the obtained various parameters were provided. Supplementary data associated with this article can be found in the online version.

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ACCEPTED MANUSCRIPT Table 1. Benesi and Hildebrand plot analysis of Rh6G-Au NPs with BSA at pH 7.2 R2

Kapp (M-1)

Rh6G-Au NPs

0.9979

1.14×101

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Probe

ACCEPTED MANUSCRIPT Table 2. Stern-Volmer plot analysis of Rh6G-Au NPs with BSA at pH 7.2 R2

Ksv (L mol-1)

kq (L mol-1 s-1)

Rh6G-Au NPs

0.9904

4.07×104

4.0653×1012

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Probe

ACCEPTED MANUSCRIPT Table 3. Modified Stern-Volmer plot analysis of Rh6G-Au NPs with BSA at pH 7.2 R2

Ka (L mol-1)

∆G (kJ/mol)

Rh6G-Au NPs

0.9908

1.04×104

-23.07

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ACCEPTED MANUSCRIPT Table 4. Scatchard plot analysis of Rh6G-Au NPs with BSA at pH 7.2 R2

Kass (M-1)

n

Rh6G-Au NPs

0.9901

4.77×109

2

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ACCEPTED MANUSCRIPT Table 5. Cooperative interaction analysis of Rh6G-Au NPs with BSA at pH 7.2 R2

Kd (M-1)

nH

Rh6G-Au NPs

0.9912

5.21×109

2.1368

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ACCEPTED MANUSCRIPT Table 6. Energy transfer parameters on the interaction of Rh6G-Au NPs with BSA at pH 7.2

E (%)

J (J)

R0 (nm)

r (nm)

kET (s-1)

Rh6G-Au NPs

90

2.6175×1014

1.65

1.15

8.72×108

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ACCEPTED MANUSCRIPT Table 7. Relative fluorescence and average fluorescence lifetime values of BSA–Rh6G-Au NPs complex formation under physiological condition (λex = 278 nm). τ values

Sample Name

BSA+ Rh6G-Au NPs (10 µL) 2 BSA+ Rh6G-Au NPs (50 µL) 3 BSA+ Rh6G-Au NPs (100 µL)

τ 1 – 5.752491×10-9 s τ 2 – 7.546198×10-9 s

τ 1 – 3.517146×10-9 s τ 2 – 6.360870×10-9 s

AN M ED PT CE AC

Average life time value

0.9682982

6.40×10-9 s

1.146189

6.32×10-9 s

B1 – 78.46 B2 – 21.54

0.8638052

6.30×10-9 s

B1 – 9.62 B2 – 90.38

1.00247

6.31×10-9 s

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4

B1 – 0.18 B2 – 17.86 B3 – 81.96 B1 – 20.54 B2 – 79.46

CHISQ

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1

τ 1 – 9.510474×10-9 s τ 2 – 3.111893×10-9 s τ 3 – 6.702507×10-9 s τ 1 – 4.047457×10-9 s τ 2 – 6.629466×10-9 s

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BSA (triexponential)

Relative Amplitude

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S.No

ACCEPTED MANUSCRIPT Figure and Scheme Captions

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Fig. 1. X-ray diffraction profile of newly prepared Rh6G-Au NPs.

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Figure Captions

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Fig. 2. HR-TEM images of Rh6G-Au NPs in 50, 5 and 2 nm magnification (a-c); selected area diffraction pattern of Rh6G-Au NPs (d).

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Fig. 3. Absorption spectral titration of BSA (1.0×10−5 M) with increasing concentration of Rh6G-Au NPs (1.0×10−4 M; 0 – 100 µL) at pH 7.2 (a); corresponding Benesi and Hildebrand

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plot (b).

Fig. 4. Fluorescence emission intensity changes of BSA (1.25×10−6 M) upon increasing the

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concentration of Rh6G-Au NPs (1.0×10−5 M; 0 – 56 µL) at pH 7.2 (slit width 5 nm; λex 278 nm).

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Fig. 5. Stern-Volmer plot of Rh6G-Au NPs with BSA at pH 7.2.

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Fig. 6. Modified Stern-Volmer plot of Rh6G-Au NPs with BSA at pH 7.2. Fig. 7. Scatchard plot of Rh6G-Au NPs with BSA at pH 7.2 (a); Job’s plot analysis for the

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interaction between BSA and Rh6G-Au NPs (b).

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Fig. 8. Hill plot of Rh6G-Au NPs with BSA at pH 7.2.

Fig. 9. Energy transfer plots of BSA with Rh6G-Au NPs at pH 7.2. green and red color indicate the emission spectrum of the donor and absorption spectrum of the acceptor molecule, respectively. Fig. 10. Time resolved fluorescence decay of BSA (1.25×10−6 M) in the absence and presence of Rh6G-Au NPs (1.0×10−5 M; 0, 10, 50 and 100 µL) (λex = 278 nm).

ACCEPTED MANUSCRIPT Fig. 11: Synchronous fluorescence spectra of BSA (1.25×10-6 M) in the absence and presence of Rh6G-Au NPs (1.0×10−5 M; 0-100 µL) in the wavelength difference of Δλ = 60 nm at pH 7.20. Fig. 12: Three-dimensional fluorescence emission spectra of BSA (1.25×10-6 M) in absence (a) and presence (b) of Rh6G-Au NPs (1.0×10−5 M; 50 µL) at pH 7.2; corresponding contour map (c

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and d).

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Fig. 13: Raman spectra of BSA alone (1.25×10-6 M) (a) and BSAwith Rh6G-Au NPs (50 µL) (b)

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at pH 7.2.

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Fig. 14. Far-UV circular dichoism spectra of BSA (1.25×10-6 M ) up on addition of Rh6G-Au NPs (1.0×10−5 M; 50 and 100 µL) at pH 7.2.

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Fig. 15. Fluorescence microscope images of HeLa cell with Rh6G-Au NPs (1.0×10−5 M; 100 μL)

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upon addition of BSA (1.25×10-6 M). (images a→e: HeLa cell only; HeLa cell+BSA; HeLa cell+ Rh6G-Au NPs; HeLa cell+ BSA+ Rh6G-Au NPs through blue channel; HeLa cell+ BSA+

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Scheme Captions

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Rh6G-Au NPs through green channel).

Scheme 1. Formation of ground-state complex during the interaction between Rh6G-Au NPs and

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BSA at pH 7.2.

Scheme 2. Schematic representation of Förster Resonance Energy Transfer mechanism between BSA and Rh6G-Au NPs.

ACCEPTED MANUSCRIPT Research Highlights

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Intrinsic fluorescence emission of BSA was quenched >80% in physiological condition

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Positive cooperative binding with 1:2 stoichiometry (BSA:Rh6G-Au NPs) will be helpful

First bounded Rh6G-Au NPs molecule influences the affinity for the second Rh6G-Au

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to develop a good oxygen carrier drug

Shortest effective binding (r 1.15 nm) and critical energy transfer distances (R 0 1.65 nm) assists maximum FRET efficiency (90%)

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Rh6G-Au NPs can be useful to detect the role of BSA in cancer cells

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NPs molecule

Graphics Abstract

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