Comprehensive Multispectroscopic Analysis on the Interaction and

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A Comprehensive Multispectroscopic Analysis on the Interaction and Corona Formation of Human Serum Albumin with Gold/Silver Alloy Nanoparticles Arumugam Selva Sharma, and Malaichamy Ilanchelian J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b00436 • Publication Date (Web): 24 Jun 2015 Downloaded from http://pubs.acs.org on June 30, 2015

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The Journal of Physical Chemistry

A Comprehensive Multispectroscopic Analysis on the Interaction and Corona Formation of Human Serum Albumin with Gold/Silver Alloy Nanoparticles Arumugam Selva Sharma and Malaichamy Ilanchelian* Department of Chemistry Bharathiar University, Coimbatore – 641046, India.

* Corresponding author E-mail: [email protected] Tel: +91 422 2428317; Fax: +91 - 422 2422 387 ABSTRACT

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In the present investigation, we have systematically studied the binding mechanism of

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model protein human serum albumin (HSA) with Gold/Silver alloy nanoparticles (Au/Ag

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NPs) using multi spectroscopic techniques. Absorption spectral studies of Au/Ag NPs in the

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presence of increasing concentrations of HSA resulted in slight red shift of Au/Ag NPs

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surface plasmon resonance band (SPR), suggesting changes in the refractive index around the

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nanoparticle surface owing to the adsorption of HSA. The results from high resolution

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transmission electron microscopy (HR-TEM), dynamic light scattering (DLS) together with

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zeta potential analysis substantiated the formation of dense layer of HSA on the surface of

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Au/Ag NPs. The formation of ground state complex between HSA and Au/Ag NPs was

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evident from the outcome of the steady state emission titration experiments of HSA-Au/Ag

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NPs system. The binding parameters computed from corrected emission quenching data

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revealed that HSA exhibited a significant binding affinity towards Au/Ag NPs. The identical

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fluorescence life time values of HSA and HSA-Au/Ag NPs from time resolved fluorescence

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spectroscopic analysis further authenticated the findings of steady state emission

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measurements. The formation of HSA corona on Au/Ag NPs surface was established on the 1 ACS Paragon Plus Environment


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basis of experimental quenching data and theoretical values. The occurrence of partial

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unfolding of HSA upon its interaction with Au/Ag NPs surface was established by using an

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extrinsic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS). Absorption, Fourier

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transform infra red (FT-IR), Raman, circular dichroism (CD) and excitation-emission matrix

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(3D) spectral studies were also carried out to explore Au/Ag NPs induced tertiary and

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secondary conformational changes of HSA. The influence of Au/Ag NPs on the esterase like

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activity of HSA was established by probing the hydrolysis of p-nitrophenyl acetate.

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Key words: HSA, Au/Ag NPs, emission quenching, multi spectroscopic studies.

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1. INTRODUCTION

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In recent years, considerable attention has been devoted to the synthesis and

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characterisation of Au/Ag bimetallic NPs.1 These NPs have emerged at the frontier

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between materials chemistry and many other fields, such as electronics, biomedical,

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pharmaceutical, optics, catalysis and biosensors. It is understood that the versatility of

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bimetallic nano sized materials may be ascribed to their size and shape-dependent

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properties,2 however, the combination of the component metals and their fine

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structure, either as an alloy or a core-shell structure are tuneable factors that also

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contribute to the bimetallic systems.1,2 In recent years, Au/Ag NPs have been widely

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used for the enhancement of optical properties in the destruction of bacteria3-10,

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advancement in the detection of cancer11 and DNA.12 In all of these applications,

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interfacial interactions between nanoparticle (NP) surfaces and biomacromolecules,

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particularly proteins are involved.13 In a recent report, Au/Ag alloy NPs was shown to

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induce some adverse effects on mammalian gametes with significant inhibition of

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cumulus-oocyte maturation.14 In particular, silver rich Au/Ag NPs are known to

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produce considerable antibacterial and cytotoxic effects in several biological medium

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owing to the release of Ag+ ions from silver rich Au/Ag NPs.15-17

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NP interactions with biological systems are often initiated through proteins

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covalently attached or physically adsorbed onto the NP surface. Target specific

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therapeutic nanomaterials rely on surface bound biomolecules to carry out their

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function and most of the metal nanoparticles will typically adsorb plasma proteins

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upon introduction to an in vivo environment.18,19 The various specific and nonspecific

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interactions of nanoparticles with amino acid residues of proteins produce significant

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impact on various biological functions of proteins. The conjugation of proteins with

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NPs affords stabilization to the system and it also imparts biocompatible

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functionalities into these NPs for further biological applications. The use of protein NP

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bioconjugates, for applications in biosensing, assembly, imaging and control has

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substantially advanced.20-26 The information gained from protein-NP interactions is

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crucial for their successful use in nanomedicine and nanotoxicology.27-29 The proteins

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present in a biofluid compete for the NP surface to form a bio-nano interface, called

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“protein corona”.30,31 The formation of protein corona around the NP surface occurs

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through a dynamic and competitive process. Protein corona is typically composed of

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an inner layer of selected proteins with a lifetime of several hours involving slow

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exchange with the surrounding medium which is called as hard corona and a soft

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corona consisting of an outer layer of weakly bound proteins which are characterized

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by a faster exchange rate with the free proteins.30-32 Owing to its long life time, the

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hard corona present on the NP surface was shown to interact with cellular receptors

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directly rather than the pristine NP surface. As a result, the fate of the NPs in

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biological medium is entirely governed by protein corona formed around NP surface.

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Accordingly, the knowledge on the exact life time and conformations of the proteins



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associated with NP surface can divulge vital implications for successful designing of

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safe

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Comprehensive studies on protein-NP interactions can help in assessing the possible

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safety concerns pertaining to the use of NP in biomedical applications. Moreover,

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these studies effectively predict NP-cell interactions and thereby contribute to the

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development of novel NPs that are safe-by design.32

nanomedicines

and

other

nanomaterials

based

consumer

products.

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Among the various classes of proteins, albumin (or serum albumin) is the most

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abundant protein in blood plasma and it is chiefly responsible for maintaining ≈80% of

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the colloidal osmotic pressure.33,34 Albumin being a multifunctional transport protein,

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has a broad affinity towards wide variety of ligands; for example, albumin acts as a

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carrier for many insoluble fatty acids in the circulatory system.35 Moreover, it plays a

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key role in the transport and deposition of many substances in therapeutic studies and

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in regulating the blood pH.33,36-39 Furthermore, it is widely used as a model protein for

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many biochemical and biophysical studies.27,40-43

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Human serum albumin (HSA), is the most abundant protein constituent in human

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blood plasma (40 kg m−3 or 0.6 mM) and has many physiological functions. HSA, a

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globular protein of Mr 66 kDa, consists of 585 amino acids. The amino acid sequence

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of HSA consists of 6 methionines, 18 tyrosines (Tyr), 1 tryptophan (Trp 214),

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17 disulfide bridges and one free thiol (Cys 34) group. The disulfides are positioned in

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a repeating series of nine loop link loop structures centered on eight sequential Cys-

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Cys pairs. The three homologous domains present in HSA viz, I, II and III assemble

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together to form a heart shaped structure. Each domain contains two subdomains

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namely A and B which share a similar structural pattern.44-46 The hydrophobic pockets

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located at subdomains IIA and IIIA are the principle ligand binding sites in HSA, with

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subdomain IIIA having the superior affinity.40 Many ligands are known to possess site


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specific binding to HSA, either in site I (located at subdomain IIA) or in site II

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(located at subdomain IIIA).46 The interaction of ligands with active sites of the

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proteins can modify their structure and functions, which in turn may leads to

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detrimental effects.47 The lone Trp residue (214) in HSA is often used as a fluorescent

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reporter group for studying ligand-protein interactions and conformational aspects.48-50

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The adsorption of proteins on the surface of NPs have been reported earlier,

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however, extensive analysis on NP-protein interactions are still scarce. It has been

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reported that metal NPs show strong affinity towards common blood proteins, where

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the binding strength of protein to nanoparticles surface is governed by various factors

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such as particle size, NP composition, capping ligand and native protein structure etc.,.

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Further, upon its association with NP surface model proteins tends to lose its native

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structure.50 While many current biophysical studies focus primarily on studying the

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binding affinity of metal nanoparticles with serum proteins, the nature of protein

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interaction with Au/Ag NPs is yet to be studied. Since, Au/Ag NPs found many

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biological applications,11-12 it is vital to understand and quantify their effects on

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structure and functions of surface bound serum proteins. In view of the above

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objectives, the binding mechanism between HSA and Au/Ag NPs was methodically

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studied by using multi spectroscopic techniques viz., absorption, steady state emission,

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time resolved fluorescence, FT-IR, CD and Raman spectroscopy. The physical

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characterisation of Au/Ag NPs in the absence and presence of HSA were done using

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DLS, zeta potential measurements and HR-TEM with line scan EDX measurements.

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The interaction of Au/Ag NPs with HSA has been elucidated in detail by steady state

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emission titration experiments. The various binding parameters for HSA-Au/Ag NPs

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system viz., binding constant, quenching constant and Gibbs energy were also

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subsequently evaluated from the emission spectral studies. We have monitored the


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dissociation of 1-anilino-8-naphthalenesulfonic acid (ANS) probe from the protein

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binding site under varying concentrations of Au/Ag NPs to explore the effect of

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Au/Ag NPs interactions on the secondary structure of serum protein. The multilayer

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formation of HSA on Au/Ag NPs surface was predicted by comparing the

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experimental results of emission quenching with those obtained by theoretical

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calculations. Absorption, FT-IR, Raman, CD and 3D emission spectroscopic

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techniques were employed to study the Au/Ag NPs induced tertiary and secondary

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conformational changes of HSA. The esterase like activity of HSA was studied in the

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absence and presence of Au/Ag NPs to unravel the influence of nanoparticles on the

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enzyme activity of HSA.

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2. EXPERIMENTAL SECTION

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2.1. Chemicals. Human serum albumin (HSA), tetrachloroauric acid (HAuCl 4, 99%),

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sodium borohydride (NaBH 4, 99%) and p-nitrophenyl acetate were purchased from

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Sigma-Aldrich Chemicals, USA and are used as received. Silver nitrate (AgNO3, 99%)

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and tri-sodium citrate were obtained from SD-Fine Chemicals, India.

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2.2. Equipments and Spectroscopic Measurements. The HSA concentration was

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determined from the molar extinction coefficient (44,000 dm3 mol-1 cm-1) at 278 nm.49

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The various concentrations of Au/Ag NPs solution (0.08  10-10 to 0.72  10-10 mol

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dm-3) were prepared by pipetting an aliquot of the stock solution into a 5 mL standard

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measuring flask containing 1 mL of HSA (1.00 × 10-5 mol dm-3) and then the solutions

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were made up to the mark with phosphate buffer saline (PBS) (pH=7.40, 0.1 M).

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Requisite concentrations of HSA and Au/Ag NPs solutions are mixed uniformly and

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allowed to equilibrate for 15 minutes, prior to recording the spectral data. Absorption

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spectral analysis were performed using JASCO V-630 UV-visible spectrophotometer

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with quartz cuvettes of path length 1 cm. Emission spectral measurements were 6 ACS Paragon Plus Environment

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carried out using JASCO FP-6600 spectrophotometer with quartz cuvette of path

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length 1 cm. Emission spectra of HSA were monitored in the wavelength range of

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300-500 nm by selectively exciting the lone tryptophan residue (Trp-214) at 295 nm.

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Emission and excitation slit widths were set at 5 nm and 2 nm, respectively. 3D

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emission spectra were recorded from 200 nm to 500 nm with a scan speed of 2000

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nm/min. Circular dichroism (CD) spectra were measured using JASCO-180

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spectropolarimeter equipped with a 0.1 cm path length quartz cell. CD spectra were

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collected over the wavelength region of 200-300 nm with 0.1 nm step resolution and

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averaged over two scans at a speed of 50 nm min-1. The α-helical content of HSA in

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the absence and presence of HSA were computed from the molar ellipticity value. The

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obtained spectral data were baseline subtracted for buffer solution. All the spectral

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analysis was carried out at room temperature (25οC). Freshly prepared stock solutions

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of HSA and Au/Ag NPs were used for all the experiments.

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Time resolved fluorescence lifetime analysis were measured by means of time-

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correlated single-photon counting (TCSPC) method using an excitation light source of

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picosecond diode (IBH Nanoled) at 280 nm to generate the fluorescence of the sample

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and the emission signals were collected at a magic-angle polarization using a

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Hamamatsu micro channel plate photomultiplier (2809U). The resulting decay profiles

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were analyzed by nonlinear least-squares fitting procedure using IBH DAS6 decay

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analysis software. The quality of the fits was assessed by the χ2 values and distribution

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of residuals.

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FT-IR scanning of HSA and HSA-Au/Ag NPs were performed using JASCO FT-

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IR-V 400 series spectrophotometer with KBr pellets over the range of 4000-400 cm-1.

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Raman spectral data were recorded using R-3000-QE Raman spectrometer (Agiltron

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Inc, USA) using a 785 nm laser excitation source. The laser beam with a laser power



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of 250 mW was focused onto the liquid samples of HSA and HSA-Au/Ag NPs kept in

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quartz cuvette.

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2.3. HR-TEM, DLS and Zeta Potential Measurements. HR-TEM data of Au/Ag

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NPs dispersion in the absence and presence of HSA was performed in a JEOL JEM

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2100 microscope instrument at an operating voltage of 200 kV. A drop of aqueous

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dispersion of Au/Ag NPs and HSA-Au/Ag NPs solutions were placed on to two

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separate carbon coated copper grids and the solutions were evaporated under ambient

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conditions. The average particle size was obtained by analysing the size of more than

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50 particles using ImageJ software (version 1.45S). DLS and Zeta potential analysis

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were performed to evaluate the hydrodynamic diameter and surface charge of Au/Ag

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NPs in the absence and presence of HSA at room temperature using Malvern Zetasizer

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Nano, ZS with 633 nm He-Ne laser, equipped with a MPT-2 Autotitrator. The

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measured data is average of at least twenty runs. The average hydrodynamic diameter

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and mean zeta potential of each sample was computed using the software provided by

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the manufacturer.

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2.4. Preparation of Au/Ag nanoparticles. Au/Ag NPs were prepared by taking

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aqueous solutions of HAuCl 4 [1.50 × 10-4 mol dm-3] and AgNO3 [4.00 × 10-4 mol dm-3]

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in 47.5 mL double distilled water with a final gold molar ratio of 0.27. The solution

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containing HAuCl 4 and AgNO3 is refluxed under constant stirring with subsequent

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addition of 2.5 mL of 1% tri-sodium citrate. The reduction of gold and silver ions by

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citrate ions are completed after 15 min. The resulting colloidal solution is then left to

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cool at room temperature (25οC). The Au/Ag NPs showed a surface plasmon band at

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410 nm in its absorption spectrum. The formation of alloy nanoparticles is obvious

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from the appearance of a single peak in the UV-vis absorption spectrum.

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Homogeneous alloy nanoparticles are stated to exhibit a single resonance peak with a


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wavelength between that observed for individual constituent elements, whereas

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core/shell type nanoparticles or phase segregation will generate a twin peak resonance

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corresponding to the different elements in the core and shell structure.15,16,17,51 Thus,

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the appearance of single SPR peak at 410 nm is indicative of the alloy nature of the

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synthesized Au/Ag NPs (for an Au molar ratio of 0.27). Moreover, the observed SPR

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peak value is in good agreement with the previously reported value. 51 The

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concentration of stock dispersion of Au/Ag NPs [2.00  10-9 mol dm-3] was estimated

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from the molar extinction coefficient of 2.50  109 dm3 mol-1 cm-1 at 410 nm.52

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2.5. Esterase-like Activity Measurements. The influence of Au/Ag NPs on the

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esterase like activity of HSA was examined with the synthetic substrate p-nitrophenyl

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acetate (p-NPA) by following the formation of p-nitrophenol at 405 nm, using fixed

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wavelength absorption spectral measurements. The reaction mixtures contained

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2.0 × 10-9 mol dm-3 HSA and 5.00 × 10-4 mol dm-3 p-nitrophenyl acetate in PBS, pH

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7.40 at 298 K. The concentration of p-nitrophenol was determined by absorption

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measurements using a molar extinction coefficient value of 17,700 dm3 mol-1 cm-1 at

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405 nm.47 One unit of esterase like activity were defined as the amount of enzyme

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required to liberate 1 × 10-6 mol dm-3 of p-nitrophenol per minute at 25οC.53

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3. RESULTS AND DISCUSSION

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3.1. Analysis on Physicochemical Changes of HSA-Au/Ag NP Bioconjugates.

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Physicochemical changes of Au/Ag NPs in the presence of HSA were analyzed using

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different techniques namely, absorption spectral measurements, HR-TEM, DLS and

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zeta potential studies.

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Absorption Spectral Behaviour of Au/Ag NPs in the Presence of HSA. The

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adsorption characteristic of HSA on the surface of Au/Ag NPs was characterized by

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absorption spectroscopy. Au/Ag NPs in the absence of HSA exhibit a strong surface 9 ACS Paragon Plus Environment


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plasmon resonance band (SPR) at 410 nm (Figure 1a).52 The absorption spectra of

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Au/Ag NPs in the presence of increasing concentrations of HSA are shown in Figure

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1(b-e). Upon the addition of increasing concentration of HSA to Au/Ag NPs solution,

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the SPR band of Au/Ag NPs was red shifted towards a longer wavelength with a slight

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increase in its absorption intensity (Figure 1 (a-e)). The red shift in SPR band of

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Au/Ag NPs is considerably strong for the addition of HSA (1.00 × 10-6 mol dm-3).

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However, subsequent addition of HSA to the Au/Ag NPs solution did not bring about

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any significant shift in SPR band. The initial red shift of SPR band is attributed to the

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changes in refractive index of Au/Ag NPs owing to the interaction of HSA molecules

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with Au/Ag NPs surface. It should be noted that the shift in SPR band of Au/Ag NPs

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is not accompanied by any peak broadening, nor a significant rise in the baseline of the

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spectrum, indicating that the change in the position of the SPR band is mainly due to

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binding of HSA, rather than aggregation of Au/Ag NPs.54 Recently, an identical result

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has been observed in the case of BSA-Au nanorods system and interaction of HSA

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with Au NPs.54,55 Moreover, the absence of significant shift in SPR band of Au/Ag

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NPs upon further addition of HSA clearly suggests that the adsorbed HSA protein acts

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as a good capping agent and it prevents the individual Au/Ag NPs from aggregation.55


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250 251 252

Figure 1. Absorption spectra of Au/Ag NPs [3.50  10-10 mol dm-3] at different HSA concentrations. [HSA]: (a) 0.0, (b) 1.00  10-6, (c) 2.00  10-6, (d) 3.00  10-6 and (e) 4.00  10-6 mol dm-3. Inset shows the enlarged image of SPR band shift in absorption spectra of Au/Ag NPs upon HSA addition.

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3.2. Morphological Properties and Zeta Potential Measurements of HSA-Au/Ag

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NPs System. To further validate the formation of homogenous alloy NPs, the

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elemental distribution and nanoparticle composition of Au/Ag alloy NPs was anlaysed

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by line scan TEM-EDX measurements. The morphological properties of Au/Ag NPs

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in the absence and presence of HSA were also examined by HR-TEM. HR-TEM

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images of Au/Ag NPs before and after the addition of HSA are presented in Figure 2

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(A(i) & B(i)). It is evident from the HR-TEM image that Au/Ag NPs in the absence of

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HSA are spherical in shape with an average diameter of 20 nm and showed a relatively

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narrow size distribution (Figure 2A (iii)). The HR-TEM images clearly (Figure 2A (i

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& ii)) suggest a high-degree of crystallinity with well-defined fcc lattice fringes.

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Similar kind of fcc lattice pattern have been reported for homogeneous Au/Ag alloy

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NPs.56

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measurements of nanoparticles are shown in (Figure 2A (iv & v)). The outcome of the

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line scan results, suggest that both Au and Ag elements are homogenously distributed

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in the analysed nanoparticles. The obtained results clearly ruled out the formation of

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phase boundaries or segregation of the elements and it is in good agreement with the

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recently reported work for homogenously distributed Au/Ag alloy NPs.56 The HR-

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TEM image of HSA-Au/Ag NPs bioconjugates displayed in Figure 2B (i), clearly

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shows that HSA-Au/Ag NPs remain isolated without any aggregation. Corona

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formation of HSA on nanoparticle surface was established by analysing the

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interparticle separation distance of HSA coated Au/Ag NPs that are connected

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together via HSA with an effective inter particle linkage. The measurement of

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interparticle distance yielded a value of 6±1 nm, indicating the formation of thick layer

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of HSA over the nanoparticle surface. It has been previously shown that BSA attaches

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to nanoparticle surfaces, forming a monolayer of approximately 7 nm thickness.57,58

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Recently, Miclaus et al., reported the formation of densely packed monolayer of HSA

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on the surface of Au NPs with a thickness of 7 nm.59 The structure constitution of

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Au/Ag NPs before and after conjugation with HSA was studied by selective area

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energy dispersion (SAED) pattern. As shown in Figures 2A (III) and 2B (II), SAED

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patterns of both Au/Ag NPs and HSA-Au/Ag NPs bioconjugates are characterised by

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well defined Debye-Scherrer rings suggesting the crystalline nature of the

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nanoparticles.

The

representative

line

scan

image

and

selected-area

composition

285

DLS measurement was performed to study the influence of HSA on the

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hydrodynamic radius of Au/Ag NPs. The hydrodynamic radii of Au/Ag NPs (2.00 ×


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10-9 mol dm-3) in the absence and presence of HSA (4.50 × 10-7 mol dm-3) are

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displayed in Figure 2C (i & ii), respectively. As shown in Figure 2C (i & ii), the

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hydrodynamic radii of Au/Ag NPs in the presence of HSA showed an increase from an

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initial value of 24±2 nm to 31±2 nm. The increase in hydrodynamic diameter of 7 nm

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signifies that a densely packed adsorption of HSA has occurred on the surface of

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Au/Ag NPs. The average particle size calculated from HR-TEM and DLS

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measurement showed appreciable difference and this deviation is attributed to the fact

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that DLS gives a measure of hydrodynamic radii of nanoparticles in solution, whereas,

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HR-TEM provides the size of the nanoparticle in dried condition.60 Owing to its low

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resolution and lack of robustness relative to cumulants analysis, DLS measurements

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are often used with other characterisation methods for qualitative comparison.27 As a

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result in the present work, the outcome of the DLS studies were compared with

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absorption and TEM measurements. The upshot of DLS measurements clearly

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indicated an increase in the hydrodynamic radii of Au/Ag NPs upon incubation with

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HSA. This slight increase in particle size suggests that possible HSA adsorption onto

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the surface of Au/Ag NPs and it conforms to the results obtained from absorption

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spectral studies.

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In order to gain insight into the HSA induced variations in net surface charge of

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Au/Ag NPs, zeta potential measurements were carried out under identical

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experimental conditions as described above. It is well known that surface charge of

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nanoparticles is directly related to the magnitude of electrical potential at the surface

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of nanoparticles and thickness of the double layer which in turn depends on the surface

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bound protein molecules.20 Zeta potential analysis of initial Au/Ag NPs dispersion

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exhibited a value of -11.60 mV. The observance of negative zeta potential is indicative

311

of negative surface charge of citrate stabilised Au/Ag NPs. Upon incubating Au/Ag



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

312

NPs dispersion with HSA, the zeta potential value of Au/Ag NPs showed a

313

considerable decrease from -11.60 mV to -5.30 mV. The observed decrease in surface

314

charge is attributed to the screening of negatively charged Au/Ag NPs by surface

315

bound protein molecules, which may establish electrostatic interactions between the

316

surface citrate ions and the positively charged amino acid residues of the protein

317

(lysine, histidine, and arginine).57,28 In addition, the role of multiple hydrogen bonds,

318

van der Waals interactions and steric contacts could have also played a crucial role in

319

the binding of HSA to Au/Ag NPs surface. It has been reported earlier that zeta

320

potential measurements of Au NPs and Ag NPs-HSA coronas exhibited a negative

321

surface charge in the range of -7.91 to -12.7 mV with no major variations in zeta

322

potential even after 24 hours of incubation. 61 However, the presence of bulky ligands

323

like proteins on the nanoparticle surface are shown to significantly interfere with the

324

zeta potential measurements. It has been reported that the presence of complex ligands

325

produce hydrodynamic drag force (Darcy drag force) around the nanoparticle surface

326

thereby increasing the electrophoretic retardation force.51,62 Owing to the above

327

mentioned shortfall the variation in zeta potential values must be interpreted as a

328

measure of ligand induced surface modifications by comparing it with other reliable

329

tools. The results from HR-TEM and DLS analysis helped us to unambiguously

330

establish the rapid adsorption of HSA on to Au/Ag NP surface.


Page 14 of 50

Page 15 of 50

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331

332



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

Page 16 of 50

333 334 335 336 337 338

Figure 2. TEM analysis of (A) (i). Au/Ag NPs, (ii). Particle size distributions, (iii). SAED pattern, (iv). Representative line scan image and (v). Selected-area composition measurements of nanoparticles. (B) (i). HSA-Au/Ag NPs conjugates and (ii). SAED pattern. (C) Hydrodynamic radii obtained from DLS measurements for Au/Ag NPs (i) in the absence and (ii) presence of HSA.

339

3.3. Steady State Fluorescence Spectroscopic Studies of HSA in the Presence of

340

Au/Ag NPs. Emission spectroscopy is an ideal tool to elucidate the binding

341

mechanism of protein with NPs.54 It is well established that protein-NP interactions

342

are often associated with changes in the intrinsic emission intensity of biomelecules.55

343

Moreover, metal nanoparticles are shown to display high quenching efficiency

344

towards many chromophores. Therefore, intrinsic emission property of protein is

345

widely explored to get better understanding on the adsorption characteristics of protein

346

on to the NP surface. The influence of Au/Ag NPs on the intrinsic emission property

347

of HSA was monitored by emission spectral studies. The emission spectra of HSA in

348

the presence of increasing concentrations of Au/Ag NPs are displayed in Figure 3. The

349

emission spectra of native HSA exhibit an emission maximum at 348 nm, upon

350

excitation at 295 nm. The choice of 295 nm as the excitation wavelength was to avoid

351

contribution from tyrosine residues. 63 It can been observed from Figure 3, that the

352

emission intensity of HSA showed a progressive decrease upon the incremental

353

addition of varying concentrations of Au/Ag NPs to HSA solution. In the present case, 16 ACS Paragon Plus Environment

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354

emission spectrum of HSA is solely due to the lone Trp residue and the perturbation in

355

Trp emission intensity of biomolecules have been previously utilised to quantify the

356

interaction of HSA with organic molecules and nanoparticles. Adsorption of HSA

357

results in exposure of amino acid residues to the proximity of Au/Ag NPs surfaces,

358

thereby, resulting in efficient emission quenching. Consequently, the decrease in

359

maximum emission intensity of HSA upon Au/Ag NPs addition is attributed to direct

360

complexation of HSA with Au/Ag NPs. Quenching of fluorescent macromolecule can

361

occur by wide variety of process namely, by static or binding related quenching,

362

dynamic quenching and inner-filter effect etc.,. Static quenching is further subdivided

363

into two types viz., ground-state complex formed between the quencher and the

364

fluorescent macromolecule, quenching arising out of excited-state energy transfer in

365

the complex or quencher induced microenvironment and conformational changes in

366

the vicinity of the fluorophores.64 In the preceding section, the mechanism involved in

367

the Au/Ag NPs induced quenching of HSA is investigated. It has been previously

368

reported that in emission titration experiments, NPs are shown to adsorb intrinsic

369

emission intensity of protein even in the absence of any direct contact with NPs

370

surface owing to high absorbance characteristics of NPs.54 Thus, in the present

371

investigation, we believe that the addition of Au/Ag NPs (even at low concentration)

372

to HSA solution may lead to scattering of the incident light which in turn may reduce

373

the emission intensity.54 To account for the reduced emission intensity of HSA due to

374

competitive absorption and re-absorption by Au/Ag NPs at the excitation and emission

375

wavelength of HSA, the following correction factor was applied to the measured

376

emission spectra, eqn. (1):64

F

= F

(

×e

)

− (1)



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

377

where, Fcorr and Fobs is the corrected and observed emission intensities, respectively,

378

Aexi and Aemi are the solution absorption at the excitation and emission wavelengths,

379

respectively. The data obtained from corrected emission spectra were used for further

380

analysis.

Page 18 of 50

381 382 383 384 385

Figure 3. Emission spectra of HSA [1.00 × 10-5 mol dm-3] at different Au/Ag NPs concentrations. [Au/Ag NPs]: (a) 0.00, (b) 0.08 × 10-10, (c) 0.16 × 10-10, (d) 0.24 × 10-10, (e) 0.32 × 10-10, (f) 0.40 × 10-10, (g) 0.48 × 10-10, (h) 0.56 × 10-10, (i) 0.64 × 10-10 and (j) 0.72 × 10-10 mol dm-3.

386

3.4. Intrinsic Emission Quenching of HSA by Au/Ag NPs. The quenching

387

mechanism involved in the HSA-Au/Ag NPs system was evaluated by Stern-Volmer

388

equation (eqn. 2).64 F = 1 + k τ [ Q] = 1 + k F

− (2)


Page 19 of 50

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389

where, F0 and F denotes the maximum emission intensities (corrected) in the

390

absence and presence of Au/Ag NPs, respectively. Kq is the apparent bimolecular

391

quenching constant of the biological macromolecule; τ0 is the unquenched lifetime of

392

the protein, [Q] is the molar concentration of quencher and Ksv is the Stern-Volmer

393

quenching constant which indicates the sensitivity of the fluorophore to a quencher.

394

The plot of Stern-Volmer equation in the presence of varying concentrations of Au/Ag

395

NPs is depicted in Figure 4.

396 397

Figure 4. Stern-Volmer plot for HSA-Au/Ag NPs system.

398

As evidenced from Figure 4, the plot of F0/F vs [Au/Ag NPs] obeys by a linear

399

regression. The Stern-Volmer quenching constant Ksv value was determined from the slope

400

and intercept of the linear curve and it is listed in Table 1. Further, the value of bimolecular

401

quenching constant (Kq) can be obtained from the equation Kq = Ksv/τ0 by making use of the 19 ACS Paragon Plus Environment


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Page 20 of 50

402

fluorescence life time value of native protein (~10-8 s) and Ksv value obtained from the Stern-

403

Volmer plot. The calculated Kq value of 4.29 × 1017 dm3 mol-1 s-1 for HSA-Au/Ag NPs system

404

is much greater than the previously available literature values for maximum scatter collision

405

quenching constant (2.00 × 1010 dm3 mol-1 s-1).64 The obtained results clearly ruled out the

406

likelihood of collisional quenching mechanism and affirms the possible existence of static

407

quenching of lone Trp residue of HSA by Au/Ag NPs.

408

3.5. Determination of binding constant for HSA-Au/Ag NPs. The formation of

409

HSA-Au/Ag NPs ground state complex was further evaluated by assessing the

410

emission quenching data of HSA-Au/Ag NPs system. In protein-nanoparticle

411

interaction, biomolecules can have multiple associative interactions with the

412

nanoparticle surface, thereby resulting in multiple cooperativity during the binding

413

process. This complex phenomena can be delineated using double logarithmic

414

equation (eqn. 3),28,58

log

F −F = log K + n log [Q] − (3) F

415

where, F0 and F are same as mentioned under eqn. 2, Kb is the binding constant.

416

The Hill coefficient, n, is regarded as the degree of cooperativity in protein binding to

417

a surface.28,58,65 In the case of positive cooperative interactions n is greater than 1 and

418

it signifies that once a protein molecule is adsorbed on to nanoparticle surface, the

419

binding of other protein molecules to NP surface is enhanced in a super linear way.

420

For interactions having negative cooperativity n is lesser than 1 and it implies that the

421

binding strength of protein is decreased progressively as further proteins bound to the

422

surface. For non cooperative interaction n = 1 and the binding of protein is

423

independent of other proteins present at the surface.28,58 The double logarithmic plot

424

for HSA-Au/Ag NPs is displayed in Figure 7. The linear dependence of emission


Page 21 of 50

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425

intensity (log [(F0-F)/F]) with respect to nanoparticle concentration (log [Au/Ag NPs])

426

is obvious from Figure 7 with an intercept (Kb) and slope value (n). The cooperativity

427

of binding n for the binding of HSA to Au/Ag NPs is approximately equal to 1.12. The

428

Hill coefficient in the present case is greater than 1 thereby suggesting the existence of

429

positive cooperative binding. The results thus obtained makes it clear that the presence

430

of HSA molecule on Au/Ag NP surface enhances the binding of other HSA molecules

431

thereby resulting in the formation of dense protein layer around the surface of Au/Ag

432

NPs. The calculated binding parameters of HSA-Au/Ag NPs system are given Table 1.

433 434

Figure 5. Double logarithmic plot for HSA-Au/Ag NPs system.

435 436 437 21 ACS Paragon Plus Environment


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438

Table 1. Binding parameters of HSA-Au/Ag NPs system

439 440 441 442 443 444

Page 22 of 50

a

Binding Parameters

Ksva (dm3 mol-1)

Kqa (dm mol-1s-1)

Kba (dm mol-1)

n

HSA-Au/Ag NPs

(4.29 ± 0.15) × 109

(4.29 ± 0.15) × 1017

(2.56 ± 0.12) × 109

1.12 ± 0.08

3

3

Represents the mean value obtained from three separate experiments. The Gibbs energy associated with the interaction of HSA and Au/Ag NPs can be

estimated by using the eqn 4,66 ∆G = −RT ln k − (4)

445

where, R represents the universal gas constant, T is the temperature and Kb is the

446

binding constant value obtained from double logarithmic plot.

447

The Gibbs energy change ΔG for the interaction of HSA-Au/Ag NPs is calculated

448

as ΔG = -52.58 kJ mol-1. The negative ΔG value clearly showed a favourable process

449

of interaction between HSA and Au/Ag NPs surface and the obtained value agrees

450

well with the previously reported values.67

451

3.6. Time Resolved Fluorescence Spectroscopic Analysis of HSA in the Presence

452

of Au/Ag NPs. To confirm the static quenching mechanism involved in the HSA-

453

Au/Ag NPs system, we have performed excited state life time measurements of HSA

454

(1.00 × 10-5 mol dm-3) in the absence and presence of Au/Ag NPs. The experimental

455

time-resolved fluorescence decays P(t) were analysed using the following expression,

456

(eqn 5),64

P( ) = b +

α exp −

t − (5) τ

457

where, t is time, n is the number of discrete decay components, b is an offset, αi is

458

the pre-exponential factor and τi corresponds to excited-state fluorescence lifetimes

459

associated with the ith component.


Page 23 of 50

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460 461 462 463 464

Figure 6. Time resolved fluorescence decay profile of [●] HSA alone c[HSA] = 1.00 × 10-5 mol dm-3 and [Δ] HSA in the presence of Au/Ag NPs. c[Au/Ag NPs] = 0.72  10-10 mol dm-3. (exi = 280 nm and emi = 348 nm).

465

The influence of Au/Ag NPs (0.72  10-10 mol dm-3) on the excited-state life time of

466

HSA (1.00 × 10-5 mol dm-3) was analysed and the representative lifetime decay is

467

displayed in Figure 6. As evidenced from Figure 6, the fluorescence decay profile of

468

HSA in the absence and presence of Au/Ag NPs is best fitted to a multi-exponential

469

decay curve with average life time values of 5.11 ns and 5.14 ns, respectively (Table

470

2). The absence of significant reduction in life time values of HSA upon the addition

471

of Au/Ag NPs is attributed to the formation of non-fluorescent ground state complex

472

between HSA and Au/Ag NPs. It has been well established that the formation of static

473

ground-state complexes do not decrease the decay time of the uncomplexed

474

fluorophores because only the life time of unquenched fluorophores are observed in 23 ACS Paragon Plus Environment


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Page 24 of 50

475

time resolved spectroscopic analysis. Whereas, dynamic quenching is a rate process

476

acting on the entire excited-state population and thus decreases the mean decay time of

477

the entire excited-state population.64 On the basis of time resolved fluorescence

478

spectroscopic analysis it is inferred that the emission quenching of HSA by Au/Ag

479

NPs is initiated by static quenching mechanism. Further, this conclusion is in good

480

agreement with the outcome of the steady state emission quenching studies.

481

Table 2. Time resolved fluorescence decay parameters of HSA and HSA-Au/Ag NPs.

System

α1

τ1 (ns)

α2

τ2 (ns)

a (ns)

χ2

482

HSA 0.35 2.44 0.65 6.56 5.11 1.09 alone HSA0.26 2.04 0.74 6.21 5.14 1.14 Au/Ag NPs a = ± 2% and = τ1 α1+ τ2 α2. The magnitude of χ2 denotes the goodness of the fit.

483

3.7. Determination of Protein Corona Formation on the Surface of Au/Ag NPs.

484

The formation of protein corona around the nanoparticle surface plays a crucial role in

485

determining their interactions with other biological matter. Therefore, it is important to

486

study the protein corona formation to get better understanding on how exposure to

487

NPs affects its biological responses of cells and organisms.70 An essential parameter

488

that characterizes a protein corona is the average number of proteins that are bound to

489

the surface of a NP under a given experimental condition.69,70 On the basis of

490

theoretical calculation it is possible to estimate approximate number of proteins on the

491

nanoparticle surface. Previous literature study has reported a triangular prism shaped

492

structure of serum albumin in solution with sides of 8.4 nm and a height of 3.15 nm.44

493

On the basis of the triangular prism shaped model for HSA,69 the average number, N,

494

of the bound protein molecules for monolayer coverage on a spherical NP is calculated

495

from eqn. (6).69,70


Page 25 of 50

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=

496 497

− 1 ×

− (6)

where, RN, R0 Vp and V0, are the volume of the bound protein molecule, the volume of NP without protein and the radii of NP with and without protein.

498

The volume of HSA molecule and Au/Ag NPs was calculated as 96.3 nm3 and

499

4180 nm3, respectively. If the HSA molecule is assumed to retain a “flat-on” or “end-

500

on” conformation over the Au/Ag NP surface, the radius of HSA-Au/Ag NPs

501

bioconjugate approximates to 13.15 nm (flat-on) and 17.24 nm (end-on), respectively.

502

To attain complete monolayer coverage over Au/Ag NPs surfaces, the required

503

number of HSA molecules per Au/Ag NPs are approximately equal to about 63 (flat-

504

on) and 179 (end-on) respectively. The formation of dense layer of HSA around

505

Au/Ag NPs surface was predicted by comparing the data obtained from emission

506

quenching experiments with those obtained by theoretical calculations. In the emission

507

quenching experiment, it is assumed that the quenching occurs only for those HSA

508

molecules that have direct contact with Au/Ag NPs surfaces and HSA molecules that

509

are quenched is assumed to be restricted to full monolayer coverage on the Au/Ag NP

510

surface. The data obtained from Au/Ag NPs induced emission quenching of HSA was

511

utilised to compute the number of HSA molecules bound to single Au/Ag NP surface

512

and it was calculated as ∼ 3.3 × 104. Based on the emission quenching studies, it has

513

been previously reported that Au nanorod can accommodate ∼ 1 × 103 - 3 × 103 HSA

514

molecules. This difference in number of surface bound HSA molecules could be

515

attributed to the fact that Au/Ag NPs are smaller in size with high surface to volume

516

ratio when compared with gold nanorod. 54 The estimated average number of HSA

517

molecules per Au/Ag NP surface showed a significant deviation from that of the

518

theoretically computed value for both “flat-on” and “end-on” conformation of HSA, 25 ACS Paragon Plus Environment


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Page 26 of 50

519

thereby, demonstrating the formation of thick layered structure of HSA on Au/Ag NPs

520

surface. Moreover, the outcome of the analysis clearly indicated that the interaction of

521

HSA to Au/Ag NPs surface continues beyond the formation of an initial monolayer

522

and that the existence of cooperative binding between HSA molecules might have

523

played a crucial role in enhancing the protein corona formation. It is well known that

524

the introduction of colloidal nanoparticles into a biological medium often result in the

525

rapid formation of protein corona around the NP surface.68,69,70 It should be noted that

526

the Hill coefficient for HSA-Au/Ag NPs system is greater than 1, thereby indicating

527

the existence of cooperative binding. The Hill coefficient value is in good accord with

528

the average number of surface bound HSA molecules, which gives compelling

529

evidence that dense layers of HSA is formed on the surface of NPs.

530

To analyze the effect of nanoparticle core material on protein corona composition,

531

we have carried out emission quenching studies of HSA with citrate capped gold

532

nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) under identical

533

experimental conditions. The emission spectral data of HSA under increasing

534

concentrations of citrate capped Au NPs and Ag NPs are displayed in Figure S1

535

(A&B). As shown in Figure S1 (A&B), the emission intensity of HSA is quenched

536

upon the addition of varying concentration of citrate capped Au NPs and Ag NPs

537

suggesting the prevalence of protein-nanoparticle interactions. The outcome of the

538

experimental results was compared by plotting the normalised corrected emission

539

intensity curve of Au NPs, Ag NPs and HSA-Au/Ag NPs (Figure S2). The results

540

revealed an identical quenching behaviour of HSA with slight variations in the

541

presence of Au NPs, Ag NPs and Au/Ag NPs with no obvious inference to divulge the

542

influence of core metal composition. It is difficult to ascertain the influence of

543

nanoparticle core material solely on the basis of emission quenching experiments. It


Page 27 of 50

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544

has been reported that the core composition of metal nanoparticles exhibit a significant

545

influence on protein corona formation when compared to the size or surface functional

546

group. For example, it was shown that the formation of serum protein corona around

547

Ag NPs showed a similarity of 36.9% with Au NPs modified with same surface ligand

548

suggesting the possible influence of core material on corona formation. The above

549

finding may be counterintuitive as it is well known that nanoparticle core is covered

550

by surface ligands. However, even in the absence of direct contact with proteins the

551

importance of core material composition on protein corona formation is further

552

supported by the fact that the nature of the metal core holds the key in determining the

553

density, arrangement and surface orientation of surface capping ligands. To get better

554

understanding on the influence of nanoparticle core in protein corona formation,

555

thorough assessment of nanoparticle surface by X-ray photoelectron spectroscopy,

556

time-of-flight secondary ion mass spectrometry or atomic force microscopic studies

557

must be carried out.71

558

3.8. Red-edge excitation shift of native HSA and HSA-Au/Ag NPs. When solvent

559

relaxation is not complete the emission spectra of polar fluorophores shift to longer

560

wavelengths when the excitation is on the long-wavelength edge of the absorption

561

spectrum. Accordingly, Red edge excitation shift (REES) is defined as the shift in the

562

emission maximum towards the red end of the absorption spectrum of fluorophore. 64

563

This phenomenon which violates Kasha’s rule for electronic transition was first

564

discovered in the year 1970. 72 REES is a wavelength dependent sensitive tool widely

565

used for monitoring the microenvironment around the fluorophore in a complex

566

biological system and the salvation dynamics in an organized medium.64 Hence, REES

567

can be used to extract information regarding the dynamics and motion of lone Trp

568

residue of HSA. In the present work, we investigated the REES effect of HSA in the



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

Page 28 of 50

569

absence and presence of Au/Ag NPs by exciting the Trp residue at 295 nm and 310

570

nm, respectively and the results are given in Table 3. The difference in maximum

571

emission wavelength of HSA upon excitation at 295 nm and 310 nm gives the measure

572

of Δλem-max value. As evidenced from Table 3, native HSA exhibits an REES value of

573

5 nm implying that the lone Trp residue in HSA is confined to a motionally restricted

574

microenvironment. Upon the addition of Au/Ag NPs, REES value of HSA showed a

575

slight increase to 6 nm suggesting that the surface adsorption of HSA has imparted

576

substantial restriction to the orientation of solvent dipoles around the excited state Trp

577

residue. An identical REES trend has been reported for the binding of BSA to Au NPs

578

and it was proposed that the interaction of BSA to Au NPs has brought about

579

significant restrictions in the mobility of Trp residues in BSA.73 The outcome of the

580

above experimental results explicitly suggest the possibility of Au/Ag NPs induced

581

tertiary structural changes in HSA. Moreover, REES result was found to be in good

582

accord with the emission quenching experiment of HSA under varying Au/Ag NPs

583

concentrations.

584 585

Sample λex

λem-max (nm) 295 nm λex 310 nm

Δλem-max (REES) (nm)

586

HSA

348

353

5

HSA-Au/Ag NPs

346

352

6

587 588 589

Table 3. REES effects of HSA-Au/Ag NPs system (λex 295 nm and λex 310 nm).

590

3.9. Effect of Au/Ag NPs on the Extrinsic Emission Property of HSA bound ANS.

591

1-anilinonaphthalene-8-sulfonate (ANS) is an important organic probe employed in

592

the emission spectral studies of proteins. ANS possess high fluorescent activity when

593

bound to hydrophobic sites of the protein. However, when exposed to polar

594

environments fluorescence intensity of ANS decreases significantly.55,74 The 28 ACS Paragon Plus Environment

Page 29 of 50

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595

perturbation in the emission intensity of ANS probe can be exploited to study the

596

tertiary structural changes of protein upon its conjugation with NPs. The interaction of

597

ANS with protein sites have been shown to take place through ion pair formation

598

between the sulfonate group and proximate positively charged side chains and on the

599

other hand, through hydrophobic interactions in sites already present or induced in the

600

protein by accommodating the naphthalene and aniline moieties.55,74

601

To ascertain the influence of Au/Ag NPs on the tertiary structure of HSA, we have

602

undertaken emission quenching studies of HSA-ANS complex under varying

603

concentrations of Au/Ag NPs (Figure 7). ANS being a hydrophobic dye exhibits a

604

very weak emission in buffer solution at 520 nm when excited at 350 nm. As shown in

605

Figure 7, HSA bound ANS showed a strong emission property at 470 nm, upon

606

excitation at 350 nm. Previously, it has been reported that the main and most active

607

binding site of ANS on HSA is located in subdomain IIIA of the protein molecules.75

608

The binding site at subdomain IIIA of HSA is a hydrophobic pocket formed by the

609

spatial combination of the nonpolar residues.75 The enhanced emission intensity of

610

ANS in the presence of HSA clearly indicates that ANS is bound to the hydrophobic

611

pocket of HSA. To the ANS bound HSA solution, increasing concentration of Au/Ag

612

NPs was added and the corresponding emission spectral data is displayed in Figure

613

7(a-j). As depicted in Figure 7, the addition of Au/Ag NPs resulted in the decrease of

614

emission intensity of protein bound ANS. The data obtained from emission quenching

615

study was utilized to determine the quenching constant from the fractional corrected

616

emission intensity plot of HSA-ANS complex in the presence of increasing

617

concentrations of Au/Ag NPs. The plot of F0/F vs [Au/Ag NPs] is shown in Figure

618

8(A). As depicted in Figure 8(A), the fractional emission intensity of HSA bound ANS

619

exhibited a good linearity within the investigated concentration of Au/Ag NPs yielding



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

620

a quenching constant value of 4.47 × 10 9 dm3 mol-1. It is to be noted that a similar

621

quenching constant value is obtained for the intrinsic emission quenching of HSA by

622

Au/Ag NPs. Therefore, from the above observation it is inferred that Au/Ag NPs

623

quench the emission intensity of HSA bound ANS in a similar way as the intrinsic

624

emission of HSA. To compute the binding constant value for the interaction of ANS

625

bound HSA with Au/Ag NPs, the emission quenching data from the above experiment

626

is subjected to analysis by double logarithmic equation (eqn. 3) and the resulting plot

627

is shown in Figure 8(B). As shown in Figure 8(B), double logarithmic plot obeys by a

628

linear regression and the binding constant value calculated from the plot is Kb = 7.2 ×

629

1010 dm3 mol-1. The Kb value thus obtained is 10 fold higher than the binding constant

630

obtained for HSA-Au/Ag NPs system (in the absence of ANS). This finding along

631

with the identical quenching constant value clearly indicates that the presence of ANS

632

molecule in site IIIA produces a strong complex of HSA-Au/Ag NPs. Recently, an

633

identical observation has been made by Canaveras et al, for the binding of citrate

634

capped Au NPs with HSA protein.55 It has been proposed that the interaction of

635

sulfonic acid moiety in ANS with Lys414 of HSA through a salt bridge increases the

636

overall negative charge of the conjugates, thereby, resulting in stronger binding

637

affinity of HSA-Au NPs.55 From the above observations, it is proposed that the

638

interaction of Au/Ag NPs have brought about partial unfolding of the protein and this

639

in turn resulted in the exposure of ANS probe to the aqueous solution.


Page 30 of 50

Page 31 of 50

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640 641 642 643 644

Figure 7. Emission spectra of HSA-ANS [1.00  10-5 mol dm-3] [1:1] at different concentrations of Au/Ag NPs. [Au/Ag NPs]: (a) 0.00, (b) 0.08  10-10, (c) 0.16  10-10, (d) 0.24  10-10, (e) 0.32  10-10, (f) 0.40 x 10-10, (g) 0.48  10-10, (h) 0.56  10-10, (i) 0.64  10-10 and (j) 0.72  10-10 mol dm-3.

645 646 647

Figure 8. (A) Stern-Volmer plot and (B) Double logarithmic plot for HSA-ANS-Au/Ag NPs system.

648



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

Page 32 of 50

649

4. Investigations on HSA conformations

650

4.1 Absorption Spectral Behaviour of HSA in Presence of Au/Ag NPs. Au/Ag NPs

651

induced secondary structural changes in native HSA was ascertained by absorption

652

spectral studies. Native HSA exhibit intense absorption maximum at 230 nm owing to

653

π-π* transition of the polypeptide backbone structure and a weak absorption peak

654

around 278 nm arising from aromatic amino acid residues, viz., Trp, Tyr and Phe.

655

Absorption spectral studies are shown to possess excellent sensitivity towards

656

alterations in the microenvironment adjacent to Trp and Tyr residues with significant

657

spectral shifts in the absorption maximum of native HSA.76,77 In view of the above

658

advantages, we monitored the absorption spectral changes of native HSA in the

659

absence and presence of Au/Ag NPs. The outcome of the absorption spectral studies

660

are displayed in Figure 9 and it is obvious from Figure 9 that the addition of Au/Ag

661

NPs to HSA solution has brought appreciable increase in the absorption intensity of

662

HSA at 278 nm (The possibility of Au/Ag NPs contribution to the increase in

663

absorption intensity was ruled out by performing appropriate blank measurements).

664

Zhao et al., reported an analogous absorption spectral behaviour for the binding of

665

BSA with carbon nanotubes. 78 It was proposed that the observed spectral changes is

666

directly related to modifications in the surrounding micro-environment of Trp and Tyr

667

residues present in BSA protein.78 In the present case, the observed hyperchromism at

668

278 nm makes it clear that the molecular arrangement around the lone Trp-214 residue

669

of HSA have undergone slight modification and the intrinsic fluorophore was exposed

670

to a more polar aqueous environment.78 Consequently in the present work, it is

671

believed that the increase in absorption maxima of HSA at 278 nm is solely due to the

672

interaction of Au/Ag NPs with HSA.


Page 33 of 50

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673

Furthermore, the outcome of the absorption spectral studies was found to be in good

674

accord with previously reported studies for the formation of non-fluorescent ground

675

state complex between the protein and nanoparticle.20 It is well known that the ground

676

state complex formation involving protein and ligands frequently results in

677

perturbation of protein absorption spectrum.20,76-78 From the above observation it is

678

inferred that Au/Ag NPs induced emission quenching of HSA is solely attributed to

679

static quenching mechanism. Moreover, this conclusion complement the findings

680

derived from steady state and time resolved fluorescence spectral studies.

681 682 683 684 685

Figure 9. Absorption spectra of HSA [1.00  10-5 mol dm-3] at different Au/Ag NPs concentrations. [Au/Ag NPs]: (a) 0.00, (b) 0.32  10-10 mol dm-3 and (c) 0.72  10-10 mol dm-3. Inset shows the enlarged image of HSA absorption spectral changes.



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Page 34 of 50

686

4.2. FT-IR Spectral Studies. The vibrational transitions associated with protein structure can

687

be effectively utilised by FT-IR spectroscopy to derive information on the ligand/NPs

688

induced conformational changes in proteins. FT-IR measurements has been used to analyse

689

the interaction of proteins with nanoparticles and to study the surface properties of NP-

690

protein bioconjugates.79,80 The secondary structure of proteins can be estimated on the basis

691

of the band at amide I region between 1600 cm-1-1700 cm-1. The carbonyl stretching

692

vibrations corresponding to peptide backbone of HSA at amide I region is highly sensitive to

693

ligand or NPs induced structural changes when compared to the amide II or amide III bands

694

and it is frequently used to determine protein conformation.81 FT-IR spectral analysis of

695

native HSA and HSA-Au/Ag NPs were carried out to study Au/Ag NPs induced

696

conformational changes in HSA (displayed in supporting information S4). As shown in

697

supporting information S4, the FTIR spectra of native HSA (panel A) exhibits a strong band

698

at 1655 cm-1 in the amide I region, suggesting rich α-helical conformation in native HSA.

699

When compared with the native HSA, the amide I band in the HSA-Au/Ag NPs bioconjugate

700

system shows obvious difference in both shape and peak position (1648 cm-1), which

701

suggests alteration in secondary structure of HSA in the bioconjugate system. To gain deep

702

insight on the conformational changes in the secondary structures of HSA, the second-

703

derivative spectrum of amide I was acquired and analyzed (Figure 10 A&B). The band

704

centered at 1665 cm-1 in the amide I region is attributed to the α-helix structure of native HSA

705

at pH 7.40 (Figure 10 A).20 The band around 1644 cm-1 is assigned to β-sheet structure for the

706

native HSA. As evidenced from Figure 10 B, the second-derivative spectrum of HSA-Au/Ag

707

NPs bioconjugate is characterised by a slight shift in the position of the band corresponding

708

to α-helix content of HSA i.e. from 1665 cm-1 to 1660 cm-1. In contrast, the intensities of the

709

band (at 1644 cm-1) pertaining to β-sheet structure of native HSA showed an increase in the

710

bioconjugate system. The observed changes in the FTIR spectrum of HSA after conjugation


Page 35 of 50

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711

with Au/Ag NPs indicated that the secondary structure of HSA undergoes obvious changes in

712

the HSA-Au/Ag NPs bioconjugate system. The second derivative spectra further indicate the

713

rise of unordered and β-sheet conformations with simultaneous decrease in α-helical content.

714

The observed modifications in the protein conformations indicate that HSA would retain a

715

more native-like structure when adsorbed on the surface of Au/Ag NPs. An identical FT-IR

716

spectral behaviour was observed for Haemoglobin-Au NPs and HSA-Au NPs

717

intercations.20,28 Moreover, it has been previously reported that HSA molecules tends to

718

retain a more native-like structure when adsorbed onto Au nanorods with high surface

719

curvature.57,28,82 Based on the FT-IR spectral studies, we arrived at a conclusion that the

720

interaction of Au/Ag NPs with HSA have brought out significant conformational changes in

721

HSA.

722

724 725

Figure 10. Second derivative FTIR spectrum of (A) HSA and (B) HSA-Au/Ag bioconjugates. The concentrations of HSA and Au/Ag NPs were 1.0 × 10-5 and 0.72  10-10 mol dm-3, respectively.

726

4.3. Raman Spectral Studies. Raman spectroscopy is often used to delve important

727

information on the protein structural changes upon its association with NPs.83 Raman

728

spectrum of native HSA and HSA-Au/Ag NPs system are displayed in Figure 11. The

729

oscillations of Raman bands at amide I and amide III region are used to estimate the

730

structural changes in polypeptide chain of protein molecules.84 Raman spectral

723



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

731

analysis of HSA in the range of amide I and amide III bands helps in evaluating the

732

extent of secondary structural changes in protein conformation.83,84 Raman spectrum

733

of native HSA showed two characteristic band around 1650 cm-1 and 1680 cm-1

734

corresponding to amide I region. The 1650 cm-1 band from amide I is mainly

735

attributed to high α-helical content in HSA. The band corresponding to amide III of

736

HSA is observed around 1230 cm-1. In the presence of Au/Ag NPs the Raman

737

spectrum of HSA showed considerable decrease in Raman intensity with simultaneous

738

change in the band positions. The reduction in α-helical content of HSA upon its

739

association with Au/Ag NPs is obvious from the decreased Raman intensity at 1654

740

cm-1. Moreover, the observed decrement in amide III band at 1230 cm-1 makes it clear

741

that the interaction of HSA with Au/Ag NPs have resulted in considerable structural

742

alterations in the protein conformation.83-85

743 36 ACS Paragon Plus Environment

Page 36 of 50

Page 37 of 50

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744 745 746

Figure 11. Raman spectra of HSA in the absence and presence of Au/Ag NPs. The concentrations of HSA and Au/Ag NPs were 1.0 × 10-5 and 0.72  10-10 mol dm-3, respectively.

747

4.4. Circular Dichroism Spectroscopy of HSA-Au/Ag NPs Conjugates. Circular

748

dichroism (CD) spectroscopy provides an experimentally very convenient means of

749

assessing secondary structural changes in proteins by monitoring the CD signals in

750

different spectral regions.86 The peptide structure in proteins are dominated by the

751

n-π* and π*-π* transitions of amide groups in the far ultraviolet region and are

752

influenced by the geometries of the polypeptide backbones. 86 Thus, CD spectrum of

753

proteins can be used to gain in depth information on the protein secondary structures.

754

The ellipticity values at 208 nm and 222 nm from CD spectral experiments can be

755

utilised to estimate the α-helical content of proteins.86 To evaluate the influence of

756

Au/Ag NPs on the secondary structure of HSA, CD spectral measurements of HSA in

757

the absence and presence of Au/Ag NPs were carried out. The total α-helical content

758

of HSA was estimated using the following eqns. (7-8) and the CD spectral results were

759

articulated as mean residual ellipticity (MRE).86

MRE =

ObservedCD(mdeg) − (7) [C nl x 10]

α − Helix (%) =

−MRE − 4000 x 100 − (8) 33000 − 4000

760

where, CP denotes molarity of the protein, n represents the number of amino acid

761

residues in HSA (585 amino acids) and l is the path length of the cell in centimetres.

762

MRE208 is the observed ellipticity value at 208 nm, 4000 and 33000 donates the total

763

ellipticity value of β-form and pure α-helix form of the protein at 208 nm,

764

respectively.



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

765

As shown in Figure 12, the CD spectrum of native HSA exhibited a characteristic

766

band with two minima at 208 nm (π-π*) and 222 nm (n-π*) corresponding to α-helical

767

structure of protein.86 Upon the addition of Au/Ag NPs, the CD signal of HSA showed

768

a decrease at all wavelength regions without any noticeable shift in the peak position

769

(Figure 12). It has been previously reported that the lowering in the negative ellipticity

770

points towards a decrease in the α-helical content of the protein with slight unfolding

771

of the peptide strand. By utilizing the eqns. (9-10), the α-helical content of native HSA

772

and HSA-Au/Ag NPs was computed. The α-helical content in native HSA showed an

773

appreciable decrease from 59.72(±2) % to 54.88(±2) % upon its association with

774

Au/Ag NPs. It is apparent from the CD spectral analysis that the interaction of HSA

775

with Au/Ag NPs has resulted in the decrease of total α-helical content with slight

776

conformational changes in the native structure of HSA.

777 38 ACS Paragon Plus Environment

Page 38 of 50

Page 39 of 50

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778 779 780


Figure 12. CD spectra of HSA in the absence and presence of Au/Ag NPs. Conditions: [HSA] = 1.00 × 10-5 mol dm-3; [Au/Ag NPs]: (a) 0.00 × 10-10 mol dm-3 and (b) 0.72  10-10 mol dm-3.

781 782

4.5. 3D Emission Spectral Studies. Excitation-emission matrix spectroscopy or 3D

783

emission spectroscopy is regarded as an important technique in unravelling the ligand

784

or binding induced conformational changes in proteins by delineating the emission

785

spectral characteristics of intrinsic fluorophore by simultaneously varying the

786

excitation and emission wavelengths.76,77 We performed 3D emission spectral analysis

787

to investigate Au/Ag NPs induced conformational changes in HSA. It is well known

788

that information obtained from characteristic 3D emission spectral data of lone Trp

789

residue can give detailed information on the secondary structural changes of HSA.76,77

790

The 3D emission spectra of HSA in the absence and presence of Au/Ag NPs is

791

displayed in Figure 13(A&B). From Figure 13(A) it is obvious that HSA exhibits two

792

peaks, namely, Rayleigh scattering peak 1 (λem=λex) and peak 2, which corresponds to

793

the spectral behaviour of Trp, Tyr residues in HSA and it is directly related to

794

microenvironmental polarity around Trp, Tyr residues, respectively.76,77 As depicted in

795

Figure 13(B) the emission intensity of peak 2 is decreased in the presence of Au/Ag

796

NPs to HSA. The obvious change in the 3D emission intensity of peak 2 is indicative

797

of conformational changes in secondary structure of HSA. Similar kind of change in

798

3D emission profile has been observed for BSA-ANS system in the presence of Ag

799

NPs.87 It is proposed that the decrease in 3D emission intensity of BSA-ANS complex

800

by Ag NPs is primarily attributed to the slight alterations happening in the BSA

801

structure.87 Based on the outcome of the 3D spectral studies, it is inferred that the

802

binding of HSA to Au/Ag NPs led to considerable conformational changes in the



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

803

native structure of HSA and this conclusion is in accordance with the results obtained

804

from absorption, FTIR, Raman and CD analysis.

Page 40 of 50

805 806 807 808

Figure 13. Three dimensional emission spectra of HSA in the absence (A) and presence (B) of Au/Ag NPs. Conditions: [HSA] = 1.00 × 10-5 mol dm-3 and [Au/Ag NPs]: 0.72  10-10 mol dm-3.

809

4.6. Evaluation of Esterase-like Activity of HSA in the Presence of Au/Ag NPs.

810

HSA is known to exhibit catalytic functions like hydrolytic and esterase-like

811

activity.47,88 The esterase-like activity of HSA is mainly facilitated by Arg-410 and

812

Tyr-411 residues located in the subdomain IIIA (Sudlow’s site II) of HSA.89 The

813

retention of the HSA activity is of particular importance for any biological application

814

involving NPs.90 Therefore, the catalytic activity of HSA on p-NPA was investigated

815

by monitoring the formation of p-nitrophenol at 405 nm. The relative esterase activity

816

of HSA in the absence and presence of Au/Ag NPs is shown in Figure 14. It is evident

817

from Figure 14 that the binding of HSA to Au/Ag NPs surface is associated with slight 40 ACS Paragon Plus Environment

Page 41 of 50

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818

decrease in esterase like activity of the protein. The outcome of the experiment

819

indicated that addition of Au/Ag NPs into HSA solution had an obvious impact on the

820

enzyme like activity of HSA towards p-NPA. However, it is to be noticed that in the

821

presence of Au/Ag NPs, HSA retained 76% of its original activity. This observation

822

complies with an earlier work on BSA-Au NPs, where 88% retention of esterase

823

activity is reported.88 The outcome of this experiment clearly implies that HSA

824

maintains most of its esterase activity in HSA-Au/Ag NPs conjugates.

825 826 827

Figure. 14. Effect of Au/Ag NPs on the esterase like activity of Conditions: [HSA] = 2.00 × 10-9 mol dm-3 and [Au/Ag NPs]: 0.72  10-10 mol dm-3.

828

5. CONCLUSIONS

HSA.

829

In conclusion, we have demonstrated the binding interaction of HSA with Au/Ag

830

NPs by various spectral techniques. Absorption spectral analysis of Au/Ag NPs in the

831

presence of varying concentrations of HSA induced slight red shift in the SPR band of 41 ACS Paragon Plus Environment


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

832

Au/Ag NPs owing to the rapid association of HSA to the NP surface. The formation of

833

dense layer of HSA on the surface of Au/Ag NPs was confirmed by HR-TEM, DLS

834

and zeta potential analysis. The outcome of the steady state emission titration

835

experiments of HSA-Au/Ag NPs clearly revealed the existence of ground state

836

complex between HSA and Au/Ag NPs. The estimated binding constant and Gibbs

837

energy values suggest that the binding of HSA to Au/Ag NPs surface occurs

838

spontaneously with substantial binding affinity. The formation of HSA corona on

839

Au/Ag NPs surface was confirmed by assessing the experimental emission quenching

840

data with theoretically computed value. The results from REES experiments revealed

841

that the association of HSA to Au/Ag NPs have brought about significant restriction in

842

the mobility of Trp residue of HSA. The partial unfolding of HSA upon its interaction

843

with Au/Ag NPs was confirmed by monitoring the emission spectral behaviour of an

844

extrinsic fluorescence probe (ANS). FT-IR, Raman and CD spectral studies

845

unambiguously helped us to conclude Au/Ag NPs induced conformational changes in

846

HSA. Absorption and Excitation-emission matrix spectral analysis (3D) suggested that

847

the microenvironment in the vicinity of lone Trp residue of HSA was altered due to

848

the binding of Au/Ag NPs. The esterase activity of HSA was inhibited slightly in the

849

presence of Au/Ag NPs and it is revealed that HSA retains most of its esterase activity

850

even after its binding to Au/Ag NPs. This in vitro study could act as a precedent in

851

understanding the interaction mechanism of biomolecules with bimetallic NPs.

852

However, further in vivo study needs to be done with Au/Ag NPs to achieve a deeper

853

understanding on the biological effects of bimetallic NPs. The studies on the effect of

854

core particle composition and varying Ag:Au molar ratio of alloy NPs on the binding

855

affinity of HSA is currently under way in our laboratory.

856


Page 42 of 50

Page 43 of 50

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857

Supporting information

858

Preparation of citrate capped gold and silver nanoparticles. Emission spectral data of HSA in

859

the presence of citrate capped gold and silver nanoparticles. FT-IR spectrum of HSA and

860

HSA-Au/Ag NP bioconjugates. This material is available free of charge via the Internet at

861

http://pubs.acs.org.

862

Acknowledgements

863

The authors’ gratefully acknowledges Department of Science and Technology (DST-SERC-

864

FAST Track scheme, project No.SR/FT/CS-015/2009 and DST-SERB project No.

865

SB/EMEQ-062/2013) and University Grants Commission (UGC-MRP, Project No. 41-

866

309/2012 (SR)), India for the financial support. We thank Prof. R. Ramaraj and Prof. A.

867

Ramu of Madurai Kamaraj University, India for access to CD spectrophotometer. We are

868

thankful to Prof. P. Ramamurthy, University of Madras, India for allowing us to utilise the

869

TCSPC facility. We also acknowledge Mr. A. Ariharan, Research Scholar, IIT-Madras, India

870

for his help in Line scan measurements.

871

Notes

872

The authors declare no competing financial interest.



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Page 44 of 50

References 1.Uppal, M.A.; Ewing, M.B.; Parkin, I.P. One-Pot Synthesis of Core-Shell Silver-Gold Nanoparticle Solutions and Their Interaction with Methylene Blue Dye, Eur. J. Inorg. Chem. 2011, 29, 4534-4544. 2.Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M.A. Chemistry and Properties of Nanocrystals of Different Shapes. Chem. Rev. 2005, 105, 1025-1102. 3.Walters, G.; Parkin, I.P. The Incorporation of Noble Metal Nanoparticles into Host Matrix Thin Films: Synthesis, Characterisation and Applications. J. Mater. Chem. 2009, 19, 574-590. 4.Nath, S.; Kaittanis, C.; Tinkham, A.; Perez, J.M. Dextran-coated Gold Nanoparticles for the Assessment of Antimicrobial Susceptibility. Anal. Chem. 2008, 80, 1033-1038. 5.Gil-Tomas, J.; Tubby, S.; Parkin, I.P.; Narband, N.; Dekker, L.; Nair, Wilson, M.; Street, C. Lethal Photosensitisation of Staphylococcus Aureus Using a Toluidine blue O–Tiopronin–Gold Nanoparticle Conjugate. J. Mater. Chem. 2007, 17, 3739-3746. 6.Noimark, S.; Dunnill, C.W.; Wilson, M.; Parkin, I.P. The Role of Surfaces in Catheter-Associated Infections. Chem. Soc. Rev. 2009, 38, 3435-3448. 7.Decrane, V.; Rampaul, A.; Parkin, I.P.; Petrie, A.; Wilson, M. Enhancement by Nanogold of the Efficacy of a Light-Activated Antimicrobial Coating. Curr. Nanosci. 2009, 5, 257-261. 8.Page, K.; Wilson, M.; Parkin, I.P. Antimicrobial Surfaces and their Potential in Reducing the Role of the Inanimate Environment in the Incidence of Hospital-Acquired Infections. J. Mater. Chem. 2009, 19, 3819-3831. 9.Narband, N.; Tubby, S.; Wilson, M.; Parkin, I.P.; Gil-Thomas, J.; Ready, D.; Nair S. P.; Wilson, M. Gold Nanoparticles Enhance the Toluidine blue-Induced Lethal Photosensitisation of Staphylococcus Aureus. Curr. Nanosci. 2008, 4, 409-414. 10. Perni, S.; Piccirillo, C.; Pratten, J.; Prokopovich, P.; Chrzanowski, W.; Parkin, I.P.; Wilson, M. The Antimicrobial Properties of Light-Activated Polymers Containing Methylene Blue and Gold Nanoparticles. Biomaterials 2009, 30, 89-93. 11. Wu, P.; Gao, Y.; Zhang, H; Cai, C. Aptamer-Guided Silver-Gold Bimetallic Nanostructures with Highly Active Surface-Enhanced Raman Scattering for Specific Detection and Near-Infrared Photothermal Therapy of Human Breast Cancer Cells. Anal. Chem. 2012, 84, 7692-7699. 12. Kim, J.Y.; Lee, J-Se. Multiplexed DNA Detection with DNA-Functionalized Silver and Silver/Gold Nanoparticle Superstructure Probes. Bull. Korean Chem. Soc. 2012, 33, 221-226. 13. Pan, H.; Qin, M.; Meng, W.; Cao, Y.; Wang, W. How Do Proteins Unfold upon Adsorption on Nanoparticle Surfaces? Langmuir 2012, 28, 12779-12787. 14. Tiedemann, D.; Taylor, U.; Rehbock, C.; Jakobi, J.; Klein, S.; Kues, W.A.; Barcikowski, S.; Rath. D. Reprotoxicity of Gold, Silver, and Gold–Silver Alloy Nanoparticles on Mammalian Gametes. Analyst 2014, 139, 931-942. 15. Grade, S.; Eberhard, J.; Jakobi, J.; Winkel, A.; Stiesch, M.; Barcikowski, S. Alloying Colloidal Silver Nanoparticles with Gold Disproportionally Controls Antibacterial and Toxic Effects. Gold Bull. 2014, 47, 83-93. 44 ACS Paragon Plus Environment

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Au/Ag alloy NPs


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Comprehensive Multispectroscopic Analysis on the Interaction and

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