Unexpected Hard Protein Behavior of BSA on Gold Nanoparticle

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Unexpected Hard Protein Behavior of BSA on Gold Nanoparticle Caused by Resveratrol Diego Coglitore, Nicoletta Giamblanco, agne Kizalaite, Pierre-Eugene Coulon, Benoit Charlot, Jean-Marc Janot, and Sebastien Balme Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01365 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Langmuir

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Unexpected Hard Protein Behavior of BSA on Gold

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Nanoparticle Caused by Resveratrol

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Diego Coglitore1, Nicoletta Giamblanco1, Agné Kizalaité1, Pierre Eugene Coulon2, Benoit

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Charlot3, Jean-Marc Janot1 and Sébastien Balme1*

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1

Institut Européen des Membranes, UMR5635, Université de Montpellier CNRS ENSCM, Place

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Eugène Bataillon, 34090 Montpellier, France 2

Laboratoire des Solides Irradiés, École polytechnique, Université Paris-Saclay, Route de Saclay,

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91128 Palaiseau Cedex, France 3

Institut d’Electronique et des Systèmes, Université de Montpellier, 34095 Montpellier Cedex 5,

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France

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Keywords : resveratrol, nanopore, FCS, gold nanoparticle

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ABSTRACT

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The understanding of the interactions between nanomaterials, biomolecules and polyphenols is

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fundamental in food chemistry, toxicology and new emerging fields such as nanomedicine. Here

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we investigated the effect of the resveratrol, a principal actor in drug delivery application on the

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interaction between BSA, employed as a vector for the delivery of polyphenol drugs, and gold

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nanoparticle (gNP), the most promising tool in theranostic applications. Through a combination

ACS Paragon Plus Environment

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of experimental techniques, which includes an initial evaluation by dynamic light scattering and

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surface plasmon resonance spectroscopy, we were able to evaluate the evolution of the gold

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nanoparticle aggregation with increasing ionic strength and the consequences of the BSA and

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resveratrol addition. In order to investigate the mechanisms of the interactions, we pursued at the

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single-molecule level using solid-state nanopore and fluorescence correlation spectroscopy. Our

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results show that without resveratrol the BSA is adsorbed on the gNP in water or saline solution.

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In presence of resveratrol, the BSA is normally absorbed on gNP in water but the salt addition

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leads to its desorption. The resveratrol clearly plays a fundamental role, changing the protein

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behavior and making the BSA adsorption a reversible process in presence of salt.

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INTRODUCTION

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Nanoparticles are increasingly considered in nanomedicine providing new solutions due to

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their unique physico-chemical properties1. Together with other therapeutic agents, they can be

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formulated in a single hybrid nanocomposite, incorporating both diagnostic and therapeutic

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functions2, leading to the so-called theranostic system. Promising theranostic nanoconstructs can

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be prepared by incorporating a specific functionality into a single and efficient anticancer agent,

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for a specific organ or patient for potential personalized medicine. Gold nanoparticles (gNP) are

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one of the most promising candidate for theranostic applications3–6. They are widely studied and

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employed as imaging agent for diagnosis, imaging and monitoring, but also in the treatment of

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malignant diseases and drug delivery systems because of their biocompatibility, high binding

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affinity, high selective targeting properties and low toxicity compared with inorganic

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nanoparticles7–9. The oscillation of the conduction electrons upon interaction with light, i.e. the


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surface plasmon resonance, make them easily detectable in the visible and near infrared spectral

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regions10,11. Gold nanoparticles require a functionalization to prevent self-aggregation or non-

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specific protein adsorption when passing through various physiological environment12. When

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circulating in blood plasma one of the most common strategies used to maintain their stability is

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to graft a layer of polyethylene glycol (PEG) onto the nanoparticle surface, increasing the steric

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repulsion against opsonization13.

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Proteins also can be used as a stabilizer, physically or chemically bound to the gold

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nanoparticle surface under different conditions of pH and ionic strength, at which the

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nanoparticles tend to aggregate14. The protein adsorption on nanoparticle is a complex

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phenomenon which involves different kinds of interactions, such as electrostatics, Van der

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Walls, hydrophobics and H-bonds. In the native conformation, the protein hydrophobic residues

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allow a close packaging of the cores15. The interactions with the nanoparticle surface can induce

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a change of

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exposes the hydrophobic moieties of the polypeptide chain to the nanoparticle surface17. The

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gold nanoparticles can directly modify the performance of redox enzyme activity such as

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glucose-oxydase18,19.Nanoparticles travelling in the blood plasma can encounter more than 3500

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different proteins, with a wide range of different concentrations

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where, initially, the most abounding proteins will be absorbed on the surface and subsequently

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replaced by proteins with higher affinitiy21,22. Proteins binding with higher affinity, directly on

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the nanoparticle surface, are imputed for the formation of the “hard” corona, on the top of which

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there is a “soft” corona, consisting of less tightly bound proteins, interacting mostly by weak

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protein-protein interactions23 , and showing much higher exchange rates24.

conformation, with a consequent denaturation16, so that the unfolded protein

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, leading to a competition


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Bovine serum albumin (BSA), one of the most abundant proteins in blood plasma, is often

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used for the stability of gold nanoparticles suspensions, preventing their aggregation25. As a soft

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protein, with a week internal energy, the BSA adsorption process induces its unfolding in order

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to minimize its energy and, consequently, to optimize its interactions with the material. BSA was

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found to be the most abundant protein to be absorbed by gold nanoparticles when injected in the

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bloodstream, even though a huge varieties of biomolecules contribute to the formation of the

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corona, including the more rigid fibrinogen beta-chain and beta-globin 26.However, the debate on

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the interactions between BSA and gNP is still open, aiming to solve issues such as if (i) the

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proteins completely or partially cover the nanoparticle or if (ii) the adsorption is irreversible or

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not. Recently, BSA has been used as a shuttle to deliver polyphenol drugs to targeted tissues27.

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The use of resveratrol (RESV) as a drug has attracted interest among researchers for its

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antioxidant and chemical cancer inhibition activities28. The binding of the resveratrol with BSA

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does not induce significant structural modification on the secondary structure29, compared to the

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effect of fatty acids 30 At high concentration of resveratrol, a conformational change of the BSA

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occurs, typically a reduction of the α-helix and increase of the β-sheet and random coil content31.

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It was recently suggested that the conformation of the adsorbed BSA on a clay mineral changes

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in presence of RESV32 . According to these reports, we can assume that the RESV modifies the

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type of interactions which govern the BSA adsorption on gNP and therefore their aggregation

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induced by the salt.

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Here we investigated the role of the RESV on the BSA adsorption by gold nanoparticles. We

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initially evaluated the impact of RESV binding the BSA on the gNP aggregation, in presence of

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salt, by surface plasmon resonance. After, we investigated the mechanism of interaction between

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the BSA and gNP by the combination of two single-molecule techniques. First, we used solid-


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state nanopore technology for sensing the populations present in solution in presence of salt. We

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pursued the investigation by fluorescence correlation spectroscopy, a particularly suitable

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technique to track proteins after labeling. Combining different techniques, we were able to

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characterize different aspects of the absorption of bovine serum albumin to the gold nanoparticle

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surface and the impact of the polyphenol on the protein-particle interaction.

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EXPERIMENTAL SECTION

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Materials

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Gold nanoparticles suspended in water with a nominal diameter of 10 nm (ref 752584 lot

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MKBX1673V),

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SLBS8228) sodium chloride (S753), sulfuric acid ACS reagent 95%- 98% (32051), hydrogen

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peroxide wt 30% (216763) were purchased from Sigma-Aldrich. Alexa-fluor 594®

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succinimidyl-ester tri(ethylamine) salt (A37572) was purchased from Molecular Probes. Silicon

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nitride grids (SiN) (10 nm thick and 50 x 50 µm windows) were purchased from Nanopore

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solution. Ultra-pure water was produced from a Q-grad®-1 MilliQ system (Millipore).

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albumin from bovine serum (BSA, A2153), resveratrol (ref 5010 lot

Preparation of the gold nanoparticle-protein and gold nanoparticle-protein-polyphenol mixtures

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BSA was directly added to the gNP dispersed in water at 6x1012 particles mL-1. Different

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proteins to nanoparticle ratios (1:1, 5:1, 10:1, 20:1 and 50:1) were investigated to test the effect

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of the BSA on the colloidal stability in a range of NaCl concentrations from 0 to 200 mM. To

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study the effect of the RESV on the BSA-nanoparticle interaction, the polyphenol was directly

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injected in the BSA solution (REVS-BSA ratio 5:1) and, afterwards, the REVS-BSA solution

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added to the gold nanoparticles, repeating the experiments with the same protein-nanoparticle

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ratios as mentioned before.


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Surface Plasmon Resonance Spectroscopy

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The spectra generated by the light absorbed in the visible region by bare gNP, BSA/gNp and

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RESV-BSA/gNp have been measured using Surface Plasmon Resonance Spectroscopy (SPRS)

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(JASCO UV/VIS/NIR spectrophotometer model V-570). Experiments were performed at room

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temperature (T = 25°).

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Dynamic Light Scattering Measurements

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The diffusion coefficients of bare gold nanoparticle, BSA/gNp and RESV-bsa/gNP have been

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measured using Photon Cross-Correlation Spectroscopy (PCCS) (Nanophox Sympatec, France).

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The experiments were performed at 25 °C. The diffusion coefficients were obtained fitting the

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raw data with the Quickfit software. The experimental data were fitted with a Liverberg-

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Marquardt non-linear algorithm without the constraint box.

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Confocal fluorescence spectroscopy

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The kinetics of the protein adsorption and the fluorescence correlation spectroscopy (FCS)

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were performed using a lab-made confocal fluorescence setup previously described [24], [25].

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The light beam was provided by a laser (SuperK EXTREME laser NKT Photonics, model EXR-

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15, wavelength 594, power 100 to 500 nW,; Dream Lasers Technology) and an objective

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(UPlanaApo 60x/1.20 w, Olympus). The confocal volume was about one femtolitre. The detector

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was an HPM-100-40 Becker& Hickl PM tube; the absence of after pulses for this detector

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permitted a better resolution for the FCS studies. The BSA was prior labeled with alexa fluor

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succinimidyl ester 594 following the method suggested by the manufacturer. The BSA/alexa

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fluor labeling ratio was 0.33 and it was calculated from the absorption spectra of the solution as

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well as verified by FCS.

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Measurements of the diffusion coefficient by fluorescence correlation spectroscopy

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The diffusion coefficients of the labeled proteins and the labeled proteins interacting with gold

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nanoparticles and resveratrol have been measured by fluorescence correlation spectroscopy

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(FCS). The measurements have been performed labeling all the proteins with the alexa fluor 594,

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at a concentration around 5 nM, in 100 mM NaCl buffer. Data were recorded for 400 s at a count

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rate of 2-4 kHz (6 µW laser excitation). The coefficients of diffusion were calculated from the

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autocorrelation curve using the Quickfit software. The confocal volumes and the calibration of

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the coefficients were determined using the alexa fluor 594 as standard (D=370 µm2 s-1).

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Kinetics of protein adsorption on SiNx surface

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The kinetics of the protein adsorption was obtained from the fluorescence profile of the labeled

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BSA solution, recorded step by step (Detector APD id100-50 from IDQ, electronics SPC-130EM

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Becker & Hickl) from the SiNx interface to the bulk solution (at least 50 µm from interface) by

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normal scanning (100 nm to 1 µm steps, collection time 50 to 100 ms). From the analysis of the

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profile, the amount of the adsorbed protein was extracted34. The adsorption experiments were

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performed on a SiNx layer (100 nm) deposited on a microscope cover-glass (diameter 25 mm)

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by PECVD35,36 after immersion in piranha solution (H2SO4/H2O2 with a ratio 3/1) for 30 min,

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rinsed with milliQ water and dried at 60°C. The protein concentration was the same than the one

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used for the nanopore experiments. A typical experiment consisted in adding 200 µl solution of

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the labeled proteins to a Teflon cuvette (6 mm diameter) obstructed at its bottom by the studied

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glass interface.

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Nanopore drilling

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The SiNx membrane (thickness 30 nm) of a TEM grid was drilled by the electron beam (beam

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current 11 nA) of a transmission electron microscope (JEOL 2010F) to obtain a nanopore with a


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diameter of about 20 nm. The process to obtain the nanopore consisted of two steps. The

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membrane was first drilled with a 1 nm probe during 60 seconds. Afterwards, a 20 nm electron

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beam was employed during 120 seconds to illuminate and enlarge the previous hole until

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reaching the desired diameter. The resulting nanopore had a diameter of 19.5 nm ± 2 nm. The

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silicon surface was cleaned in a piranha solution (H2SO4/H2O2 with a ratio 3/1 for 30 min at

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room temperature) to ensure the hydrophilicity and to remove potential organic contaminants,

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then rinsed with milliQ water and dried at 60°C for 5 min to remove residual water droplets.

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BSA-nanoparticle and RES-BSA-nanoparticle detection through SiNx nanopore

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The single nanopore, previously cleaned by piranha, was placed in a Teflon cell containing 100

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mM NaCl solution. Two Ag/AgCl electrodes were used to measure the current due to the

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presence of the ionic medium. One electrode was plugged to the positive end of the amplifier

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(trans chamber) and the other electrode connected to the ground (cis chamber). Initially, the

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BSA/gNp or REVS-BSA/gNp (nanoparticle concentration of 1.5 x 1018 particles m-3, a BSA:gNP

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ratio 20:1) were injected in the cis chamber just before the transport experiment through the

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nanopore. A constant voltage (from 100 to 500 mV) was applied to generate an electric field

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between the trans and cis chamber and favor the translocation of the BSA-nanoparticle

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complexes through the nanopore. The ionic current was recorded using a patch-clamp amplifier

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(EPC 800, HEKA electronics, Germany) at a sampling frequency of 100 kHz. A 10 kHz filter

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was applied. The data acquisition was performed with a HEKA LIH 8+8 acquisition card using

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patch master software (HEKA electronics, Germany). The event analysis was performed using

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the Matlab (Matworks, USA) code developed by Plesa et al.37. The events detection was carried

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out defining a threshold by multiplying a peak detection factor and the rms noise level calculated

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by the global standard deviation methods. In this work, the peak detection factor has been fixed


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at about 5. The SiNx nanopore was cleaned again with the piranha solution to remove residuals

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and reuse it for further experiments.

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RESULTS AND DISCUSSION

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Influence of BSA and resveratrol on nanoparticle aggregation

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The surface plasmon resonance of gNPs generates an extinction spectrum which depends on

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the size, shape, and aggregation of gold nanoparticles38. Since the change in the optical

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properties of the gNPs is a consequence of their aggregation, we monitored it with UV–VIS

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absorbance spectroscopy and diffusion light scattering. When the NaCl concentration increases,

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the bare gNPs in solution (6 x 1012 particles mL-1) tend to aggregate and the characteristic

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surface plasmon band at ∼520 nm (Fig SI-1) shifts up to∼670 nm. Consequently, the color of the

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solution changes from red to blue (Figure 1c-d). The autocorrelation functions of bare gNPs

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obtained by DLS confirm the aggregation, already observed in the spectra analysis (Figure 1a-

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b), even at low ionic strength.

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As demonstrated in previous studies, BSA can be used to stabilize gNP suspensions25,39. We

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tested different protein to nanoparticle ratios to find the best condition to prevent the nanoparticle

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aggregation. Upon contact, the BSA changes its structure to minimize its internal energy40,41.

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The BSA was directly added to the nanoparticle suspension at different protein to particle ratios

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(Figure SI-1a). At a ratio 1:1 the BSA does not guarantee a full coverage of the 10 nm gNP,

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avoiding the particle aggregation only at 10nM NaCl. Similar situations are observed for protein

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to nanoparticle ratios of 5:1 and 10:1. The optimal ratio was found at protein/particle 20:1. In this

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case the absorbance spectra, as well as the autocorrelation functions, do not shift to higher

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wavelengths or longer decay times, respectively. Thus, the nanoparticle suspension remains


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stable over the whole range of NaCl concentrations. Here, we assumed the optimal coverage of

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the 10 nm gNP by the unfolded BSA, that eludes the particle aggregation.

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The following step was the study of the effect of the RESV binding the BSA on the gNP

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aggregation. RESV was directly added to the BSA solution, and subsequently the RESV-BSA

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solution was injected in the nanoparticle suspension. UV spectra collected for different

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RESV/BSA ratios, in a range from 0.2 to 10, show that there are no significant differences on the

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aggregation of the gold nanoparticles (Figure SI-2). The protein-nanoparticle ratio was fixed to

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20:1, which represents the best situation to avoid the nanoparticle aggregation. On the contrary

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than the BSA, the complex RESV-BSA induces the nanoparticle aggregation by NaCl, as shown

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by the autocorrelation function and absorbance curves. The plot in Figure 2 shows a summary of

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the absorbance experiments. Following the shift of wavelength, dependent on the increase of

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ionic strength, two regions can be identified to describe the protein/nanoparticle interactions and

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the effect of the resveratrol. In the low-ionic-strength region no aggregation occurs, probably

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because of the steric repulsion. Out of this area, the increase of ionic strength generally led to

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aggregation, which can be avoided only reaching an optimal coverage of the nanoparticle by the

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protein. When resveratrol is added to BSA, it causes the nanoparticle aggregation even at the

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concentration that guarantees the optimal coverage. A similar trend was reported for the

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Lysozyme which is a hard protein42.

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One explanation to interpret our results is that the binding of the resveratrol to the BSA

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increases the energy required by the protein to unfold. Thus the conformation of the BSA

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adsorbed onto gNP is non-optimal to minimize its energy. However, the mechanism has to be

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clarified and several assumptions can be proposed. The first is that the resveratrol inhibits the

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BSA adsorption on the nanoparticle. The second one is that the NP coverage is not optimal


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because the BSA is not unfolded. The last is that the adsorption of RESV-BSA by the

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nanoparticles occurs in water but the salt addition induces a desorption of the protein.

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Characterisation of the species by single nanopore technique

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It is essential to know which the species are in solution after salt addition, for an understanding

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of the effect of resveratrol on the BSA-nanoparticle interaction. To do so, we used single

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nanopore for the detection of proteins and nanoparticles in solution (SI-3). The experiments were

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performed for a NaCl concentration of 100 mM. Two sets of experiments were performed to

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consider the effect of resveratrol. In the first place, gold nanoparticles (c = 1.5 x 1012 particles

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mL-1) with BSA (ratio 20:1) were directly injected, in the cis chamber before the beginning of the

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transport experiment. As previously shown, under this experimental condition, no aggregation of

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gNP occurred. In the second set of experiments, the gold nanoparticles, together with RESV-

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BSA complexes, were added in the cis chamber. In this case, as shown in the previous section,

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the nanoparticle aggregation occurred. In Figure 3, selected current traces recorded after

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addition of BSA-nanoparticle and RESV-BSA-nanoparticle are reported. Current blockades

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generated from the translocation across the nanopores were detected. The intensity of the relative

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current blockade ∆I/I0, the dwell time ∆t and the capture rate f were extracted. The histograms

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and events maps are reported in Figure 4 and SI-4.

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The relative current blockade (∆I/I0) is the ratio between the nanopore conductance with and

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without object inside. The histograms can be represented by Gaussian distributions, where the

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position of the center depends on the volume of the object passing through the single pore

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according to equation 1 43. The model assumes that the relative current blockade is only due to

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the exclusion of mobile ions due to the presence of the object in the channel, neglecting its


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charge and the one of the nanopore inner surface; however, it efficiently predicts the current

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blockage caused by the protein translocation.

246 247 248 249

୼ூ ூబ

ସఊஃ

= గௗమ ൫௟

೛ ೛ ା଴.଼ௗ೛ ൯

௥೛

ܵௗ

ಾ

(1)

Where dpand lp are the diameter and length of the pore, Λ is the volume of the nanoparticle, ߛ is a form factor equal to 1.5 for a spherical geometry, and ܵ ௗ௥ಾ೛ is a correction factor ௥೛

ௗಾ

=

ଵ య ோ ଵି଴.଼ቀ ೓ൗ௥೛ ቁ

≈ 1.

250

At 300 mV (Figure 4) the ∆I/I0 distributions are centered on 0.030 and 0.013, without and

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with resveratrol. The two values correspond to translocation of spherical objects with diameter 8

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nm and 6 nm, respectively. These results suggest that when only BSA is added on gNP, the

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complex BSA/gNp is detected. Indeed, the expected value of ∆I/I0 for a object with a diameter

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around 10 nm is close to the experimental result (0.036). The discrepancy could be explained by

255

the surface charge of BSA/gNp which is not taken into account in equation 1 44. When RESV is

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added to the BSA at the first stage, the diameter of the object which translocates through the

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nanopore nicely fits with the protein hydrodynamic radius. According to that, we can assume that

258

only REVS-BSA complex is passing through the nanopore, while the nanoparticle aggregates do

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not, because of their charge and their size. The dwell time in absence of resveratrol (8.7 ms) was

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found to be slightly longer than with it (6.5 ms). We detected events of various lengths in both

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cases. These values are longer than the ones expected for a single BSA or single gNP, which are

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estimated to be around 16 µs and 20 µs, assuming that only diffusion governs the transport. As

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previously demonstrated, the long dwell time can be attributed to the protein interaction with the

264

surface 36.


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The capture rate is dependent on the force driving the translocating object, the diffusion and

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the electric field. When the object is smaller than the nanopore diameter, it can be predicted by

267

equation 2:

268

݂ = 2ߨܿ‫∗ ݎܦ‬

(2)

269

where r∗ is the radius at the vicinity of the nanopore, which depends on the voltage, c and D

270

are the concentration and the diffusion coefficient of the nanoparticle. Assuming that only free

271

BSA is detected in the presence of the RESV, the expected capture rate (f) calculated from

272

equation 2 is about 102 events per second 45. This is lower than the one experimentally obtained

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at 100 mV (6.5 events s-1). Without BSA, the expected capture rate (4.2 events s-1) for a 10nm

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gNP, taking in account the nanoparticle concentration and the diffusion coefficient (4,5 µm2 s-1),

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is close to the experimental one (3.9 events s-1) at 100 mV. In presence of resveratrol, which

276

leads to the nanoparticle aggregation, the higher capture rate can be explained by smaller objects

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(BSA and RESV-BSA complex) than the BSA/gNp complex translocating through the pore.

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The experiments performed by single nanopore suggest that the complex BSA/gNp translocate

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through the nanopore when the RESV is not involved. This is in good agreement with the results

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from absorbance and DLS. When RESV binds the BSA, only the proteins seem to be detected.

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In both experiments the dwell times are longer than expected, that can be explained by the

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protein adsorption on the SiN inner wall as previously demonstrated. In order to confirm this

283

assumption, we have studied the adsorption of BSA on SiN with (and without) RESV and gNPs

284

(Figure SI-5), under the same condition than the one used for nanopore experiments. At the

285

equilibrium, the interfacial concentration reaches 600 BSA µm-2 for the BSA/gNP and up to 300

286

BSA µm-2 with resveratrol under 100 mM NaCl. Without gNP the interfacial concentration of

287

BSA reaches 90 BSA µm-2 and 73 BSA µm-2 with resveratrol. These results show an affinity

288

between the SiN and the BSA and can explain the discrepancy observed for the dwell time.


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Measurements of the diffusion coefficient by fluorescence correlation spectroscopy

290

The nanopore experiments show that without RESV the BSA is adsorbed on the gNP, while

291

with resveratrol BSA seems free in solution. However, the detection by single nanopore did not

292

solve the query on the role of the RESV-BSA complex preventing the adsorption on the gNP or

293

if the BSA is desorbed from the gNP after NaCl addition. To answer this question, we employed

294

fluorescence correlation spectroscopy, often used to efficiently characterize the nanoparticle-

295

protein complex 24464748.

296

Since gold nanoparticles are non-fluorescent objects, our strategy was the labeling of the BSA

297

with Alexa fluor 594 to observe the difference between the protein autocorrelation functions and

298

between the measured diffusion coefficients with and without RESV, with and without gNP ,

299

with and without NaCl. The experiments were performed maintaining a BSA to nanoparticle

300

ratio of 20:1 and a resveratrol to BSA ratio of 5:1. We first verified that the fluorescence lifetime

301

of the Alexa did not change significantly in presence of gNP and resveratrol. For all the

302

experiments, the best fit was obtained for a combination of two components: (i) a fast one at

303

about 370 µm2 s-1, which corresponds to the free Alexa in solution and (ii) a slower one, which

304

was assigned to the dye bound to the BSA. If the BSA is adsorbed on the nanoparticle, we expect

305

the Brownian motion to slow down. The differences between the BSA free in solution and the

306

BSA/gNp complex are clear from the fluorescence correlation functions in Figure 6a. The

307

values of the diffusion coefficients are reported in Table 1. Under water or 100 mM NaCl

308

solution, the BSA and RESV-BSA exhibit a diffusion coefficient of ~52 µm2 s-1. After

309

adsorption on the gNP in water, the diffusion coefficient decreases to 45 µm2 s-1 confirming the

310

BSA adsoprtion. It is interesting to notice that a similar result is obtained when the resveratrol

311

binds the BSA, so that it is likely it does not inhibit the protein absorption in water. The most


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312

remarkable difference occurs after salt addition. Indeed for the BSA/gNp the diffusion

313

coefficient stays similar after NaCl addition. On the contrary, the diffusion coefficient of the

314

RESV-BSA/gNP increases to 52 µm2 s-1 with salt, which corresponds to the one of the free BSA.

315

This suggests that the binding of the RESV with the BSA favors the protein desorption with the

316

increase of the ionic strength. In order to illutrate the displacement of REVS-BSA from gNP to

317

the solution, we have followed the kinetic of RESV-BSA/gNp asdorption (Figure 6b). The

318

equilibrium in water reaches a low interfacial concentration (about 6 proteins µm-2) while

319

without gNP the intercfacial concentration was measured at 204 proteins µm-2. After salt

320

addition, the interfacial concentration of protein increase to reach 175 proteins µm-2 which is in

321

the same order of magnitude of the RESV-BSA without gNP.

322

From our results, we can elucidate the scenario which occurs when the RESVinteracts with the

323

BSA. Without salt the complex RESV-BSA is adsorbed by the gNP and released after NaCl

324

addition, inducing the NP aggregation. The protein desorption with the ionic strength was

325

previously reported for the toxin Bt

326

unfolding to optimize its internal energy during the adsorption process. Here the binding of

327

RESV seems to partially maintain the integrity of the BSA structure and to increase its internal

328

energy. It appears the BSA having a “hard” protein behavior when interacting with RESV.

49

on mica but never for BSA, which is prone to the

329

Conclusion

330

To sum up, our work aimed to elucidate the impact of resveratrol on the BSA/gNp interactions.

331

Our result show that without salt the BSA and the complex RESV-BSA are adsorbed on the

332

gNP. Increasing the salt concentration, the BSA prevents the gNP aggregation, due to its

333

unfolding properties leading to the optimization of the interactions with the gNP. On the

334

contrary, the presence of RESV on the BSA does not avoid the nanoparticle aggregation when


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Page 16 of 32

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dispersed in NaCl solution. Using several single molecule techniques, we have shown that the

336

addition of salt induces the RESV-BSA complex desorption which is never reported in literature.

337

More interestingly, our result show for the first time that the resveratrol can strongly modify the

338

BSA “hardness”, leading to a reduction of the α-helix and increase of the β-sheet, as previously

339

mentioned, with consequences on the protein rigidity and thus changing its interaction with the

340

nanoparticle. This understanding can be useful to optimize new therapeutic approaches such as

341

theranostics and generally in nanodrugs design. In addition, the impact of polyphenols on

342

protein-nanoparticle interactions has a key role in food chemistry and toxicology, where

343

nanoparticles are commonly used.

344 345 346 347


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348 349

Figure 1 – DLS curves and UV-Vis absorbance spectra at increasing NaCl concentrations for

350

bare gNPs (a-b), BSA/gNp (e-f), RESV-BSA/gNP (i-j). When gNP, BSA/gNP or RESV-

351

BSA/gNP are dispersed in water, they present the tipical red wavlength (c-g-k), while, when

352

dispersed in a 100mM NaCl solution, only the BSA/gNp (h) does not shift to blue compared to

353

bare gNP (d) and RESV-BSA/gNP (l). The experiments were performed with a gNP

354

concentration of 6 x1012 particles mL-1, BSA (or RESV-BSA):gNP ratio 20:1 and a RESV:BSA

355

ratio of 5:1


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Page 18 of 32

356 357

Figure 2 – General picture of the changes of the gNP in the visible spectra, at different

358

protein/nanoparticle ratios, increasing the NaCl concentration. The experiments present in this

359

figure were performed with a gNP concentration of 6 x1012 particles mL-1, BSA (or RESV-

360

BSA):gNP ratio 20:1 and a RESV:BSA ratio of 5:1.Two regions can be identified. Only a

361

BSA/gNP avoids the nanoparticle aggregation with increasing the ionic strength; under the same

362

condition, the addition of resveratrol inhibits this behaviour causing the nanoparticle

363

aggregation.

364 365 366


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Figure 3 – Current traces recorded at 300mV and current blockades recorded for the BSA/gNp

369

(red) and for the RESV-BSA/gNP (blue) translocation through the SiNx nanopore, for a

370

nanoparticle concentration of 1.5 x 1012 particles mL-1, a BSA:gNP ratio of 20:1 and a

371

BSA:resveratrol ratio of 1:5.

372 373 374 375 376 377 378 379


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Page 20 of 32

380 381

Figure 1 – Event map (a), current blockade (b) and dwell time (c) distributions at 300 mV, for a

382

nanoparticle concentration of 1.5 x 1012 particles mL-1, a BSA:gNP ratio of 20:1, BSA:RESV

383

ratio of 1:5. The results for the BSA/gNp complex are in red, in blue for the RESV-BSA/gNP.


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384 385

Figure 2 – Capture rate without (red) and with resveratrol (blue) at different voltage in the range

386

from 100 mV to 500 mV, for a gNP concentration of 1.5 x 1012 particles mL-1, a BSA:gNP ratio

387

of 20:1 and a BSA:RESV ratio of 1:5.

388 389 390 391 392 393


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Page 22 of 32

394 395

Figure 3 – (a) Fluorescence correlation spectroscopy curves for labelled BSA (red circles) and

396

BSA/gNp (black squares), for which the protein/nanoparticle ratio was 20:1(b) Kinetic of RESV-

397

BSA/gNp asdorption in water during about 20 minutes and after 100 mM NaCl addition, for a

398

BSA:RESV ratio of 1:5 and a BSA:gNP ratio of 20:1.


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399 400 401

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Table 1 – Diffusion coefficients in water and 100 mM NaCl measured by fluorescence correllation spectroscopy. The BSA was labelled with Alexa fluor. The BSA/gNP ratio was 20:1, the RESV/BSA ratio was 5:1. Diffusion coefficient in water

Diffusion coefficient in

(µm2 s-1)

100mM NaCl (µm2 s-1)

BSA

54±3.5

51.8±0.9

BSA+gNP

45±0.5

47±0.9

RESV+BSA

51.4±1

53.7±0.8

RESV+BSA/gNP

46.8±0.8

52.2±1

402 403 404

Supporting Information

405

Additional results from experiments on gold nanoparticle plasmon resonance, the

406

characterization of the SiN nanopore, additional results from the nanopore translocation

407

experiments and the kinetic of BSA adsorption on SiN are available free of charge in the

408

Supporting Information at...

409

Corresponding Author

410

E-mail: [email protected]

411 412

ACKNOWLEDGMENT


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Page 24 of 32

413

This work was supported by the “chercheur d’avenir” grant co-funded by Région Languedoc-

414

Roussillon and European Union (FEDER) – project “NanoDiag”. N.G. was supported by a

415

fellowship from LabEX Chemisyst (ANR- 10-LABX605-UI) and Université de Montpellier –

416

project “AmyDiag”.

417 418

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Unexpected Hard Protein Behavior of BSA on Gold Nanoparticle

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