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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
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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.
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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
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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
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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
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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
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equation 2:
268
݂ = 2ߨܿ∗ ݎܦ
(2)
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where r∗ is the radius at the vicinity of the nanopore, which depends on the voltage, c and D
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are the concentration and the diffusion coefficient of the nanoparticle. Assuming that only free
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BSA is detected in the presence of the RESV, the expected capture rate (f) calculated from
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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
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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
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assumption, we have studied the adsorption of BSA on SiN with (and without) RESV and gNPs
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(Figure SI-5), under the same condition than the one used for nanopore experiments. At the
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equilibrium, the interfacial concentration reaches 600 BSA µm-2 for the BSA/gNP and up to 300
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BSA µm-2 with resveratrol under 100 mM NaCl. Without gNP the interfacial concentration of
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BSA reaches 90 BSA µm-2 and 73 BSA µm-2 with resveratrol. These results show an affinity
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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
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The nanopore experiments show that without RESV the BSA is adsorbed on the gNP, while
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with resveratrol BSA seems free in solution. However, the detection by single nanopore did not
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solve the query on the role of the RESV-BSA complex preventing the adsorption on the gNP or
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if the BSA is desorbed from the gNP after NaCl addition. To answer this question, we employed
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fluorescence correlation spectroscopy, often used to efficiently characterize the nanoparticle-
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protein complex 24464748.
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Since gold nanoparticles are non-fluorescent objects, our strategy was the labeling of the BSA
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with Alexa fluor 594 to observe the difference between the protein autocorrelation functions and
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between the measured diffusion coefficients with and without RESV, with and without gNP ,
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with and without NaCl. The experiments were performed maintaining a BSA to nanoparticle
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ratio of 20:1 and a resveratrol to BSA ratio of 5:1. We first verified that the fluorescence lifetime
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of the Alexa did not change significantly in presence of gNP and resveratrol. For all the
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experiments, the best fit was obtained for a combination of two components: (i) a fast one at
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about 370 µm2 s-1, which corresponds to the free Alexa in solution and (ii) a slower one, which
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was assigned to the dye bound to the BSA. If the BSA is adsorbed on the nanoparticle, we expect
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the Brownian motion to slow down. The differences between the BSA free in solution and the
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BSA/gNp complex are clear from the fluorescence correlation functions in Figure 6a. The
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values of the diffusion coefficients are reported in Table 1. Under water or 100 mM NaCl
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solution, the BSA and RESV-BSA exhibit a diffusion coefficient of ~52 µm2 s-1. After
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adsorption on the gNP in water, the diffusion coefficient decreases to 45 µm2 s-1 confirming the
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BSA adsoprtion. It is interesting to notice that a similar result is obtained when the resveratrol
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binds the BSA, so that it is likely it does not inhibit the protein absorption in water. The most
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remarkable difference occurs after salt addition. Indeed for the BSA/gNp the diffusion
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coefficient stays similar after NaCl addition. On the contrary, the diffusion coefficient of the
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RESV-BSA/gNP increases to 52 µm2 s-1 with salt, which corresponds to the one of the free BSA.
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This suggests that the binding of the RESV with the BSA favors the protein desorption with the
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increase of the ionic strength. In order to illutrate the displacement of REVS-BSA from gNP to
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the solution, we have followed the kinetic of RESV-BSA/gNp asdorption (Figure 6b). The
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equilibrium in water reaches a low interfacial concentration (about 6 proteins µm-2) while
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without gNP the intercfacial concentration was measured at 204 proteins µm-2. After salt
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addition, the interfacial concentration of protein increase to reach 175 proteins µm-2 which is in
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the same order of magnitude of the RESV-BSA without gNP.
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From our results, we can elucidate the scenario which occurs when the RESVinteracts with the
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BSA. Without salt the complex RESV-BSA is adsorbed by the gNP and released after NaCl
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addition, inducing the NP aggregation. The protein desorption with the ionic strength was
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previously reported for the toxin Bt
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unfolding to optimize its internal energy during the adsorption process. Here the binding of
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RESV seems to partially maintain the integrity of the BSA structure and to increase its internal
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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
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Conclusion
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To sum up, our work aimed to elucidate the impact of resveratrol on the BSA/gNp interactions.
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Our result show that without salt the BSA and the complex RESV-BSA are adsorbed on the
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gNP. Increasing the salt concentration, the BSA prevents the gNP aggregation, due to its
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unfolding properties leading to the optimization of the interactions with the gNP. On the
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contrary, the presence of RESV on the BSA does not avoid the nanoparticle aggregation when
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dispersed in NaCl solution. Using several single molecule techniques, we have shown that the
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addition of salt induces the RESV-BSA complex desorption which is never reported in literature.
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More interestingly, our result show for the first time that the resveratrol can strongly modify the
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BSA “hardness”, leading to a reduction of the α-helix and increase of the β-sheet, as previously
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mentioned, with consequences on the protein rigidity and thus changing its interaction with the
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nanoparticle. This understanding can be useful to optimize new therapeutic approaches such as
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theranostics and generally in nanodrugs design. In addition, the impact of polyphenols on
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protein-nanoparticle interactions has a key role in food chemistry and toxicology, where
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nanoparticles are commonly used.
344 345 346 347
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Figure 1 – DLS curves and UV-Vis absorbance spectra at increasing NaCl concentrations for
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bare gNPs (a-b), BSA/gNp (e-f), RESV-BSA/gNP (i-j). When gNP, BSA/gNP or RESV-
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BSA/gNP are dispersed in water, they present the tipical red wavlength (c-g-k), while, when
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dispersed in a 100mM NaCl solution, only the BSA/gNp (h) does not shift to blue compared to
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bare gNP (d) and RESV-BSA/gNP (l). The experiments were performed with a gNP
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concentration of 6 x1012 particles mL-1, BSA (or RESV-BSA):gNP ratio 20:1 and a RESV:BSA
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ratio of 5:1
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356 357
Figure 2 – General picture of the changes of the gNP in the visible spectra, at different
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protein/nanoparticle ratios, increasing the NaCl concentration. The experiments present in this
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figure were performed with a gNP concentration of 6 x1012 particles mL-1, BSA (or RESV-
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BSA):gNP ratio 20:1 and a RESV:BSA ratio of 5:1.Two regions can be identified. Only a
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BSA/gNP avoids the nanoparticle aggregation with increasing the ionic strength; under the same
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condition, the addition of resveratrol inhibits this behaviour causing the nanoparticle
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aggregation.
364 365 366
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Figure 3 – Current traces recorded at 300mV and current blockades recorded for the BSA/gNp
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(red) and for the RESV-BSA/gNP (blue) translocation through the SiNx nanopore, for a
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nanoparticle concentration of 1.5 x 1012 particles mL-1, a BSA:gNP ratio of 20:1 and a
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BSA:resveratrol ratio of 1:5.
372 373 374 375 376 377 378 379
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380 381
Figure 1 – Event map (a), current blockade (b) and dwell time (c) distributions at 300 mV, for a
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nanoparticle concentration of 1.5 x 1012 particles mL-1, a BSA:gNP ratio of 20:1, BSA:RESV
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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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Figure 2 – Capture rate without (red) and with resveratrol (blue) at different voltage in the range
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from 100 mV to 500 mV, for a gNP concentration of 1.5 x 1012 particles mL-1, a BSA:gNP ratio
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of 20:1 and a BSA:RESV ratio of 1:5.
388 389 390 391 392 393
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Figure 3 – (a) Fluorescence correlation spectroscopy curves for labelled BSA (red circles) and
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BSA/gNp (black squares), for which the protein/nanoparticle ratio was 20:1(b) Kinetic of RESV-
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BSA/gNp asdorption in water during about 20 minutes and after 100 mM NaCl addition, for a
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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
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Additional results from experiments on gold nanoparticle plasmon resonance, the
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characterization of the SiN nanopore, additional results from the nanopore translocation
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experiments and the kinetic of BSA adsorption on SiN are available free of charge in the
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Supporting Information at...
409
Corresponding Author
410
E-mail:
[email protected] 411 412
ACKNOWLEDGMENT
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This work was supported by the “chercheur d’avenir” grant co-funded by Région Languedoc-
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Roussillon and European Union (FEDER) – project “NanoDiag”. N.G. was supported by a
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fellowship from LabEX Chemisyst (ANR- 10-LABX605-UI) and Université de Montpellier –
416
project “AmyDiag”.
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