# Concanavalin A adsorption on gold nanoparticles: Influence of citrate ions on nanoparticle-protein interactions

Sephora Lahouari, Jérémie Gouyon, Grégory Francius, Fabien Picaud, Roberta Albino dos Reis, Amedea B Seabra, Igor Clarot, Ariane Boudier

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Sephora Lahouari, Jérémie Gouyon, Grégory Francius, Fabien Picaud, Roberta Albino dos Reis, et al. Concanavalin A adsorption on gold nanoparticles: Influence of citrate ions on nanoparticle-protein interactions. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2025, 723, pp.137370. 10.1016/j.colSURFA.2025.137370. hal-05101824

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Concanavalin A adsorption on gold nanoparticles: Influence of citrate ions on nanoparticle-protein interactions

Sephora Lahouari, Jérémie Gouyon, Grégory Francius, Fabien Picaud, Roberta Albino dos Reis, Amedea B. Seabra, Igor Clarot, Ariane Boudier

<figure><img src="image_2.png" /></figure>

PII: S0927-7757(25)01273-7

DOI: https://doi.org/10.1016/j.colsurfa.2025.137370

Reference: COLSUA137370

To appear in: *Colloids and Surfaces A: Physicochemical and Engineering Aspects*

Received 18 March 2025

date:

Revised date: 9 May 2025

Accepted 29 May 2025

date:

Please cite this article as: Sephora Lahouari, Jérémie Gouyon, Grégory Francius, Fabien Picaud, Roberta Albino dos Reis, Amedea B. Seabra, Igor Clarot and Ariane Boudier, Concanavalin A adsorption on gold nanoparticles: Influence of citrate ions on nanoparticle-protein interactions, *Colloids and Surfaces A: Physicochemical and Engineering Aspects*, (2025)
doi:https://doi.org/10.1016/j.colsurfa.2025.137370

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# Concanavalin A adsorption on gold nanoparticles: Influence of citrate ions on nanoparticle-protein interactions

Sephora Lahouari<sup>a,b</sup>, Jérémie Gouyon<sup>a,c</sup>, Grégory Francius<sup>c</sup>, Fabien Picaud<sup>d</sup>, Roberta Albino dos Reis<sup>e</sup>, Amedea B. Seabra<sup>c</sup>, Igor Clarot<sup>a,b</sup>, Ariane Boudier<sup>a,b,f*</sup>

a: Université de Lorraine, CITHEFOR, F-54000 Nancy, France

b : Université de Lorraine, CNRS, LRGP, F-54000 Nancy, France

c : Université de Lorraine, CNRS, LCPME, F-54000 Nancy, France

d : Université Marie et Louis Pasteur, SINERGIES, F-25030 Besançon, France

e : Federal University of ABC, Center for Natural and Human Sciences, Santo andré 09210-580, SP, Brazil

f : Institut Universitaire de France (IUF)

*Corresponding author at: Université de Lorraine, CITHEFOR, F-54000 Nancy, France ariane.boudier@univ-lorraine.fr

## Abstract

We report in this article a dynamic overcoating of a protein on gold nanoparticles (AuNP). By exploring in depth the experimental and theoretical aspects of the interactions between AuNPs, stabilized by a layer of sodium citrate (AuNP–cit), and a lectin, Concanavalin A (ConA), we highlighted a dynamic interaction between these two entities. Experiments (physicochemical characterization, fluorescence quenching of tryptophan residues of ConA, and atomic force microscopy in force mode) were run in parallel with a numerical approach using molecular dynamics (MD) to explain in detail the different aspects of the [AuNP–cit ConA] interactions. Our results were compared to the ones obtained using human serum albumin (HSA) and bare AuNP (without the sodium citrate coating) obtained via MD. The complex [AuNP–cit ConA] was of low energy non-covalent binding via electrostatic interactions (with ionic binding) and low affinity between the two partners. Additionally, ConA was not able to displace the sodium citrate corona from AuNP–cit, leading to an important electrostatic competition between the different species in the solvent. This phenomenon suggested that a dynamic overcoating of the sodium citrate corona of the AuNPs took place rather than a ligand exchange, in which sodium citrate is displaced from the surface of AuNPs, which is more classically described in the case of NP – protein interactions.
Keywords

Concanavalin A, gold nanoparticles, dynamic complex, low energy binding, molecular dynamics.
Graphical abstract

<figure><img src="image_3.png" /></figure>
Introduction

Lectins are a class of proteins found in plants, mushrooms and animals, that can reversibly bind carbohydrates *via* hydrogen bonds and hydrophobic interactions[1]. Uses are reported for bacterial[2], [3] and cancer cells detection[4], [5]. Concanavalin A (ConA) is a lectin obtained after purification from the Jack Bean (*Canavalia ensiformis*) and is part of the legume lectin family, which is the most described and used in the literature. It is a homo-tetrameric protein bearing 4 recognition sites that specifically binds mannosyl and glucosyl residues[6]. Lectins are often coupled with nanoparticles (NP) to enable a wider range of detection methods and applications. The NP functionalized by lectins are already used in breast cancer bioimaging[4], [7] and bacteria detection[8], [9], [10] with very good detection thresholds, down to tens of colony forming unit (CFU) per milliliter [11]. In these studies, NP functionalization by lectin is performed either in an undirect[4], [7], [8] (*via* covalent binding using a spacer) or in a direct manner (direct incubation of lectin and NP) [9], [10], [12].

Interactions between NP and proteins have been extensively studied in the literature [13], [14], [15]. The protein corona, the phenomenon by which a NP is covered by proteins when incubated in a serum or a solution containing a purified protein, is fast (within seconds) and ruled by intricate equilibria, creating complexes *i.e.* [NP protein] [16]. These equilibria are often governed by low energy binding [17], [18], [19]. They can be modified in a dynamic manner depending on the presence and concentration of other proteins, the [NP protein] affinity or the NP surface chemistry [20], [21], [22]. All proteins are concerned by this phenomenon. One of the main phenomena taking place is the Vroman effect which is a continuous adsorption and desorption of plasma proteins on the surface of NP [23]. In this mechanism, the protein corona changes according to the abundance and the association kinetics of the different proteins in the medium. The NP properties strongly affect the protein corona. For example, the increase of AuNP size (from 5 to 100 nm) was shown to increase the protein binding constant [24]. The surface chemistry of NP is also responsible for the different behavior and composition of the protein corona. In fact, charged surfaces of NP are reported to form more stable complexes with proteins compared to neutral charged NP [16]. Additionally, the isoelectric point (pI) of the protein is to be considered when describing the protein corona formation. Indeed, a protein with a pI lower than 5.5 may show higher affinity for positively charged NP, whereas a protein with a pI higher than 5.5 tends to prefer negatively charged NP [16].

Our study will specifically focus on gold nanoparticles (AuNPs) which are frequently used for lectin coupling, due to their plasmonic properties [4], [7], [8], [11], [12] among others. AuNPs synthesis protocols, such as the Turkevich's one, require the use of sodium citrate. Sodium citrate provides the stabilization of the suspension through charge repulsion by creating a negatively-charged layer around the AuNPs, *i.e.* AuNP-cit [25]. The resulting sodium citrate corona is a key structure in the surface functionalization of AuNPs. It is currently accepted that, when adding a ligand to the NP, the sodium citrate corona will be modified. If sodium citrate molecules are displaced from the gold core to the medium by thiol groups for example, a ligand exchange occurs [26]. On the contrary, if the sodium citrate corona is covered by ligands containing, for example, amine groups at the surface of AuNP-cit, an overcoating takes place [27], [28]. Since proteins bear a great variety of functional groups it becomes more complex to interpret the mechanisms by which the NP-protein interactions occur. For instance, it was demonstrated that for Human Serum Albumin (HSA, a heart-shaped, globular protein with a pI of ~5 [29]), citrate ions were displaced by the protein and not overcoated, as proved by Surface-Enhanced Raman Scattering [27]. Our group previously showed that HSA has a strong affinity towards AuNP with an affinity constant of more than 10¹³, involving H bonds and Van Der Waals bindings [21]. However, the
mechanisms behind the interaction of proteins and the discussions regarding citrate ions possibly being displaced (and how many) remain to be addressed. The displacement of sodium citrate ions finds its importance in discussions regarding NP design and stability. Indeed, modifying the surface of a NP directly influences the stability: either a stabilization of the NP takes place, which was the case in our previous work [21] or a subsequent aggregation is observed [25]. Many discussions in the literature focus on the citrate corona of AuNP, proving that the role of sodium citrate is not limited to a stabilizing agent of AuNP nor an actor to be exchanged to benefit to a ligand of better affinity [25], [30], [31], [32]. Therefore, understanding the behavior of the sodium citrate corona of AuNP in contact with proteins is extremely important to properly design and understand the behavior and fate of AuNP.

In this article, we studied the mechanisms of the interaction between AuNP-cit and ConA using two different experimental approaches: the study of the quenching of fluorescence of the tryptophan residues in ConA in presence of AuNP-cit and the study of the adhesion forces between AuNP-cit and ConA through atomic force microscopy (AFM) in force mode. These experimental approaches were compared to numerical results obtained by molecular dynamics (MD). The data were confronted to the ones obtained studying the interaction between HSA (from our previous work [21]) and AuNP-cit completed, in the present paper, by MD and AFM in force mode analyses. Performing these experiments with this new methodology allowed us to further solidify our previous findings as well as confirm and justify the use of HSA as a reference. The behavior of the sodium citrate layer was analyzed by the comparison of the results obtained by MD using bare AuNP (bAuNP) i.e. AuNP without sodium citrate ions adsorbed at the surface of the gold core, vs AuNP-cit. The dynamics of the binding, the involved energy, and the possibility of sodium citrate displacement or overcoating were addressed.
# Experimental section

## Reagents and standards

All reagents were of analytical grade and used without further purification. ConA from *Canavalia ensiformis* (Jack bean) type IV lyophilized powder, HSA, sodium borohydride, sodium citrate, tetrachloroauric acid trihydrate (HAuCl₄·3H₂O), H₃PO₄, chloroform, sodium hydroxide, ethanolamine, triethylamine, and sodium cyanoborohydrate were obtained from Sigma-Aldrich. KCl was purchased from Merck. NaCl and Dimethylsulfoxide (DMSO) were obtained from VWR. Na₂HPO₄, 12 H₂O was purchased from Carlo Erba. KH₂PO₄ was purchased from Labosi. Acetal-polyethyleneglycol-N-hydroxysuccinimide (Acetal-PEG-NHS) was purchased from Linz University (Austria). Phosphate buffer (PBS 0.148 M) was prepared by combining Na₂HPO₄ 6.68 mM, KH₂PO₄ 1.47 mM, NaCl 138.00 mM and KCl 2.67 mM. The final pH was adjusted to 7.4 with NaOH 0.1 M. Ultrapure deionized water (>18.2 MΩ cm) was used for the preparation of all solutions.

## Gold nanoparticles synthesis

AuNP-cit were synthesized according to previous work [26]. Briefly, 90 mL of water was put under inert atmosphere (N₂) and stirred, 1 mL of AuCl₄⁻ solution (10 mg/mL) was then added. A solution of 55 mM sodium citrate was added and 1 mL of NaBH₄ 19.5 mM was added after 1 min. The resulting colloidal suspension was kept under stirring for 5 min and then stored in the dark at 4°C. The molar concentration of AuNP-cit (expressed in NP and not in gold molar concentration [33]) was determined by Beer-Lambert's law.

$$A_{AuNPs} = \varepsilon_{AuNPs} \times l \times c_{AuNPs}$$

With ε<sub>AuNPs</sub> the absorbance value of AuNPs previously determined to be 1.2 ×10⁷ M⁻¹.cm⁻¹ (acquired at the Surface Plasmon Resonance (SPR) band) [33].

## Incubation of ConA with AuNP-cit

[AuNP-cit ConA] were obtained by a direct incubation with AuNP-cit. The method was carried out by adding a solution of ConA in a citrate sodium solution of 1.53 mM, with AuNP-cit. [AuNP-cit ConA] was obtained at a molar ratio of 1:1, calculated through the determination of the molar concentration of AuNP-cit and the preparation of ConA solution, accordingly. For this ratio and in this study, the exact molar concentration was 30 nM for both components, which corresponds to 1.8 x 10¹³ NP/L. The suspension was kept in the dark at 4°C.

## Physicochemical characterization

After synthesis, AuNP-cit and [AuNP-cit ConA] were characterized without any dilution step. A UV-Vis spectrophotometer (Shimadzu UV-2600) was used to obtain spectra recordings and absorbance measurements. The hydrodynamic diameter (Dh) and zeta potential (ζ) measurements were carried out at 20°C using a Zetasizer Nano ZS (Malvern Instruments UK). Images from Transmission Electron Microscopy (TEM) were recorded using a Philips CM20 instrument with a LaB₆ cathode at 200 kV and with CF200-CU carbon covered copper grids. ImageJ software was used to determine AuNPs cores diameters (Dc). Atomic force microscopy (AFM) measurements were performed at room temperature on a Bioscope Resolve (Bruker) device using the Quantitative Nanomechanical Mapping (QNM) method. A Peakforce HIRS-SSB probe (Bruker) was used after thorough cleaning in piranha solution (H₂SO₄/H₂O₂ 3/1). The experiments were conducted using a freshly cleaved mica surface on which the samples were deposited before
being dried with nitrogen (5 x 100 µL). AFM allowed to obtain the AFM Diameter (Da) after data analysis. Aside from TEM and AFM force results, all the results are given as a mean of three measurements performed on three independent samples related to their standard deviation.

# AFM force measurements

## Cantilever functionalization

AFM force measurements were carried out using a Bruker MLCT cantilever (non-conductive, silicon nitride) and an ultra-flat silicon wafer from Tedpella. An amino-functionalization protocol was followed to functionalize the cantilevers [34], [35] with either ConA or HSA. Functionalization of AFM tips was carried out by washing the cantilevers in chloroform and ethanol before generating amino groups on the tip surface by immersing them overnight in a solution of ethanolamine hydrochloride in DMSO (3.3 g in 6 mL). The amino groups were reacted with the N-hydroxysuccinimide (NHS) ester function of 1 mg Acetal-PEG-NHS dissolved in 0.5 mL chloroform and triethylamine (30 µL). After washing the cantilevers in chloroform, the acetal groups were converted into aldehydes by immersing them in a 1% citric acid solution for 10 min. To couple the proteins (ConA or HSA) on the cantilever tips, a 1 mg/mL solution in PBS was prepared and a 100 µL drop was used to cover the tip. A solution of 1 M sodium cyanoborohydride with 20 mM NaOH was prepared and 2 µL was added to the drop. After 1 h of incubation, 5 µL of a 1 M solution of ethanolamine (pH 8.0) was added to the drop and left to incubate for 10 min before washing and storing the cantilevers in PBS.

## Force adhesion data processing

For each force measurement, a 5 x 5 µm² force map was recorded which corresponds to 32 x 32 points or 1024 curves, equating to 62.5 nm for one point. The curves were analyzed on the Nanoscope analysis software to highlight the adhesive events, which correspond to the interaction force between the protein (ConA or HSA) and the substrate (with D-mannose and AuNPs on the surface for ConA and strictly AuNPs for HSA).

The substrate was composed of silicon wafer. The wafer was thoroughly cleaned first in piranha solution ($H_2SO_4/H_2O_2$ 3/1), rinsed with milli-Q water and dried under a nitrogen stream. Another wash in ethanol and drying with nitrogen is performed before washing once more in chloroform for 10 min and drying as mentioned. Lastly, the wafer was put in a UV-ozone cleaner (PSD UV4, Novascan Technologies Inc. Ames, IA) for 30 min. Deposition of D-mannose or AuNP on the wafer was conducted by depositing a 200 µL drop of the required solution on the surface and drying it with nitrogen. This deposition step was repeated 5 times. The statistical analysis of the curves was executed on all the detected peaks of each retraction curve as well as the intensity of the peak. A Kruskal-Wallis One Way Analysis Variance on Ranks was performed on SigmaPlot software to determine the significant differences between the different groups. The intensity of the peaks corresponds to the force needed to break the interaction between the protein and the substrate. The peaks are representative of the interaction between the protein and the substrate. A total of 552 curves were analyzed for HSA-AuNP, 527 for HSA-SAM-NH₃, 178 for ConA-Mannose, 801 for ConA-AuNP and 242 for ConA-SAM-NH₃.

## Fluorescence quenching study
The interaction between ConA and AuNP-cit was studied through a fluorescence quenching study performed at 20, 30, 40, 50 and 60 °C based on a previous work [21]. AuNP-cit acted as the quencher with varied concentrations (1, 2, 3 and 5 nM) while ConA concentration was kept constant (15 nM), greater than AuNP-cit and with a reasonable fluorescence signal to carry out the study. All the solutions were pre-incubated for one hour at the temperatures needed prior to mixing and measurements. After mixing AuNP-cit with ConA, the fluorescence intensity was recorded using a quartz cuvette from Hellma Analytics with a light path of 10 x 2 mm. A Jasco FP-8300 Spectrofluorometer equipped with a Xenon lamp and with a temperature-controlled chamber was used to perform the measurements. The parameters were set as follows: excitation wavelength = 280 ± 5 nm, emission wavelength = 340 ± 5 nm. The sensitivity was set on high.

The basis of the protein fluorescence quenching value relies on the interaction of ConA with AuNP-cit according to the following equation (1):

$$AuNP-cit + ConA \xrightarrow{Ka} [AuNP-cit ConA] \quad (1)$$

With Ka representing the affinity constant.

The Stern-Volmer's equation was used to determine the fluorescence quenching mechanism (2):

$$\frac{F_0}{F} = 1 + K_{SV} [AuNP-cit] \quad (2)$$

$F_0$ and $F$ are the fluorescence intensities of ConA in the absence and presence of the quencher (AuNP-cit) respectively.

$K_{SV}$ represents the Stern-Volmer's quenching constant.

The affinity constant (Ka) as well as Hill's coefficient (n) can be calculated in the case of static quenching through the following equation (3):

$$\log \frac{F_0-F}{F} = \log Ka + n\log[AuNP-cit] \quad (3)$$

The aforementioned constants were determined through the plotting of $\log \frac{F_0-F}{F}$ against $\log[AuNP-cit]$. The resulting curves allowed the determination of $\log Ka$ through the slopes and intercepts respectively.

Van't Hoff's model was used to determine the entropy (ΔS) and enthalpy (ΔH) variations. These constants were determined by plotting $\ln Ka$ as a function of $\frac{1}{T}$, with T being the temperature in K. The slopes and intercepts of the resulting curves represented $\frac{-\Delta H}{R}$ and $\frac{\Delta S}{R}$ respectively, with R as the universal gas constant equal to 8.314 J·K⁻¹·mol⁻¹.

Gibbs free energy (ΔG) was determined through the following equation (4):

$$\Delta G = - RT \ln Ka \quad (4)$$

Statistical analysis was performed on R software version 4.4.2 (http://www.r-project.org 2025) using dunn.test package.

Results were obtained by running a Kruskal-Wallis test followed by a Dunn test with Bonferroni correction. Statistical significance was determined as a p value < 0.05.

## Molecular dynamics study

### *Simulated systems*

The two proteins were studied during our investigations (i.e. ConA and HSA). Their structure was obtained through the protein data bank internet library with ID 1I3H or 6M4R for ConA-dimannose or HSA, respectively. Before
associating each protein with AuNP, we first equilibrated the protein in a water solvent with 0,15 M NaCl at room
temperature for 25 ns. Mannose molecules were extracted from the 1I3H pdb structure.

bAuNP was built according to CHARMM-GUI procedure. The chosen model was an equilibrated structure composed
of 887 Au atoms with a truncated octahedron shape [25]. Its maximum diameter was around 3 nm for a material volume
of 18,5 nm³ and the low index (111), (100) and (110) facets were all available for binding citrate molecules.

Citrate was modeled by bound and unbound interactions where parameter models were obtained through the SWISS-PARAM protocol [36], [37]. bAuNP was described only by nonbonded interactions using van der Waals (vdW) contributions for pair interaction with the other components of the systems.

To create the modelled AuNP–cit, citrate anions (fully deprotonated C₆H₅O₇³⁻) were bound near the bAuNP (90 citrate molecules to respect a local concentrated layer around the NP as reported in[38], [39]). To keep the total system neutral, sodium ions were added to an amount of three Na⁺ cations per citrate molecule. The resulting system was then minimized in vacuum. No restraint on gold atom was applied to allow it to move freely. Once optimized in vacuum, the AuNP–cit was released in a solvent bath made of water molecules (depicted in the TIP3P framework) and ions (Na⁺ and Cl⁻) at 1 M plus supplementary Na⁺ ions to compensate the citrate ion charges. Note that citrate ions were modeled in their fully deprotonated tri-anionic form (Cit³⁻), following a well-established model previously reported in the literature [38], [39] and widely used in similar studies. This approach emphasizes the electrostatic interactions between citrate and the surfaces (protein and gold) and provides a consistent framework for comparative analysis. We acknowledge that at pH 5.5, citrate may exist in partially protonated forms, as shown in structural studies [25]. However, given the widespread use of the fully deprotonated model and its capacity to capture the dominant interaction features, we believe this representation remains appropriate for the scope of our study.

Simulation protocol

MD simulations for all systems are carried out using the NAMD 12 package[40] with explicit TIP3P water molecules
and the CHARMM36 force field [41].

TIP3P water model remains consistent with the general force field depicting the protein behavior in solvent [42]. In
these simulations, a 1 fs time step is employed to increment the time simulation. The cut-off distance applied to the
vdW interactions was equal to 1.2 nm with a pair list distance of 1.3 nm. The temperature was set to 310 K in a
Langevin piston scheme [43] and pressure was fixed at 1 bar using the Nose-Hoover Langevin piston control [44].
Periodic boundary conditions were applied in all dimensions. Full electrostatic interactions were performed using the
Particle–Mesh–Ewald method [45].

Initial conformations for the MD simulations implying protein and bAuNP (or AuNP–cit) are obtained from docking
simulations performed in Hex package [46]. The best ranking positions were extracted from these simulations. To avoid
a too high constraint simulation, we separated bAuNP (or AuNP–cit) from the protein by around 1 nm before running
the MD simulations.

Each MD simulation was separated in three phases, i.e. one minimization stage lasting 3.8 ps, one equilibration stage
for 1 ns and a final production stage for 200 ns, at least. The minimization stage was calibrated according to the total
energy variation between each calculation step. The duration of the equilibration stage was set to 1 ns in order to let the
system converge to the imposed temperature, pressure and density. As shown in Fig.S1, all these data tend to their
targeted value before 1 ns or around 1 ns, explaining our choice for the system.
Different analyses were then produced to determine the stability variations of the protein when interacting with the different AuNP. For instance, we will extract and discuss the protein RMSD in order to obtain the average variations of the protein backbone positions during the simulations (Eq.5). The total unbound pair interactions (ΔE<sub>A/B</sub>) (Eq.6) between the proteins and the AuNP (or ions) were also extracted to interpret the behavior of the AuNP around the simulated protein. From these data, ΔH will be calculated as defined below in Eq.7. To compare the simulations and the experimental data extracted from the AFM measurements, we also computed the total force between the AuNP and the protein during the simulations.

$$RMSD = \sqrt{\frac{1}{N} \sum_{1}^{N} (r_{i} - r_{0})^{2}} \quad (5)$$

Where r<sub>i</sub> is the position at time t and r<sub>0</sub> is the reference position

$$\Delta E_{A/B} = E_{A/B} - E_{A} - E_{B} \quad (6)$$

With E<sub>A/B</sub>, representing the pair interaction energy between A and B species and E<sub>A</sub> (resp. E<sub>B</sub>) is the interaction energy of the isolated system A (resp. B)

$$\Delta H = E_{complex/water} + \Delta E - E_{A/water} - E_{B/water} \quad (7)$$

Where E<sub>complex/water</sub> described the pair interaction energy between the formed complex (A+B) and the water molecules and E<sub>A/water</sub> (resp. E<sub>B/water</sub>) is the interaction energy of the isolated system A (resp. B) with water molecules.
# Results and discussion

## Characterization of [AuNP–cit ConA] complex

MD was performed on bAuNP, AuNP–cit and the proteins, ConA and HSA. In parallel, AuNP–cit were synthesized and directly incubated with ConA. [AuNP–cit ConA] was prepared at a final molar ratio of 1:1 with a concentration of 30 nM each (1.8 x 10¹³ NP/L of AuNP). This 1:1 ratio came as the result of Dh and UV-visible spectra stability over 5 days, which was not observed with other ratios which had either higher concentration of AuNP or ConA (0.02:1; 0.2:1; 0.5:1; 0.6:1; 0.75:1; 1.5:1; 2:1; 4:1; 20:1; 40:1, ratios given for [AuNP-ConA] respectively, with the constant AuNP concentration at 30 nM). Meanwhile, the AuNP concentration in MD was equal to 1.6 mM. The formation of the AuNP–cit functionalized by ConA ([AuNP–cit ConA]) complex was first attempted using PBS as the medium. However, the presence of this buffer induced AuNPs aggregation. This aggregation happened as the result of a charge screening effect due to the increased ionic strength of the medium induced by the PBS buffer. The [AuNP–cit ConA] complex could not be formed in this medium and so, PBS was substituted by a sodium citrate solution (1.53 mM in water, pH 6.9) which is the AuNPs synthesis medium. We followed through with this solvent as our study was not aimed at developing a biocompatible complex but rather to understand the effect of the presence of a protein on the surface of the AuNP at a fundamental level. This method allowed for the stabilization of AuNPs in presence of ConA. The characterization results are presented in Figure 1 and Table 1.
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Visualization of the optimized structures obtained through MD simulations of a) bAuNP, b) AuNP–cit, c) ConA, and d) HSA. Gold, oxygen, hydrogen, carbon and nitrogen atoms are depicted in pink, red, white, blue and dark blue balls, respectively. ConA and HSA proteins are shown in ribbons representation for simplicity. Physicochemical characterizations of AuNP–cit and [AuNP–cit ConA] using e) spectrophotometry, f) dynamic light scattering, g) TEM of AuNP–cit and size distribution (inserted graph) and h) TEM of [AuNP–cit ConA] and size distribution (inserted graph).</figcaption></figure>

<table><caption>**Table 1.** Physicochemical characterization of [AuNP–cit ConA]. AuNP–cit 1:1 at 30 nM for each component, which translates to 1.8 x 10¹³ NP/L.</caption><thead><tr><th></th><th>λmax<br>(nm)</th><th>Absorbance<br>(a.u.)</th><th>Dh<br>(nm)</th><th>Dc<br>(nm)</th><th>Da<br>(nm)</th><th>Zeta potential<br>(mV)</th></tr></thead><tbody><tr><td>[AuNP–cit]</td><td>513 ± 1</td><td>0.674 ± 0.026</td><td>6.5 ± 0.4</td><td>3.4 ± 0.7</td><td>5.7 ± 1.9</td><td>-30.2 ± 9.1</td></tr><tr><td>[AuNP–cit ConA] 1:1</td><td>517 ± 1</td><td>0.393 ± 0.003</td><td>18.4 ± 2.4</td><td>4.0 ± 0.8</td><td>23.2 ± 3.1</td><td>-29.6 ± 4.7</td></tr></tbody></table>
The characterization of the different elements (bAuNP, AuNP-cit and the proteins, ConA and HSA) was performed using both numerical (MD simulations) and experimental techniques (except for bAuNP). To model bAuNP, 887 gold atoms were used (Figure 1 a)) and 90 citrate ions were adsorbed on the surface of the crystal [25] as explained in the Experimental section. Sodium cations were added in the solvated system in excess to compensate for the negative charge of the citrate ions (Figure 1 b)). All systems were relaxed in a solvent with [NaCl] 1 M and T = 37 °C (Figure S1). At the end of these preliminary simulations, the modelled bAuNP and AuNP-cit showed diameters of 3 and 4 nm, respectively, which was in accordance with the obtained experimental data (Figure 1 e), f) and g) and Table 1) and our previous work [21]. The size of ConA was almost the same as the AuNP while the one of HSA was twice the size of them as shown in Figure 1 c) and d), respectively. After the incubation with ConA, the AuNP-cit remained individualized without aggregation (Figure 1 e), f) and h)), which was proved by TEM and the visible spectra.

The dynamic light scattering (DLS) results provided a Dh value of 6.5 ± 0.4 nm for AuNP-cit, which was in accordance with our previously published results [47] and with the AuNP diameter from the AFM analysis Da (5.7 ± 1.9 nm) as shown in Figure 1 f) and Figure 2 a) and b). Additionally, DLS and AFM results highlighted the modification in the layers surrounding the metallic core of the NP due to ConA interaction, as demonstrated by the increase of both the plasmonic band (+ 4 nm), the Dh (+ 11.9 nm) and Da (+ 17.5 nm) (Figure 1 e) and f) and Figure 2 c). The increase in Dh after addition of a lectin to a NP is often seen and documented in the literature [9], [48], [49]. The characterization shows no significant difference after 5 days, results can be found in Table S2.
<figure><img src="image_5.png" /><figcaption>**Figure 2.** a) AFM recording of AuNP–cit (blue line shows the representative line section shown in d)) and 3D topography of [AuNP–cit] b) and [AuNP–ConA] c).</figcaption></figure>

Dynamic interaction between AuNPs and ConA

The interactions between AuNP and ConA were studied using fluorescence quenching from the innate fluorescence of the 4 tryptophan residues in ConA and MD respectively. The results illustrated in Figure 3, Table 2, Figure S3, Movie S5, and S6 can be compared to the ones obtained when incubating AuNPs with HSA from our previous study [21] (see Figure 4, Movies S7, and S8.)

The incubation of AuNP–cit with ConA led to a significant decrease of its innate fluorescence in a concentration-
dependent manner, proving that at least one of its four tryptophan residues was near the AuNP–cit (Figures 3 a) and b),
and Table 2). No modification of emission wavelength value was noticed, demonstrating that there was no modification
in the protein structure by AuNP–cit [50]. In a more mechanistical point–of–view, two kinds of quenching are described.
A static one appears via the formation of a complex in which the quencher (AuNP–cit) is within a distance ranging
between 10 to 100 Å from the tryptophan residue(s) of ConA and/or a dynamic or collisional quenching [51]. To study
this, the experiment was run at different temperatures. Using the Stern–Volmer’s equation, a good linearity was
obtained, as shown by the R² values. The deduced K<sub>SV</sub> values showed a decrease from 20 °C to 30 °C, proving that a
static complex predominates whereas these values increased from 30 °C to 50 °C proving a modification of the
quenching type: from a static one to a dynamic complex predominance [50]. This result was unexpected since our
previous results measured with HSA showed that a static complex was created in interaction with AuNP–cit [21]
whatever the temperature. However, we know that the interaction between NP and proteins highly depends on the NP (shape, core and surface chemistry) and the protein itself (pI, size, amino acid sequence).

MD was then performed on AuNP-cit and bAuNP, interacting with either ConA or HSA. For bAuNP, we observed a rapid adsorption of the NP on the ConA towards a stable site, demonstrated by a constant pair interaction energy between bAuNP and ConA and a constant distance between each center of mass (Figure 3 c) e) for T= 37 °C). This adsorption site belongs to an area of protein containing a tryptophan group. The presence of the AuNP would tend to reduce the displacement range of this group that could potentially explain the quenching observed in the fluorescence spectra. For AuNP-cit, the behavior is fundamentally different. Indeed, the AuNP-cit could not reach a stable configuration on a right place with ConA. It diffused around the protein, still remaining in its neighboring area. The evolution of the mean pair interaction energy and the mean distance between the AuNP-cit and the protein with time (Figure 3 d) and f) described well these first observations.

The protein stability was studied using the root medium square deviation of atomic positions (RMSD) analysis. As shown in Figure 3 e) and f), the RMSD values of the protein tend towards a stable value around 3.5 +/- 0.1 Å (for AuNP-cit and bAuNP). The RMSD appear to slightly fluctuate at the end of the simulation but remains in a narrow range of 3.0-3.3 Å during the final 80 ns of the simulation. Combined to the evolution of the total energy of the system which presents a small drift of 0,05% and the temperature, clearly stable over time simulation (see Figure S4 of the Supporting Information), we can conclude on the good stability of the whole system. Moreover, on its own, we found that the protein backbone is modified through a RMSD value equal to about 3.0 +/- 0.1 Å. This means that, even if they interact with the protein, the NP does not introduce important changes in the protein backbone compared to its usual solvent.

<table><caption>**Table 2.** Values obtained from the Stern Volmer model from fluorescence quenching.</caption><thead><tr><th>Temperature (°C)</th><th>K<sub>sv</sub> (10<sup>8</sup> L.mol<sup>-1</sup>)</th><th>R<sup>2</sup></th><th>Association mode</th></tr></thead><tbody><tr><td>60</td><td>6.15 ± 1.03</td><td>0.9738</td><td rowspan="3">Dynamic[52]</td></tr><tr><td>50</td><td>6.56 ± 1.45</td><td>0.8994</td></tr><tr><td>40</td><td>4.95 ± 1.94</td><td>0.8674</td></tr><tr><td>30</td><td>3.84 ± 1.60</td><td>0.9897</td><td rowspan="2">Static[52]</td></tr><tr><td>20</td><td>4.59 ± 1.84</td><td>0.9929</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Data illustrating the interaction between AuNP-cit and ConA in a), b), d) and f) using fluorescence and MD or between bAuNP and ConA in c) and e) by MD a) Fluorescence spectra of ConA after incubation with different AuNP-cit concentrations at 20 °C; b) plot of the value of fluorescence intensity of ConA at 340 nm at 20 °C as a function of the concentration of AuNP expressed as a mean of 3 measurements with their standard deviation a). Total unbonded pair interactions c) $\Delta E_{bAuNP/ConA}$ between bAuNP and ConA and d) $\Delta E_{AuNP-cit/ConA}$ between AuNP-cit and ConA at T= 37 °C. Snapshots in Figure 3 c) and 3 d) show the positions of the NP at different simulation time from Movies S5 and S6. RMSD of the protein and mean distance (inserted graph) between e) the mass centers of the bAuNP and the protein; and f) the mass centers of the AuNP-cit and the protein, as a function of time.</figcaption></figure>

The same simulations were carried out on HSA at T= 37 °C for bAuNP and AuNP-cit. In these two cases, we observed the same behavior for the two kinds of NP (bAuNP and AuNP-cit). After a rapid diffusion inside the solvent, the NP
found a stable adsorption site where it stayed for the rest of the simulations. Figure 4 illustrates these conclusions with a constant pair interaction energy obtained after 300 ns for bAuNP (200 ns for AuNP–cit). The RMSD of the HSA did not show further variations after these simulation times (as such as the distance between the centers of mass of the protein and the NP). These simulations revealed an entirely different behavior of the same NP toward different kinds of proteins. Our results obtained here for ConA, appear thus in opposition to what was described in another work in which authors proved a static complex between silver NP and jacalin lectin (extracted from *Artocarpus integrifolia*) as for HSA here [9]. However, the dual behavior of the AuNP–cit towards ConA has already been observed for titanium oxide NP with bovine serum albumin and lysozyme for which both a static and a dynamic complex were co-existing [53].

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Total unbonded pair interactions a) $\Delta E_{bAuNP/HSA}$ between bAuNP and HSA and b) $\Delta E_{bAuNP/HSA}$ between AuNP–cit and HSA at T= 37 °C. RMSD of the HSA protein (c and d) and mean distance (inserted graph) between c) the mass centers of the bAuNP and the protein; and d) the mass centers of the AuNP–cit and the protein, as a function of time.</figcaption></figure>

## Interaction via low-energy binding

To go further, the thermodynamic parameters were calculated from fluorescence quenching using Eq 4, Van't Hoff's model and MD simulations. The results are presented in Table 3 for ConA and Table 4 for HSA.
<table><caption>**Table 3.** Thermodynamic parameters obtained by calculation based on either fluorescence quenching data or on MD, on AuNPs (functionalized or not) interacting with ConA.</caption><thead><tr><th rowspan="3">Temperature (°C)</th><th colspan="4">AuNP-cit</th><th colspan="2">bAuNP</th></tr><tr><th rowspan="2">Ka</th><th colspan="3">Fluorescence quenching</th><th rowspan="2">MD</th><th rowspan="2">MD</th></tr><tr><th>ΔG (kJ.mol<sup>-1</sup>)</th><th>ΔH (kJ.mol<sup>-1</sup>)</th><th>ΔS (J.K<sup>-1</sup>.mol<sup>-1</sup>)</th></tr></thead><tbody><tr><td>60</td><td>8.04.10<sup>6</sup></td><td>- 44</td><td></td><td></td><td></td><td></td></tr><tr><td>50</td><td>2.94.10<sup>8</sup></td><td>- 52</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>3.34.10<sup>8</sup></td><td>- 51</td><td>- 32</td><td>50</td><td>- 15500 ± 5000</td><td>- 1400 ± 200</td></tr><tr><td>30</td><td>8.26.10<sup>6</sup></td><td>- 40</td><td></td><td></td><td></td><td></td></tr><tr><td>20</td><td>3.54.10<sup>8</sup></td><td>- 48</td><td></td><td></td><td></td><td></td></tr></tbody></table>

The interaction with AuNP-cit was spontaneous for both proteins as shown by the negative value of ΔG. The affinity constant Ka of [AuNP-cit ConA](3.5 x 10⁸ at 20 °C) was 10⁵ less fold than the one obtained for [AuNP-cit HSA] (1.8 x 10¹³) [21]. From the values of ΔH and ΔS, it was concluded that ionic bonds were involved in [AuNP-ConA] formation as per [52]. To confirm this, [AuNP-cit ConA] was incubated in presence of high concentrations of NaCl and the fluorescence emission spectra were recorded (Figure 5 a)). Interestingly, in the presence of a high concentration of salt, the complex seemed partially dissociated. Indeed, the fluorescence intensity of ConA was not as quenched as when incubated without NaCl. The ionic bonds contribution was therefore confirmed. This result was in opposition to what was previously obtained with other AuNPs and lectin from *Eranthis hyemalis* (EHL)[12]. In their work, the interaction of AuNP-cit by EHL was strong enough not to be destabilized by the addition of 200 mL of NaCl 2M. It should be noted, however, that this study managed to stabilize AuNP with a molar ratio not equal to 1:1 but with a high concentration in EHL. Nonetheless, the AuNP in this study possess a Dh of 18.8 nm whereas our present study uses smaller AuNP of 6.5 nm. While the difference in size of AuNP must play a role in the association dynamics between AuNP and EHL or ConA, this reference is interesting to mention as very little studies focus on the binding mechanisms between AuNP and lectins. In MD simulations, the calculations of the ΔH for bAuNP did not show differences between HSA and ConA. However, when AuNP-cit is taken into consideration, we observed negative values and a ratio of 5 between the two ΔH in favor of HSA. The interaction of AuNP-cit with ConA was weaker than the one with HSA with a 6-fold when extracted from fluorescence quenching data. These values are relatively close and reproduce qualitatively the general behavior of the AuNPs with the proteins. The presence of citrate ions and sodium counter ions that increase the global electrostatic contribution to the energy seems to be at the origin of these differences.

<table><caption>**Table 4.** Thermodynamic parameters obtained by calculation based on either fluorescence quenching data or on MD, on AuNPs (functionalized or not) interacting with HSA.</caption><thead><tr><th>AuNP-cit</th><th>bAuNP</th></tr></thead></table>
<table><thead><tr><th rowspan="2">Ka*$</th><th colspan="3">Fluorescence quenching</th><th>MD</th><th>MD</th></tr><tr><th>ΔG (kJ.mol<sup>-1</sup>)*$</th><th>ΔH (kJ.mol<sup>-1</sup>)*$</th><th>ΔS (J.K<sup>-1</sup>.mol<sup>-1</sup>)*$</th><th>ΔH (kJ.mol<sup>-1</sup>)</th><th>ΔH (kJ.mol<sup>-1</sup>)</th></tr></thead><tbody><tr><td>1.8.10<sup>13</sup></td><td>- 65</td><td>- 200</td><td>- 432</td><td>- 79000 ± 4600</td><td>- 800 ± 280</td></tr></tbody></table>

<footnote>*Values obtained from fluorescence quenching data from.[21].<sup>§</sup> Value given at 37 °C</footnote>
To further confirm these results, AFM in force mode was carried out to quantify the adhesion forces between ConA or HSA and AuNPs. For this, an AFM cantilever tip was functionalized with ConA or HSA to probe interactions on a gold amine self-assembled monolayer (SAM\_NH₃) which surface was modified according to different conditions (AuNPs or D-mannose deposition). Force curves were recorded first between the ConA-modified tips and SAM\_NH₃ with a mannose deposition to confirm that ConA retained its carbohydrate-binding activity. The analysis of the force curves showed an adhesion value of 0.5 nN for the interaction of ConA and mannose and a value of 0.25 nN for ConA and the SAM\_NH₃ surface. Interestingly, the adhesion value for the ConA tip on an AuNP-modified SAM\_NH₃ surface was lower than both previous conditions at 0.1 nN. When comparing with the same surface but with a HSA functionalized AFM cantilever tip, we obtained a 90 times more important adhesion value of 9 nN. These new original data were also estimated by MD simulations by which the mean interaction forces between the protein (HSA or ConA) and the AuNP-cit were calculated. We found in these different cases, values ranging of 49 ± 5 nN for HSA with AuNP-cit compared to 8 ± 0 nN for ConA with AuNP-cit, i.e., a factor of 6.3 between the two proteins. These computational data confirmed once again our results. While the systems (experimental and modelling) are not strictly the same ones, we can, however, observe, for both, a strong tendency toward a stronger interaction between the AuNP-cit and HSA compared to the one obtained for AuNP-cit and ConA. This has been demonstrated with an enthalpy ratio that clearly shows preference for HSA and with the adhesion force ratio, that confirms this result. Note also that orders of magnitude are well respected between experiment and simulations, especially for the adhesion forces that remain very challenging to compute precisely for comparison with experiments due to complexity in experimental setups. In experiments, measurements have been performed between a protein and a surface of AuNP while in simulation, only one protein and one NP have been considered. Nevertheless, the tendency and order of magnitude remain satisfying.
<figure><img src="image_8.png" /><figcaption>**Figure 5.** a) Experimental data representing the low energy, non-covalent interactions of [AuNP–cit ConA] by showing the emission spectra of fluorescence in presence or absence of NaCl to confirm the presence of ionic bonds and b) violin plot showing the adhesion force values obtained in AFM force mode for [AuNP–cit ConA] and [AuNP–cit HSA]. A Kruskal-Wallis One Way Analysis of Variance on Ranks test was performed with * representing *p* < 0.001.</figcaption></figure>
ConA can displace neither citrate anions nor sodium cations from the surface of AuNP–cit

Lastly, we tried to understand even further the mechanisms occurring at the surface of the AuNPs. Depending on the
type of protein submitted to AuNP–cit, we observed different behaviors during the MD simulations. Indeed, as shown
in Movies can conclude that no citrate ion left AuNP–cit surface during its interaction with ConA, while several ones
are liberated from the AuNP–cit when it approaches the HSA. This allows the HSA to adsorb on AuNP–cit on a specific
place while it was not possible when the AuNP–cit interacted with ConA. These observations are demonstrated by
plotting in Figure 6 a) the distribution of sodium and citrate ions around the proteins. From this graph, we can deduce
several information on the direct surrounding of the AuNP–cit. First, as it can be expected, the number of sodium ions
is about 3 times larger than the number of citrate ions in order to modulate the global charges around the AuNP–cit.
Second, by comparing the ions number between the ConA vs. HSA systems, we clearly show that the number of ions
is decreasing slightly when AuNP–cit is interacting with HSA compared to its interaction with ConA (full curve remains
higher than dashed ones).

<figure><img src="image_9.png" /><figcaption>**Figure 6.** a) Distribution number of sodium (red) and citrate (black) ions as a function of the distance from the center of mass of the AuNP–cit. Full curves have been computed for ConA proteins while dotted ones are for HSA. b) Pair interaction energy between sodium ions and ConA (yellow curves); AuNP–cit and ConA (red curves) and AuNP–cit surrounded by its sodium ions and ConA (blue curves) at T= 37 °C.</figcaption></figure>

This modification of behavior is also clearly highlighted in Tables 3 and 4. bAuNP was adsorbed on both proteins with the same enthalpy energy (for which only gold atoms interacted with the proteins), while the modification of the AuNP by sodium citrate creates a major imbalance when comparing the interactions of the AuNP–cit with the 2 proteins. This situation is not easy to understand, as the proteins are globally negatively charged (-7 for ConA and -28 for HSA at pH 5). We would even have expected to observe the opposite situation: a repulsive tendency for HSA under these conditions. But the surface modification of AuNP–cit (departure of citrate and sodium ions) and its specific interaction on a given area of HSA changes these interpretations. When we look at the contact zones between AuNP–cit and HSA (over 10 Å² around the contact), the total charge carried by AuNP–cit in this zone is +5, while that proposed by HSA in the same zone is -7. We can therefore see that there is a significant electrostatic affinity in this precise zone between the protein and AuNP–cit freed from these few surface ions.

For ConA, this is made much more difficult by the fact that the size of ConA and AuNP–cit are almost equivalent. This
makes it more difficult for AuNP–cit to meet an attractive area of the protein if no surface ions are removed.
Based on these initial findings, it is fair to assume that ionic interactions are responsible for this difference in behavior. To prove this, we plotted in Figure 6 b) the interaction energies between sodium ions in solution and ConA (or between AuNP-cit and ConA) at 37 °C. We then obtained a curve corresponding to the interaction energies of AuNP-cit surrounded by its sodium counterions. Although no specific adsorption was seen, at 37°C, thermal agitation makes the diffusion of sodium ions much more significant. This diffusion disturbs the direct environment of the AuNP-cit, itself surrounded by static sodium ions, through electrostatic repulsion. The result fluctuations that are almost symmetrical between the direct interaction of the NP with the protein (red curve) and those caused by sodium ions (yellow and blue curves). These fluctuations alternate between moments for which the interaction becomes favorable (attractive) and moments when the interaction is unfavorable (repulsive). This was not observed with HSA for which the protein released citrate and sodium ions as soon as it approached the protein to adsorb it. ConA is thus not able to displace the sodium citrate corona from AuNP-cit, suggesting a dynamic overcoating of the sodium citrate corona of the AuNPs. Once more, these findings are in accordance with the ones from our experimental data as we are able to draw the same conclusions.

## Conclusions

To sum up, this work helped to understand the mechanism of the interaction between ConA and AuNPs. NP-protein interactions are well-documented in the literature and many discussions regarding the fate of the citrate capping layer of AuNPs can be found. Studying an environment that contains both NP and proteins is particularly challenging. This study highlighted, through experimental and theoretical methods, that a dynamic interaction occurs between AuNP-cit and ConA. The nature of these interactions was also determined to be non-covalent and of low energy through electrostatic bonds, with confirmation of ionic bonds, and with a low affinity between the two partners of the complex. These low energy non-covalent bonds were confirmed through AFM in force mode which allowed for the quantification of these interactions, confirming once more the previously obtained results. MD simulations further validated the experimental results and provided more information regarding the behavior of the [AuNP-cit ConA] complex according to the presence of sodium counter ions. Through all these experiments, we concluded that ConA is not able to displace the sodium citrate corona from AuNP-cit which rather suggests a dynamic overcoating of the sodium citrate corona of the AuNPs. Further work on the impact of environmental factors (i.e. the different components of the medium, different media, etc.), the protein hydrophobicity, structure and flexibility will be able to enrich our findings and provide an overall better understanding of the binding mechanism of a protein to a NP. The strength of this study relies on the complementarity of the used methods. These three methods support the originality of this work as they are performed to investigate the behavior of proteins on top of AuNP as opposed to molecules or ligands. Moreover, all the collected data from the experimental physicochemical characterization, fluorescence quenching, AFM in force mode and from the theoretical chemistry via MD were consistent and converged towards the same conclusions.
## Author contributions

**Sephora Lahouari:** Writing – original draft, Data curation, Formal analysis, Investigation, Validation. **Jérémie Gouyon:** Conceptualization, Supervision, Investigation, Validation, Methodology, Writing – review & editing. **Grégory Francius:** Data curation, Formal analysis, Writing – review & editing. **Fabien Picaud:** Data curation, Formal analysis, Writing – review & editing. **Roberta Albino dos Reis:** Data curation, Writing – review & editing. **Amedea B. Seabra:** Writing – review & editing. **Igor Clarot:** Writing – review & editing. **Ariane Boudier:** Writing – original draft, Supervision, Funding acquisition, Project administration, Conceptualization, Methodology, Investigation, Validation.

All authors have given approval to the final version of the manuscript.

## Conflicts of interest

There are no conflicts to declare.

## Data availability

Data will be made available on request

## Acknowledgements

The authors would like to thank COFECUB/CAPES program (Ph-C983/23, 49445SA, 88881.711920/2022-01), ANR NanoBioID (ANR-22-CE09-001) and IUF for funding. ABS and RAR thank the Coordination for the Improvement of Higher Education Personnel (Capes, Brazil) – Finance Code 001 and Capes 88881.711920/2022-01. Support from the National Council for Scientific and Technological Development (CNPq, Brazil, grant no. 313117/2019-5), São Paulo Research Foundation (FAPESP, Brazil, grant no. 2023/16363-7 and grants no 2022/14645-2; 2024/12112-2). The authors thank the spectroscopy and microscopy Service Facility (SMI) of LCPME (UMR 7564, Université de Lorraine - CNRS, 405 rue de Vandoeuvre 54600 Villers-lès-Nancy, France, https://www.lcpme.ul.cnrs.fr) where AFM experiments were performed. Computations have been performed on the supercomputer facilities of the *Mésocentre de calculs de Franche-Comté*. The authors acknowledge also the national Jean Zay supercomputer for its access to the HPC resources under the allocation AD010713746R1.

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**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:

**Highlights**

* AuNP functionalized by Concanavalin A are prepared and characterized.

* Fluorescence quenching and Force Mode AFM showed high affinity and low interaction energy.

* Molecular Dynamics showed Concanavalin A cannot displace citrate ions of AuNP.