Lysozyme–aunps interactions: Determination of binding free energy

nanomaterials Article Lysozyme–AuNPs Interactions: Determination of Binding Free Energy Axel Gomes 1, Jose M. Carnerero 1, Aila Jimenez-Ruiz 1, Elia Grueso 1, Rosa M. Giráldez-Pérez 2 and Rafael Prado-Gotor 1,*   Citation: Gomes, A.; Carnerero, J.M.; Jimenez-Ruiz, A.; Grueso, E.; Giráldez-Pérez, R.M.; Prado-Gotor, R. Lysozyme–AuNPs Interactions: Determination of Binding Free Energy. Nanomaterials 2021,11, 2139. https://doi.org/10.3390/ nano11082139 Academic Editor: Yurii K. Gun'ko Received: 29 July 2021 Accepted: 19 August 2021 Published: 22 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Physical Chemistry, Faculty of Chemistry, University of Seville, 41012 Seville, Spain; [email protected] (A.G.); jcarnerer[email protected] (J.M.C.); [email protected] (A.J.-R.); [email protected] (E.G.) 2Department of Cellular Biology, Physiology and Immunology, Faculty of Science, University of Córdoba, 14014 Cordoba, Spain; r[email protected] *Correspondence: [email protected] Abstract: Investigation and optimization of lysozyme (Lys) adsorption onto gold nanoparticles, AuNPs, were carried out. The purpose of this study is to determine the magnitude of the AuNPs– lysozyme interaction in aqueous media by simple spectrophotometric means, and to obtain the free energy of binding of the system for the first time. In order to explore the possibilities of gold nanoparticles for sensing lysozyme in aqueous media, the stability of the samples and the influence of the gold and nanoparticle concentrations in the detection limit were studied. ζ potential measurements and the shift of the surface plasmon band showed a state of saturation with an average number of 55 Lys per gold nanoparticle. Lysozyme–AuNPs interactions induce aggregation of citratestabilized AuNPs at low concentrations by neutering the negative charges of citrate anions; from those aggregation data, the magnitude of the interactions has been measured by using Benesi–Hildebrand plots. However, at higher protein concentrations aggregation has been found to decrease. Although the nanocluster morphology remains unchanged in the presence of Lys, slight conformational changes of the protein occur. The influence of the size of the nanoclusters was also investigated for 5, 10, and 20 nm AuNPs, and 10 nm AuNPs was found the most appropriate. Keywords: binding free energy; gold nanoparticles; lysozyme; nanoparticle size; surface plasmon resonance 1. Introduction Nanoparticles are characterized by the properties of the metal cluster core, but also by the organic molecules that constitute the monolayer, that is, the capping agents. Gold colloids (AuNPs) are the most stable metal nanoparticles with promising applications because of their electrical, optical, magnetic, and catalytic properties [ 1 ]. Besides, gold nanoparticles can be functionalized with a wide variety of structural units by using simple chemical transformations [ 2 – 5 ]. From the first preparation of gold colloids by Faraday in 1857, research on the use of gold nanoparticles has been an area of broad interest, a fact which is reflected by an exponential growth in the number of publications in the recent years. They are considered largely nontoxic, even though this non-toxicity is strongly dependent on their size and recent publications have reported conflicting data [ 6 ]. They are also stable, conductive, catalytically active, and electron dense [ 7 ]. Among their many qualities, one stands out greatly: AuNPs possess unique optical properties in terms of extremely high extinction coefficients, distance–dependent color, and outstanding fluorescence quenching ability, popularizing them for biosensor development [ 8 ]. Owing to those inherent optical properties (AuNPs possess extinction coefficients which are over 1000 times larger than those of organic dyes) certain chemical interactions lead to aggregation-induced color changes allowing for visual detection and quantification of those interactions [ 9 ]. Works dealing with protein detection using AuNPs aggregation-based assays, where both label Nanomaterials 2021,11, 2139. https://doi.org/10.3390/nano11082139 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021,11, 2139 2 of 17 free and functionalized approaches have been explored, are constantly emerging. The simplicity of these methods should not be ignored; one of their strongest points is the low cost that stems from not requiring sophisticated equipment. In aqueous solution AuNPs can appear with different colors (red, blue, purple) and have a broad absorption peak in the range 500–550 nm, depending on the size of the cluster. This peak is called the surface plasmon resonance (SPR) band [ 10 ]. This band depends on several factors and therefore making an analysis is not easy. However, for particles smaller than 30 nm it can be simplified since the phenomenon of resonance when light of adequate wavelength reaches the surface of the nanomaterial produces dipoles and not multipoles, as happens with larger ones. The shape, size, and chemical nature of the nanoparticles are factors that affect the plasmon band, as well as the physical–chemical conditions of the medium in which they are found [ 11 ]. This fact makes it possible to analyze the interaction of AuNPs with a ligand in solution if aggregation of the clusters takes place. Among studies using gold nanoparticles as biosensors, those directed toward the detection of proteins are particularly important. The number of possible proteins that in aqueous media can and have shown affinity for gold nanoparticles, and that are able to induce aggregation and the aforementioned color change is vast and fast-growing. Among them, lysozyme (lys) has proven to be a specially interesting case given its medical relevance: it is virtually absent in the finally excreted urine of healthy subjects, but a urinary excretion of elevated levels of lysozyme (lysozymuria) suggests the existence of tubular disfunction [ 12 ]. Increased Lys concentration in urine and serum is associated with leukemia [ 13 ], renal diseases [ 14 ], and meningitis [ 15 ]. Although the present study does not focus on biological systems, instead being limited to lysozyme solutions in water, we believe that a good understanding of the phenomena being reported is essential in order to allow for a future extrapolation of the system to more complex cases, where interferents such as ions or other proteins can be present, as are human body fluids. Only by having a good knowledge of the effects that are directly and exclusively caused by lysozyme on gold nanoparticle systems can the influence of the aforementioned interferents be accounted for and compensated, and to our knowledge no such studies have been carried out as of today. Therefore, we have developed a rigorous study of the interaction of lysozyme with the surface of gold nanoparticles, paying special attention to those factors that can directly impact the performance of the gold colloid systems in potential lysozyme detection systems, such as the stability of the samples, the influence of the nanoparticle size, and the correct way to express gold concentration in the system in order to achieve the most precise and reproducible results. Besides, as a result of our studies, the free energy of binding for the 10 nm AuNPs/Lysozyme interaction in water was determined by surface plasmon resonance (SPR) and a deconvolution method, a procedure that to our knowledge has never been described. 2. Materials and Methods 2.1. Materials All chemicals were of Anal. R. Grade. Gold colloid 5 nm (G1402), gold colloid 10 nm (G1527), gold colloid 20 nm (G1652), and lysozyme (L6876) were purchased from Sigma and NaCl from Merck. Solutions were prepared with deionized water, its conductivity being less than 10−6Sm−1. 2.2. UV-Vis Spectra The spectra of the AuNPs in the presence and in the absence of lysozyme (Lys) were recorded with a Cary 500 spectrophotometer at 298.2 K from 400 to 700 nm. Nanoparticle concentrations were: [AuNPs] = 8.22 × 10 −10 M or [AuNPs] = 3.28 × 10 −9 M (exceptions are indicated in the figure legends). Lysozyme concentrations ranged from 10 −8 M to 10 −3 M. Total of 200 µ L (or 800 µ L, see Results and Discussion) of AuNPs 8.22 × 10 −9 M was added into a clean quartz cuvette (optical path 10 mm). A timer was started simultaneously as 1800 µ L (or 1200 µ L) of protein solution of variable concentrations was mixed with Nanomaterials 2021,11, 2139 3 of 17 the nanosystem solution in the cuvette, which was then reversed several times and then inserted into the cuvette holder in the UV-vis spectrometer. Photograph corresponding to Figure S6 was taken with a Canon 5-50 digital camera. 2.3. Zeta Potential Experiments Zeta potential measurements were carried out at 298.2 K with a Zetasizer Nano ZS Malvern Instruments Ltd (UK). A DTS 1060 polycarbonate capillary cell was used. Figure S1 depicts zeta potential distributions of the 10 nm gold colloids with a negative charge of about − 33.1 ± 1.6 mV in water, which is sufficient to keep the particles from interacting with each other and therefore prevent aggregation of the sample, maintaining a stable particle size. Measurements were performed in Lys concentrations range from 10 −8 M to 3×10−4M. [AuNPs] = 8.22 ×10−10 M in each case. 2.4. Circular Dichroism (CD) Spectra Electronic CD spectra were recorded in a BioLogic Mos-450 spectropolarimeter. A standard quartz cell of 10 mm path length was used. The spectra were expressed in terms of ellipticity. Scans were taken from 190 nm to 250 nm and for each spectrum 5–10 runs were averaged at a constant temperature of 298.2 K with a 5 min equilibration before each scan. [AuNPs] = 8.22 ×10−10 M. [Lys] = 10−8M. Deconvolution of CD spectra was done by using CDNN software (version 2) with the standard basis set of 33 spectra [16]. 2.5. Kinetic Measurements Kinetic runs were carried out in a stopped-flow spectrophotometer from Applied Photophysics. The reaction was monitored by following the changes in absorbance of the surface plasmon band, SPR, at 565 nm, in the presence of [Lys] = 4 × 10 −7 M and [AuNPs] = 3.28 × 10 −9 M. A solution of the same AuNPs concentration in water was used as reference. The temperature was maintained at 298.2 ±0.1 K. 2.6. TEM Measurements For TEM examinations, a single drop (10 µ L) of the aqueous solution of the gold nanoparticles was placed on a carbon film-coated copper grid which was then left to dry in air for several hours at room temperature. TEM analysis was carried out in a Philips CM 200 electron microscope working at 200 kV. [AuNPs] = 8.22 × 10 −10 M; [NaCl] = 0.1 M; [Lys] = 4 ×10−8M. 2.7. Deconvolution Deconvolution of experimental spectra was carried out by fitting to Voigt functions, which best reproduced the SPR of free nanoparticles. Fityk software (0.9 version) was used to carry out the fit [ 17 ]. Spectra were fitted to a three-band model, where the free AuNPs band was always found at around 521 nm; the linked AuNPs band appeared at 561 nm for almost all cases except those where the aggregation degree was too small to be significant. A third broad band was fixed at 300 nm in order to account for both residual Au 3+ absorbance and for the light scattering phenomena linked to the presence of nanoparticles in solution [18]. 3. Results and Discussion 3.1. Stability and Detection Limit Considering the Red Shift of the SPR Figure 1shows the surface plasmon band for two different batches of 5 nm colloidal gold (ref. G1402). The spectra correspond to a mixture of 200 µL of commercial gold with 1800 µ L of distilled water. The black solid line corresponds to the batch Lot. SLBB2962V with an average size of 4.8 nm and a gold concentration of 67 µ g/mL. The red dashed line corresponds to the batch Lot. 061M6031 with an average size of 5.1 nm and a gold concentration of 45 µ g/mL. In order to highlight the influence of gold concentration and Nanomaterials 2021,11, 2139 4 of 17 nanoparticle size over the SPR band, gold concentrations have not been corrected. It can be clearly seen that a small difference in size (0.3 nm) promotes a change in the maximum of the SPR, (∆λ= 9 nm), and in the optical density (O.D): ∆(O.D) ~ 18%. Nanomaterials 2021, 11, x FOR PEER REVIEW 4 of 18 with an average size of 4.8 nm and a gold concentration of 67 µg/mL. The red dashed line corresponds to the batch Lot. 061M6031 with an average size of 5.1 nm and a gold concentration of 45 µg/mL. In order to highlight the influence of gold concentration and nanoparticle size over the SPR band, gold concentrations have not been corrected. It can be clearly seen that a small difference in size (0.3 nm) promotes a change in the maximum of the SPR, (Δλ = 9 nm), and in the optical density (O.D): Δ(O.D) ~ 18%. λ / nm 400 450 500 550 600 650 700 O.D 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 512 nm 521 nm Figure 1. Experimental spectra of AuNPs solutions on the visible region. Commercial gold nanoparticles were used as received. Black solid line corresponds to 4.8 nm AuNPs and gold concentration of 67 µg/mL. Red dashed line corresponds to 5.1 nm AuNPs and gold concentration of 45 µg/mL. Figure 2 shows the SPR stability corresponding to 10 nm AuNPs in the presence of lysozyme 3 × 10−4 M. When compared to the SPR of lone AuNPs, λmax shifts to slightly higher values over time when the protein is present in solution; those changes are accompanied by a slight darkening of the red tint of the nanoparticles. Although during the first minutes λmax changes some nanometers, as the optical density does, after the first 10 min. and at least during 1 hour the system’s changes take place at a very slow speed, which allows for the 10 minute mark to be taken as a reference in all measurements. For this reason, in further experiments, the values of λmax and O.D. where obtained 10 min after the addition of the protein to the colloidal gold. In order to control the stability of the aggregates, measurements were made at multiple fixed [AuNPs]/[Lys] relationships. Figure 3 reflects compliance with the Lambert–Beer law in the range [AuNPs]/[Lys] = 4.10 × 10−9 M/2.50 × 10−7 M - 8.22 × 10−10 M/5.00 × 10−8 M. This implies that while we worked at low concentrations of gold nanoparticles so as to determine the potential lowest detection limits of a lysozyme-sensing system in water, the optical density of the solutions also provides valuable information to this end, and could be explored as an alternate mean for lysozyme quantification. Figure 1. Experimental spectra of AuNPs solutions on the visible region. Commercial gold nanoparticles were used as received. Black solid line corresponds to 4.8 nm AuNPs and gold concentration of 67 µg/mL. Red dashed line corresponds to 5.1 nm AuNPs and gold concentration of 45 µg/mL. Figure 2shows the SPR stability corresponding to 10 nm AuNPs in the presence of lysozyme 3 × 10 −4 M. When compared to the SPR of lone AuNPs, λmax shifts to slightly higher values over time when the protein is present in solution; those changes are accompanied by a slight darkening of the red tint of the nanoparticles. Although during the first minutes λmax changes some nanometers, as the optical density does, after the first 10 min. and at least during 1 hour the system’s changes take place at a very slow speed, which allows for the 10 minute mark to be taken as a reference in all measurements. For this reason, in further experiments, the values of λmax and O.D. where obtained 10 min after the addition of the protein to the colloidal gold. In order to control the stability of the aggregates, measurements were made at multiple fixed [AuNPs]/[Lys] relationships. Figure 3reflects compliance with the Lambert–Beer law in the range [AuNPs]/[Lys] = 4.10 ×10−9M/2.50 ×10−7M−8.22 ×10−10 M/5.00 ×10−8M . This implies that while we worked at low concentrations of gold nanoparticles so as to determine the potential lowest detection limits of a lysozyme-sensing system in water, the optical density of the solutions also provides valuable information to this end, and could be explored as an alternate mean for lysozyme quantification. Nanomaterials 2021, 11, x FOR PEER REVIEW 5 of 18 λ / nm 450 500 550 600 650 700 O.D 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 30 s 3 min 6 min 10 min 20 min 30 min 50 min Figure 2. Experimental spectra of 10 nm AuNPs in the presence of Lys 3 × 10−4 M. [AuNPs] = 8.22 × 10−10 M. [Lys] / M 050x10 -9 100x10 -9 150x10 -9 200x10 -9 250x10 -9 300x10 -9 O. D. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Figure 3. Optical density at different Lys concentrations for a constant ratio [AuNPs]/[Lys] = 0.0164 measured at 565 nm. The influence of the protein concentration in experimental absorbance λmax measurements is shown in Figure 4A. As can be seen, at low enough concentrations of lysozyme, the SPR experiences a red shift that peaks at [Lys] = 4 × 10−8 M; this effect is reflected in Table 1, which shows the values of Δλ at different Lys concentrations when keeping the gold concentration constant. Aggregation phenomena are more marked at low concentrations of the protein, yielding a detection limit in the nanomolar range, around 1.3 × 10−9 M. It is also possible to employ the O.D. and consider the degree of aggregation of the nanoclusters in function of the protein concentration (A569/A518; where Aλ stands for absorbance values at λ nm). However, the loss of accuracy can be seen in Figure 5B when compared with Figure 5A. In this study, we have worked with very low gold concentrations, even at the risk of having O.D. ≈ 0.1 in order to achieve high accuracy in the value of λmax. Our main goal is to explore the possibilities of using the pronounced λmax variations as an alternative signal instead of A569/A518 and thus improve the perception of the system changes taking place during the formation of the AuNPs/Lysozyme complex. Figure 2. Experimental spectra of 10 nm AuNPs in the presence of Lys 3 × 10 −4 M. [AuNPs] = 8.22 ×10−10 M. Nanomaterials 2021,11, 2139 5 of 17 Nanomaterials 2021, 11, x FOR PEER REVIEW 5 of 18 λ / nm 450 500 550 600 650 700 O.D 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 30 s 3 min 6 min 10 min 20 min 30 min 50 min Figure 2. Experimental spectra of 10 nm AuNPs in the presence of Lys 3 × 10−4 M. [AuNPs] = 8.22 × 10−10 M. [Lys] / M 050x10 -9 100x10 -9 150x10 -9 200x10 -9 250x10 -9 300x10 -9 O. D. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Figure 3. Optical density at different Lys concentrations for a constant ratio [AuNPs]/[Lys] = 0.0164 measured at 565 nm. The influence of the protein concentration in experimental absorbance λmax measurements is shown in Figure 4A. As can be seen, at low enough concentrations of lysozyme, the SPR experiences a red shift that peaks at [Lys] = 4 × 10−8 M; this effect is reflected in Table 1, which shows the values of Δλ at different Lys concentrations when keeping the gold concentration constant. Aggregation phenomena are more marked at low concentrations of the protein, yielding a detection limit in the nanomolar range, around 1.3 × 10−9 M. It is also possible to employ the O.D. and consider the degree of aggregation of the nanoclusters in function of the protein concentration (A569/A518; where Aλ stands for absorbance values at λ nm). However, the loss of accuracy can be seen in Figure 5B when compared with Figure 5A. In this study, we have worked with very low gold concentrations, even at the risk of having O.D. ≈ 0.1 in order to achieve high accuracy in the value of λmax. Our main goal is to explore the possibilities of using the pronounced λmax variations as an alternative signal instead of A569/A518 and thus improve the perception of the system changes taking place during the formation of the AuNPs/Lysozyme complex. Figure 3. Optical density at different Lys concentrations for a constant ratio [AuNPs]/[Lys] = 0.0164 measured at 565 nm. The influence of the protein concentration in experimental absorbance λmax measurements is shown in Figure 4A. As can be seen, at low enough concentrations of lysozyme, the SPR experiences a red shift that peaks at [Lys] = 4 × 10 −8 M; this effect is reflected in Table 1 , which shows the values of ∆λ at different Lys concentrations when keeping the gold concentration constant. Aggregation phenomena are more marked at low concentrations of the protein, yielding a detection limit in the nanomolar range, around 1.3 ×10−9M. It is also possible to employ the O.D. and consider the degree of aggregation of the nanoclusters in function of the protein concentration (A 569 /A 518 ; where A λ stands for absorbance values at λ nm). However, the loss of accuracy can be seen in Figure 5B when compared with Figure 5A. In this study, we have worked with very low gold concentrations, even at the risk of having O.D. ≈ 0.1 in order to achieve high accuracy in the value of λmax . Our main goal is to explore the possibilities of using the pronounced λmax variations as an alternative signal instead of A 569 /A 518 and thus improve the perception of the system changes taking place during the formation of the AuNPs/Lysozyme complex. The detection limit for lysozyme concentrations in water has been obtained from 3S/D, where S is the standard deviation of the measurements ( ± 0.37) and D the slope of the calibration line (see Figure 5A). Meanwhile, the lower limit of quantification (10 S/D) is 4.3 × 10 −9 M. Those detection limits, which have been achieved with non-functionalized 10 nm AuNPs, are remarkable when compared to previous studies, even those that use functionalized nanoparticles [19]. Table 1. Values of ∆λ/nm at different protein concentrations. [AuNPs(10 nm)] = 8.22 ×10−10 M. [Lys]/M ∆λ/nm [Lys]/M ∆λ/nm 1.0 ×10−845.0 ×10−724 1.5 ×10−891.0 ×10−622 2.0 ×10−815 1.0 ×10−521 2.5 ×10−816 5.0 ×10−515 3.0 ×10−823 1.0 ×10−413 4.0 ×10−828 3.0 ×10−411 5.0 ×10−827 1.0 ×10−38 1.0 ×10−723 - - 3.2. Saturation of the System AuNPs/Lys: Structural Characterization If data of λmax in Table 1are carefully analyzed, it can be observed that aggregation reaches a state of saturation. Indeed, at higher concentrations of protein, aggregate size begins to decrease; this fact becomes apparent when analyzing the absorbance spectra in Figure 4B, which shows a λmax shift to lower values for lysozyme concentrations higher than 4 × 10 −8 M. At a given point the nanoparticle surface is saturated and protected all around Nanomaterials 2021,11, 2139 6 of 17 by a layer of Lys, which prevents aggregation due to steric hindrance and repulsion forces. It is important to note that the concentration of Lys from which λmax starts to decrease matches the absorbance ratio (see Figure S2 in Supporting Information). Considering the AuNPs concentration and the concentration of Lys at which saturation is observed, the average number of protein molecules per nanoparticle has been determined to be 55. Lysozyme has been found to have an estimated size of 3.0 nm ×3.0 nm ×4.5 nm [ 20 ]. If the ellipse formed by the longer and one of the shorter diameters, which would have an area of 10.6 nm 2 , is assumed to fully interact with the 10 nm AuNPs surface ( 314.2 nm2 ), then around 30 lysozyme molecules would fit. If, instead, the shorter diameters are considered, then each lysozyme would take up a circle of 7.07 nm 2 , which would allow around 44 lysozyme molecules to fit. Those theoretical approximations are in accordance with the reported experimental data, since the lysozyme molecules do not need to fully use one of their sides in order to interact with the AuNPs. Nanomaterials 2021, 11, x FOR PEER REVIEW 6 of 18 Table 1. Values of Δλ/nm at different protein concentrations. [AuNPs(10 nm)] = 8.22 × 10−10 M. [Lys] / M Δλ / nm [Lys] / M Δλ / nm 1.0 × 10−8 4 5.0 × 10−7 24 1.5 × 10−8 9 1.0 × 10−6 22 2.0 × 10−8 15 1.0 × 10−5 21 2.5 × 10−8 16 5.0 × 10−5 15 3.0 × 10−8 23 1.0 × 10−4 13 4.0 × 10−8 28 3.0 × 10−4 11 5.0 × 10−8 27 1.0 × 10−3 8 1.0 × 10−7 23 - - (A) (B) Figure 4. Experimental spectra of 10 nm AuNPs solutions on the visible region showing the red (A) and blue (B) shift in function of the Lys concentration. [AuNPs] = 8.22 × 10−10 M. λ / nm 400 450 500 550 600 650 700 O.D 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 [Lys] = 1.0 x10 -8 M [Lys] = 1.5 x10 -8 M [Lys] = 2.5 x10 -8 M [Lys] = 3.0 x10 -8 M [Lys] = 4.0 x10 -8 M λ / nm 400 450 500 550 600 650 700 O.D 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 [Lys] = 5.0 x10 -8 M [Lys] = 5.0 x10 -7 M [Lys] = 1.0 x10 -5 M [Lys] = 5.0 x10 -5 M [Lys] = 1.0 x10 -3 M Figure 4. Experimental spectra of 10 nm AuNPs solutions on the visible region showing the red ( A ) and blue (B) shift in function of the Lys concentration. [AuNPs] = 8.22 ×10−10 M. Nanomaterials 2021,11, 2139 7 of 17 Nanomaterials 2021, 11, x FOR PEER REVIEW 7 of 18 (A) (B) Figure 5. Changes of the maximum of the SPR (A) and the A569/A518 ratio (B) with Lys concentration. From Figure 5A, a value of 1.3 × 10−9 M for the detection limit (L.D) has been obtained, with L.D. = 3S/D being D the slope of the line and S the standard deviation of the measurements. The detection limit for lysozyme concentrations in water has been obtained from 3S/D, where S is the standard deviation of the measurements (±0.37) and D the slope of the calibration line (see Figure 5A). Meanwhile, the lower limit of quantification (10 S/D) is 4.3 × 10−9 M. Those detection limits, which have been achieved with non-functionalized 10 nm AuNPs, are remarkable when compared to previous studies, even those that use functionalized nanoparticles [19]. 3.2. Saturation of the System AuNPs/Lys: Structural Characterization If data of λmax in Table 1 are carefully analyzed, it can be observed that aggregation reaches a state of saturation. Indeed, at higher concentrations of protein, aggregate size begins to decrease; this fact becomes apparent when analyzing the absorbance spectra in Figure 4B, which shows a λmax shift to lower values for lysozyme concentrations higher than 4 × 10−8 M. At a given point the nanoparticle surface is saturated and protected all around by a layer of Lys, which prevents aggregation due to steric hindrance and repulsion forces. It is important to note that the concentration of Lys from which λmax starts to decrease matches the absorbance ratio (see Figure S2 in Supporting Information). Considering the AuNPs concentration and the concentration of Lys at which saturation is observed, the average number of protein molecules per nanoparticle has been determined [Lys] / M 5x10 -9 10x10 -9 15x10 -9 20x10 -9 25x10 -9 30x10 -9 35x10 -9 40x10 -9 45x10 -9 Δλ / nm 0 5 10 15 20 25 30 35 [Lys] / M 5x10 -9 10x10 -9 15x10 -9 20x10 -9 25x10 -9 30x10 -9 35x10 -9 40x10 -9 45x10 - 9 A569/A518 0.5 0.6 0.7 0.8 0.9 1.0 1.1 Figure 5. Changes of the maximum of the SPR ( A ) and the A 569 /A 518 ratio ( B ) with Lys concentration. From Figure 5A, a value of 1.3 × 10 −9 M for the detection limit (L.D) has been obtained, with L.D. = 3S/D being D the slope of the line and S the standard deviation of the measurements. Figure 6shows three arrays of TEM image photographs that help confirm that the aggregation process takes place as previously stated. It shows images corresponding to solutions in the presence of 10 nm AuNPs ( λmax = 518 nm) (A), in the presence of NaCl 0.1 M as the reference aggregation process ( λmax = 616 nm) (B), and in the presence of Lys concentration at which the observed shift of λmax is maximum ( λmax = 546 nm, see Table 1, [Lys] = 4 × 10 −8 M) (C). Figure 6C,D clearly shows the aggregation of nanoparticles in the presence of Lys without any apparent changes in nanoclusters morphology. In order to further explore the nature of AuNPs–Lys interactions, polarization spectroscopy measurements were carried out (Figure 7). Despite the clear shape difference observed between the lone lysozyme and the lysozyme–AuNPs CD spectra, the deconvolution with CDNN software did not show appreciable changes in the percentages of α -helix, β -turn, and random coil structures present in lone lysozyme in relation to those that form in the presence of AuNPs (see Table S1). Those results are not unexpected due to lysozyme presenting four internal disulphide bonds that confer it a certain degree of stiffness and greatly stabilize its secondary structure, even in the presence of a ligand such as AuNPs. Since there is no appreciable change in the secondary structure of the protein, which strongly depends on those disulphide bonds as well as on intra-molecular hydrogen bonds, we can conclude that the interaction with the AuNPs does not break nor alter either of those links. It is also to note that peptide bonds in proteins are well-known to cause two strong CD bands in the far-UV: one corresponding to π→π * electronic transitions, Nanomaterials 2021,11, 2139 8 of 17 located around 190 nm, and the other to n →π *, which appears at around 210 nm [ 21 ]. Those transitions are clearly visible in the lone lysozyme CD spectra (red line), and show a shift to higher wavelengths, implying lower transition energies, when AuNPs are added (blue line) with a fine structure appearing around 195 nm. We can conclude that even though sulfur atoms have been known to bond strongly with gold [ 22 ] the added stability of the disulphide bonds causes AuNPs to preferably interact with the peptidic bond atoms, thus not affecting the secondary structure of the protein. This effect is in agreement with previous reports; for example, 2-(10-mercaptodecyl) malonic acid functionalized 2 nm core gold nanoparticles (anionic AuDA), which form a high affinity complex with Lys, have been found to actually promote re-folding of denatured proteins by shielding them from inter-protein hydrophobic forces; denatured proteins that were treated with AuDA have been shown to present conformations that mimicked those of the native proteins, further proving the point that nanoparticles do not cause significant alterations of the secondary structure of the protein [ 23 ]. In any case, the CD results presented in the present study should be considered as an indication, not definitive proof in relation with the secondary structure of the protein. Nanomaterials 2021, 11, x FOR PEER REVIEW 8 of 18 to be 55. Lysozyme has been found to have an estimated size of 3.0 nm × 3.0 nm × 4.5nm [20]. If the ellipse formed by the longer and one of the shorter diameters, which would have an area of 10.6 nm 2 , is assumed to fully interact with the 10 nm AuNPs surface (314.2 nm 2 ), then around 30 lysozyme molecules would fit. If, instead, the shorter diameters are considered, then each lysozyme would take up a circle of 7.07 nm 2 , which would allow around 44 lysozyme molecules to fit. Those theoretical approximations are in accordance with the reported experimental data, since the lysozyme molecules do not need to fully use one of their sides in order to interact with the AuNPs. Figure 6 shows three arrays of TEM image photographs that help confirm that the aggregation process takes place as previously stated. It shows images corresponding to solutions in the presence of 10 nm AuNPs (λ max = 518 nm) (A), in the presence of NaCl 0.1 M as the reference aggregation process (λ max = 616 nm) (B), and in the presence of Lys concentration at which the observed shift of λ max is maximum (λ max = 546 nm, see Table 1, [Lys] = 4 × 10 −8 M) (C). Figure 6C,D clearly shows the aggregation of nanoparticles in the presence of Lys without any apparent changes in nanoclusters morphology. (A) (B) (C) (D) Figure 6. TEM images corresponding to 10 nm AuNPs. (A) [AuNPs] = 8.22 × 10 −10 M, in water (shown at a lesser magnification in order to better convey nanoparticle dispersion). (B) [AuNPs] = 8.22 × 10 −10 M in the presence of [NaCl] = 0.1M. (C,D) [AuNPs] = 8.22 × 10 −10 M in the presence of [Lys] = 4 × 10 −8 M. In order to further explore the nature of AuNPs–Lys interactions, polarization spectroscopy measurements were carried out (Figure 7). Despite the clear shape difference observed between the lone lysozyme and the lysozyme–AuNPs CD spectra, the deconvolution with CDNN software did not show appreciable changes in the percentages of αhelix, β-turn, and random coil structures present in lone lysozyme in relation to those that form in the presence of AuNPs (see Table S1). Those results are not unexpected due to Figure 6. TEM images corresponding to 10 nm AuNPs. ( A ) [AuNPs] = 8.22 × 10 −10 M, in water (shown at a lesser magnification in order to better convey nanoparticle dispersion). ( B ) [AuNPs] = 8.22 × 10 −10 M in the presence of [NaCl] = 0.1 M. (C,D) [AuNPs] = 8.22 ×10−10 M in the presence of [Lys] = 4 ×10−8M. Nanomaterials 2021,11, 2139 9 of 17 Nanomaterials 2021, 11, x FOR PEER REVIEW 9 of 18 lysozyme presenting four internal disulphide bonds that confer it a certain degree of stiffness and greatly stabilize its secondary structure, even in the presence of a ligand such as AuNPs. Since there is no appreciable change in the secondary structure of the protein, which strongly depends on those disulphide bonds as well as on intra-molecular hydrogen bonds, we can conclude that the interaction with the AuNPs does not break nor alter either of those links. It is also to note that peptide bonds in proteins are well-known to cause two strong CD bands in the far-UV: one corresponding to π → π* electronic transitions, located around 190 nm, and the other to n → π*, which appears at around 210 nm [21]. Those transitions are clearly visible in the lone lysozyme CD spectra (red line), and show a shift to higher wavelengths, implying lower transition energies, when AuNPs are added (blue line) with a fine structure appearing around 195 nm. We can conclude that even though sulfur atoms have been known to bond strongly with gold [22] the added stability of the disulphide bonds causes AuNPs to preferably interact with the peptidic bond atoms, thus not affecting the secondary structure of the protein. This effect is in agreement with previous reports; for example, 2-(10-mercaptodecyl) malonic acid functionalized 2 nm core gold nanoparticles (anionic AuDA), which form a high affinity complex with Lys, have been found to actually promote re-folding of denatured proteins by shielding them from inter-protein hydrophobic forces; denatured proteins that were treated with AuDA have been shown to present conformations that mimicked those of the native proteins, further proving the point that nanoparticles do not cause significant alterations of the secondary structure of the protein [23]. In any case, the CD results presented in the present study should be considered as an indication, not definitive proof in relation with the secondary structure of the protein. λ / nm 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 θ / mdegree -8 -6 -4 -2 0 2 4 6 Figure 7. CD spectra of Lys in the absence (red line) and in the presence of 10 nm gold nanoparticles (blue line). [AuNPs] = 8.22 × 10−10 M. [Lys] = 10−6 M. The stability of different kinds of nanoparticles strongly depends on the valence of the counterions in the solution due to the electrokinetic or ζ potential, this is, the difference between the compact layer potential and the diffuse potential. In this sense, the magnitude of the measured ζ potential is an indication of the repulsive force that prevents nanoparticle aggregation and actually allows the colloidal gold solutions to be stable, and as such can be used to predict the long-term stability of the nanoparticle suspension. Table 2 shows ζ potential values at different Lys concentrations when measured at a constant gold nanoparticles concentration. In the absence of the protein a value of -33 mV (see S1 in supporting information) is sufficient to keep the AuNPs away from each other, thus achieving a stable particle size. ζ potential corresponding to the Lys in the absence of AuNPs indicates that the protein is positively charged at neutral pH (+8.71 mV). As can be seen in Table 2, as the protein concentration increases, the zeta potential becomes less negative, as is to be expected in response to the association of Lys molecules on the gold Figure 7. CD spectra of Lys in the absence (red line) and in the presence of 10 nm gold nanoparticles (blue line). [AuNPs] = 8.22 ×10−10 M. [Lys] = 10−6M. The stability of different kinds of nanoparticles strongly depends on the valence of the counterions in the solution due to the electrokinetic or ζ potential, this is, the difference between the compact layer potential and the diffuse potential. In this sense, the magnitude of the measured ζ potential is an indication of the repulsive force that prevents nanoparticle aggregation and actually allows the colloidal gold solutions to be stable, and as such can be used to predict the long-term stability of the nanoparticle suspension. Table 2shows ζ potential values at different Lys concentrations when measured at a constant gold nanoparticles concentration. In the absence of the protein a value of − 33 mV (see S1 in supporting information) is sufficient to keep the AuNPs away from each other, thus achieving a stable particle size. ζ potential corresponding to the Lys in the absence of AuNPs indicates that the protein is positively charged at neutral pH (+8.71 mV). As can be seen in Table 2, as the protein concentration increases, the zeta potential becomes less negative, as is to be expected in response to the association of Lys molecules on the gold surface. The partial neutralization of the AuNPs surface decreases the repulsion forces between nanoparticles, facilitating the aggregation. At a concentration around 10 −5 M a charge reversal occurs, and ζ potential becomes positive. Now AuNPs/Lys entities have a positive average charge and actually begin to repel each other. The situation of maximum aggregation observed from the shifts of the SPR appears at a protein concentration ( [Lys] = 4 ×10−8–5 ×10−8M ) which agrees well with the Lys concentration at which the ζ potential approaches zero. These two behaviors can be described by Scheme 1. Table 2. Values of ζ potential/mV at different protein concentrations. [AuNPs (10 nm)] = 8.22 ×10−10 M. ζPotential = +8.71 mV for Lys in the absence of AuNPs. [Lys]/M ζPotential/mV 0−33.0 1.0 ×10−8−30.1 2.0 ×10−8−29.9 3.0 ×10−8−22.6 4.0 ×10−8−13.8 5.0 ×10−8−10.5 1.0 ×10−69.4 5.0 ×10−515.4 3.0 ×10−410.1 Nanomaterials 2021,11, 2139 16 of 17 sponding to the first nine Lys concentrations of Table 3in the presence of [AuNPs] = 8.22 ×10−10 M and [AuNPs] = 3.28 ×10−9M. Author Contributions: Methodology, A.J.-R.; validation, E.G.; supervision, R.M.G.-P. and R.P.-G.; validation: E.G; formal analysis, J.M.C. and R.M.G.-P.; investigation, A.G., J.M.C. and A.J.-R.; writingoriginal draft preparation, R.P.-G.; writing—review and editing, E.G.; funding acquisition, R.P.-G. All authors have read and agreed to the published version of the manuscript. Funding: This work was financed in part by the Consejería de Educación y Ciencia de la Junta de Andalucía (P08-FQM-03623). We thank European Social Fund and Junta de Andalucía for the postdoctoral fellowship “Contratación de Personal Investigador Doctor (Convocatoria 2019). PAIDI 2020”. Acknowledgments: We thank F. Varela (CITIUS, University of Seville) and J. Quispe for their assistance in obtaining TEM images and ζpotential measurements, respectively. Conflicts of Interest: The authors declare no conflict of interest. References 1. Wilcoxon, J. Optical Absorption Properties of Dispersed Gold and Silver Alloy Nanoparticles. J. Phys. Chem. B 2009 ,113, 2647–2656. [CrossRef] [PubMed] 2. Hostetler, M.J.; Green, S.J.; Stokes, J.J.; Murray, R.W. Monolayers in Three Dimensions: Synthesis and Electrochemistry of ω-Functionalized Alkanethiolate-Stabilized Gold Cluster Compounds. J. Am. Chem. Soc. 1996,118, 4212–4213. [CrossRef] 3. Ingram, R.S.; Murray, R.W.; Hill, C.; Carolina, N. Electroactive Three-Dimensional Monolayers: Anthraquinone ω -Functionalized Alkanethiolate-Stabilized Gold Clusters. Langmuir 1998,14, 4115–4121. [CrossRef] 4. Green, S.J.; Stokes, J.J.; Hostetler, M.J.; Pietron, J.; Murray, R.W. Three-Dimensional Monolayers: Nanometer-Sized Electrodes of Alkanethiolate-Stabilized Gold Cluster Molecules. J. Phys. Chem. B 1997,101, 2663–2668. [CrossRef] 5. Templeton, C.; Hostetler, M.J.; Warmoth, E.K.; Chen, S.; Hartshorn, C.M.; Kroshnamurthy, V.M.; Forbes, M.D.E.; Murray, R.W.; Hill, C.; Carolina, N. Gateway Reactions to Diverse, Polyfunctional Monolayer-Protected Gold Clusters. J. Am. Soc. 1998 ,120, 4845–4849. [CrossRef] 6. West, J.L.; Halas, N.J. Applications of nanotechnology to biotechnology: Commentary. Curr. Opin. Biotechnol. 2000 ,11, 215–217. [CrossRef] 7. Daniel, M.-C.; Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev. 2004,104, 293–346. [CrossRef] 8. Link, S.; Mohamed, M.B.; El-Sayed, M. Simulation of the Optical Absorption Spectra of Gold Nanorods as a Function of Their Aspect Ratio and the Effect of the Medium Dielectric Constant. J. Phys. Chem. B 1999,103, 3073–3077. [CrossRef] 9. Thioune, N.; Lidgi-Guigui, N.; Cottat, M.; Gabudean, A.-M.; Focsan, M.; Benoist, H.-M.; Astilean, S.; Lamy de la Chapelle, M. Study of gold nanorods—Protein interaction by localized surface plasmon resonance spectroscopy. Gold Bull. 2013 ,46, 275–281. [CrossRef] 10. Yeh, Y.; Ceran, B.; Rotello, V.M. Gold nanoparticles: Preparation, properties, and applications in bionanotechnology. Nanoscale 2012,4, 1871–1880. [CrossRef] 11. Amendola, V.; Pilot, R.; Frasconi, M.; Maragò, O.M.; Iatì, M.A. Surface plasmon resonance in gold nanoparticles: A review. J. Phys. Consens. Matter 2017,29, 203002. [CrossRef] 12. Shima, K.; Hirota, M.; Fukuda, M.; Tanaka, A. Determination of urinary lysozyme for potential detection of tubular dysfunction in diabetic nephropathy. Clin. Chem. 1986,32, 1818–1822. [CrossRef] 13. Levinson, S.S.; Elin, R.J.; Yam, L. Light Chain Proteinuria and Lysozymuria in a Patient with Acute Monocytic Leukemia. Clin Chem. 2002,48, 1131–1132. [CrossRef] 14. Harrison, J.F.; Lunt, G.S.; Scott, P.; Blainey, J.D. Urinary Lysozyme, Ribonuclease, and Low-Molecular-Weight Protein in Renal Disease. Lancet 1968,291, 371–375. [CrossRef] 15. Klockars, M.; Reitamo, S.; Weber, T.; Kerttula, Y. Cerebrospinal Fluid Lysozyme in Bacterial and Viral Meningitis. Acta Med. Scand. 1978,203, 71–74. [CrossRef] 16. Sreerama, N.; Woody, R.W. Estimation of protein secondary structure from CD spectra: Comparison of CONTIN, SELCON and CDSSTR methods with an expanded reference set. Anal. Biochem. 2000,287, 252–260. [CrossRef] 17. Wojdyr, M. Fityk: A general-purpose peak fitting program. J. Appl. Crystallogr. 2010,43, 1126–1128. [CrossRef] 18. Yang, S.; Shian, M.S.; Chang, F.Y. Evolution study of photo-synthesized gold nanoparticles by spectral deconvolution model: A quantitative approach. New J. Chem. 2006,30, 729–735. [CrossRef] 19. Chen, Y.; Yu, C.; Cheng, T.; Tseng, W. Colorimetric Detection of Lysozyme Based on Electrostatic Interaction with Human Serum Albumin-Modified Gold Nanoparticles. Langmuir 2008,24, 3654–3660. [CrossRef] 20. Kim, T.; Blanch, H.W.; Radke, C.J. Direct Imaging of Lysozyme Adsorption onto Mica by Atomic Force Microscopy. Langmuir 2002,18, 5841–5850. [CrossRef] Nanomaterials 2021,11, 2139 17 of 17 21. Da Paoli Lacerda, H.; Park, J.J.; Meuse, C.; Pristinski, D.; Becker, M.L.; Karim, A.; Douglas, J.F. Interaction of Gold Nanoparticles with Common Human Blood Proteins. ACS Nano 2010,4, 365–379. [CrossRef] [PubMed] 22. Lundqvist, M.; Stigler, J.; Cedervall, T.; Berggard, T.; Flanagan, M.B.; Lynch, I.; Elia, G.; Dawson, K. The Evolution of the Protein Corona around Nanoparticles: A Test Study. ACS Nano 2011,5, 7503–7509. [CrossRef] 23. Kelly, S.M.; Jess, T.J.; Price, N.C. How to study proteins by circular dichroism. Biochim. Biophys. Acta 2005 ,1751, 119–139. [CrossRef] [PubMed] 24. Benesi, H.A.; Hildebrand, J.H. A Spectrophotometric Investigation of the Interaction of Iodine with Aromatic Hydrocarbons. J. Am. Chem. Soc. 1949,71, 2703–2707. [CrossRef] 25. Liu, X.; Atwater, M.; Wang, J.; Huo, Q. Extinction coefficient of gold nanoparticles with different sizes and different capping ligands. Colloids Surf. B Biointerfaces 2007,58, 3–7. [CrossRef]