1. Introduction
In recent years, many studies have been conducted to establish a correlation between food-enriched dietary fibers and human well-being. Specifically, epidemiological studies demonstrated that daily intake of dietary fibers had an inverse relationship with the incidence chronical diseases [
1,
2]. Therefore, the willingness of societies to opt for a high-fiber diet approach increased significantly, and at the same time, consumers demanded the market introduction of dietary fiber supplements and functional foods enriched with these polysaccharides [
3,
4].
Based on the current definition (FAO/WHO, 2009 revised in 2010), dietary fibers are classified as “edible carbohydrate polymers naturally occurring in foods as well as isolated, modified, and synthetic polymers with proven physiological effects of benefit to health” [
5]. The fibers’ solubility in water is one of the discriminating factors for their classification. Pectin, mixed-linkage β-glucans, galactomannan, fructans, hemicelluloses, gums, and mucilage are indigestible, (at least partially) water-soluble dietary fibers (SDF). Conversely, cellulose, lignin, and resistant starch are classified as insoluble dietary fibers (IDF) [
6]. Although the latter also exert beneficial physiological effects such as increasing fecal bulk or decreasing intestinal transit, the growing attention has been focused on the soluble fraction. Solubility, water-holding capacity, viscosity, and gelling properties as well as binding ability and fermentability are the key factors that make SDFs the favored option for food processing operations and incorporation in food products [
7].
In the upper intestine, the absorption of minerals, bile acids, drugs, sugars, and toxins can be affected by the ability of SDFs to entertain water molecules and form gels or viscous solutions that can reversibly retain these compounds as they pass through the gastrointestinal tract. It became clear among researchers that this physical entrapment mechanism based on the viscosity of the fiber solution as well as the formation of a fiber-based barrier in the small intestine and reversible molecular interactions between fiber and nutritionally relevant compounds were the main processes behind how soluble dietary fibers could exert their health-supporting activity by altering the bioavailability of certain undesirable molecules [
8,
9]. However, it is challenging to find studies in the literature that demonstrate the specificity of action of soluble dietary fibers toward a specific molecule, and the supramolecular mechanism by which SDFs may exert their protective effects. Thus, this unknown mechanism of interaction between dietary fibers and components present in the upper intestine might also cause undesirable effects. For example, it is important to consider the possibility of decreased mineral bioavailability in some specific population group. Findings from in vitro studies have shown that semi-purified insoluble (cellulose, hemicelluloses, and lignin) as well as soluble fibers (gums and pectin) have mineral binding properties closely related to the type of fiber and the concentration in solution [
5]. Moreover, Douwina Bosscher and co-workers investigated the effect of increasing amounts of alginic acid, locust-bean gum, and guar gum in casein and whey-based infant formulas, discovering that SDFs inhibited iron, calcium, and zinc availability more in casein than in whey-based formulas [
10]. In addition, the therapeutic effect of some orally administered drugs might be reduced when these pharmaceutical molecules are simultaneously taken with fibers. For example, Reppas and co-workers showed that in canine models, both the rate and extent of absorption of paracetamol and hydrochlorothiazide were significantly decreased when administered together with guar gum [
11]. Currently, more and more research is supporting the hypothesis that weak chemical interactions between fiber and small molecule are additionally impacting the beneficial effect of fibers, which is strictly correlated to the physicochemical properties of the fiber and the nature of the ligand. For example, Espinal-Ruiz and Parada-Alfonso [
12] used isothermal titration calorimetry (ITC) to show that pectin associates with bile acid and calcium through hydrophobic and electrostatic interactions, respectively. Elucidation of the binding behavior between λ-carrageenan and Fe
3+/Al
3+ was done by Cao and colleagues [
13]. They discovered that the interaction was driven by an exothermic ion-binding behavior with a relatively large binding constant. In another study, response surface methodology (RSM) was used to explore the influence of different binding factors such as pH, temperature, and salt concentration on the binding nature of polyphenols and neutral cellulose. It was found that pH was the main factor affecting the nature of the interaction, which in turn was controlled by hydrophobic forces and hydrogen bonding [
14].
Although some interaction studies between charged polysaccharides and nutritionally relevant ligands are available in the literature, the chemical mechanism governing the weak interaction between neutral soluble dietary fibers and nutritionally relevant small molecules remains almost entirely unexplored. This low affinity association makes the design of an adequate high-sensitivity detection system a particularly difficult challenge. Here, we propose a unique approach exploiting the latest innovations in the field of surface engineered glyco-nanotechnology to study the association between galactomannan and concanavalin A. This interaction experiment can be considered as a positive control, which is always an important gold standard in any new method development. Con A is a lectin that has a high binding affinity to the terminal α-D-mannopyranoside residues of a branched trimannoside unit (Man)
3 [
15]. More recent studies have shown that Con A interacts with galactomannan (GM), even though the glycosidic linkages of the mannose residues exists in a β-anomeric configuration. For example, the experiments suggested by Vilaro’ and coworkers have shown that the galactose side chains and the reducing end of the GM mannose interacted weakly with the active sites of lectins, and that these interactions were magnified by hydrophobic effects between non-ionic GM and lectins [
16]. Zahang and colleagues used Quart Crystall Microbalance (QCM) technology to prove the biospecific interaction between galactomannan and Con A through a competitive binding study with Me-α-man, which is known to have a strong affinity to Con A [
17]. From these studies, it looks like not only the terminal group of a polysaccharide chain but also the other monomer units present in the polymer can take part in the interaction with con A, and that the affinity between Con A and galactomannan is susceptible to a number of factors such as flexibility/stiffness of the polysaccharide chain, length and density of the polymer linker on the nanoparticle surface, and distance between them. Here, we want to emphasize the influence of the size and surface density of the polysaccharide in the interaction with con A using TEM. Specifically, we used a well-known procedure involving thiol-mediated functionalization of gold nanoparticles to create a powerful tool that can assess, with the help of advanced analytical methods, the nature of these weak interactions (
Figure 1).
Using a bottom-up approach, sphere-like gold nanoparticles (AuNPs) were synthesized starting from a tetrachloroauric acid solution (HAuCl
4∙3H
2O) as a gold precursor [
18]. Neutral, thiolated galactomannans with two different molecular weights (
Mw) and dispersity (
Đ) were used to functionalize the gold nanoparticle surface through citrate-ligand exchange. Finally, after exposing the glyco-nanoparticles (GlycoAuNPs) to a target compound (Concanavalin A, (Con A)) as model system, potential interaction was revealed by monitoring the spatial arrangement of the GlycoAuNPs before and after the addition of the target molecules.
The synthetic strategy used in this study leads to a satisfactory covalent functionalization of the gold particles with dietary fibers of relatively high molecular weight. Therefore, these glyco-nanoparticles can be considered a versatile platform for the detection of specific interactions between fibers and molecules, which can have positive or negative consequences on human health.
2. Materials and Methods
2.1. Materials
Tetrachloroauric (III) acid trihydrate (HAuCl4∙3H2O ≥ 99%), distilled water (DI), nitric acid (HNO3 70.0%), concentrated hydrochloric acid (HCl, 37%), tri-sodium citrate dihydrate (Na3C6H5O7·2H2O ≥99.0%), sodium phosphate dibasic (Na2HPO4, 99.95%), isopropanol (i-PrOH, ≥99.7%), sulfuric acid (H2SO4, ACS reagent, 95.0–98.0%), anthrone (C14H10O, ACS reagent, 97.0%), bicinchoninic acid disodium salt hydrate (BCA, ≥98.0%), borane dimethylamine complex (DMAB, ≥97.0%), copper(II) sulfate pentahydrate (CuSO2·5H2O, ≥98.0%), sodium carbonate (Na2CO3, ReagentPlus®, ≥99.5%), sodium bicarbonate (NaHCO3, ACS reagent, ≥99.7%), sodium (meta)periodate (NaIO4, ≥99.0%), ethylene glycol ((CH2OH)2, ReagentPlus®, ≥99.0%), 4-aminophenyl disulfide (APDS, ≥99.0%), acetic acid (CH3COOH, glacial, ReagentPlus ®, ≥99%), sodium acetate (CH3COONa, ACS reagent, ≥99.0%), and Concanavalin A (Con A) from Canavalia ensiformis (Jack bean) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Deuterium oxide (D2O, 99.9%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Endo-1,4-β-Mannanase (Aspergillus niger) supplied at 600 U/mL was purchased from Megazyme (Bray, Ireland). One unit of mannanase activity is defined as the amount of enzyme required to release one µmole of mannose reducing-sugar equivalents per minute from carob galactomannan (10 mg/mL) in sodium acetate buffer (100 mM), pH 4.0 at 40 °C. Guar galactomannan (GM) high viscosity (Gal depleted, viscosity > 10 dL/g, Mw = 350 kDa, sugar ratio Gal:Man = 21:79 (Lot#10502 A), and (Lot#10502 B)) was purchased from Megazyme (Bray, Ireland). Tris-(hydroxymerhyl) aminomethane (Tris base, ≥99.8%) was purchased from Bio-Rad (Richmond, CA, USA). All aqueous solutions were prepared using Milli-Q water (Milli-Q® IQ 7000 Ultrapure Water System). Dialysis membranes made from regenerated cellulose with MWCO 12,000–14,000 Da (25 Å; 29 mm) were supplied by SERVA (Heidelberg, Germany). All reagents were used as received without further purification.
2.2. Synthesis and Characterization of Thiolated Galactomannan
2.2.1. Synthesis of Low Mw Galactomannans
Low molecular weight galactomannan was obtained by both acid and enzymatic treatment of the fiber. For the acid hydrolysis, galactomannan (GM (Lot A)) was hydrated in water at a concentration of 0.6% (
w/
v) for 1 h at 80 °C. To ensure a complete dissolution of the polysaccharide, the solution was kept at room temperature overnight under stirring. Concentrated HCl was added to reach a final concentration of 0.1 M HCl. Galactomannan was hydrolyzed for 24 h at 50 °C under stirring (800 rpm). The reaction was stopped by the addition of an equal volume of 0.2 M Na
2HPO
4. The product was purified by dialysis against water for 72 h at room temperature and freeze-dried for 48 h. The purified galactomannan sample was solubilized and separated to different molecular weight fractions with
i-PrOH according to a previous published procedure with some modifications [
19]. A 0.6% (
w/
v) solution of the purified galactomannan was dissolved for 1 h at 80 °C, and stirred at room temperature overnight. This polysaccharide solution (300 mL) was mixed with 60 mL of
i-PrOH and kept on ice for 2 h. The precipitate (GM
HCl) was collected by centrifugation at 12,000×
g for 20 min at 5 °C. The precipitate was freeze-dried for 48 h and stored in a desiccator at room temperature.
For the enzymatic treatment, 2 g of galactomannan (GM (Lot B)) was dissolved in 400 mL of water at 0.5% (
w/
v) final concentration and hydrolyzed via a procedure reported by Cheng et al. using endo-1,4-β-mannanase [
20]. Briefly, 432 µL of enzyme (supplied at 600 U/mL) was diluted into 2 mL of a 0.1 M sodium acetate/acetic acid pH = 6.0 buffer solution (2.432 mL final volume). Subsequently, 40 µL (4.3 U) of the enzyme solution was added to GM (0.01 U/mL enzyme concentration) and the mixture was reacted under stirring for 2.5 h at room temperature. After that, the enzyme was denatured by heating the polysaccharide solution for 20 min at 100 °C. Thereafter, the mixture was centrifuged at 4000 rpm for 20 min. The product was precipitated using three volumes of
i-PrOH, and freeze-dried for 48 h. The resulting sample GM
Enz was stored in a desiccator at room temperature.
2.2.2. Controlled Oxidation of Hydrolyzed Galactomannans
To facilitate the functionalization of galactomannan polymer chains with a thiol group via reductive amination, additional carbonyl groups (in addition to the one reducing end group per chain) were introduced into the polysaccharide. Dialdehyde galactomannans were prepared according to the method described by da Silvia et al. with some modifications (
Figure 2) [
21]. Briefly, 0.6% (
w/
v) of each GM
HCl and GM
Enz were hydrated in water under agitation for 24 h. After complete dissolution, 2 equiv. (with respect to the polysaccharide chains according to their
Mn) of a 1% (
w/
v) NaIO
4 solution (=47 mM) were added to each polysaccharide solution, and the mixtures were reacted for 6 h in the dark at room temperature. The reactions were quenched by addition of 2 equiv. ethylene glycol. Oxidized galactomannan solutions were purified by dialysis against water for 12 h. The products were precipitated with twice the volume of
i-PrOH l and freeze-dried for 48 h. Solid oxidized galactomannans GM
HCl/ox and GM
Enz/ox were stored in a desiccator at room temperature until further analysis.
2.2.3. Conjugation of Oxidized Galactomannans with Thiol Linker
Oxidized galactomannans GM
HCl/ox and GM
Enz/ox were reacted with 4-aminophenyl disulfide (APDS), leading to the formation of thiol derivatives through reductive amination (see
Figure 3). The synthesis was based on the procedure described by Seo and coworkers [
22]. A 1% (
w/
v) aqueous solution of GM
HCl/ox and GM
Enz/ox was prepared in an amber glass tube by overnight dissolution. Separately, a 50 mM solution of APDS was prepared in water:acetic acid = 1:1. A total of 10 mL of the polysaccharides were mixed with 10 mL of the thiol-linker solution for 1 h at 30 °C. Dimethylamine borane was added into each of the reaction chambers at 100 mM final concentration and the reaction was stirred unsealed for 1 h at room temperature. Subsequently, the tubes were resealed and heated again for 1 h at 50 °C. The products were purified by dialysis for 48 h and freeze-dried for two nights. Thiolated galactomannans GM
HCl/ox/ATP and GM
Enz/ox/ATP were stored at −20 °C in the dark.
2.2.4. Molecular Weight (Mw and Mn) and Dispersity (Đ) Analysis
Weight-average
Mw and dispersity (
Đ) of the hydrolyzed, oxidized, and thiolated galactomannans were measured using high-performance size exclusion chromatography (HPSEC) (OMNISEC, Malvern Panalytical Ltd., Malvern, UK) following the procedure described by Demuth et al. with slight modifications [
23]. Briefly, two A’6000M columns in series (8.0 × 300 mm, exclusion limit of 20,000,000 g/mol, Viscotek, parent organization: Malvern Panalytical Ltd., Malvern, UK) were used for the separation. Low and right-angle laser light scattering detector (LALS/RALS), the refractive index (RI), and a viscometer detector were employed for the analysis. OMNISEC software version v.10.30 was used for data acquisition, analysis, and reporting. A solution of 0.1 M NaNO
3 with 0.02% NaN
3 was used as the mobile phase. The temperature of both columns was kept at 30 °C and the flow rate was 0.7 mL/min with an injection volume of 100 µL. The total run time was 60 min. Samples were dissolved in the mobile phase at a concentration of 0.1% (
w/
v) and filtered through a 0.45 µm nylon filter prior to injection. For the absolute molecular weight determination, a calibration was performed using the narrow molecular weight distribution polyethylene oxide (PEO-24K) standard. The refractive index increment (dn/dc) value of the galactomannan samples was set at 0.144 mL/g as determined in the previous publication [
19]. Samples were measured in triplicate.
2.2.5. Monosaccharide Composition Analysis
The monosaccharide ratio (Gal/Man) of acid-treated (GM
HCl) and enzymatically treated (GM
Enz) galactomannan was measured by high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) after complete hydrolysis with HCl, as done previously [
19].
An amount of 50 mg of the dried samples were completely hydrolyzed in 10 mL 2 M HCl solution at 100 °C for 45 min. After cooling to room temperature, the reaction mixture was neutralized with 4 M NaOH and centrifuged for 15 min at 4000 rpm to separate debris from the sample solution. The analysis was taken on an aliquot of the supernatant, which was diluted with water to reach a concentration of 10 mg/L and filtered through a 0.45 µm PTFE filter.
The analysis was performed with the Dionex ICS-5000+ System (Thermo Scientific, Sunnyvale, CA, USA). A disposable gold electrode, an Ag/AgCl reference electrode, and a CarboPAC PA1 (4 × 250 mm) column were used for the analysis. The temperature of the column was kept at 26 °C, and the flow rate was set at 1.0 mL/min. The mobile phase consisted of two eluents: (A) 200 mM NaOH (prepared from 50% (w/w) NaOH solution) and (B) water. An isocratic method was applied for the sugar separation, namely 8% (A) and 92% (B) for the first 22.5 min, followed by 100% (A) for 8.5 min, and back to the initial conditions for 8 min. The total run time was 39 min. For the determination of the absolute monosaccharide amount, an external standard calibration was performed using a mixture of standard sugars containing galactose and mannose with a concentration ranging between 1.25–30.0 mg/L. D-Sorbitol was used as internal standard and added at a constant concentration of 10 mg/L to each sample and calibrant solution. The monosaccharide concentration was quantified relative to the internal standard signal. Data processing was carried out on Chromeleon 7 (Thermo Fischer Scientific AG, Basel, Switzerland). All samples were measured in triplicate.
2.2.6. Determination of Carbonyl Groups in the Oxidized Samples
An estimation of the total carbonyl content of the oxidized samples GM
HCl/ox and GM
Enz/ox was performed by the 2,2′-bicinchoninate (BCA) assay usually applied for the determination of reducing end group concentrations [REG] [
24,
25].
Briefly, assay solution A (pH = 9.7) was prepared by dissolving 5.43 g of Na2CO3 and 2.42 g of NaHCO3 in 80 mL of water. A total of 0.5 mmol of BCA was added to the solution and the volume was adjusted to 100 mL with water. For assay solution B, 0.5 mmol CuSO4∙5H2O and 1.2 mmol L-serine were dissolved in 100 mL of water. The working solution was freshly prepared by mixing equal volumes of solution A and solution B. GMHCl/ox and GMEnz/ox were dissolved in water at a 0.1% (w/v) concentration. Afterward, equal volumes of working solution and oxidized galactomannan were mixed and incubated at 75 °C in a water bath for 30 min. Samples were cooled down at room temperature for 20 min and their absorbance was measured at 560 nm against a blank containing no carbohydrate using disposable polystyrene cuvettes of 1 cm path length. The instrument was zeroed with water. The calibration was accomplished on the basis of mannose reducing end groups (REG) with a mannose standard curve obtained with concentrations ranging from 1 to 50 µM, and all the standard solutions were treated with the BCA assay following the same procedure described above. All samples were measured in triplicate.
2.2.7. Raman and FTIR Spectroscopy
Fourier transform infrared absorption (FTIR) and Raman scattering spectroscopy are molecular spectroscopy techniques sensitive to different types of vibration and therefore they provide complementary vibrational spectra. Thiolated polysaccharides were analyzed by Raman and FTIR spectroscopy to detect the presence of the aromatic thiol linker in the polysaccharide structure.
FTIR measurements were recorded using a Varian 640 (Palo Alto, CA, USA). A mean of 64 scans were registered in the range of 4000 to 400 cm−1 at a 4 cm−1 resolution. Samples were prepared as KBr pellets with a sample to KBr ratio of 1:100.
Raman scattering spectra were recorded in a solid state, in the range of 100–3199 cm−1 using a Horiba LabRAM HR Evol spectrometer (HORIBA France SAS, Longjumeau, France) supplied with a Nd:Yag single frequency laser source with an excitation wavelength of 532 nm.
2.2.8. Proton Nuclear Magnetic Resonance (1H-NMR)
Thiolated galactomannans were characterized using nuclear magnetic resonance (
1H-NMR) to obtain the number of 4-amino thiophenol linkers (ATP) per polymer chain. About 20 mg of GM
HCl/ox/ATP and GM
Enz/ox/ATP were solubilized overnight with 1.2 mL of D
2O, and 700 µL of this solution were transferred into NMR tubes and analyzed by a Bruker AVANCE III-400 spectrometer (Bruker, Ettlingen, Germany) operating at room temperature at 400 MHz with 16 repetitive scans and an acquisition time of 4 s. Data processing was carried out on MestReNova 14 (Mestrelab Research SL, Santiago de Compostela, Spain). To determine the number of ATP molecules per polymer chain (n°(ATP)), the following equation was used
where
Mn (PS-SH) is the number average molecular weight of the thiolated fibers, which was measured by SEC (
Mn (GM
HCl/ox/ATP) = 70.80 kDa,
Mn (GM21
Enz/ox/ATP) = 26.90 kDa). n°(Gal) and n°(Man) are the number of molecules of galactose and mannose in the polysaccharide chain. n°(ATP) is the number of molecules of thiol linker, and its molecular weight is
Mw (ATP) = 107.18 g/mol.
Mw (Gal) and
Mw (Man) are the molecular weights of the monomeric unit
Mw (Gal) =
Mw (Man) = 162.14 g/mol, considering that each hexopyranose unit lost a water molecule in the polymerization process. Knowing the ratio n°(Gal)/n°(Man) from HPAEC analysis, and the relative peak area of the 2 aromatic ring protons, it was possible to solve Equation (1) as a function of n°(ATP) considering the following steps
- (1)
- (2)
from
1H-NMR data analysis
where
A(
1H
Gal) is the relative peak area of the anomeric galactosyl proton and
is the relative peak area of the aromatic group protons. These two values were determined by setting the total integral of the repeating unit [RU] protons equal to 6 [
26]. By substituting Equation (5) into Equation (3), the number of mannose units in the polysaccharide chain can be given as
Substitution of Equations (5) and (6) into Equation (1) gives the number of thiol linkers per polymer chain (n°(ATP))
2.3. Synthesis and Characterization of Citrate-Gold Nanoparticles (Citrate-AuNPs) and Glyco-Gold Nanoparticles (Glyco-AuNPs)
2.3.1. Citrate-Functionalized Gold Nanoparticle Synthesis
Citrate-capped gold nanoparticles (citrate-AuNPs) were synthesized following the Turkevich method [
27]. Briefly, 43 mg of HAuCl
4∙H
2O were dissolved in 100 mL water. The solution was heated to 100 °C under stirring (800 rpm). As soon as the solution boiled, 10 mL of a freshly prepared sodium citrate (38.8 mM) solution was quickly added (1.0 mM HAuCl
4∙H
2O final concentration). The mixture was heated for a further 15 min at 90 °C under stirring (800 rpm). Within 10 min, a change in the solution’s color from colorless to purple was observed. The particle solution was maintained under stirring for an additional 20 min, cooled to room temperature, and transferred into an amber bottle where the colloidal solution was stored at room temperature until further analysis. Assuming that the reduction from Au
+3 to Au
0 was 100% complete, that the density of AuNPs is equal to that of gold [ρ = 19.3 g/cm
3], and that the particles are spherical in shape with an average diameter of d = 18.3 nm (see
Section 3.2.1), the number of nanoparticles in 110 mL of solution is
where m(Au
0) is the mass of gold atoms calculated from the mg of HAuCl
4∙H
2O used, and V is the volume of a sphere and from which the concentration of the particles can be derived as
where V is the volume of the particle solution and N
A is Avogadro’s number (6.022 × 10
23 mol
−1).
2.3.2. Synthesis of Glyconanoparticles
Glyconanoparticles (glyco-AuNPs) were prepared based on the procedure described by Hone and colleagues [
28]. A total of 50 mL of citrate-capped gold nanoparticle solution was prepared as described in
Section 2.3.1, and containing approximately 2.4 nM Au particles were mixed with 50 mg or 30 mg of thiolated polysaccharides GM21
HCl/ox/ATP or GM21
Enz/ox/ATP, respectively. The solutions were stirred for 48 h at room temperature in the dark where the self-assembly mechanism took place. To ensure a complete removal of unbound thiolated polysaccharides, a washing step of the glyconanoparticle solution was performed. Briefly, the particles were centrifuged at 23,710×
g for 25 min, after which the supernatant solution was discarded and replaced with 10 mL of 10 mM Tris buffer (pH = 7.6). After being strongly mixed, the glyco-AuNPs solution was centrifuged again, and the procedure described above was repeated two more times. Modified nanoparticles (GM21
HCl/ox/ATP-AuNPs and GM21
Enz/ox/ATP-AuNPs) were resuspended in 10 mM Tris buffer (pH = 7.6) solution to give a concentration of about 6–10 nM of Au particles.
2.3.3. UV–Vis Absorption Measurements
The absorbance values of citrate-AuNPs and glyco-AuNPs solutions were measured on a Cary 100 spectrophotometer (Agilent Technologies Inc., Santa Clara, CA, USA). A total of 500 µL of citrate-AuNPs were diluted with 500 µL water, vortex mixed, and placed in a 1 cm path length disposable polystyrene cuvette. UV–Vis spectra were acquired from 350 nm to 700 nm and the instrument was zeroed with water. For the derivatized nanoparticles, 500 µL of glyco-AuNPs were diluted with 500 µL 10 mM Tris buffer (pH = 7.6), vortexed, and placed into a 1 cm path length disposable cuvette. The wavelength was scanned from 350 nm to 700 nm and 10 mM Tris buffer (pH = 7.6) was used as a blank. All the measurements were performed in triplicate.
2.3.4. DLS (for dh) and Zeta Potential (ζ)
The hydrodynamic diameter (dh) and polydispersity index (PdI) of citrate-capped gold nanoparticles were measured by dynamic light scattering (DLS) using the Zetasizer Nano-ZS (Malvern Instrument Ltd., Malvern, UK). In addition, the net surface charge of the nanoparticle was determined by measuring the zeta potential (ζ) using the Zetasizer Nano-ZS. Before analysis, 1 mL of citrate-capped gold nanoparticle solution was filtered through a 0.45 µm PTFE filter. All the measurements were performed in triplicate. In addition, the dh and PdI of the glyco-AuNPs were measured. Before analysis, 1 mL of glyco-AuNPs solution was filtered through a 0.45 µm PTFE filter. All the measurements were performed in triplicate.
2.3.5. Determination of Morphology and Size Distribution
The citrate-AuNPs size (
d) and morphology were analyzed by a transmission electron microscope (FEI TalosTM F200X) operating at 200 kV. Scanning transmission electron microscope acquisition (STEM) was used to map the sample in dark-field mode (HAADF). An aliquot of about 5 µL of the sample was dropped onto copper grids coated with carbon film (300 mesh) and placed on absorbent paper to absorb the excess solution. The STEM images were analyzed using ImageJ software. A total of 454 particles were counted, and their area was measured. Approximating the shape of each particle to that of a circle, the average diameter (
d) of the analyzed particle samples were calculated by the following equation
where
A is the area of a circle and
r is the radius. Knowing
r of the particle, the diameter (
d) was calculated as
Glyco-AuNPs were also analyzed by TEM using the same procedure described above.
2.3.6. Ligand Surface Density
The anthrone/H
2SO
4 assay was used as a colorimetric method for the determination of the amount of carbohydrate ligands coupled to the surface of the AuNPs [
29]. Sulfuric acid promotes the hydrolysis of the polysaccharide chains into monoscaccharide units of which the open-form reacts with an anthrone molecule, leading to a green colored complex. A calibration curve was created using different concentrations of monosaccharide mixtures equivalent to the Gal:Man molar ratio of the respective GM. The calibration ranged from 12 µg/mL to 192 µg/mL. A 0.2% (
w/
v) anthrone solution was prepared in concentrated H
2SO
4 in iced water. A total of 0.5 mL of each standard solution was transferred inside an amber bottle and stored in iced water under stirring. One mL of the freshly prepared anthrone solution was added into the standard solutions. The samples were heated to 100 °C for 10 min under stirring. After that, the standard solutions were cooled in an iced water bath and the absorbance was measured at λ = 625 nm. The blank sample consisted of 1 mL of anthrone solution in 0.5 mL water, and the instrument was zeroed with water. All standards were measured in triplicate.
Glyco-AuNP solutions were extensively mixed and 2 mL were centrifuged at 23,710×
g for 25 min. The supernatant was discarded, and the particles were resuspended in 1.5 mL water. The particle solution was divided into three aliquots (0.5 mL each) and placed inside an amber bottle previously cooled in an iced water bath. The solutions were treated with anthrone reagent following the same procedure described for the standard solutions, with the exception that glyconanoparticles were reacted with the anthrone reagent for 20 min [
30]. The blank sample consisted of 1 mL of anthrone solution in 0.5- mL water, and the instrument was zeroed with water. A control experiment was performed by measuring the absorbance of Glyco-AuNPs at λ = 625 nm in the absence of the anthrone reagent. All samples were measured in triplicate.
2.4. Interaction Study by TEM
Transmission electron microscopy (Thermo Fisher Talos F200X and Hitachi HD-2700) operating at 200 kV was used as a preliminary interaction study method between guar galactomannan and concanavalin A (con A). Scanning transmission electron microscope (STEM) acquisition was used to map the sample in a high angle annular dark field (HAADF) mode. For the experiment, 500 µL of GMHCl/ox/ATP-AuNPs (6.0 nM), GMEnz/ox/ATP-AuNPs (5.5 nM) were incubated with 88 µL of a 2.35 µM Con A solution in 10 mM Tris buffer pH = 7.6, containing 99 mM NaCl, 1 mM CaCl2 and 1 mM MnCl2. The solutions were left under shaking for 3 h at room temperature. Afterward, 5 µL of each sample solution was placed onto a 300 mesh copper grid and the excess liquid was absorbed by paper tissue. The control sample consisted of a 500 µL glyconanoparticle solution with 88 µL of 10 mM Tris buffer pH = 7.6 (including NaCl, CaCl2, and MnCl2). A control experiment was conducted by exposing the citrate-stabilized nanoparticles to the same conditions described above.