Synthesis of Gold Nanoparticles (AuNPs) Using Salt-Soluble Wheat Flour Proteins and Their Use in the Fluorescence-based Sensing of Folic Acid

Protein-stabilized metal nanoparticles have been receiving significant attention because their optical properties could be used for sensing applications. This study describes the synthesis of gold nanoparticles (AuNPs) stabilized with salt-soluble proteins extracted from wheat flour, and their use in the fluorescence-based detection and quantification of folic acid. The salt-soluble protein extract from defatted wheat flour contained at least eight components with molecular weights ranging from 7.26 to 57.0 kDa. The extract was made alkaline and then mixed with a HAuCl 4 solution to synthesize the AuNPs. The wheat-AuNPs were red in color and had a surface plasmon resonance (SPR) peak at 519 nm. Based on the UV-visible spectrum, the AuNPs may have ~3.4 nm average size. The AuNPs exhibited fluorescence emission at 734 nm upon excitation at 365 nm. The fluorescence quenching of the AuNPs observed upon incremental addition of folic acid (0 to 10 μg mL -1 ) was fitted to the Stern-Volmer equation. From the Stern-Volmer plot, the limits of linearity (LOL), detection (LOD), and quantitation (LOQ) were determined as 22.0 μg mL - 1 , 0.92 μg mL -1 , and 3.1 μg mL -1 , respectively. Several substances, including some metal ions, were tested for their effects on the AuNPs’ fluores cence. Moreover, a proposed fluorescence-based folic acid quantification was evaluated by determining the folic acid content of several pharmaceutical formulations.


INTRODUCTION
Folic acid (FA, (2S)-2-[ (4-{[(2-amino-4-hydroxypteridin-6-yl)methyl]amino}phenyl)formamido] -pentanedioic acid), also known as vitamin B9 or folacin, is a water-soluble compound with roles in metabolic pathways and involved in cell development and maintenance (Blom et al., 2011).Deficiencies in FA can lead to higher risks of heart disease and congenital defects (Blom et al., 2011).Because of its importance, FA-containing products, such as food supplements and enriched or fortified foods had been manufactured to prevent deficiencies (Di Tinno et al., 2021).Demands for FA quantification methods, especially in pharmaceutical formulations (Zhao et al., 2006), are on the rise to assess how certain formulations or supplements should be used.This is crucial since the risks of excessive ingestion of FA could mask possible deficiencies of another important Bvitamin, vitamin B12 or cobalamin (Ribeiro et al., 2016).The standard method of determination is a microbiological assay (MA) involving the turbidimetric bacterial growth of Lactobacillus casei that is proportional to the amount of FA (Rahman et al., 2015).Though MA has high reliability and accuracy, the method can be susceptible to other growth factors and can be time-consuming.High-performance liquid chromatography (HPLC) offers a useful alternative for FA analysis (Jastrebova et al., 2003).However, HPLC-based methods could have high operating costs, sizable consumption of samples and solvents, and can generate difficult-to-dispose organic wastes.There were also proposed electroanalytical methods (Flores et al., 2005) and spectrophotometric techniques (Nagaraja et al., 2002;Anastasopoulos et al., 2007).Still, some of these methods can be laborious and have low sensitivity.
Metal nanoparticles have been receiving attention because of their interesting size-dependent optical properties appropriate for a range of applications.Metal nanoparticles are composed of clusters of metal atoms with sizes of 1.0 nm to 100 nm (Ghosh and Pal, 2007).The particles exhibit a characteristic absorption in the visible region due to surface plasmon resonance (SPR), which is the oscillation of electrons between the conduction and valence bands of the metal caused by the interaction of electrons with the electric fields of light (Kelly et al., 2013).On the other hand, because of their discontinuous band structures and quantum confinement, metal nanoparticles can fluoresce in the visible region of the electromagnetic spectrum when excited with nearultraviolet radiation (Zheng et al., 2012).Gold is commonly used in preparation due to its stability and non-reactivity relative to other metals such as silver and copper (Zheng et al., 2007).AuNPs tend to aggregate into larger clusters, hence capping agents are used to ensure long-term stability (Housni et al., 2008).Proteins had been demonstrated to be capable of reducing metal ions and stabilizing metal nanoparticle preparations (Xie et al., 2009).Protein-stabilized AuNPs are biocompatible and are frequently utilized in biomedical applications such as drug delivery (Rastogi et al., 2012), diagnostic imaging (Zu et al., 2016), and cancer therapy (Chiu et al., 2018).They are also employed as sensors for the determination of various chemical species, from metal ions (Lee and Huang, 2011) and small compounds (Huang et al., 2012) to large macromolecules (Cui et al., 2008).
Wheat proteins are important in relation to the baking quality of wheat, mainly attributed to their viscoelastic properties (Veraverbeke and Delcour, 2002).Wheat proteins constitute two distinct protein fractions: non-gluten and gluten proteins.Non-gluten fractions include albumins and globulins, which consist of enzymes, enzyme inhibitors, structural proteins, and storage proteins.Gluten fractions include gliadins and glutenins, which are largely responsible for dough viscoelasticity and are divided based on solubilities in alcoholic solutions.These four major protein types are typically separated sequentially using a series of appropriate solvents (Lookhart and Bean, 1995).Generally, wheat proteins can be acquired by using inexpensive and readily accessible reagents such as 2-propanol, sodium chloride, and water (DuPont et al., 2005).Most of the wheat proteins also have considerable stability to heat and in weak acids and bases, which contributes to ease in preparation and storage.With the availability and relatively low costs of wheat flour, the usage of wheat proteins in applied biochemistry may help reduce costs in general research.
This study aimed to provide a simple, inexpensive, and environment-friendly approach to the synthesis of AuNPs stabilized with salt-soluble wheat proteins, and their use in the detection and quantification of folic acid through fluorescence quenching.Preliminary observations on the responses of these fluorescent protein stabilized AuNPs to folic acid and several other chemical species revealed the potential in the development of a method for folic acid determination.

Materials and Equipment.
The wheat flour sample used was the SM Bonus All-Purpose Flour brand.Gold (III) chloride trihydrate and folic acid were purchased from Sigma-Aldrich.Sample pharmaceutical formulations used for FA determination were Folicap®, Biogesic®, and Centrum® Advance.Absorption measurements for the Bradford assay were obtained using the Milton Roy Co. Spectronic 21D UV-Visible Spectrophotometer.The UV-Visible absorption spectrum of wheat-AuNPs was obtained using the Thermo Scientific™ Multiskan™ FC Microplate Photometer.Fluorescence emission spectra and measurements for wheat-AuNPs were obtained using the Shimadzu RF-5301PC Fluorescence Spectrophotometer.
Extraction of Salt-Soluble Wheat Proteins.Extraction of albumin and globulins was adapted from Sandiford et al. (1997), with modifications.Wheat flour was defatted with n-hexane for about 1 h.Albumins and globulins were extracted by stirring 10 g defatted wheat flour in 50 mL 0.5 M NaCl for 1 h at room temperature.Supernatants containing the wheat proteins were obtained by centrifugation (10000 g, 25 C, 15 min) of the flour-salt mixture.
Quantification of Salt-Soluble Wheat Proteins.The Bradford assay (1976) was employed for protein quantification.Protein standards used were 0 to 2.0 mg mL -1 BSA solutions.The assay was performed by mixing 20 µL protein extract or standard with 1.0 mL Bradford reagent, incubating the solutions for 5 min, and measuring their absorbances at 595 nm.The concentration of the protein extract was determined by interpolation from an absorbance () vs. concentration ([protein]) calibration curve constructed using the BSA standards, in reference to the Beer-Lambert law, shown below.Molar absorptivity () and path length () were kept constant.

𝐴 = 𝑎𝑏[𝑝𝑟𝑜𝑡𝑒𝑖𝑛]
Characterization of Salt-Soluble Wheat Proteins.Laemmli's (1970) SDS-PAGE protocol was performed for the electrophoresis of wheat proteins.A 15% polyacrylamide resolving gel was cast in the electrophoresis setup followed by a 4% polyacrylamide stacking gel, and a running buffer was poured into the setup.Protein extracts (25 µL) were mixed with a sample buffer (25 µL).The mixtures were shaken vigorously, heated to 95 C, and loaded to the wells of the stacking gel, along with protein markers.The electrophoretic run was done at room temperature and 100 V. Coomassie Blue gel staining was performed to visualize the protein bands.Molecular weights (MWs) of the protein samples were estimated by interpolation from a retention factor (Rf) vs. log MW calibration curve constructed using the protein markers.

𝑅𝑓 =
ℎ     ℎ   Synthesis of Wheat-AuNPs.The synthesis of AuNPs stabilized by salt-soluble wheat proteins, designated as wheat-AuNPs, was adapted from the synthesis of BSA-stabilized gold nanoclusters of Xie et al. (2009), with modifications.The wheat protein extract (10 mL, 1.132 mg mL -1 ) was first stirred with 3 M NaOH (226 µL) at room temperature for 5 min.The resulting solution was then stirred vigorously with 200 mM HAuCl4 (113 µL) at room temperature for 30 min.The final mixture was allowed to stand overnight in a dark place at room temperature.Figure 1 shows the scheme for the extraction of salt-soluble proteins from wheat followed by wheat-AuNP synthesis.Characterization of Wheat-AuNPs.The UV-visible absorption spectrum of the wheat-AuNPs mixture was acquired from 400 nm to 800 nm.The fluorescence emission spectrum of a solution of wheat-AuNPs, diluted to 20 volumes with distilled water, was acquired from 500 to 900 nm with 305 nm to 395 nm excitation wavelengths at 15 nm intervals and 15-nm excitation and emission slit widths.
Fluorescence Quenching of Wheat-AuNPs by Folic Acid.The wheat-AuNPs were diluted to 20 volumes with distilled water prior to analysis.Standard FA solution (1000 µg mL -1 ) was added to the diluted wheat-AuNPs (2.0 mL) at 4.0-µL increments.The fluorescence spectrum from 685 to 785 nm was acquired for each FA addition (0 to 20 µL), with 365 nm as the excitation wavelength.The procedure was repeated three times on different days.Another identical experiment was performed with the FA addition increased up to 100 µL.For each experiment, the initial ( 0 ) to final () ratio of fluorescence intensities at 734 nm (365 nm excitation) were plotted against the FA concentrations ([]), in reference to the Stern-Volmer equation, shown below.Slopes or Stern-Volmer constants (  ) were determined by simple linear regression.
The limits of detection () and quantitation () of the fluorescence quenching experiment were calculated from the standard deviation of the lowest concentration signal () and slope of the calibration curve (), shown below.

Folic Acid Determination in Pharmaceutical
Fluorescence intensities of the wheat-AuNPs at 734 nm (365 nm excitation) were measured before and after the addition of the samples.The FA contents of the formulations were determined by the method of external calibration using a standard calibration curve produced using FA standards, with the Stern-Volmer equation as the working equation.Analysis for recovery was performed on the Folicap® samples by spiking them with standard FA (1000 µg mL -1 ) at increasing concentrations (0 to 6.0 µg mL -1 ), performing the same fluorescence analysis, and computing the percentage of found FA concentration relative to expected FA concentration.

RESULTS AND DISCUSSION
Extraction of Salt-Soluble Wheat Proteins.The salt extraction of defatted wheat flour produced a clear pale-yellow solution having a final volume of approximately 42 mL.Wheat flour consists of four distinct components: starches, lipids, non-starch polysaccharides, and wheat proteins (Goesaert et al., 2005).Starches are insoluble in aqueous media due to their polymeric structures.
Lipids are also insoluble due to their hydrophobic nature.Non-starch polysaccharides include arabinoxylans, β-glucan, cellulose, and arabinogalactan-peptides, where some of which are soluble in aqueous media (Goesaert et al., 2005).The salt-soluble extract obtained may contain the non-gluten proteins, albumins and globulins, and some non-starch polysaccharides.
The protein concentration of the extract was 1.131 mg mL -1 while the total protein extracted was 47.51 mg, which was 0.4751% by mass of defatted wheat flour.The reported composition of wheat flour included 10 to 12% protein, where 15 to 20% of these proteins are albumins and globulins (Goesaert et al., 2005).Based on this report, non-gluten proteins should constitute about 1.5 to 2.4% of the flour mass.The amount of total protein extracted was about three to five times lower than the amounts of non-gluten proteins reported.To increase the efficiency of extraction, it may be necessary to use smaller portions of the 0.5 M NaCl solvent multiple times (Sandiford et al., 1997).

Characterization of Salt-Soluble Wheat Proteins.
The salt-soluble wheat protein extract was found to contain at least eight components based on the SDS-PAGE result shown in Figure 2. Molecular weights of the components ranged from 7.26 kDa to 57.0 kDa.The SDS-PAGE profile also provided a good estimation of relative protein amounts, which are proportional to the band intensity.Among the bands seen in Figure 2, the 57.0-kDa band displayed the greatest intensity, followed by the components having MWs of 7.26 kDa and 10.7 kDa.These results suggest that these three components had the highest concentrations in the extract.
Using the estimated MWs, probable identities of the extracted wheat proteins were proposed and presented in Table 1.The UniProt (uniprot.org)database was searched for wheat proteins that were closest to the estimated MWs.The probable identities may include amylase inhibitors, lipid transfer proteins, gliadin precursors, and other enzymes.The SDS-PAGE profile was found to be similar to the profile in Sandiford et al. (1997) where they suggested that the salt-soluble extract contained albumins and globulins.Singh et al. (2001) reported that the water-soluble proteins from Chinese spring flour included amylase inhibitors, gliadin precursors, lipid transfer proteins, globulins, and albumins, determined through N-terminal sequencing.Mass spectrometry could provide more accurate identities of the proteins in the salt-soluble extract.Altenbach et al. (2020) reported that the KCl-soluble fraction of US wheat Butte 86 flour contained some gluten proteins and about 57 different types of non-gluten proteins.Synthesis of Wheat-AuNPs.The AuNP synthesis initially produced a pale-yellow solution, which turned into a red colloidal mixture (Figure 3a, inset), after overnight incubation.The study of Xie et al. (2009) on the green synthesis of protein-stabilized gold nanoclusters included a gold to protein ratio of 1 mM gold to 5 mg protein.With a 1.131 mg mL -1 calculated protein concentration, 113 µL 200 mM HAuCl4 were used, along with 226 µL 3 M NaOH, per 10 mL protein solution for wheat-AuNP synthesis.The characteristic red color of the resulting mixture indicated the existence of AuNPs and hinted at the successful conjugation between the AuNPs and the saltsoluble wheat proteins.The observed color agreed with AuNPs synthesized using citrate (Bastús et al., 2011) and using proteins or peptides (Aldeek et al., 2013;Matei et al., 2019).
The formation of protein-stabilized AuNPs follows a general mechanism, by which wheat-AuNPs could be hypothesized to also follow.The first step is the dispersion of the wheat proteins and gold ions in one mixture allowing the formation of protein-AuCl4 -complexes (Tan et al., 2010).Upon pH adjustment by NaOH addition, Au 3+ ions are reduced to Au + and Au 0 species by tyrosine residues, developing Au 0 clusters via nucleation (Xie et al., 2009).After a short incubation, there would be the creation of Au + -S bonds between the cysteine residues of the wheat proteins and gold ions (Xie et al., 2009;Bolaños, 2019).Amino acids such as tryptophan may also participate in the reduction of the gold ions, while cysteine, histidine and methionine may play roles in complex formation (Tan et al., 2010).Following long incubation, further aggregation of the gold clusters, prompted by the need to shoulder lower energy configurations, finally materializes the nanoparticles (Tan et al., 2010).
Characterization of Wheat-AuNPs.Characterization of wheat-AuNPs was accomplished by UV-Visible and fluorescence spectrophotometry.
UV-Visible Spectrophotometry.The UV-Vis absorption spectrum of the wheat-AuNPs is displayed in Figure 3a.The UV-Vis spectrum shows a peak around 519 nm.The presence of this SPR peak in the absorption spectrum, in addition to the red color of the wheat-AuNP mixture, confirmed the existence of AuNPs and their successful production.SPR is the oscillation of electrons between conduction and valence bands of metal when electric fields of light rays interact with them (Amendola et al., 2017).For AuNPs, an absorption at around 515 nm to 570 nm in their UV-Vis spectra gives AuNPs a distinct red color (Haiss et al., 2007).Information about the shapes (Amendola et al., 2017) and sizes (Haiss et al., 2007) of gold nanoparticles can be determined from their UV-visible spectra.The single SPR peak at 519 nm suggested that the wheat-AuNPs should conform to a spherical geometry, which is consistent with several studies (Housni et al., 2008;Garcia-Hernandez et al., 2019).Based on the proposed model of Haiss et al. (2007) that described the relationship between particle size and SPR absorbances, the estimated sizes of the wheat-AuNPs should be around 3.4 nm.To establish the shapes and sizes, analysis by scanning or transmission electron microscopy (SEM, TEM) must be performed.
From the spectroscopic data, it is plausible that the as-prepared AuNPs consisted of particles with a variety of sizes.
Fluorescence Spectrophotometry.The fluorescence emission spectra of the wheat-AuNPs (20x diluted) are shown in Figure 3b.The emission spectra showed a series of peaks from 613 nm to 792 nm upon excitation with radiation from 305 nm to 395 nm.Maximum intensity (  ) was seen at 734 nm upon 365 nm excitation.The spectra demonstrated the photoluminescence of the wheat-AuNPs within the visible region.Multiple peaks can be seen on several emission spectra in Figure 3b.Apart from the sharp peak at 734 nm, which can be ascribed to the fluorescence of wheat-AuNPs, there are other signals around 600 and 800 nm.These signals may be attributed to the wheat proteins left in the colloidal mixture from wheat-AuNP synthesis (Mathew and Joseph, 2017), whose fluorescence was said to be caused by their aromatic residues.The fluorescence of protein stabilized AuNPs could be due to much smaller particles or nanoclusters with sizes ˜0.8 nm (Xie et al., 2009).Larger particles (>20 nm) were reported to not exhibit fluorescence (Xie et al., 2009).
Fluorescence of wheat-AuNPs can be associated with electronic transitions that occur between the lowest unoccupied molecular orbitals (LUMOs) in the sp band and the highest occupied molecular orbitals (HOMOs) in the d band (Zhou et al., 2010).Thiol groups of the wheat protein cysteine residues are π-acceptor ligands, and their p orbitals have higher energy than the d orbitals of Au + ions (Zhou et al., 2010).Overlap of these orbitals can lead to the formation of metalto-ligand charge transfer excited states.As a result, excitation can occur between the hybrid MOs, followed by non-radiative vibrational relaxations toward the LUMO of the sp band (Zheng et al., 2012).After which, emission can take place between there and HOMOs of the d band (Zheng et al., 2012).
Fluorescence Quenching of Wheat-AuNPs by Folic Acid.Standard FA was added in incremental amounts to a 20x diluted wheat-AuNP solution to determine the effects on the fluorescence of wheat-AuNPs.In Figure 4a, the fluorescence intensity of wheat-AuNPs at 365 nm excitation decreases as the concentration of FA increases, indicating the quenching effect of FA on the fluorescence of wheat-AuNPs.
A plausible mechanism for the fluorescence quenching of wheat-AuNPs by FA may be based on FA-tryptophan interactions.From Table 1, the extract could contain proteins that, except for nonspecific lipid transfer proteins, have at least two tryptophan residues.FA molecules can bind to the tryptophan residues of the wheat proteins via charge-transfer complex formation (Liang and Subirade, 2010), hydrogen bonding, and hydrophobic forces (Shi et al., 2017).The resulting wheat-AuNP-FA complex becomes non-fluorescent owing to a non-radiative energy transfer from wheat-AuNPs to FA (Mote et al., 2011).
The fluorescence experiment was replicated on different days to determine the reproducibility of the quenching phenomenon.Initial ( 0 ) to final () fluorescence ratios of wheat-AuNPs at 734 nm (365 nm excitation) were plotted against concentrations of FA in the mixture ([]), in reference to the Stern-Volmer equation.The plots constructed in Figure 4b presented a consistent linear relationship between the wheat-AuNP fluorescence ratios and FA concentrations.
Stern-Volmer constants (  ) or slopes of the best fit lines and coefficients of determination ( 2 ) were obtained from simple linear regression (Table 2).The Stern-Volmer constants ranged from 0.0920 to 0.1001 mL µg -1 with an RSD of 3.72% and the coefficients of determination ranged from 0.9659 to 0.9906 with an RSD of 1.15%.The data indicated the repeatability of the linear relationship between the wheat-AuNP fluorescence ratios and FA concentrations (Fajgelj and Ambrus , 2000).Hence, the proposed method for FA determination was subjected to further validation and figures of merit were calculated.The limit of linearity () was determined from the data after FA addition was increased up to 50 µg mL -1 .The LOL is the FA concentration where the resulting best fit line gives  ≥ 0.99 (Green, 1996).In the quenching experiment, a continuous decrease in the fluorescence ratio of wheat-AuNPs was observed, even when FA concentration was increased up to 50 µg mL -1 (Figures 4c  and 4d).An acceptable linear relationship was observed to be lost starting at around 22 µg mL -1 (Figure 4d), which indicated the .From the four-day experiment, values of  and  were calculated to be 0.92 µg mL -1 and 3.1 µg mL -1 , respectively.All estimated limits are presented in Table 2. Earlier studies using fluorescence-based analysis using nanoparticles had lower LODs.Hemmateenejad et al. (2014) reported an LOD of 18.3 ng mL -1 folic acid using BSA-stabilized gold nanoclusters.Moreover, in the study of Fereja et al, 2021, the use of BSA-capped Au-Ag bimetallic nanoclusters for folic acid analysis had an LOD of 0.47 nM (0.21 ng mL -1 ).Compared to the previous studies, the as-prepared wheat-AuNPs are highly likely to contain a variety of sizes, unlike the highly uniform nanoparticles or nanoclusters synthesized using highly purified proteins as capping agents.
Effect of Other Chemical Species on Wheat-AuNP Fluorescence.Twelve biological compounds and nine metal ions were subjected to the same fluorescence quenching experiment to test their effects on the fluorescence of wheat-AuNPs.Initial ( 0 ) to final () fluorescence ratios of wheat-AuNPs for each ion or compound in comparison to FA were plotted (Figures 5) to show their relative magnitudes of fluorescence change.Quenching is observed when  is less than  0 ( 0 / > 1) while an enhancing effect is observed when  is greater than  0 ( 0 / < 1).From Figure 5a, the wheat-AuNPs showed little to no fluorescence changes upon the addition of the biological compounds.Among the substances, starch, gallic acid, and acetic acid had some noticeable effects on the wheat-AuNP fluorescence.From Figure 5b, the wheat-AuNPs displayed substantial fluorescence increases upon the addition of some metal ions.Cu 2+ , Ti 4+ , Co 2+ , Ni 2+ , and Fe 3+ , had enhancing effects on wheat-AuNP fluorescence.These metal ions may interfere with FA determination if they are present in the samples for analysis.Results indicate that the fluorescence of wheat-AuNPs can be altered not only by FA but also by other chemical species.Possible interfering substances should be considered in FA determination via fluorescence quenching of wheat-AuNPs.The addition of ethylenediaminetetraacetic acid (EDTA), a heavy metal antagonist, to sequester metal ions could work as a part of sample preparation before the FA determination (Hemmateenejad et al., 2014).Isolation of FA from samples using organic solvent extraction and thin layer chromatography should also be considered in sample preparations.
Folic Acid Determination in Pharmaceutical Formulations using Wheat-AuNPs.The method for FA determination through wheat-AuNP fluorescence quenching was used in the analysis of various pharmaceutical formulations.This aimed to evaluate the applicability of the proposed method to actual samples.For the experiment, three drugs of different constituents (Folicap®, Biogesic®, and Centrum® Advance) were used.
The FA content of each formulation was estimated via interpolation from a calibration curve prepared using standard solutions of the analyte (Table 3).Calculations returned 4205 µg of FA per Folicap® capsule compared to the 5000 µg of FA declared, with a 15.89% absolute error and 5.55% RSD.For Centrum® Advance and Biogesic®, the fluorescence of wheat-AuNPs was observed to increase after each sample was added, indicating enhancing effects.FA amounts from the data could not be obtained since fluorescence enhancement was not the expected response for the wheat-AuNP and FA interactions.The abundance of components in Centrum® Advance affected the FA determination.The presence of several transition metal salts (notably cupric sulfate, CuSO4) likely caused a significant increase in wheat-AuNP fluorescence.Acetaminophen in Biogesic® may have fluorescence enhancing effects due to the compound also acting as a stabilizing agent for the gold nanoclusters (Rajendran et al., 2019).In Table 4, recoveries in Folicap® samples were 84.10% to 106.1% with 1.89% to 5.55% RSD upon addition of 0 to 5.96 µg mL -1 FA.The % recoveries fall within acceptable values (Fajgelj and Ambrus, 2000).However, the FA recoveries were observed to increase when concentrations of the spike were increased.This suggested matrix effects of the drug extract slightly interfering with the detection of FA (Mathew and Joseph, 2017).The matrix effects can be deemed as a possible source of error in the FA determination in the Folicap® samples.Additional washing procedures of the solid residue during FA extraction can help better recover FA from the pharmaceutical preparations and improve its determination (Hemmateenejad et al., 2014).The matrix effects could also be addressed by improving the sample preparation protocols.

CONCLUSIONS
This study demonstrated the synthesis of AuNPs using salt-soluble proteins from wheat flour.The as-prepared AuNPs exhibited the distinct and established optical properties of AuNPs such as presence of the SPR absorption, and the fluorescence properties.Utilizing the fluorescence properties of the wheat-AuNPs, a method for the determination of folic acid based on fluorescence quenching was proposed.The proposed method had decent limits of linearity, detection, and quantitation but may encounter some measurement errors in the presence of potentially interfering species such as heavy metal ions.Therefore, it is highly recommended to conduct more work to establish the optimum conditions for a protocol using the wheat-AuNPs for FA determination.

Figure 1 .
Figure 1.Simplified schematic diagram for the extraction of salt-soluble proteins from wheat flour and the synthesis of wheat-AuNPs.

Figure 2 .
Figure 2. SDS-PAGE profile of the salt-soluble protein extract from wheat flour.The protein extract contained at least eight components with MWs between 7.26 kDa to 57.0 kDa.Electrophoresis run was performed using a 15% polyacrylamide gel SDS-PAGE gel under 100 V potential.
) a Values are means of three replicates.b [|Declared FA -Calculated FA|/Declared FA] *100% c [Standard deviation of Calculated FA values/Mean of Calculated FA values] * 100%

Table 1 . Molecular Weights and Some Probable Identities of the Extracted Wheat Proteins.
a Values were calculated based on relative mobilities compared to the protein MW markers.b Values were based on the complete sequence of the mature protein.c [ |Theoretical MW -Experimental MW|/Theoretical MW] * 100%

Table 2 . Simple Linear Regression and RSD Analysis of Wheat-AuNP Fluorescence Ratios vs. Folic Acid Concentrations (0 to 10 µg mL -1 ) at Different Time Periods and Figures of Merit of the Fluorescence Quenching of Wheat-AuNPs by Folic Acid.
[Standard deviation of   values/Mean of   values] * 100% b

Table 3 . Folic Acid (FA) Determination in Different Pharmaceutical Formulations Through Fluorescence Quenching of Wheat-AuNPs.
*n.d.-not determined (negative concentrations calculated

Table 4 . Recoveries of Folicap® Upon Addition of Increasing Amounts of Folic Acid (FA) Standard.
[Found FA /Expected FA] *100% c [Standard deviation of Found FA values/Mean of Found FA values] * 100% a Values are means of three replicates.b