Silver and gold nanoparticles based colorimetric assays for the determination of sugars and polyphenols in apples

Food Research International 119 (2019) 359–368

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Silver and gold nanoparticles based colorimetric assays for the determination of sugars and polyphenols in apples Annalisa Scroccarello, Flavio Della Pelle, Lilia Neri, Paola Pittia, Dario Compagnone

T ⁎

Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64023 Teramo, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: Rapid methods Sugars assay Polyphenols assay Apple samples Antioxidant capacity Metal nanoparticles Ag nanoparticles Au nanoparticles

In this work, the exploitability of rapid and easy to use methods for the evaluation of the antioxidant capacity (AOC) and sugars content (SC), through metal nanoparticles (MNPs) formation, has been proved and applied to apples. In particular, an AgNPs-based sugar quantification assays and an AuNPs-based polyphenols antioxidant capacity assay have been used as tools for the evaluation of apple extracts composition. Both assays are based on the ability of the analytes (sugars and polyphenols) to reduce the source of metal (Au3+ and Ag+), stabilizing, at the same time, the resulting MNPs colloidal suspensions. The AuNPs and AgNPs formation depends on the analyte structure and concentration, resulting in red (AuNPs) and yellow (AgNPs) colored suspensions. Both assays require an initial mixing step, followed by MNPs formation under mild conditions (10 min, room temperature or 45 °C), and the colorimetric response is easily acquired at a fixed wavelength (AgNPs: 430 nm and AuNPs: 540 nm). The analytical performance of both assays has been proven, obtaining good reproducibility (RSD ≤ 6%,), sensitivity (LODs ≤8.7 μmol L−1) and recoveries (91% −113.7%). The produced MNPs (AgNPs and AuNPs) have been characterized using UV–Vis spectroscopy and TEM, and the cross-reactivity between assays, as well as the possible endogenous interferents, studied. The assays have been tested on 42 apple samples, and the data obtained compared with those obtained by conventional methods (i.e. FC, ABTS, and ion chromatography). The proposed AgNPs-sugars assay gives results comparable (R = 0.915) to those determined by ion chromatography in terms of total sugars, and the AuNPs-polyphenols assay results able to assess the polyphenols antioxidant capacity, being correlated with those obtained by the ABTS method (R = 0.922). This MNPs-based approach demonstrated to be an excellent tool for rapid and facile analysis of sugars and polyphenols in apple samples.

1. Introduction

products. Despite, their substantial difference in terms of implication and impact on health, both classes of compounds affect the quality and stability of the food, their functional and sensory characteristics as well as their technological suitability towards processing. PCs are a heterogeneous class of compounds characterized by a phenolic hydroxyl group(s); they are ubiquitous secondary metabolites present in plant foods (El Gharras, 2009). These compounds have been received exceptional attention in the last decades; in fact, PCs provide foods an added value for their well-known health benefits, their technological role and also marketing (Della Pelle & Compagnone, 2018). In these last years, main efforts have been devoted to assess the real PCs health effect through in vitro and particularly in vivo studies, with particular attention to bioavailability issues (Del Rio et al., 2013; Pandey & Rizvi, 2009; Peluso, Miglio, Morabito, Ioannone, & Serafini, 2015; Serafini & Peluso, 2016; Zhang & Tsao, 2016). It has been proven that long term consumption of diets rich in plant polyphenols is

Nowadays the accessibility of easy and fast analytical methods for quality assurance is highly required; in fact, rapid quality control plays a key role along the entire food production chain. In addition to conventional instrumental analytical protocols, there is the need for complementary screening assays, possibly environment-friendly (e.g. small amount of solvent employed, low waste generation), easy to be used even by unspecialized staff. In recent years, the growing awareness of the relationship between food and health has increased consumer concern about the composition, processing and health properties of food products. This encouraged the food industry to explore new food sources, technology and formulations in order to improve and diversify the food productions and to meet consumers' dietary needs. In this context, particular attention has been reserved to sugars (SGs) and polyphenols (PCs) in food

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Corresponding author. E-mail address: [email protected] (D. Compagnone).

https://doi.org/10.1016/j.foodres.2019.02.006 Received 3 November 2018; Received in revised form 3 January 2019; Accepted 3 February 2019 Available online 06 February 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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of soluble solids related to the sugars content is based on the use of a refractometer. It's a rudimental technique commonly used for Brix degree determination in horticultural products and beverages. Nowadays, chromatography approaches, in particular, ion chromatography (IC) and capillary electrophoresis, coupled with different detectors are commonly employed for the SGs qualitative and quantitative analysis (Downes & Terry, 2010; Lacourse, 2002; Ma et al., 2014; Martínez Montera, Rodríguez Dodero, Guillén Sánchez, & Barroso, 2004; Peters, Levine, & Jones, 2001). Besides nuclear magnetic resonance (NMR) (Pospíšilová, Polášek, Šafra, & Petriška, 2007) can be used for the SGs determination, whereas near-infrared spectroscopy (NIRs) analysis can be employed for fruits ripening evaluation during their growing/cultivation steps (Magwaza et al., 2012; Magwaza & Opara, 2015). Moreover, the determination of total reducing sugars can be performed by Fehling reaction and other optical assays (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956; Quisumbing & Thomas, 1921). The reported methods to assess PCs and SGs in foods can results time-consuming, require several steps (extraction, clean up, running time, titrations at high temperature, precipitations, etc.) and large amounts of solvents, as well as expensive instrumentation. Recently analytical nanomaterials-based approaches have become a valuable strategy to assess a plethora of analytes using different analytical approach, allowing improvement of sensitivity and reducing the time of analysis, and, then, resulting in sample and reagents significant volumes reduction (Blandón-Naranjo et al., 2018; Capoferri, Della Pelle, Del Carlo, & Compagnone, 2018; Del Carlo et al., 2012; Della Pelle et al., 2016; Della Pelle et al., 2017; Della Pelle et al., 2018; Della Pelle & Compagnone, 2018; Della Pelle, Del Carlo, Sergi, Compagnone, & Escarpa, 2016; Pérez-Lòpez & Merkoci, 2011; Rojas et al., 2018; Valdés, Angela, Calzón, & Díaz-garcía, 2009). Among nanomaterials, noble metal nanoparticles (MNPs) possess a peculiar feature named ‘localized surface plasmon resonance’ (LSPR), that allows the interaction with electromagnetic radiation (Jain, Huang, El-Sayed, & El-Sayed, 2007; Mayer, Hafner, & Antigen, 2011; Petryayeva & Krull, 2011; Willets & Van Duyne, 2007). In fact, under the influence of electromagnetic radiation in the visible range, the MNPs electrons of surface atoms can easily move through vacant orbitals generating absorption at a particular wavelength. This MNPs property results in a typical absorption spectrum, widely exploited to realized plasmonic-based methods and assay (Della Pelle & Compagnone, 2018; Jain et al., 2007; Özyürek, Güngör, Baki, Güçlü, & Apak, 2012; Palazzo et al., 2012; Petryayeva & Krull, 2011; Scarano, Pascale, & Minunni, 2017; Vasilescu, Sharpe, & Andreescu, 2012; Vilela, González, & Escarpa, 2012; Vilela, González, & Escarpa, 2015; Willets & Van Duyne, 2007). MNPs-based methods turn out to be extremely modular and tunable towards specific analytes. In fact, changes in metal source, MNPs shape, MNPs dimensions, or capping agent employed and reaction media, lead to substantial reactivity changes able to elicit (towards target analytes) or avoid (towards potential interfering compounds) a specific class of compounds reactivity. Recently, our group have pointed out the capacity of PCs (Della Pelle et al., 2015; Della Pelle et al., 2015; Della Pelle et al., 2018) and SGs (Della Pelle, Scroccarello, Scarano, & Compagnone, 2018) to form MNPs, thanks to their reducing power and capability to support/stabilize the MNPs formation. The study here reported aims to demonstrate the suitability of two assays based on the selective formation of different metal nanoparticles, to assess the polyphenols antioxidant capacity and the total sugars in apples. The reactivity of AgNPs selectively synthesized by sugars and AuNPs produced by polyphenols has been studied and the formed MNPs characterized. Hence, an AgNPs-based sugar quantification assays and an AuNPs-based polyphenols antioxidant capacity assay have been optimized and tested on 42 apple samples. The MNPs-based data were in good agreement with the analysis of sugar content and antioxidant capacity carried out by conventional methods. The final aim of this work is to demonstrate the real samples applicability of these MNPs-

inversely associated with the risk of developing diseases linked to oxidative stress (Del Rio et al., 2013; Di Mattia, Sacchetti, Mastrocola, & Serafini, 2017; Pandey & Rizvi, 2009; Zhang et al., 2015; Zhang & Tsao, 2016), offering, thus, protection against the development of cancer, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases (Del Rio et al., 2013; Joseph, Edirisinghe, & Burton-Freeman, 2016; Pandey & Rizvi, 2009; Serafini & Peluso, 2016). Beside the biological activity, PCs play other important roles in food due to their coloring properties, stabilizing effect towards oxidation reactions (Shahidi & Ambigaipalan, 2015) and antimicrobial properties against pathogenic and spoilage bacteria (Hayek, Gyawali, & Ibrahim, 2013). However, PCs content, depending on food type, processing and storage conditions, and end users, can account for either positive or negative features. PCs have been shown to contribute to the definition of food sensory characteristics, to strongly affect the rate of enzymatic browning (Persic, Mikulic-Petkovsek, Slatnar, & Veberic, 2017), to influence the emulsification processes and the physical and chemical stability of dispersed emulsified systems (Di Mattia et al., 2015; Giacintucci, Di Mattia, Sacchetti, Neri, & Pittia, 2016) and to interact with proteins. This latter phenomenon resulting from interactions of tannins with salivary proteins is responsible for astringency perception, formation of haze and precipitates in beverages, and inhibition of enzymes and reduced digestibility of dietary proteins (El Gharras, 2009; Soares, Brandão, Mateus, & de Freitas, 2017). Moreover, PCs in some plants can be used as maturation index due to their accumulation during ripening (Garrido et al., 2016). Official methods for the PCs quantification and their antioxidant capacity (AOC) evaluation, being the latter needed due to the multiple mechanisms of action of bioactive compounds, are not officially established, probably because a single method cannot cover all the desired functional proprieties that need to be assessed for a particular purpose (Della Pelle & Compagnone, 2018). PCs qualitative and quantitative evaluation is performed classically by high-performance liquid chromatography (HPLC) coupled with Uv-Visible (Uv-Vis), diode array (DAD), or mass spectrometry (MS) detector (Ignat, Volf, & Popa, 2011; Simeoni et al., 2018). Very often the PCs evaluation aim is to give an estimation of the PCs content or an overall AOC evaluation. Thus, FolinCiocalteu (FC) assay (Singleton & Rossi Jr, 1965), evaluation of the scavenging activity against radical compounds (DPPH• and ABTS• assays) (Brand-Williams, Cuvelier, & Berset, 1995; Re et al., 1999), reduction of metal ions (FRAP and CUPRAC assays) (Apak, Güçlü, Özyürek, & Karademir, 2004; Benzie & Strain, 1996) and competitive methods (ORAC and TRAP) (Cao, Verdon, Wu, Wang, & Prior, 1995; Miller, Rice-Evans, Davies, Gopinathan, & Milner, 1993), as well as electrochemical approaches (Del Carlo et al., 2012; Della Pelle & Compagnone, 2018; Hoyos-Arbeláez, Blandón-Naranjo, Vázquez, & Contreras-Calderón, 2018; Hoyos-arbeláez, Vázquez, & Contrerascalderón, 2017), are widely employed (Antolovich, Prenzler, Patsalides, McDonald, & Robards, 2002; Ignat et al., 2011; López-Alarcón & Denicola, 2013). Presence and concentration of SGs can also be used as a quality and process marker in foods. The quantity of SGs gives information on the suitability of cultivation practices, fruit maturation at harvest, postharvest and overall fruit quality (Alonso-Salces et al., 2005; Beckles, 2012; Carbone, Giannini, Picchi, Lo Scalzo, & Cecchini, 2011; Khanizadeh et al., 2008; Ma et al., 2014; Magwaza & Opara, 2015; Neri et al., 2016; Palazzo, Facchini, & Mallardi, 2012; Wu et al., 2007). Moreover, several reactions driven by sugars (e.g. fermentation, hydrolysis, caramelization, Maillard reactions, crystallization) may occur in food processing and affect products shelf-life. In addition, SGs content is strictly related to foods sweetness, caloric content and glycemic index or glycemic load with the latter repeatedly and independently associated with diabetes and other chronic diseases (Dhurandhar & Thomas, 2015; Scazzina et al., 2016). Much effort has been devoted to developing sugars content evaluation methods in foods. A classical empiric method for the evaluation 360

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desiccator in the dark until analysis. An aliquot of 0.5 g of freeze-dried sample was weighted and 5 mL of acetone/water solution (70:30 v/v) added. The mixture was homogenized with an Ultra-Turrax T18 basic homogenizer (IKAR Werke GmbH & Co. KG, Staufen, Germany) for 1 min at 13500 rpm. Thus, the solution was centrifuged for 5 min at 3000 rpm at +4 °C. After centrifugation, the supernatant was separated from the solid pellet and the recovered solution was filtered with a 0.45 μm MDI cellulose filters (Ambala, India). Finally, the supernatant was dried by rotavapor and recovered with 1 mL of methanol. Extracts from apples collected in 2018 have been prepared as follow: apples were preliminarily peeled, the core removed by a corer and diced (~1 cm3). An aliquot of 20 g was weighted and 100 mL of acetone/water solution (70:30 v/v) was added. The mixture was homogenized with Ultra-Turrax T18 basic homogenizer for 1 min at 13500 rpm and centrifuged for 5 min at 3000 rpm at +4 °C. The supernatant was, then, separated from the solid pellet and filtered with an MDI 0.25 μm PTFE filter (Ambala, India). Finally, the supernatant was dried by rotavapor and recovered with 1 mL of methanol. All the extracts were stored at −20 °C°C in the dark until analysis.

based assays, overcoming definitely the proof of applicability, in order to provide to the ‘foods science community’ new effective and easy to use tools able to asses sugars and polyphenols in apple samples. 2. Materials and methods 2.1. Reagents and stock solutions All the chemicals were of analytical reagent grade. Epicatechin, catechin, and phlorizin were purchased from Extrasynthese (Genay, France). Gallic acid, chlorogenic acid, fructose, glucose, sucrose, inositol, trehalose, mannitol, raffinose, sorbitol, xylitol, and xylose, Cetyltrimethylammonium chloride (CTAC; 25% in water), hydrogen tetracholoroaurate (HAuCl4·3H2O, 99.9%), silver nitrate (AgNO3, > 99%), 2,2-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), sodium hydroxide(NaOH), sodium carbonate (Na2CO3), Folin & Ciocalteu's reagent, sodium phosphate mono basic monohydrate ACS reagent (NaH2PO4·H2O) sodium phosphate dibasic anhydrous (Na2HPO4), methanol, acetonitrile and formic acid were purchased from Sigma-Aldrich (St Louis, MO, USA). Polyphenol standards stock solutions were prepared in methanol at a concentration of 1.0 × 10−2 mol L−1 and stored at −20 °C in the dark. Stock solutions of sugar standards were prepared in water at a concentration of 1.0 × 10−2 mol L−1 and stored at +4 °C in the dark. Glucose stock solutions were prepared 24 h before use and stored at +4 °C in the dark to allow mutarotation. Milli-Q water (18.2 MΩ) was used for all the experiments and stock solutions preparation.

2.5. Preparation of extracts for sugars analysis Extracts from apple samples were obtained according to Neri et al. (2016) with some modification. Apples were preliminarily peeled, the core removed by a corer and diced (~1 cm3). Each sample was weighted (5 g) and diluted with 20 mL of distilled water. The dispersion was finely ground and homogenized with an Ultra-Turrax T18 basic homogenizer (IKAR Werke GmbH & Co. KG, Staufen, Germany) for 2 min. The dispersion was preliminarily shaken for 20 min at +4 °C, then centrifuged at 5000 rpm for 10 min at +4 °C in a refrigerated centrifuge. Then, the supernatant was filtered through a 0.45 μm nylon filter (MDI, Ambala, India) and stored at +4 °C under dark conditions until use.

2.2. Apparatus Samples were shaken and centrifuged with an orbital shaker (SSL1, Stuart equipment, Belfast, UK) and an ALC4237R refrigerated centrifuge (ALC Intl., Cologno Monzese, Italy), respectively. For extracts drying a Rotavapor LABOROTA 4000, Heidolph Instruments (Schwabach, DE) was used. For silver nanoparticles assay the thermostated mixing was carried out with a VDRL 711/CT orbital shaker from Asal (Florence, Italy). For the gold nanoparticles assay, the reaction mix was heated in a water bath using a 720 D thermostat digital group (Asal, Italy). Absorbance measurements were performed using a JENWAY 6400 spectrophotometer from Barloworld Scientific (Staffordshire, UK,). Sugars were determined by ICS 3000 Ionic Chromatograph Dionex (San Donato Milanese, Italy) equipped with an ICS 3000 SP pump and an ICS 3000 ED detector. Transmission electron microscopy measurements (TEM) were carried out using a Zeiss EM10C transmission electron microscope. Apple samples were brought to dry in a ScanVac CoolSafe 95–15 Pro freeze dryer (Lynge, Denmark).

2.6. Total polyphenols determination (Folin-Ciocalteu method) 20 μL of a properly diluted apple extract or phenolic standard was added to 20 μL of Folin-Ciocalteu reagent and stirred for 3 min. Then 400 μL of sodium carbonate (Na2CO3, 7.5%) and deionized water were added up to the final volume of 1000 μL. The solution was stirred for 60 min, at room temperature in the dark and the total polyphenol was determined with a spectrophotometer by reading the absorbance at 760 nm. Gallic acid standard solutions were used to calibrate the method. 2.7. Radical scavenging activity determination (ABTS)

2.3. Samples

The total AOC was evaluated by the ABTS method. ABTS reagent stock solution was prepared according to Re et al. (1999). The radical ABTS·+ solution was diluted to reach 0.70 ( ± 0.02) absorbance value at 734 nm and directly used for the assay. An appropriate volume of sample or standard/phenolic compounds was diluted up to the final volume of 2000 μL with ABTS reagent; then the reaction mix was stored in the dark, at room temperature for 5 min. Afterward, the absorbance at 734 nm was determined and the sample or standard antioxidant mediated ABTS·+ shutdown was compared with the respective blank/ control prepared without phenolic compounds or sample addition. Gallic acid standard solutions were used to calibrate the method.

Apples of two different varieties (‘Golden Delicious’ and ‘Royal Gala’) were purchased in local markets from different batches and seasons. The sampled batch was selected in order to randomize the apple composition, trying to maximize the differences of color, size, weight and ripening degree. A total of 42 samples was collected in the year 2017 (samples from 1 to 28, Golden Delicious) and 2018 (samples from 29 to 35, Golden Delicious; samples from 36 to 42 Royal Gala). 2.4. Preparation of extracts for polyphenols analysis Extracts from apples collected in 2017 have been prepared as follows: apples were preliminarily peeled, the core removed by a corer and diced (~1 cm3) and immediately freeze-dried. To this aim, aliquots of apple cubes were preliminarily arranged in Petri dishes and frozen at −40 °C for 24 h. Apple freeze-drying was carried out for 72 h at a pressure of ≈ 0.5 hPa and by setting the shelves temperature at 0 °C. After freeze-drying samples were stored at room temperature in a

2.8. Ion chromatography Chromatographic analyses of glucose, fructose, sorbitol, sucrose, trehalose, and maltose were conducted by an ICS 3000 Ionic Chromatograph Dionex (San Donato Milanese, Italy). The chromatographic separation was performed with a CarboPac PA20 column (3 mm × 150 mm, Dionex) equipped with a guard column (CarboPac 361

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Fig. 1. (A) MNPs spectra obtained with fructose (50 μmol L−1), glucose (50 μmol L−1) and sorbitol (40 μmol L−1) subjected to the AgNPs-sugars assay. (B) MNPs spectra obtained with gallic acid (10 μmol L−1), chlorogenic acid (10 μmol L−1) and epicatechin (5 μmol L−1) subjected to the AuNPs-polyphenols assay. Inset to each figure is reported the colorimetric response obtained with increasing concentration of fructose (A) and chlorogenic acid (B), corresponding to AgNPs (A) and AuNPs (B) colloidal solutions, respectively.

PA20, 3 mm × 30 mm, Dionex) according to Neri, Hernando, PérezMunuera, Sacchetti, and Pittia (2011) with some modifications. The following work condition was used: NaOH 5.0 × 10−2 mol L−1 as mobile phase, a flow rate of 0.5 mL min−1, a 35 min run at a column temperature of 30 °C, and a volume of injection of 10 μL. The sugar detection was performed using the time/potential waveform A, as recommended by the Dionex technical manual. Sugars identification and quantification were carried out using retention times and the related sugar calibration curve, respectively.

with an orbital shaker, followed by heating for 10 min in a water bath at 45 °C; the reaction was then blocked in ice for 5 min. Finally, the absorbance of the newly formed AuNPs was recorded at 540 nm. All the measures were conducted against a blank (reaction mix without the reducing agent). Moreover, for each sample extract, a dose-response curve was obtained by adding an increasing amount of extract and reading the absorbance at the LSPR maximum. Gallic acid standard solutions were used to obtain a calibration curve and quantification purposes.

2.9. Silver nanoparticles assay (AgNPs-sugars)

2.11. AgNPs-sugars and AuNPs-polyphenol assays recovery and potential interfering compounds

An AgNPs-based colorimetric assay (AgNPs-sugars) was used for the total sugars quantification, according to Della Pelle, Scroccarello, Scarano, and Compagnone (2018) with some modifications. All the reagents of the reaction were used at room temperature. For the AgNPs formation were used 5 μL of CTAC (1.0 × 10−3 mo L−1), 25 μL of an AgNO3 solution (2.0 × 10−2 mol L−1), appropriate dilution of sugars standard or sample and finally 10 μL NaOH (5.0 mol × L−1) to start the reaction. Then, the reaction mix was brought to 500 μL with water. The AgNPs formation was then carried out at 25 °C for 10 min under stirring in a thermostated orbital shaker. Finally, the reaction was stopped in ice where it was kept for 10 min. The absorbance due to the AgNPs formed was recorded at 430 nm. All the measurements were carried out against the blank (reaction mix without standard or sample reacted). For each sugar standard and sample extract, a dose-response curve was obtained by adding increasing amounts of standards/extract and reading the absorbance at the LSPR maximum. Glucose standard solutions were used to obtain a calibration curve and quantification purposes.

A mix of the two apple samples extract of a different variety was spiked with SGs and PCs standards in order to obtain solutions of 20, 40 and 60 μmol L−1 and 5, 10 and 10 μmol L−1, respectively. The fortified samples were separately subjected to the AgNPs-sugars and AuNPspolyphenols assay and compared with the relative non-spiked samples. Recoveries were calculated using the following eq. (1): [(analyte concentration fortified sample − analyte concentration unfortified sample)/analyte concentration added] × 100. The possible interference effects of apple extract components such as sugars, polyols, polyphenols, and organic acids were studied. To this aim different amounts of the possible interfering compounds were spiked on samples to evaluate the effect on the respective MNPs assay. The concentration of the interferents tested were chosen according to literature data (Alonso-Salces et al., 2005; Carbone et al., 2011; Hecke et al., 2006; Khanizadeh et al., 2008; Ma et al., 2014; Neri et al., 2016; Palazzo et al., 2012; Wu et al., 2007). 3. Results and discussion

2.10. Gold nanoparticles assay (AuNPs-polyphenols) 3.1. Reactivity of sugars (AgNPs) and polyphenols (AuNPs) standards towards metal nanoparticles formation

AuNPs based colorimetric assay (AuNPs-polyphenols) was used for the polyphenols AOC evaluation, according to Della Pelle, Vilela, et al. (2015), with some modifications. For the AuNPs formation 10 μL of CTAC (8.0 × 10−4 mol L−1) as capping agent, 25 μL of HAuCl43H2O solution (2.0 × 10−2 mol L−1) as gold source and an appropriate amount of polyphenol standard or sample extract in a final volume of 500 μL, reached with phosphate buffer solution (PB pH 8.0; 1.0 × 10−2 mol L−1) were used. The reaction mix was stirred for 2 min

The aim of this study was to use colorimetric assays based on AuNPs and AgNPs formation for the determination of SGs and polyphenolic AOC in apples. Thus, the reactivity of standard polyphenols and sugars was initially investigated. An example of the visible spectra obtained, following the AgNPsassay (Section 2.9) and AuNPs-assay (Section 2.10) protocols are 362

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Table 1 Analytical parameters obtained by sugars and polyphenols standards dose-response curves by using the AgNPs-sugars and AuNPs-polyphenols assays. Linear range

Linear equationa

Slopea

Intercepta

Determination coefficient

μM

y = Abs and x = μM

std.dev.

std.dev.

R2

AgNPs-sugars assay Glucose Fructose Sorbitol

10–90 10–90 20–80

y = 0.0098x + 0.0639 y = 0.0101x + 0.0421 y = 0.0100x + 0.0319

± 4.2 × 10−4 ± 5.3 × 10−4 ± 4.4 × 10−4

± 2.7 × 10−3 ± 2.2 × 10−3 ± 1.3 × 10−3

0.998 0.996 0.999

AuNPs-polyphenols assay Chlorogenic acid Epicatechin Gallic acid

3–25 1–7.5 1–15

y = 0.0204x − 1.813 y = 0.0229x − 1.012 y = 0.0216x − 0.9237

± 1.2 × 10−3 ± 1.3 × 10−3 ± 1.1 × 10−3

± 1.1 × 10−1 ± 6.1 × 10−2 ± 4.6 × 10−2

0.992 0.995 0.991

a

Mean value of three dose-response curves.

Fig. 2. MNPs spectra obtained with increasing amount of apple extract subjected to the AgNPs-sugars (A) and AuNPs-polyphenols (B) assays. Inset to each figure at the left is reported the colorimetric response corresponding to each spectrum. Apple extracts volume employed: (A) 5 (a), 10 (b), 15 (c) μL of sample 11 diluted 100fold; (B) 20 (a), 40 (b), 60 (c) μL of sample 11 diluted 2-fold. Inset to each figure at the right is reported a TEM micrograph corresponding to the AgNPs (A) and AuNPs (B) formed with the respective assay.

and easier, the usability of rapid and effective assays, based on the MNPs formation, have been demonstrated. Thus, to prove the exploitability of the AgNPs-sugars and the AuNPs-polyphenols assays, 42 different apple samples (Section 2.4) and different procedures of extraction/pretreatment (2.5 and 2.6) have been carried out. The TEM micrographs reported corresponding in Fig. 2 are related to AgNPs (Fig. 2A, right inset) and AuNPs (Fig. 2B, left inset) formed using directly apple extracts, following the reported procedures. Sugars from apples were able to generate well-defined and dispersed spherical AgNPs (ϕ < 10 nm); phenolic compounds, as well, gave rise to welldefined and dispersed spherical AuNPs with a ϕ < 25 nm. Fig. 2 also reports the absorbance spectra and the corresponding colloidal suspensions obtained by increasing amounts of apple extracts, for both the AgNPs (Fig. 2A) and the AuNPs assay (Fig. 2B). These spectra clearly demonstrate that sugars and polyphenols in apples are able to generate quantitatively AgNPs and AuNPs, respectively. The apple extracts produced AgNPs and AuNPs having the maximum absorbance intensity band (LSPRmax) at 430 ± 9 nm and 540 ± 7 nm, respectively, thus, totally in accordance with the LSPRmax obtained by the pure compounds reported in Section 3.1. This allowed fixing the reading at 430 (AgNPs-sugars) and 540 nm (AuNPs-polyphenols) avoiding the run of the absorbance spectrum for each sample. To further confirm the ability of the proposed MNP-based assays to assess, in a quantitative way, SGs and PCs compounds, a mix of different extracts of apples have been spiked with different amount of fructose and glucose in the 20–60 μmol L−1 range and with chlorogenic

reported in Fig.1, together with a picture of the suspensions obtained by increasing concentrations of fructose and chlorogenic acid (insets). The LSPR maximum (LSPRmax) obtained (employed as analytical signal) for the AgNPs and AuNPs generated was in all the cases 430 ± 10 nm (A430) and 540 ± 10 nm (A540), respectively, in accordance with the literature (Della Pelle, Scroccarello, Scarano, & Compagnone, 2018; Della Pelle, Scroccarello, Sergi, et al., 2018). Table 1 reports the analytical parameters obtained by the dose-response curves, achieved with an increasing concentration of SGs and PCs vs. the respective MNPs LSPRmax. In all the cases, a reproducible (RSD ≤ 6%, n = 3) linear dose-response range was recorded, with a good determination coefficient (R2 ≥ 0.991). Both assays exhibited a quite low limit of detections (LOD) of ≤8.7 μmol L−1 and ≤ 3.3 μmol L−1 for the AgNPs-sugar and AuNPs-polyphenols assays, respectively. Moreover, the MNPs suspensions result stable for 60 min at room temperature, instead if stored in ice the stability reach the 6 h. This can be attributed to the synergistic stabilization of analytes and capping agent resulted in MNPs formation in mild conditions, with no aggregation, collapse, and precipitation. These phenomena are typical drawbacks MNPs-based assays when absorbance at LSPRmax is used as analytical signal. 3.2. AgNPs-sugars and AuNPs-polyphenols assays applied to apple extracts: MNPs characterization, recovery and interferents studies In order to make the apple polyphenols and sugars analysis faster 363

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Table 2 Results of phenolic content and antioxidant properties evaluated by the AuNPs-polyphenols assay, ABTS and FC method of 42 apple extracts. Sample

AuNPS-polyphenols (mg Kg

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 a

−1 a

)

131.6 ± 6.0 127.8 ± 6.3 97.3 ± 9.5 130.0 ± 9.2 109.8 ± 5.0 120.0 ± 5.7 138.5 ± 6.6 94.1 ± 4.3 129.4 ± 7.0 128.8 ± 6.9 155.8 ± 7.1 110.1 ± 5.1 127.9 ± 6.2 132.6 ± 7.1 162.9 ± 23.0 172.2 ± 9.8 143.4 ± 7.8 184.2 ± 9.9 152.0 ± 12.3 164.8 ± 7.5 157.7 ± 12.7 91.0 ± 7.4 142.3 ± 8.2 144.2 ± 6.5 136.6 ± 7.3 141.9 ± 9.5 164.8 ± 9.6 108.2 ± 5.9 112.3 ± 7.1 107.6 ± 5.4 100.8 ± 6.4 134.6 ± 4.7 128.9 ± 3.3 149.6 ± 3.6 156.3 ± 2.9 170.8 ± 3.9 160.0 ± 5.8 99.1 ± 5.3 125.8 ± 3.6 127.6 ± 11.7 154.5 ± 11.3 128.9 ± 4.2

ABTS

Folin-Ciocalteu −1 a

RSD (%)

(mg Kg

)

3.0 5.9 7.7 2.4 6.0 7.2 4.2 7.9 5.1 2.9 6.4 2.4 3.1 5.0 3.6 2.4 2.9 3.1 3.9 1.5 3.4 2.6 1.9 3.1 2.4 1.7 1.6 2.6 1.1 0.5 0.5 1.4 3.8 2.4 1.8 2.3 1.2 2.5 0.3 0.7 2.0 2.2

155.8 ± 4.7 120.1 ± 7.1 92.3 ± 7.1 142.0 ± 3.4 111.4 ± 6.7 118.3 ± 8.6 132.9 ± 5.6 83.5 ± 6.6 144.5 ± 7.4 155.5 ± 4.5 174.3 ± 11.1 105.8 ± 2.5 159.1 ± 4.9 144.8 ± 7.3 201.6 ± 7.2 197.9 ± 4.7 174.4 ± 5.1 201.2 ± 6.3 161.1 ± 6.3 197.3 ± 2.9 159.8 ± 5.4 98.4 ± 2.6 162.2 ± 3.1 153.2 ± 4.7 145.7 ± 3.5 171.9 ± 2.9 165.7 ± 2.6 111.9 ± 2.9 103.5 ± 1.1 106.5 ± 0.6 87.9 ± 0.5 119.2 ± 1.7 114.7 ± 4.3 153.2 ± 3.7 157.7 ± 2.0 182.0 ± 4.2 166.4 ± 2.0 102.0 ± 2.6 118.6 ± 0.3 126.7 ± 0.9 148.3 ± 3.0 126.7 ± 2.8

RSD (%)

(mg Kg−1)a

RSD (%)

4.5 4.9 9.7 7.1 4.5 4.8 4.7 4.6 5.4 5.3 4.6 4.6 4.9 5.4 14.1 5.7 5.5 5.4 8.1 4.6 8.1 8.1 5.7 4.5 5.3 6.7 5.8 5.5 6.3 5.0 6.3 3.5 2.5 9.8 1.8 2.3 3.6 5.3 2.9 9.2 7.3 3.2

167.8 ± 5.4 117.4 ± 8.3 73.6 ± 6.0 171.2 ± 11.5 156.3 ± 10.6 138.5 ± 6.5 137.6 ± 9.6 83.3 ± 4.4 162.4 ± 1.6 177.9 ± 10.9 193.5 ± 11.6 153.9 ± 1.6 177.9 ± 11.5 170.1 ± 9.5 261.0 ± 13.7 194.4 ± 11.4 229.8 ± 6.4 227.2 ± 5.1 155.1 ± 5.1 206.0 ± 9.5 204.6 ± 9.0 78.8 ± 2.8 155.4 ± 6.4 143.1 ± 4.1 172.4 ± 12.4 184.5 ± 6.7 229.0 ± 11.8 91.4 ± 4.7 106.1 ± 6.8 103.3 ± 0.3 95.2 ± 1.7 96.0 ± 1.3 87.6 ± 2.7 107.2 ± 6.3 204.7 ± 5.3 177.7 ± 13.3 207.6 ± 7.7 90.8 ± 2.4 84.3 ± 3.5 85.0 ± 3.2 159.3 ± 5.8 82.1 ± 1.5

3.2 7.1 8.1 6.7 6.8 4.7 7.0 5.3 1.0 6.1 6.0 1.1 6.4 5.6 5.2 5.9 2.8 2.2 3.3 4.6 4.4 3.6 4.1 2.9 7.2 3.6 5.1 5.1 6.4 0.3 1.8 1.4 3.1 5.8 2.6 7.5 3.7 2.6 4.1 3.8 3.6 1.9

Mean value, n = 3. The results are expressed with respect to the weight of fresh apple.

acid and epicatechin in the 5–10 μmol L−1 range. Satisfactory recovery values (from 91% to 113.7%) were obtained. Selectivity of the assays was evaluated by testing different potential interfering compounds. The type and concentration of the interfering compounds was chosen according to literature data (Alonso-Salces et al., 2005; Carbone et al., 2011; Hecke et al., 2006; Khanizadeh et al., 2008; Ma et al., 2014; Neri et al., 2016; Palazzo et al., 2012; Wu et al., 2007). The AuNPs-polyphenols assay was tested against different sugars (fructose, glucose, sucrose, inositol, trehalose, mannitol, raffinose, sorbitol, xylitol, and xylose) in a broad concentration range (from 5.0 × 10−4 mol L−1 to 1.5 mol L−1). None of the reported sugars was able to form AuNPs in the experimental conditions used. In addition, the possible sugars ‘perturbation’ during the AuNPs formation was also studied using real samples. To this aim, two apple extracts belonging to different varieties, were mixed and spiked with chlorogenic acid to obtain a final concentration of 50 μmol L−1; the mix was further fortified with fructose, glucose, sucrose and their mix (60% fructose, 30% glucose, 10% sucrose) in a 5.0 × 10−4 mol L−1 to 1.5 mol L−1 concentration range. None effect, on the obtained colorimetric response, was again observed on the AuNPs assay. The selectivity of the AuNPs-based assay towards polyphenols was achieved using the selected medium of reaction. In fact, at pH 8.0 sugars are not ionized; ionization in strong alkaline conditions, for these compounds,

is required to reduce the Au3+ and at the same time surround/stabilize the formed AuNPs, in the times required by this assay. This behavior is related to the synergic action of sugars and capping agent (CTAC), that are able to act as ‘charge carriers’, around the AuNPs, because of their ionizable moieties. These results confirm the data reported by different authors (Della Pelle, Scroccarello, Sergi, et al., 2018; Filippo, Serra, Buccolieri, & Manno, 2010; Palazzo et al., 2012). It should be also noticed that above pH 10 in the medium used for the assay, the AuNPs are formed without the use of external reducing compounds, impairing the possible quantification of sugars. Ascorbic acid in principle can form MNPs due to the reducing activity, however, both AuNPs and AgNPs based assays, have been optimized in order to react, with sugars and polyphenols, respectively, and, thus, the effect is minimal. Preliminary experiments run with AuNPs assay have demonstrated that reactivity of ascorbic acid is about 250 times lower than polyphenols (e.g. chlorogenic acid). The content of ascorbic acid in our sample extracts (measured on 10 random samples) was found in each case below 50 ppm. Thus, considering concentration and reactivity ascorbic acid does not interfere in the AuNPs based analysis of polyphenols. The same considerations apply for AgNPs assay for sugars since the difference in concentration of analytes is much larger. The AgNPs-sugars assay was tested against polyphenols. 364

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3.3. Comparison with ABTS, FC and ion chromatography

Table 3 Results of sugars content by the AgNPs-based assay and ion chromatography of 42 apple extracts. Sample

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

AgNPS-sugars

Ion chromatography

Relative error

(g Kg−1)a

RSD (%)

(g Kg−1)a

RSD (%)

(%)b

146.3 ± 2.4 129.3 ± 2.7 60.1 ± 1.2 155.4 ± 2.2 129.0 ± 4.1 166.9 ± 0.5 181.2 ± 3.4 129.2 ± 0.8 89.3 ± 4.7 122.9 ± 3.5 87.5 ± 1.3 141.2 ± 4.0 212.0 ± 3.2 133.9 ± 1.1 149.8 ± 0.4 107.6 ± 6.2 131.1 ± 3.3 140.7 ± 2.1 182.9 ± 2.4 189.9 ± 1.4 150.2 ± 2.5 185.8 ± 0.4 119.1 ± 2.3 121.4 ± 3.8 144.0 ± 1.9 161.4 ± 3.3 184.8 ± 0.5 98.5 ± 0.5 198.4 ± 2.3 173.5 ± 3.4 250.1 ± 1.5 135.2 ± 4.2 170.3 ± 2.1 165.4 ± 1.9 124.8 ± 3.7 88.1 ± 7.7 109.5 ± 2.7 181.3 ± 1.7 164.4 ± 1.1 177.7 ± 3.6 108.5 ± 2.4 191.8 ± 3.3

3.5 3.5 0.7 3.4 5.4 0.8 6.1 1.0 4.2 4.3 1.0 5.7 6.9 1.5 0.6 6.7 4.3 3.0 4.4 2.6 3.7 0.8 2.7 4.6 2.7 5.4 0.9 0.5 4.7 5.9 3.8 5.6 3.5 3.2 4.6 6.8 2.9 3.1 1.9 6.4 2.6 6.2

136.5 ± 1.6 130.2 ± 1.8 77.6 ± 4.1 146.7 ± 1.4 128.7 ± 0.9 149.6 ± 1.6 179.5 ± 1.1 122.1 ± 3.0 85.4 ± 5.1 147.8 ± 2.9 105.3 ± 6.4 165.3 ± 2.4 198.5 ± 3.1 127.0 ± 2.3 150.3 ± 2.2 97.5 ± 4.1 122.6 ± 2.1 137.8 ± 1.5 193.3 ± 1.7 195.5 ± 1.5 138.2 ± 1.0 168.5 ± 1.2 144.1 ± 1.4 148.7 ± 1.7 177.7 ± 1.7 161.0 ± 2.5 179.0 ± 0.6 107.7 ± 2.4 206.4 ± 1.3 184.0 ± 1.7 245.3 ± 1.1 149.3 ± 2.0 168.4 ± 1.1 198.3 ± 1.4 126.8 ± 2.5 124.6 ± 2.0 129.5 ± 2.5 195.7 ± 1.1 149.8 ± 1.8 152.4 ± 1.7 107.2 ± 2.0 201.3 ± 0.8

2.2 2.4 3.2 2.0 1.1 2.5 1.9 3.7 3.3 1.0 1.7 2.6 2.6 3.0 3.3 4.0 2.6 2.0 3.2 3.0 1.4 2.0 2.0 2.5 3.0 4.0 1.0 2.5 2.7 3.2 2.6 3.0 1.8 2.8 3.2 2.5 3.3 2.1 2.8 2.6 3.2 1.6

7.2 +0.7 −22.6 +5.9 +0.3 +11.6 +1.0 +5.8 +4.5 −16.8 −26.4 −14.6 +6.8 +5.4 −0.3 +10.3 +6.9 +2.1 −5.4 −2.9 +8.6 +10.2 −17.3 −18.4 −18.9 +0.3 +3.2 −8.5 −3.9 −5.7 +2.0 −9.4 +1.2 −16.6 −1.6 −29.3 −15.4 −7.3 +9.8 +16.6 +1.3 −4.7

The quantification of the sugars and the polyphenolic AOC evaluation was then carried out on 42 samples of apples by applying both the MNPs-based proposed assays and the conventional methods. Table 2 reports the concentration of polyphenols obtained from the analyses performed by the different methods expressed as gallic acid equivalents. According to the AuNPs-based assay the polyphenolic content ranged from 91.9 to 230.8 mg Kg−1; good reproducibility has been obtained for all the samples (RSD ≤ 7.9%). Using the FC assay, usually used to obtain the total PCs content, data ranged from 73.5 to 261.0 mg Kg−1 (RSD ≤ 8.1%). The ABTS assay, related to the scavenging activity of the apple extracts, gave data from 83.5 to 201.6 mg Kg−1 with acceptable reproducibility (RSD ≤ 14.1). Table 3 reports the data obtained with the AgNPs-sugars assay and the ion chromatographs, being in the first case the sugar content expressed as glucose equivalents. The analysis carried out by the ion chromatography highlighted that in all the samples the sugars found in higher concentration are glucose and fructose (their sum 75–85%); lower amounts were determined for sucrose (10–20%), trehalose and sorbitol (their sum 2–5%) (data not shown). In Table 3 the data have been expressed as the sum of the concentration of all the mono- and disaccharides, each one calculated by the relative calibration curve. The data of the AgNPs-sugars assay and the ion chromatography ranged between 60.1 and 250.1 g Kg−1 and 77.6–245.3 g Kg−1, respectively. The proposed AgNPs-sugars method results reproducible (RSD ≤ 6.9%), as well as, as expected, ion chromatography (RSD ≤ 4%). Overall, the obtained data are in agreement with the SGs and PCs content in apples reported in the literature (Alonso-Salces et al., 2005; Carbone et al., 2011; Khanizadeh et al., 2008; Ma et al., 2014; Neri et al., 2016; Wu et al., 2007). Linear correlations among the methods, employed for the polyphenols and sugars evaluation, are reported in Fig. 3. The AuNPspolyphenols assay results well correlated with the ABTS method (R = 0.922) (Fig. 3A), while a lower correlation was found with the FC method (R = 0.764). Hence, the AuNPs-assay gives an estimation of the antioxidant capacity, and this ability is related to the intrinsic mechanism of formation and stabilization of the AuNPs, that results related to the polyphenols quantitative and qualitative (intrinsic antioxidant power) composition. The relationship of the results of the FC method vs. those obtained by the AuNP-based assay, as well as for the ABTS assay, results in a slight data overestimation and this behavior was already reported in the literature (Escarpa & Gonzalez, 2010; Escarpa & González, 2001; Sanchez-Rangel, Benavides, Heredia, Cisneros-Zevallos, & Jacobo-Velazquez, 2013). FC is definitively nonselective and in apples, the overestimation can be attributed to the reducing sugars presents. Fig. 3B shows the linear correlation trend between the data obtained by AgNPs-based assay and those by ion chromatography. The AgNPssugars assay presented a good correlation with the ion chromatographic data (R = 0.915), as well as a good quantitative correspondence between the obtained data with a relative error between +11.6 and −29.3% (Table 3). Despite the different principle of the methods, the AgNPs-assay is able to allow a quantitative estimation of the total sugars present in samples. Among the MNPs-based methods, none of them can be used for sugars contents evaluation. Several methods, both for polyphenols and sugars analysis need high temperatures, long times, large volumes of sample and solvent. Moreover, many methods require a seed-mediated MNPs growth that requires different steps, which greatly complicates the application. For these reasons, very few are the methods easily exploitable directly for the evaluation of food constituents by not expert personnel. Moreover, the employed MNPs-based methods require low amount of sample (from 2.5 to 60 μL of apple extract), do not require particular solvents (apart the solvent used for the extraction) neither the use of radicals. The equipment needed is very simple as a photometer and the colorimetric output allows easily a

a Mean value, n = 3. The results are expressed with respect to the weight of fresh apple. b Relative error calculated using the data obtained with ion chromatography as the true value.

Chlorogenic acid, catechins and phlorizin, and their equimolar mix have been tested, with a similar approach on the apple sample extract (5.0 × 10−4–3.0 mmol L−1 range); also in this case, no interference was found. Indeed, the ratio between sugars and antioxidant compounds in apples results strongly in favor of sugars (750/1000 fold) resulting in high selectivity vs. polyphenols. These results confirm the ability of AgNPs to be formed by sugars in a selective way even in a complex matrix (Della Pelle, Scroccarello, Scarano, & Compagnone, 2018). Eventually, the influence of organic acids present in apples has been evaluated in both assays by adding malic acid, succinic acid, citric acid, tartaric acid, oxalic acid, quinic acid, and shikimic acid. In all the cases these compounds resulted not able to form MNPs or to interfere with the assays proposed, taking into account the concentrations found in apples and reported in the literature (Atona & Ova, 2004; Hecke et al., 2006; Samukelo et al., 2006; Wu et al., 2007).

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Fig. 3. (A) Correlation curve between the data obtained, analyzing the 42 apple extracts, with the proposed AuNPs-polyphenols assay and ABTS method (y = 0.662x + 40.69; R = 0.922). (B) Correlation curve between the data obtained, analyzing the 42 apple extracts, with the proposed AgNPs-sugars assay and ion chromatography (y = 0.986x − 2.17; R = 0.915).

qualitative assessment. Furthermore, the AgNPs-based sugars evaluation requires 5 min vs. ion chromatography that needs around 1 h. On the other hand, the AuNPs-based method for polyphenols evaluation requires an analysis time of 10 min, comparable with the ABTS assay (5 min), and is significantly faster with respect to FC (about1 h). Furthermore, in term of sensitivity, robustness, and reproducibility the proposed assays appear totally comparable with the existing methods or result even more sensitive as for the AgNPs-based assay with respect to ion chromatography. Finally, it should be pointed out that the whole assays take place directly in an Eppendorf tube and that the reagents used are stable and non-volatile. These features can allow the development of partially automated assay kits for on-site detection.

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4. Conclusions In this study, the ability of metal nanoparticles to act as optical nanoprobes, able to allow rapid and efficient detection of the total sugars and polyphenol antioxidant capacity in apples has been demonstrated. In particular, the evaluation of SCs and polyphenols AOC have been carried out by using the formation of AuNPs and AgNPs with no problems of selectivity. MNPs assays reactivity has been compared with the results obtained by conventional methods, and the AgNPssugars assay gives comparable results (R = 0.915) to ion chromatography when the total sugars concentration is taken into account while the AuNPs-polyphenols assay is able to assess the polyphenols antioxidant capacity of apple extracts, with a significant correlation with the results obtained by the ABTS method (R = 0.922). The ease of formation of these nanoprobes (mild condition, 5–10 min) and the simplicity of the detection (absorbance read at a fixed wavelength), makes these probes attractive in industrial applications for quality control. In addition, the proposed MNPs do not require radical compounds, and the solvent use and waste produced are significantly lower compared to classical methods. This approach appears, thus, promising for rapid monitoring of AOC and SCs in food. References Alonso-Salces, R. M., Herrero, C., Barranco, A., Berrueta, L. A., Gallo, B., & Vicente, F. (2005). Classification of apple fruits according to their maturity state by the pattern recognition analysis of their polyphenolic compositions. Food Chemistry, 93(1), 113–123. https://doi.org/10.1016/j.foodchem.2004.10.013. Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods for testing antioxidant activity. The Analyst, 127, 183–198. https://doi.org/ 10.1039/b009171p. Apak, R., Güçlü, K., Özyürek, M., & Karademir, S. E. (2004). Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion

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