A sensitive triple colorimetric sensor based on plasmonic response quenching of green synthesized silver nanoparticles for determination of Fe2+, hydrogen peroxide, and glucose

Accepted Manuscript Title: A sensitive triple colorimetric sensor based on plasmonic response quenching of green synthesized silver nanoparticles for determination of Fe2+ , hydrogen peroxide, and glucose Authors: Sedigheh Basiri, Ali Mehdinia, Ali Jabbari PII: DOI: Reference:

S0927-7757(18)30142-0 https://doi.org/10.1016/j.colsurfa.2018.02.053 COLSUA 22305

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

16-12-2017 5-2-2018 20-2-2018

Please cite this article as: Basiri S, Mehdinia A, Jabbari A, A sensitive triple colorimetric sensor based on plasmonic response quenching of green synthesized silver nanoparticles for determination of Fe2+ , hydrogen peroxide, and glucose, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2010), https://doi.org/10.1016/j.colsurfa.2018.02.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A sensitive triple colorimetric sensor based on plasmonic response quenching of green synthesized silver nanoparticles for determination of Fe2+, hydrogen peroxide, and glucose

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Sedigheh Basiria, Ali Mehdiniab,*, Ali Jabbaria

Department of Chemistry, Faculty of Science, K. N. Toosi University of Technology, Tehran,

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Department of Marine Living Science, Ocean Sciences Research Center, Iranian National

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Institute for Oceanography and Atmospheric Science, Tehran, Iran

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Corresponding author. Tel.: +98 21 66944873; fax: +98 66944869.

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Graphical abstract

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E-mail address: [email protected] (A. Mehdinia).

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Abstract A new strategy based on Fenton reaction coupled by silver nanoparticles (AgNPs) was designed to establish a multifunctional platform for highly sensitive colorimetric determination of Fe2+, H2O2 and glucose. For this purpose, AgNPs were synthesized in a green manner using agar and ascorbic acid and then utilized as a colorimetric sensor.

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The UV-Vis spectroscopy, DLS, FTIR, EDX, XRD and TEM were used to characterize the green synthesized AgNPs. Addition of H2O2 to the mixture of Fe2+ and AgNPs caused the production of hydroxyl radical (OH•), oxidation of AgNPs and discoloration of the solution. Changing the amount of Fe2+ affected on discoloration of the solution, while other metallic ions did not show such this effect. So, a selective colorimetric sensor with a detection limit of 0.54 µM was provided for Fe2+ measuring. On the other hand, the concentration of H2O2 affected both on the

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oxidation of AgNPs and discoloration of the solution. These conditions was used to measure H2O2 with a LOD as

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low as 0.032 µM. Owing to the fact that glucose oxidase produces H2O2 from glucose, this system was also applied for determination of glucose. LOD of the method for glucose detection was 0.29 µM. Glucose determination in

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human blood serum sample was performed using the fabricated sensing system.

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Keywords: Silver nanoparticles, colorimetric sensor, Fenton reaction, Iron (II), glucose

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1. Introduction It is broadly admitted that Fe is one of the most abundant detected elements that belongs to the fundamental transition metal ions, and has a critical role in cellular metabolism, DNA synthesis, oxygen transport and formation of some neurotransmitters and hormones. Nevertheless, excess amount of free iron in the cells can result oxidation

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and damage of the proteins, nucleic acids and lipids. On the other hand, deficiency of Fe can cause anemia, liver and kidney injuries, heart illnesses, and diabetes. Hence, it is essential to develop the facile and accurate methods for detection of iron [1-6].

According to the previous studies, determination of iron has been presented using various analytical techniques, which include high performance liquid chromatography [7], anodic stripping voltammetry [8], inductively coupled

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plasma-mass spectrometry [9, 10], atomic absorption spectrometry (AAS) [11, 12], fluorescent spectroscopy [13,

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14] and etc. Although these techniques represented good accuracy and sensitivity, their application is limited

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because of their inescapable restrictions such as high cost, complex instrumentations, complicated pretreatment and

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severe interferences of different cations [15]. Additionally, many of these analytical techniques determine the total iron amount. However, it is clear that the distinction of Fe2+ and Fe3+ is highly important in order to apperceive the

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biological activities of iron, because the Fe2+/Fe3+ pairs are one of the valuable redox states in the biological systems. Therefore, as adequate information to perceive the biological activity of iron is not presented by using total

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iron amount, speciation techniques are necessary to specifically recognize Fe2+ [16-21].

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Due to the mentioned deficiencies of the instrumental analytical methods, colorimetric sensors based on plasmonic AgNPs demonstrate an efficient substitute for determination of metal ions [22-26]. They have achieved increasing

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consideration owing to benefits such as simplicity of construction, quickness, easily data attainment by naked eyes, cost-effectiveness, no necessity to any complicated instrument and multi-analyte detection capability [27-29]. It

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should be considered that although colorimetric sensors have been highly developed, to the best of our knowledge, the sensitive determination of Fe2+ and H2O2 based on plasmonic AgNPs has not been reported yet, and the previous works have been focused on other types of sensors for Fe2+ determination [30, 31]. From the other point of view, Fe3+ interferes in the detection of Fe2+ was not studied in many of the works and only a few sensors have been reported which allow selective determining of Fe2+ [15, 30, 32]. Hence, developing the probes with Fe2+ determination capability is favorable and as yet considered as a challenge.

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Hydrogen peroxide (H2O2) is an essential significant chemical and it is broadly used in different industrial processes including the textile industry, food processing, water treatment and medical and pharmaceutical aims. Additionally, H2O2 has a substantial role in various cellular biochemical procedures [33]. High amounts of H2O2 can cause cellular harm [34, 35], therefore the precise determination of H2O2 in trace concentrations is essential. Various analytical

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methods, such as spectrophotometry [36], spectrofluorometry [37], chemiluminescence [38], chromatography [39] and electrochemistry [40], were commonly used for detection of H2O2. In spite of the sensitivity and selectivity of these instrumental methods, they are usually complex, costly and not portable. Therefore, a facile, fast, inexpensive, and also portable approach for H2O2 detection is preferable. Moreover, H2O2 can be produced as a main product of the oxidation of glucose with glucose oxidase. As a result, this reaction demonstrates a chance for indirectly quantification of glucose [41, 42]. Glucose is a very important molecule for living cells, with a main role in the

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metabolism. The blood or urine glucose level is one of the important indicators of diabetes. Thus, rapid glucose

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detection has a critical importance in medical and food industries [43]. Therefore, glucose dtetermination has been

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done by some methods such as electrochemical approaches [44, 45].

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Recently, it has been shown that H2O2 cannot lead to oxidation and color change of AgNPs, and only can regenerate AgNPs [46, 47]. On the other hand, in 1894, a chemist, named Fenton, discovered that the simultaneous presence of

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H2O2 and Fe2+ could oxidize contaminants or waste waters as follows: 𝐻2 𝑂2 + 𝐹𝑒 2+ → 𝑂𝐻 • + 𝑂𝐻 − + 𝐹𝑒 3+

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During this reaction, the hydroxyl radical (OH•) is created [48, 49], that has the oxidation potential of 2.73 V. This high redox potential causes the oxidizing ability of OH • to be higher than H2O2 [50, 51]. In this regard, it is possible

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that the generated OH• radicals from Fenton reagents can oxidize AgNPs, change the color of AgNPs solution, and consequently detect Fe2+ and H2O2.

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Recently, green synthesis methods using biological materials have been used for the preparation of metal nanoparticles which have three main stages including (1) selection the type of solvent, (2) selection of an ecofriendly reducing agent, and (3) selection of a nontoxic stabilizer [22]. Although, many bacteria, fungi, and plant extracts are known as the main agents for metal NPs synthesis [52], biopolymers, especially the polysaccharides, have emerged in this field as a new chance [53]. Agar, a polysaccharide extracted from the marine red algae, such as

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Gelidium and Gracilaria spp, is a promising natural biopolymer [54, 55]. There are a few available reports for the production of AgNPs using biopolymer agar. In the first study, AgNPs had an intense band at 421 nm, but conglomeration of AgNPs was observed in the synthesized nanoparticles at 100 °C. In addition, although conglomeration was not seen in the synthesized nanoparticles at room temperature, the synthesis needed a 48 h time-

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consuming process [53]. In another work, AgNPs, at first, were synthesized using agar as the stabilizing medium and the xenon lamp as the reductant. These AgNPs had the average size of 100 nm with maximum absorption band of 440 nm. Then, the average size of AgNPs decreased to about 10 nm by photolysis induced by ultra-short laser pulses [55].

However, in this study, in agreement with the prospects of the green synthesis, the agar was used as the stabilizing

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medium. Ascorbic acid (ASC) was also used as a stronger reducing agent, to accelerate the reduction of Ag + ions to Ag atoms. In addition, water was utilized as an eco-friendly solvent throughout the preparation process.

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Accordingly, a new, simple, rapid and green strategy was introduced to directly produce of monodisperse and highly

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stable AgNPs. Besides, unlike previous time-consuming methods [53, 55], monodisperse AgNPs with average size

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of 20.7 ± 3 nm was obtained during 1h and showed the plasmonic peak at 400 nm. Furthermore, the nanoparticles were stable six months. The synthesize AgNPs were used for determination of Fe2+, H2O2, and glucose, based on a

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2. Experimental

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colorimetric sensor.

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2.1. Chemicals and materials

All chemicals and solvents were of analytical grade and commercially available. Silver nitrate, thiourea and GOx

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were purchased from Sigma-Aldrich (Shanghai, China). Iron (II) chloride tetra hydrate, hydrogen peroxide (H 2O2, 30 wt %), di-Sodium hydrogen phosphate, glucose, sulfuric acid (H2SO4), sodium hydroxide (NaOH), L(+)-ascorbic

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acid, L-cysteine, agar and metal salts including NaCl, KCl, Cu(SO4)2.5H2O, Zn(NO3)2.6H2O, CaCl2, FeCl3.6H2O, Mg(NO3)2.6H2O, Hg(NO3)2.H2O, Ni(NO3)2, Pb(NO3)2, CrCl3, Co(NO3)2.6H2O and Cd(NO3)2.4H2O were all obtained from Merck (Darmstadt, Germany). Deionized water was used in all experiments. All glassware were cleaned in a bath of fresh HNO3-HCl (1:3, v/v) solution, rinsed wholly in deionized water, and dried in air prior to usage.

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2.2. Instrumentation The UV-Vis experiments were performed using a Lambda-45 spectrophotometer (Perkin-Elmer, USA). To verify the presence of agar in the structure of AgNPs, FTIR bands were obtained by a FTIR instrument (Shimdazu, Japan) with combining of about 3 mg of pure agar or freeze-dried AgNPs and KBr. X-ray diffraction spectrum was

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achieved by a X-ray diffractometer (XRD) model: X’Pert PRO MPD (PANalytical Company, Netherlands), with Cu Kα radiation (2θ = 5–80, K = 1.1546 A ̊). The energy-dispersive X-ray spectrum (EDX) was obtained using 3 mg of freeze-dried AgNPs on a TESCAN Vega model instrument that is operated at 200 kV. The average particle size was obtained using Malvern Zetasizer Nano ZS (UK). Transmission electron microscopy (TEM) was used to study the morphology and nanostructure of AgNPs by a Zeiss - EM10C - 80 KV (Germany).

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2.3. Synthesis of AgNPs

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AgNPs were synthesized through the following protocol: First of all, 5 mL of the aqueous solution of ASC with the

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concentration of 0.75 mM was added into 40 mL water, and then the pH of the solution was adjusted at 11 by NaOH

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(1 M). Next, the solution was allowed to be heated under stirring until boiling (T=100°C). At the same time, 0.045 g of agar was added to 4 mL of water and the mixture heated until the complete dissolution of agar (T=40°C). Then, 1

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mL of AgNO3 (4.5 mM) was added to the agar solution, followed by mixing 1 min on the heating and stirring

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(T=40°C). Finally, the mixture of agar solution and AgNO3 was added to the ASC boiling solution. The color of the reaction solution quickly changed from colorless to light brown. The reaction solution was further boiled for 1 h

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under stirring to guarantee the formation of AgNPs (T=100°C). After completing the reaction, the light yellow solution of AgNPs was obtained. The AgNPs solution was stored at 4 °C in a dark place until use.

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2.4. Colorimetric determination of Fe2+

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For Fe2+ sensing, H2SO4 was added to 1200 μL phosphate buffer solution (PBS, 20 mM, pH = 7.4) containing 62.5 µM H2O2 to adjust the pH of solution at 3.0. Then, the above solution were added to the mixture of AgNPs (1200 μL) and Fe2+ (100 μL) solutions, containing certain amounts of Fe2+, and the final mixture was incubated at room temperature for 15 min. Finally, the UV-Vis spectrum of the final solution was obtained using a spectrophotometer. 2.5. Colorimetric determination of H2O2

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For determination of H2O2 concentration, at first, H2SO4 was added to 1200 μL PBS (20 mM, pH = 7.4) containing different concentrations of H2O2 to adjust the pH at 3.0. Then, the above solutions were added to the mixture of 1200 μL AgNPs and 100 μL Fe2+ (2.5 mM), and the final mixture was incubated at room temperature for 15 min. Finally, the UV-Vis spectrum of the final solution was obtained using a spectrophotometer.

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2.6. Colorimetric determination of glucose

Glucose was determined as the following procedure. At first, 150 μL of 0.1 mg.mL-1 GOx was added to 1050 μL PBS (20 mM, pH = 7.4) containing different concentrations of glucose, and the solution was incubated at room temperature for 30 min. Next, H2SO4 was added to adjust the pH to 3.0. After that, the above solution was added to the mixture of 1200 μL AgNPs and 100 μL Fe 2+ (2.5 mM), and the final mixture was incubated at room temperature

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for 15 min. Finally, the UV-Vis spectrum of the final solution was measured using spectrophotometer.

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2.7. Real sample analysis

To assess the applicability of the method in real media, it was used for the analysis of glucose in human blood

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serum. The human blood serum samples were obtained from a local hospital and pretreated by ultra-filtration to remove conceivable interferences from the proteins. Thereupon, the pretreated samples were diluted in PBS solution

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(20 mM, pH = 7.4) and glucose concentrations were determined.

3. Results and discussion

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3.1. Optical and structural characteristics of agar-stabilized AgNPs

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As explained in section 2.3, the synthesis of AgNPs was successfully obtained via a one-step procedure without complicated pretreatment. The precursors, AgNO3 and agar, were dissolved in water. Before the addition of this mixture to the boiling ASC solution, Ag+ can react with the agar to form agar-Ag+ complex. When the agar-AgNO3

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mixture was added to the boiling solution of the reducing agent (ASC), Ag+ was reduced to Ag (0). The nucleation occurred and the agar-stabilized AgNPs were synthesized. The capping agent, agar, was easily soluble in water, which it caused the AgNPs showed an excellent water-solubility and capability for determination of metal cations.

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As mentioned, the methods previously used agar for the synthesis of AgNPs had some disadvantages such as being time-consuming, requiring to laser sources or producing large-size AgNPs [53, 55], but the green synthesis procedure, introduced here, provided highly stable AgNPs with the average size of 20.7 ± 3 nm. The AgNPs were characterized by UV–Vis absorption spectroscopy (Fig.1A), and the related photograph was

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recorded (Fig.1B). The appearing of a single, strong and sharp surface plasmon resonance (SPR) band at λmax =400 nm confirmed the formation of AgNPs [56]. The stability of the agar-stabilized AgNPs was investigated by recording the UV–Vis spectrum in different interval times. Comparison of the UV–Vis bands of the freshly prepared AgNPs and the AgNPs stored for six months (Fig. 1A) showed minor change that indicates the high stability of the agar-stabilized AgNPs.

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FT-IR spectra of pure agar and agar-stabilized AgNPs are shown in Fig. 2. The characteristic absorption peak of

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pure agar (Fig. 2A) can be observed at 3445.91 cm−1 which is the sign of the OH stretching bonds. The peak of

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2909.35 cm−1 stands for C–H stretching, while 1644.18 cm−1 and 1381.14 cm−1 indicate C–C stretching and the C–H

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vibration, respectively. In addition, the peak at 1066.82 cm−1 is related to the stretching vibration of C-O [57, 58]. Also, such characteristic peaks are observed in the FT-IR spectrum of the agar-stabilized AgNPs (Fig. 2B). It

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demonstrates that agar has been successfully coated on the surface of AgNPs.

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To verify the successful synthesis of the agar-stabilized AgNPs, EDX and XRD analyses were further performed to characterize the elemental composition and atoms state in the AgNPs. Fig. 2C shows the EDX analysis graph of the

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agar-stabilized AgNPs. Based on the EDX data, elemental Ag is identified in the agar-stabilized AgNPs. Besides, the presence of C and O can be attributed to agar structure. The XRD survey spectrum is shown in Fig. 2D. The first

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sharp peak was observed commonly at 2θ value of 27.95º in the agar-stabilized AgNPs which is attributed to the agar [54]. In addition, diffraction peaks at 38.26º, 44.35º, 64.62º and 77.44º can be indexed to diffractions from the

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(111), (200), (220), and (311) planes of face-centered cubic silver crystals, respectively [56]. All of these results illustrate the formation of AgNPs and successful loading of agar onto the surface of AgNPs. The morphology and size of the AgNPs were characterized using TEM micrograph, displayed in Fig. 3A-a. The spherical AgNPs with an appropriate dispersion, and a mean particle size of 20.7 ±3 nm were observed. According

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to the DLS measurements (Fig. 3B-a), a narrow size distribution of AgNPs with an average size of 20 nm was obtained that it was in agreement with the TEM results. 3.2. Strategy of the colorimetric sensing

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The AgNPs were utilized as a colorimetric probe for sensitive determination of Fe2+, H2O2 and glucose. At first, the change of UV-Vis spectrum of the AgNPs in the presence of Fe2+ was studied. As can be seen in Fig. 4A-b and 4Bb, the AgNPs containing 100 μM Fe2+ did not show any significant variation in color or absorption intensity. Then, the effect of H2O2 on the absorption spectrum was investigated. Based on the results (Fig. 4A-c and 4B-c), UV-Vis band intensity and color of the AgNPs solution demonstrated no evident variation in the presence of H2O2 (30 µM). Nevertheless, when H2O2 (30 μM) and Fe2+ (100 μM) were simultaneously present in the AgNPs solution, the UV-

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Vis spectrum intensity decreased significantly (Fig. 4A-d), and the AgNPs solution turned to colorless (Fig. 4B-d).

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Consequently, it seems that the simultaneous presence of H2O2 and Fe2+ results in the discoloration and quenching

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of the plasmonic response of the AgNPs solution without any red or blue shifts (Fig. 4A-d).

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Based on these results, it can be suggested that discoloration and decrease in the absorption strength originates from decreasing the size of the AgNPs which is resulted from AgNPs oxidation. To confirm this suggestion, TEM, DLS

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and UV–Vis data were utilized. After adding Fe2+ and H2O2 to the AgNPs solution, no growth or aggregation can be

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observed for the nanoparticles in the TEM image (Fig. 3A-b) and DLS histogram (Fig. 3B-b). Moreover, it can be found from the TEM images that the size of AgNPs in the presence of Fe2+ and H2O2 ( 7.7 nm) are lower than that

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in the absence of Fe2+ and H2O2 ( 20 nm). Additionally, the SPR band of the AgNPs-Fe2+-H2O2 solution exhibited no characteristic absorption spectrum related to the large size AgNPs (Fig. 4A-d). These data suggested that no

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aggregation of AgNPs was happened after adding of Fe2+ and H2O2 into the solution. Also, the only presence of H2O2 can not cause changing in the size or color of the solution of AgNPs (Fig. 4A-c, 4B-c).

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It has been represented that AgNPs catalyze the decomposition of H 2O2 through the regeneration of AgNPs [46, 47]. However, in Fenton reaction, Fe2+ reduces H2O2 and produces OH•. This radical (with the redox potential of 2.73 V) is a very stronger oxidizer than H2O2 [50, 51] and possibly could oxidize AgNPs, and lead to convert atomic Ag to Ag+. It results in the discoloration of AgNPs solution and reducing the absorption peak intensity.

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To investigate whether generated OH• can cause the oxidation of AgNPs and discoloration of the solution, thiourea, an effective OH• scavenger [59], was added to the AgNPs-based sensing probe. The results displayed that the discoloration and reduction of the absorption bands of AgNPs were greatly decreased in the presence of 300 μM thiourea (Fig. 4A-e), disclosing that OH• actually has a considerable role in discoloration of the AgNPs solution.

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According to these findings, a new detection mechanism for Fe2+ and H2O2 was proposed here. Simultaneous presence of Fe2+ and H2O2 in the agar-stabilized AgNPs solution can cause the decomposition of AgNPs by producing OH• radicals. By increasing the concentrations of Fe2+ and H2O2, the gradual decrease in SPR strength was observed.

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3.3. Colorimetric detection of Fe2+ based on the agar-stabilized AgNPs and the Fenton reaction

As mentioned in section 3.2, after the introduction of Fe 2+ to the agar-stabilized AgNPs, no considerable changes

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were observed in the solution color or absorption strength of AgNPs (Fig. 4A-b and 4B-b). After addition of 30 µM

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H2O2 to the agar-stabilized AgNPs solution, containing Fe2+, a sever color change was observed (Fig. 4B-d). Hence,

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the AgNPs-based assay for successful detection of Fe2+ in the presence of H2O2 was optimized. The influences of solution pH and reaction time were investigated. The absorbance strength change, ΔA, (ΔA = A0﹣At, where A0 and

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as a signal for Fe2+ detection.

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At are the absorption strengths of the AgNPs solution at 400 nm before and after the reaction, respectively) was used

One of the fundamental parameter affecting the Fenton reaction is pH of the solution. It has been proven that the

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precipitation of Fe3+ to the hydroxide form at high pH values inhibits the conversion of Fe2+/Fe3+, and the Fenton reaction generally is occurred at low pH [60]. As a result, the effect of pH on the ΔA amounts was firstly

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investigated. Fig. 5A displays that ΔA increased with increasing the pH from 1 to 3, achieved a maximum at pH 3.0, and then decreased from 3 to 7. According to the results, pH of 3.0 was used in next experiments. Moreover, Fig. 5B

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shows that ΔA is increased from 5 to 15 min and became approximately constant after 15 min. Therefore, the reaction time was set at 15 min. Accordingly, a simple colorimetric probe based on oxidation of AgNPs is developed for monitoring of Fe2+. A gradual decrease in the SPR band intensity of the AgNPs solution containing H 2O2 and different amounts of Fe2+ (the final concentration: 0 to 100 µM) was observed (Fig. 6A), while the color of the solution underwent a

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remarkable change from pale yellow to colorless (Fig. 6B: inset). To evaluate the sensitivity of the sensor towards Fe2+, the ΔA was plotted against the different concentrations of Fe2+ (Fig. 6B). This curve showed a good linear relationship in the concentration range of 1-90 μM (R2 = 0.9963). The LOD for Fe2+ determination (S/N = 3, n=5) was 0.54 μM. The relative standard deviation (RSD) for three replicate analysis of 70 μM solution of Fe2+ was 1.76

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%. The selectivity of the sensor for Fe2+ was evaluated by recording UV-Vis spectra of the AgNPs in the presence of other of metal ions (Hg2+, Ca2+, Fe3+, Na+, Cu2+, K+, Ni2+, Co2+, Pb2+, Mn2+, Mg2+, Zn2+, Cr3+ and Cd2+ at the same concentration of 100 µM). Fig. 7A indicates no significant interferences observed from most of the metal ions for Fe2+ detection. However, Hg2+ could also result in the considerable decrease of the absorption intensity. The redox

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potential of Hg2+/Hg (0.851 V) is higher than that of Ag +/Ag (0.7996 V), and it causes oxidation of the AgNPs and reducing the intensity of the absorption band [61]. To circumvent this challenge, L-cysteine was added to the

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solution as a masking agent, because Hg2+ has a great tendency to L-cysteine and hence, it can exhibit a great

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sheltering effect for Fe2+. Fig. 7B displays the ΔA values at 400 nm, in the presence of interfering ions, before and

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after addition of L-cysteine in the solution. Obviously, in the presence of 200 µM L-cysteine, Hg2+ has little effect on the sensing efficiency of Fe2+. Comparison of Figs. 8A and 8B depicts that upon the introduction of different

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metal ions into the AgNPs solution, only Fe2+ can result in discoloration. It shows the high selectivity of the sensing

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system to Fe2+.

Different sensing systems for Fe2+ determination are summarized in Table 1. As can be seen, most of the systems

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showed poor selectivity, so they are undesirable for routine usage. However, the introduced AgNPs probe not only demonstrated an appropriate LOD and linear range, but also was selective for Fe2+, and prevents Fe3+ interference in

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compared with the other methods. Moreover, after optimizing the sensing parameters, the semi-quantitative selective

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determination of Fe2+ could be achieved using naked-eyes by the present AgNPs assay. 3.4. Colorimetric detection of H2O2 based on the agar-stabilized AgNPs and the Fenton reaction It was found that the color changes of the AgNPs solution is affected by both the concentration of Fe2+ and H2O2. By increasing the amount of Fe2+ or H2O2, discoloration of AgNPs became stronger. Consequently, the AgNPs–Fe2+– H2O2 system could be utilized to determine Fe2+ at a certain H2O2 concentration or to determine H2O2 at a certain

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Fe2+ concentration. Here, by using 100 µM Fe2+ and in the presence of H2O2, AgNPs solution became completely colorless as a result of Fenton reaction (Fig. 4B-d), and the color did not show any significant changes at 100 µM of Fe2+. Accordingly, to avoid the formation of iron sludge [23], 100 µM Fe2+ was selected for further experiments. Under the optimal conditions (solution pH of 3.0, 100 μM Fe2+, and reaction time of 15 min), the gradual decrease

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of absorbance intensity vs. concentrations of H2O2 (the final concentration: 0-15 μM) is shown in Fig. 9A. Additionally, the color of AgNPs solution changed from pale yellow to colorless at a specified reaction time, proportional to the concentration increasing of the H2O2 added (Fig. 9B: inset). In order to investigate the analytical performance of SPR-based colorimetric probe for H2O2 detection, the calibration curve was plotted based on the absorbance strength change (ΔA) at 400 nm vs. concentrations of the introduced H2O2 (Fig. 9B). As could be

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observed, ΔA decreases at 15 min with increasing of H2O2 concentration. In addition, the synthesized AgNPs by

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agar and ASC established a linear correlation over a wide concentration range of H2O2 (0.05-7.5 µM, R2=0.9979). The obtained LOD (S/N=3, n=5) was 0.032 µM, which demonstrates that the Fenton reaction-based system is more

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sensitive in compared with the most reported H2O2 colorimetric assays (Table 2). RSD of H2O2 determination at

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concentration of 3.75 µM was 2.24% (n=3). Interestingly, no clear decrease in the absorption intensity of AgNPs was observed in the presence of other typical reactive oxygen species (ROS) or reactive nitrogen species (RNS) such

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as 𝑂2−• , NO•, ClO• and ONOO•. It demonstrates that the sensor had high selectivity for H2O2 detection (Fig. S1). 3.5. Analytical application of the agar-stabilized AgNPs sensor for the determination of glucose

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Because of producing H2O2 during the oxidation of glucose by GOx (Eq. 2) [66, 67], Fenton reaction-mediated discoloration of AgNPs solution was used for determination of glucose at a two-step process. First, H2O2 was

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produced using glucose oxidation by GOx, and then the pH of the solution was adjusted to 3.0. Next, the prepared solution was added to the AgNPs and Fe2+ mixture, to start the Fenton reaction and the absorption intensity is

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recorded. In order to investigate the sensitivity of the platform to the glucose concentration under the optimized conditions, different amounts of glucose were examined. Regular increasing of glucose caused the gradual decreasing of the SPR peak of AgNPs (Fig. 10A). The curve of ΔA against the glucose concentrations was linear within 1.5-30 μM (Fig. 10B, R2= 0.9946). The LOD was measured as 0.29 μM (S/N = 3, n=5). Comparison of the analytical performance data of the method with other colorimetric glucose determination approaches was presented

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in Table 3. Obviously, the colorimetric assay based on AgNPs–Fe2+–H2O2 system demonstrates an excellent sensitivity for the glucose determination. To evaluate the selectivity of the Fenton reaction-based system to glucose detection (40 μM), different possible interferences including lactose (200 μM), maltose (200 μM) and fructose (200 μM) were tested at the same

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conditions. As can be observed in Fig. 10C, these species did not interfere the glucose determination owing to the high substrate selectivity of GOx. Accordingly, a new colorimetric sensor was established for glucose detection, based on discoloration of the AgNPs solution using Fenton reaction and GOx-mediated oxidation of glucose. 𝐺𝑂𝑥

𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 + 𝐻2 𝑂 →

𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐻2 𝑂2

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To evaluate the feasibility of the introduced method to detect glucose in human blood serum, standard addition

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experiments were designed. In addition, the applicability of the glucose recognition system was verified using the standard clinical method. Table 4 represents the analytical performance data of our sensing platform and the other

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methods. It can be seen that no remarkable differences were observed between three methods. Appropriate

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recoveries guarantee that the AgNPs–Fe2+–H2O2 system has the potential of application for glucose determination in

D

clinical samples.

TE

4. Conclusion

Generally, a multi-functional colorimetric probe for Fe2+, H2O2 and glucose detections was developed based on

EP

Fenton reaction and bio-synthesized AgNPs. The key benefit is that this sensor is prepared by a typical green synthesis approach using ASC as a reducing agent and agar as a stabilizing agent. This novel system provides a

CC

colorimetric bioassay for glucose through H2O2 production by catalyzed oxidation of glucose using GOx. Moreover, this new probe allows the selective detection of Fe2+ with no interference of Fe3+. Also, the method was tested

A

successfully for the analysis of glucose in human serum samples, indicating that it is a promising method for the clinical applications. The advantages of the fabricated system depict that the conjugated nanostructures have an excellent potential for development of sensors.

Acknowledgements This work was financially supported by the Khajeh Nasir Toosi University of Technology, Tehran, Iran.

13

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[61] A. Mehdinia, S. Basiri, A. Jabbari, A novel label-free method for determination of inorganic mercury in environmental aqueous media using BSA-modified silver nanoparticles. Int J Environ Sci Technol.13 (2016) 26632674. [62] Y. Zhang, J. Tian, S. Liu, L. Wang, X. Qin, W. Lu, G. Chang, Y. Luo, A.M. Asiri, Abdulrahman Al-Youbi O,

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of H2O2 and glucose using silver nanoparticles induced by H2O2 and UV. Chin Chem Lett. 24 (2013) 1053-1058. [66] E. Zor, M.E. Saglam, I. Akin, A.O. Saf, H. Bingol, M. Ersoz, Green synthesis of reduced graphene

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EP

[68] G. Darabdhara, B. Sharma, M.R. Das, R. Boukherroub, S. Szunerits, Cu-Ag bimetallic nanoparticles on reduced graphene oxide nanosheets as peroxidase mimic for glucose and ascorbic acid detection. Sens Actuators B

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Chem. 238 (2017) 842-851.

[69] C. Radhakumary, K. Sreenivasan, Naked Eye Detection of Glucose in Urine Using Glucose Oxidase

A

Immobilized Gold Nanoparticles. Anal Chem. 83 (2011) 2829-2833.

20

Figure captions Fig. 1 (A) UV–Vis absorption bands of the freshly prepared agar-stabilized AgNPs and the stored AgNPs for six months; (B) The photographic image of the freshly prepared agar-stabilized AgNPs.

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Fig. 2 FTIR spectrum of (A) pure agar and (B) the agar-stabilized AgNPs; (C) EDX spectrum of the agar-stabilized AgNPs; (D) XRD pattern of the agar-stabilized AgNPs.

Fig. 3 (A) TEM image and (B) lateral size distribution histogram of the agar-stabilized AgNPs (a) before and (b) after incubation with Fe2+ (20 µM) + H2O2 (62.5 µM).

Fig. 4 (A) UV–Vis absorption intensity of (a) AgNPs, (b) AgNPs + 100 μM Fe2+, (c) AgNPs + 30 μM H2O2, (d)

U

AgNPs +100 μM Fe2+ + 30 μM H2O2, and (e) AgNPs + 100 μM Fe2+ + 30 μM H2O2 + 300 μM thiourea; (B)

N

Coresponding photographs of the above solutions.

A

Fig. 5 The effect of (A) pH and (B) incubation time on the ΔA values of the agar-stabilized AgNPs.

M

Fig. 6 (A) Changes of the SPR absorbance intensity of the agar-stabilized AgNPs by varying the concentrations of Fe2+ (0-100 μM) in the presence of 30 µM H2O2; (B) Correlation between the change in the absorption intensity of

D

the agar-stabilized AgNPs solution (ΔA) and the Fe2+ concentration (Inset: The color change of agar-stabilized

TE

AgNPs by different concentrations of Fe2+).

EP

Fig. 7 (A) UV–Vis absorption bands and (B) the changes of the SPR absorbance intensity of the agar-stabilized AgNPs solution in the presence of different metal ions (100 μM). In panel (B), the red and blue bars demonstrate the

CC

change in the absorption intensity of the agar-stabilized AgNPs solution (ΔA) solution with and without addition of 200 µM L-cysteine.

A

Fig. 8 Photographs of the agar-stabilized AgNPs solution containing 200 µM L-cysteine (A) before and (B) after addition of various metal ions (100 μM). Fig. 9 (A) Changes of the SPR absorbance intensity of the agar-stabilized AgNPs with increasing amounts of H2O2 (0-15 µM) in the presence of 100 µM Fe2+; (B) The function curve of the change in the absorption intensity of the

21

agar-stabilized AgNPs solution (ΔA) versus the concentrations of H2O2 (Inset: The color change of the agarstabilized AgNPs by different concentrations of H2O2). Fig. 10 (A) Corresponding absorption spectra of the agar-stabilized AgNPs with concentrations of glucose (0-30 µM) in the presence of 100 µM Fe2+; (B) The relationship between the absorption intensity of the agar-stabilized

SC RI PT

AgNPs solution (ΔA) and glucose concentrations; (C) The ΔA of the glucose detecting probe in the presence of

A

CC

EP

TE

D

M

A

N

U

glucose (40 μM), maltose (200 μM), lactose (200 μM), and fructose (200 μM).

22

23

D

TE

EP

CC

A

SC RI PT

U

N

A

M

24

D

TE

EP

CC

A

SC RI PT

U

N

A

M

25

D

TE

EP

CC

A

SC RI PT

U

N

A

M

26

D

TE

EP

CC

A

SC RI PT

U

N

A

M

Table 1 The comparison between the performances of the proposed sensor and the other sensors for Fe 2+ detection Detection mechanism

Linear range for Fe2+ (µM)

Detection limit for Fe2+ (µM)

Selectivity to Fe2+

Reference

Optical sensor for Fe2+ and Fe3+

sol-gel as a support and TPTZ as a reagent

0.07-1.54

0.02

No selective

[15]

visual strip sensor for Fe2+

sol-gel as a support and TPTZ as a reagent

0.36

Selective

[30]

SC RI PT

Detection assay

0.358-35.8 Chelate-type Schiff base acting

-

0.19

No selective

[31]

Colorimetric sensor for Fe2+

based on the partition of phenanthroline complexes into polymeric hydrogels

-

0.18

Selective

[32]

Agar-stabilized AgNPs for Fe2+

colorimetric sensor

1-90

0. 54

Selective

This work

A

CC

EP

TE

D

M

A

N

U

colorimetric sensor for Fe2+ and Fe3+

27

Table 2 The comparison of different AgNPs-based colorimetric sensors for the determination of H 2O2

Linear range (µM)

Detection limit (µM)

Reference

CoFe-LDHsa

colorimetric

1-20

0.4

[62]

PtPdNDs/GNsb

colorimetric

0.5-150

Ag nanoparticles

colorimetric

10-7-10-1 M

Ag nanoparticles

colorimetric

0.5-60.0

Ag nanoparticles

colorimetric

0.05-7.5

CoFe layered double hydroxide PtPd nanodendrites on graphene nanosheets

A

CC

EP

TE

D

M

A

N

U

b

Detection mechanism

SC RI PT

a

Detection assay

28

0.1

[63]

0.035

[64]

0.2

[65]

0.032

This work

Table 3 The comparison of the analytical operation of the proposed assay with other colorimetric probes for glucose detection

Detection mechanism

Linear range (µM)

Detection limit (µM)

Reference

Au nanoparticle-anchored nitrogen-doped graphene

seed-assisted growth method

40 µM to 16.1 mM

12

[43]

CoFe-LDHs

colorimetric

1-10

citrate-capped Ag nanoparticles

colorimetric

0.06-4.0 mM

Cu-Ag bimetallic nanoparticles

colorimetric

1-30

citrate-capped Au nanoparticles

colorimetric

55.51-499.57

Agar-stabilized AgNPs

colorimetric

1.5-30

U

N A M D TE EP CC A

29

SC RI PT

Detection assay

0.6

[62]

18

[67]

3.82

[68]

28

[69]

0.29

This work

Table 4 Determination of glucose in human serum

Standard clinical method (mM)

Detected (mM)

Added (mM)

Founda (mM)

Recoveryb %

RSD % (n=3)

1

4.88

4.76

3

7.68

97.3

3.1

2

5.23

5.17

3

8.27

10.3

2.6

3

4.1

3.97

3

6.85

a

96

The mean concentration of three replications % Mean recovery=(mean of found concentration- detected concentration) /added concentration

A

CC

EP

TE

D

M

A

N

U

b

SC RI PT

Sample

30

2.5