Ultrasonic synthesized Konjac gum/PEG-silver nanoparticles for colorimetric detection of hydrogen peroxide

Green and low-cost synthesis strategy for ultrasonic preparation of polymer blend matrix based silver nanoparticles (Ag NPs) and the development of rapid and high sensitive detection route have a great attention in biomedical applications. Therefore, in this study, we investigated the hydrogen peroxide detection performance of Konjac gum (KG)/PEG-Ag NPs. The KG/PEG-Ag NPs was synthesized via an ultrasonic process and characterized by different techniques such as ultraviolet–visible spectroscopy (UV–Vis), Fourier-Transform Infrared spectroscopy (FT-IR), Scanning Electron Microscope (SEM) and Energy Dispersive X-ray spectroscopy (EDX). Furthermore, we determined the experimental optimization on the effect of the rheological parameters of nanostructure with the highest correlation constant (R 2 : 0.989-0.996), and the intrinsic viscosity (14.71-26.77 dl/g). To provide the miscible polymer blends and homogeneous dispersion of the nanostructure, we compared the rheological parameters with the experimental results. The response time was less than 5 s and the lower limit of detection was 0.071 μM. This novel highly sensitive, rapid, and naked-eye colorimetric biosensor based Ag NPs which are prepared ultrasonic manufacturing approach, opens up a green approach of development facile and rapid detection of hydrogen peroxide in practical biomedical applications.


Introduction
Hydrogen peroxide (H2O2) is a major biological reactive and the main product of reaction of industrial enzymes such as oxidases, peroxidases, and catalases [1]. It has a role on the cellular damage, apoptosis, pathogen defense mechanism, and mutagenesis in a low concentration in the presence of hydroxyl radicals (OH•) [2][3]. Accordingly, the rapid, lowcost, practical, selective, and sensitive determination of H2O2 is very important in different applications such as chemistry, environmental, and biomedical applications [4][5]. In the literature, it was reported that different costly and not portable techniques such as spectrophotometer, chemiluminescence, electrochemical, and electrochemiluminescence were used with complicated procedures for the determination of H2O2 [6][7][8][9].
In recent years, economical, simple applicable and portable systems such as colorimetric methods have come to the fore in order to eliminate the restrictions on the application [10][11][12][13][14].
Various metal nanoparticles (NPs) such as Ag NPs, gold (Au) NPs, palladium (Pd) NPs, copper (II)-coated Fe3O4 NPs, titanium dioxide (TiO2) NPs, and rhodium NPs have been preferred in the development of H2O2 sensors due to the effects of localized surface plasmon resonance energy [15][16][17][18][19]. The localized surface plasmon resonance energy is related to the morphology, composition, size, shape, and dielectric environment of NPs [20]. In this context, Carbone et al. prepared a novel colorimetric sensor based on silver-poly (methyl methacrylate) NPs for the detection of H2O2 [21]. Teodoro et al. prepared a colorimetric sensor based on cellulose nanowhiskers and Ag NPs with a low limit of detection of 0.014 μM for the detection of H2O2 [22]. Nguyen et al. prepared Ag NPs and graphene quantum dots (AgNPs/GQDs) consisted a colorimetric sensor for H2O2 [23]. Huang et al. fabricated a novel dual mode colorimetric and fluorometric sensor with an average diameter of NPs 15.2 nm, which was employed for H2O2 detection [24]. In the literature, several green methodologies for use in the synthesis of a biodegradable polymer matrix-based nanostructure have been investigated [25][26][27][28][29]. Chen et. al reported that KG is a polysaccharide form the class of macromolecular carbohydrates and could be used as a reducing and stabilizing agent in the synthesis of Ag NPs [30]. However, there is no study on the ultrasonic synthesized KG/PEG-Ag NPs based colorimetric sensor to be used for detection of H2O2.
In this study, the ultrasonic synthesized KG/PEG blend was used as a polymer matrix and the Ag NPs was prepared by a green the ultrasonic method in a liquid at ultrasonic frequencies and characterized via several techniques. Furthermore, we examined the experimental parameters such as the blend of ratios of KG/PEG (wt./wt.) Ag NPs, the sonication time, and the amplitude of the sonication on the viscosities of the ultrasonic synthesized KG/PEG-Ag NPs were investigated to optimize designing conditions. We calculated the intrinsic viscosity of synthesized KG/PEG-Ag NPs. Also, we investigated the effect of H2O2 concentration on the colorimetric biosensor.

Preparation of KG/PEG matrix
KG/PEG-Ag NPs were prepared using a simple and green sonication method. Firstly, 2.5 g of KG was dissolved in 250 mL distilled water. Then, it was kept in the dark for 5 days to gel the solution without stirring. The solution of KG was stirred at 50 °C and filtered using a coarse filter paper and a sterile syringe filter with a 0.45 um pore size, respectively. Thereafter, 12,5 mL of PEG was added into the KG solution and then sonicated for 10 min at 45% amplitude frequency at room temperature.

Preparation of KG/PEG-Ag NPs
0.84 g of AgNO3 was dissolved in 500 mL distilled water. The Ag solution was slowly added to the KG solution and was stirred at 25 °C for 10 min. 0.1 g of NaOH was dissolved in 250 mL distilled water. The solution of NaOH was slowly added to the KG/PEG blend with AgNO3 solution until it was stable at pH 8. The solution was sonicated for 10 min at 45% amplitude frequency at 25 °C. Finally, the solution was filtered using a sterile syringe filter with a 0.22 um pore size.

Rheological analysis of KG/PEG-Ag NPs
We used a simple, easy, and low-cost Dilute Solution Viscometer (DSV) method to characterize the stability of the Ag NPs. We analyzed the rheological parameters of the nanostructure in different experimental conditions and the intrinsic viscosities ([η]), Huggins constant (k), the voluminosity ( ), and the shape factor (γ) were calculated using Equations 2.1-2.4 (Table 1) [31][32][33].
Where C: the concentration of the solution, η sp : the spesific viscosity, η rel : the relative viscosity, t0: the flow time of the solvent, t: the flow time of the solution, k: Huggins constant, : the voluminosity, and γ: the shape factor.

Statistical Analysis
All experimental results were performed in triplicate and are given as the mean for each experiment. The analysis of variance (ANOVA) procedure was conducted in SPSS (version 16, Chicago) with a significance level of P < 0.05.

Characterization of KG/PEG-Ag NPs
SEM images of (a) pure KG and (b) KG/PEG-Ag NPs with different magnifications (x10.000 and x40.000) and EDX result of the KG/PEG-Ag NPs (at accelerating voltage of 20 kV) were given in Fig. 1-2. According to obtained SEM results, we observed that KG has a regular fibrous shape and tightly arranged branched structure [34]. Also, the SEM images of Ag NPs were shown that the structure had a spherical shape and small-sized with an average size of 48.8 ± 2 nm [30]. Fig. 3 showed the EDX spectra of the KG/PEG-Ag NPs, in which Ag o were observed at characteristic absorption peaks in the range of 1-2 and 3 keV. The elemental EDX analysis also revealed the presence of C, O, and Ag [35]. Consequently, compared to the SEM images of KG with NPs were observed significant differences. The predicted reason for this is the interaction of Ag NPs with the KG/PEG polymer blend matrix, which had a role as a reducing and stabilizing agent. In addition, the ultrasonic synthesized KG/PEG-Ag NPs exhibited a homogeneous distribution showing the interaction between the polymer blend matrix and the Ag NPs. We assumed that this interaction played a critical role in easily detecting of H2O2 with the naked eye in the output signals of a colorimetric biosensor. formation of Ag NPs interacting due to -OH and -COOH functional groups have a role on the reduction of Ag + ion to Ag 0 [38][39]. We assumed that the formation of Ag NPs was due to the electrostatic interaction between the KG/PEG with Ag.

Viscosity measurement of KG/PEG-Ag NPs
In this study, we prepared KG and PEG polymer blends with Ag NPs using a simple and  sonication times (4-10 min) were calculated and these results were 14.71 dl/g for 4 min, 18.48 dl/g for 6 min, and 26.77 dl/g for 10 min. We found the minimum viscosity was 14.71 dl/g for at amplitude 45% (Fig. 6). When the experimental data were compared, we observed that the sonication time and the values of amplitude were important parameters in determining the viscosity at the optimum conditions, from the low [η] value. through intercept of plotting γ versus C. As it is known, υ is a parameter to show the shape of the structure as υ> 2.5 it indicates ellipsoidal and υ <2.5 it indicates spherical shape. Our results proved that the nanostructures had a spherical shape since the values of υ of KG / PEG-Ag NPs were less than 2.5 [48].

Colorimetric measurement of KG/PEG-Ag NPs for the detection of H 2 O 2
For determination of selectivity of the novel colorimetric biosensor, we compared the color changes of the sample in the presence of Omega 3, testosterone, progesterone, and H2O2 was taken photographed using a Nikon D5100 + 18-55mm lens digital SLR camera (Fig. 7). All of the colorimetric experimental runs were carried out in the presence of phosphate buffered saline (pH  The transformation of the yellow KG/PEG-Ag NPs to a transparent color in the presence of H2O2 analyte enabled it to be perceived with the naked eye in the detection of H2O2. This possible mechanism was due to the oxidation of Ag 0 to Ag + ions with the electrocatalytic ability of KG/PEG-Ag NPs [21]. These results of the novel colorimetric biosensor were appreciable from the comparison with results of previous studies in the literature (Table 3). 60-600 μM [22] Ag and Au NPs Chemical reduction 1.25 μM to 1250 μM [49] Wheat straw extracted lignin in Ag NPs Chemical reduction 1-100 μM [50] Acacia lignin stabilized Ag NPs Chemical reduction 5-500 μg/ml [51] Silver chloride NPs (AgCl-NPs) Solution 1-120 μM [20] KG/PEG-Ag NPs Ultrasonic 1-100 μM This study

Stability of KG/PEG-Ag NPs
In this study, the stability of KG/PEG-Ag NPs solution (5.0 mL) was evaluated under the ultraviolet light (365 nm) for 1 h. We measured the amount of remaining sample every 10 min. In order to evaluate the effect of the ultraviolet factor on KG/PEG-Ag NPs (1:5), the stability of KG/PEG-Ag NPs was examined by their intrinsic viscosity value. As a result, it was seen that the nanostructure had a stable structure in Fig. 9. Also, it was proved that the viscosity value was slightly affected by ultraviolet light. It was found that there was an 8.51%  highly sensitive, rapid, and naked-eye colorimetric biosensor based on KG/PEG-Ag NPs has a potential sensor facile and rapid detection of hydrogen peroxide in practical applications.