Encapsulation Capacity of β-Cyclodextrin Stabilized Silver Nanoparticles towards Creatinine Enhances the Colorimetric Sensing of Hydrogen Peroxide in Urine

The β-cyclodextrin shell of synthesized silver nanoparticles (βCD-AgNPs) are found to enhance the detection of hydrogen peroxide in urine when compared to the Horse Radish Peroxidase assay kit. Nanoparticles are confirmed by the UV-Vis absorbance of their localized surface plasmonic resonance (LSPR) at 384 nm. The mean size of the βCD-AgNPs is 53 nm/diameter; XRD analysis shows a face-centered cubic structure. The crystalline structure of type 4H hexagonal nature of the AgNPs with 2.4 nm β-CD coating onto is confirmed using aberration corrected high-resolution transmission electron microscopy (HRTEM). A silver atomic lattice at 2.50 Å and 2.41 Å corresponding to (100) and (101) Miller indices is confirmed using the HRTEM. The scope of βCD-AgNPs to detect hydrogen peroxide (H2O2) in aqueous media and human urine is investigated. The test is optimized by examining the effect of volumes of nanoparticles, the pH of the medium, and the kinetic and temperature effect on H2O2 detection. The βCD-AgNPs test is used as a refined protocol, which demonstrated improved sensitivity towards H2O2 in urine compared to the values obtained by the Horse Radish Assay kit. Direct assessment of H2O2 by the βCD-AgNPs test presented always with a linear response in the nM, μM, and mM ranges with a limit of detection of 1.47 nM and a quantitation limit of 3.76 nM. While a linear response obtained from 1.3 to 37.3 nmoles of H2O2/mole creatinine with a slope of 0.0075 and regression coefficient of 0.9955 when the βCD-AgNPs is used as refined test of creatinine. Values ranging from 34.62 ± 0.23 nmoles of H2O2/mole of creatinine and 54.61 ± 1.04 nmoles of H2O2/mole of creatinine when the matrix is not diluted and between 32.16 ± 0.42 nmoles of H2O2/mole of creatinine and 49.66 ± 0.80 nmoles of H2O2/mole of creatinine when the matrix is twice diluted are found in freshly voided urine of seven apparent healthy men aged between 20 and 40 years old.


Introduction
Reactive oxygen species (ROS) are very reactive unstable products of oxygen metabolism in all organisms, formed through different mechanisms. They are generated in mitochondria as result of energy production, or also as part of an antimicrobial or antiviral responses [1]. The process of generating ROS can be favored under exogenous stimuli such as environmental pollutants' exposures, UV radiation, air pollution, and cigarette smoking [2]. ROS are used as biomarkers to gain information about the status of biological redox system, progression, and state of diseases and the status of health enhancement by enzymatic and non-enzymatic antioxidants in humans [3]. ROS are found to have a direct role in cell signaling namely: gene expression, apoptosis, and overall activation of cell signaling cataracts, and could serve as intra and intercellular messengers [4,5]. Mitochondrial electron transport process is found to be responsible for generating superoxide (O •− 2 ) which is converted to hydrogen peroxide (H 2 O 2 ) under the effect of superoxide dismutase or even under spontaneous dismutation [3]. In metal catalyzed reactions ROS are necessary intermediates that host electrons released by the oxidation reactions. Superoxide, for instance, is produced by electron transfer to O 2 at Q o site of ubiquinol-cytochrome c oxidoreductase, a complex formed from large number of polypeptides, three heme groups, and a Fe-S center [6]. Atomic oxygen has two lone pairs of electrons that occupy two different orbitals, which makes it more vulnerable to changes especially radicals' formation, the consecutive acceptance of electrons by oxygen lead to the formation of many ROS species, namely: superoxide (O •− 2 ); hydrogen peroxide (H 2 O 2 ); hydroxyl radical (HO • ); and hydroxyl ion (HO − ). Other oxygen-containing ROS includes: alkoxy radicals (RO • ), nitric oxide (NO • ), peroxyl radical (HOO • ), sulfate radical (SO •− 4 ), and lipids hydroperoxides (LOOH), all these ROS share the property of being able to cause oxidation of essential components of the cell [7]. The imbalance between the produced and removed ROS is referred to as "oxidative stress". All survival organisms detoxify ROS through defense mechanisms that provide a balance between produced and removed ROS [8]. Oxidoreductase enzymes, such as superoxide dismutase catalyze the reaction of conversion of two superoxide molecules to one hydrogen peroxide molecule and one molecular oxygen [9]. Hydrogen peroxide is considered as important biomarker between other ROS due to superoxide's dismutation by the superoxide dismutase (SOD) which acts as first line defense against oxidative stress [10]. In addition of being a major product of oxidase catalyzed reactions, H 2 O 2 level is responsible for many cellular damages [11][12][13]. Sensing of H 2 O 2 is significant in many fields, mainly medical and environmental, measuring the concentration of H 2 O 2 in micro and nano levels is essential to control quantities of many important biomolecules in our diet such as carbohydrate, lipids, and proteins, and their monomeric molecules [14]. Many analytical assays have been used to quantify the concentration of H 2 O 2 such as electrochemical [15][16][17][18], enzymatic [19][20][21], and colorimetric assays are still the most attractive due to their simplicity and cost effective [22][23][24][25][26]. The disadvantage of the analytical methods based on colorimetric detection of H 2 O 2 , is mainly due to their low sensitivity. A satisfactory reproducibility and high sensitivity for H 2 O 2 detection is therefore difficult to obtain.
Recently there has been a lot of focus on metals nanoparticles characteristics for ROS detection methods [27,28]. This is due to their small size and extraordinary optical, magnetic, catalytic, and powered properties of nanoparticles that are not found in bulk materials [29,30]. Based on the hypothetical attributes of the nanomaterials such as nanoparticles, nanotubes and nanofibers these are used in the form of quantum dots (QDs) or noble metal nanoparticles (NPs); for instance, gold and silver for the development of chemical sensors and biosensors [31,32].
On the other hand, cyclodextrins are macromolecule oligosaccharides that occur naturally, and consist of six, seven, and eight glucose cycles that are joined by glycosidic bonds. They are created from enzymatic starch conversion [33]. Cyclodextrins are arranged like cones, and they can host small molecules, such as aromatic molecules and steroids inside their cavities. The interior cavity has a lipophilic character. However, the external surface of cyclodextrins is very soluble in water. This is what explain their highest solubility in water [34].
The encapsulation character of cyclodextrins lead to their wide use in drug delivery. β-cyclodextrin is occupying a middle position because of its moderated size cavity of 0.6 nm compared to α and γ-cyclodextrins [35]. Herein, a novel strategy based on mediated creatinine encapsulation by the β-cyclodextrin organic shell of the nanoparticles is developed. Due to the hosting ability of the β-cyclodextrin, the creatinine could easily be hosted by the shell and improves the stability and reproducibility of H 2 O 2 concentration in urine. The test results are validated through comparison with previous assays and a commercially available assay kit.

Chemicals
All chemicals are purchased from Sigma Aldrich through a local company (Labcoltd-Dubai, UAE), they are used without any further purification as received unless otherwise stated. Soluble β-cyclodextrin (C 42

Preparation of β-CDAgNPs
β-cyclodextrin capped nanoparticles are prepared subsequent the modified procedure described by Yingju et al. [36]. In a 500 mL one neck round bottom flask, 200 mL of saturated solution of β-cyclodextrin (0.2% w/v) is stirred for a period of 30 min, then 3.0 mL of a 0.1 M solution of AgNO 3 as source of Ag + and 6.0 mL of 0.1 M of newly prepared NaBH 4 are added to the mixture. A color change occurs instantly after the addition of NaBH 4 from uncolored to greenish. This is a strong signal of the creation of silver nanoparticles. The solution is stirred for an additional time of 30 min. The produced nanoparticles are stable for a period of more than 6 months; nanoparticles are centrifuged to ×4000 rpm for 10 min preceding any utilization. The β-CDAgNPs are brought to analysis and afterward are used for the detecting of H 2 O 2 . Because the absorbance of the bare nanoparticles is very high, nanoparticles are diluted 3 times in all experiments.

Optimization of the H 2 O 2 Detection by β-CDAgNPs
The detection of H 2 O 2 by β-CDAgNPs is optimized using batch equilibrium experimentations at room temperature.

H 2 O 2 Detection in Urine and Limit of Detection
The βCD-AgNPs test is compared with a peroxidase assay test as reference. The peroxidase assay allows the development of a pink color when Horse Radish peroxidase (HRP) is mixed with the OxiRed probe and H 2 O 2 sample. The working standards of peroxidase assay are made ranging from 1. Midstream urine samples were collected from seven healthy-looking men volunteers between 20 and 40 years in age. The collected samples were stored at 4 • C for a period of 12 h followed by centrifugation for 15 min at ×1000 rpm before use. H 2 O 2 concentrations are measured in triplicates in the samples' supernatants. To reduce the effect of the matrix, analysis of H 2 O 2 was studied in three sets: the pure urine samples, twice and ten times diluted urine samples. Stock solutions and working standards of H 2 O 2 are prepared from the 30% concentrated H 2 O 2 , from which 0.88 mM and 0.2 mM are gradually diluted using MilliQ ultra-pure water with a resistivity of 17.5 MΩ·cm (Fisherbrand Accu20 Ultrapure Water System, Loughborough, UK). Then, working standards ranging from 1.0 to 10.0 nmoles/microplate are freshly prepared from 0.2 mM using 0.1 M buffer solution supplied with the assay kit. The standards as well as urine samples were treated similarly as mentioned in Abcam Assay Kit's procedure: 46 µL of 0.1 M buffer, 2 µL of OxiRed probe, and 2 µL of HRP are blended, forming a reaction mixture to which 50 µL of the standard or sample is added. The working standards and urine samples are incubated for 3 h, prior to absorbances recording versus the reaction mixture blank for the peroxidase assay kit at 570 nm. However, for the βCD-AgNPs assay, 150 µL of the working standard or sample are mixed with 150 µL of the nanoparticles. Samples and working standards are incubated for 40 min, and absorbances are recorded at 384 nm against the 0.1 M buffer as a blank. The limit of detection (LOD) of βCD-AgNPs assay test is verified using the standard deviation of the absorbance and slope of the calibration curve after the equation LOD = 3.3 σ/S [29], where σ is the standard deviation of three reading of the lowest possible concentration, made by mixing 0.75 mL of βCD-AgNPs with 100 µL of H 2 O 2 to give a total volume of 0.95 mL. The limit of quantitation (LOQ) is defined using the equation LOQ = 10 L v /S, where L v , is the lowest possible concentration identified by the test. S signifies the slope of the calibration curve in the nM range. Conversion between molar concentration and moles of H 2 O 2 is made by multiplying the concentration by 300 µL, representing the volume of the microplate's well. Measurements are made in triplicates and presented as Mean ± SD.

Instrumentation
The XRD analysis are made on a Bruker D8 Analytical diffractometer operated by the software DIFFRAC.SUITE at 40 kV and 40 mA, equipped with a copper anode and a graphite monochromator to select CuK α1 radiation (λ = 1.540 Å). Diffractions are recorded from 2θ = 5.0 • to 80 • with step increase of 0.03 • and time of 0.5 s/step. To determine the abundance of elements in βCD-AgNPs, EDS is made concomitantly with SEM using a Tescan VEGA XM electron microscope machine provided with an X-Max 50 EDS detector's Oxford Instruments (Tescan, Brno, Czech Republic), at 125 eV resolution and managed by the AZtecEnergy analysis software. The software VEGA TC is used to record SEM images of the βCD-AgNPs using an accelerating voltage of 30 kV. TEM images are recorded by transferring the βCD-AgNPs onto the TEM grid (Electron Microscopy Sciences Nanomaterials 2021, 11, 1897 5 of 14 300 mesh grid made of a carbon layer deposited on copper). The aberration corrected TEM (Hillsboro, Oregon, USA, Thermo Fisher Scientific formerly FEI, Titan G2) is used for the structural characterization of the βCD-AgNPs, run at 300 kV and using spherical aberration)-correction. The hydrodynamic sizes of the nanoparticles are measured using the Nanotrac Wave II DLS analyzer (Microtrac, Pennsylvania, USA) working between 0.8 nm and 6.5 and is run for 15 cycles at a 90 • angle and 26 • C. Figure 1a represents the UV-Vis absorbance of the surface plasmon peaks of βCD-AgNPs with a characteristic peak at 384 nm. This peak is found to decrease as the concentration of H 2 O 2 increases. The color of the nanoparticles faded from a green olive color to uncolored with the increase of the concentration of H 2 O 2 ( Figure S1, Supplementary Materials), suggesting a proportionality between the color change of the βCD-AgNPs and concentration change of H 2 O 2 . Consequently, color change is exploited as an optical test for the hydrogen peroxide detection.  [40]. An enlargement of the βCD-AgNPs peaks in comparison to metallic silver (PDF no. 04-0783), is assigned to a nanosized Ag in the βCD-AgNPs material [41]. The Ag atomic lattice of the nanoparticles is also confirmed using HRTEM ( Figure S5     The color change of the βCD-AgNPs, from green olive to uncolored, is accompanied by a gradual decrease in the absorption intensity of the characteristic plasmonic peak of the βCD-AgNPs at 384 nm due to atomic dissolution of βCD-AgNPs in the presence of H 2 O 2 , and this explanation is in accordance with the proposed mechanism by which the βCD-AgNPs are oxidized in the presence of H 2 O 2 to release Ag + to the medium. H 2 O 2 may induce aggregation of the AgNPs, as is indicated by Wang et al. [37] during the regeneration of AuNPs from an aromatic thiol (ArSH) dissolved AuNPs solution. However, aggregation cannot be an overall mechanism of βCD-AgNPs interaction with H 2 O 2 because the resulting solution does not have any absorbance.

Results and Discussion
The first parameter to check when optimizing a test is its selectivity, the βCD-AgNPs are mixed volume by volume with Ca 2+ , Co 2+ , Cu 2+ , Mn 2+ , Ni 2+ , Pb 2+ , Zn 2+ , F − , urea, glucose, and ascorbic acid as possible interfering species with H 2 O 2 . Little changes are assessed in the case of all interfering species, with the most pronounced change found for H 2 O 2 , results of this analysis are presented in Figure S2 (Supplementary Materials). Energy Disperse Spectroscopy (EDS) presented in Figure S3 (Supplementary Materials) demonstrates the presence of silver, carbon, and oxygen, and proves that β-cyclodextrin macromolecules successfully capped the silver metal. The weight percent of the silver is found to be the highest at 44.1 wt% followed by oxygen and carbon.
The βCD-AgNPs size distribution is determined using the dynamic light scattering (DLS) and the aberration-corrected high-resolution transmission electron microscopy (HRTEM) represented in Figure 1b,d (and Figure S4 in Supplementary Materials), respectively. The DLS size is found to be 52.0 nm/dimeter, however the HRTEM shows regular spherical shaped nanoparticles with average size varying between 19.4 and 53 nm/dimeter, and the high diameter presented by DLS has been attributed to the DLS measuring the hydrodynamic size of the particle which is the size of the βCD macromolecules shell and the liquid layer all around the particle. Shells may be involved in various forms of intermolecular forces (van der Waals forces and hydrogen bonding), while the HRTEM gives the actual size of the nanoparticle [38]. Zheng et al. demonstrated, however, that the hydrodynamic diameters of the nanoparticles can significantly vary with nanoparticles' concentration and incident beam used in the instrument [39].  [40]. An enlargement of the βCD-AgNPs peaks in comparison to metallic silver (PDF no. 04-0783), is assigned to a nanosized Ag in the βCD-AgNPs material [41]. The Ag atomic lattice of the nanoparticles is also confirmed using HRTEM ( Figure S5 [42]. Figure 2a represents the evolution of absorbances of different samples at different volumes of βCD-AgNPs. An increase in the concentration of βCD-AgNPs (volume increase) is faced with a decrease in the sample's absorbance, ∆Abs represents the difference between the reference unreacted βCD-AgNPs minus the absorbance of the sample, which is found to increase with concentration increase of H 2 O 2 . Even though the volume increase of βCD-AgNPs nanoparticles is expected to increase the number of active sites of interaction with H 2 O 2 , however, the number of H 2 O 2 molecules circumvent the sites of interaction's number provided by the material. At 2.0 mL of βCD-AgNPs, enough active sites of interactions have been provided by the material to interact in a mole-to-mole ratio with the H 2 O 2 in the medium. After this point, the number of active sites provided exceed the required number and a saturation is observed on words. The kinetic of H 2 O 2 interaction with βCD-AgNPs is presented in Figure 2b, it can be observed that increasing the time of the reaction lead to direct increase in the ∆Abs until it reaches a plateau. In the beginning, the material Nanomaterials 2021, 11, 1897 7 of 14 has many sites of interaction, with time increase these sites are occupied by H 2 O 2 , while the time proceeding these sites are fully occupied by H 2 O 2 and therefore saturation is achieved. The kinetic of the reaction is found to be very fast, equilibrium is attained at time less than 0.5 min, the sites of interaction of βCD-AgNPs almost consumed after 0.5 min to attain equilibrium. lower in alkaline pH compared to neutral pH = 7.0 and acidic pH = 4.0, lower ∆Abs corresponds to a higher Abs, which is indicative that the nanoparticles are more stable in alkaline mediums, this behavior could be explained based on hydroxide anions could enhance aggregation of the nanoparticles. The acidic pH = 4 is still presenting a ΔAbs lower (high Abs) than the neutral pH = 7.0 which may be explained by competition reaction between the hydronium ion and H2O2. From Figure 2d, it can be concluded that βCD-AgNPs operate very well at high temperature, since 45 °C is presented with lowest ∆Abs (highest Abs). Thirty-seven degrees, the physiological temperature where most of the biological samples are extracted, presented with the second lowest ∆Abs when adopted for biological analysis. The temperature increases the speed of collision between the H2O2 and sites of interactions on the βCD-AgNPs. Increasing the temperature slows down the process. The reaction is therefore classified as exothermic.  Figure 2c shows an increase in ∆Abs by increasing the concentration of H 2 O 2 until ∆Abs reaches a plateau for the three pH (pH = 4.0, pH = 7.0, and pH = 10.0). The ∆Abs is lower in alkaline pH compared to neutral pH = 7.0 and acidic pH = 4.0, lower ∆Abs corresponds to a higher Abs, which is indicative that the nanoparticles are more stable in alkaline mediums, this behavior could be explained based on hydroxide anions could enhance aggregation of the nanoparticles. The acidic pH = 4 is still presenting a ∆Abs lower (high Abs) than the neutral pH = 7.0 which may be explained by competition reaction between the hydronium ion and H 2 O 2 . From Figure 2d, it can be concluded that βCD-AgNPs operate very well at high temperature, since 45 • C is presented with lowest ∆Abs (highest Abs). Thirty-seven degrees, the physiological temperature where most of the biological samples are extracted, presented with the second lowest ∆Abs when adopted for biological analysis. The temperature increases the speed of collision between the H 2 O 2 and sites of interactions on the βCD-AgNPs. Increasing the temperature slows down the process. The reaction is therefore classified as exothermic.
The mechanism of interaction of βCD-AgNPs, with H 2 O 2 represented in Figure 3, is based on the oxidative dissolution of the nanoparticles to release Ag + and other reactive oxygen species such as OH • which will further dissolute the nanoparticles. Molleman and Hiemstra [43] demonstrated that the shape of the AgNPs plays a big role in their reactivity, and they found that nanorods and nanoprisms are more reactive than nanospheres because they exhibit exposed facets and therefore react faster. The mechanism of interaction of βCD-AgNPs, with H2O2 represented in Figure 3, is based on the oxidative dissolution of the nanoparticles to release Ag + and other reactive oxygen species such as OH • which will further dissolute the nanoparticles. Molleman and Hiemstra [43] demonstrated that the shape of the AgNPs plays a big role in their reactivity, and they found that nanorods and nanoprisms are more reactive than nanospheres because they exhibit exposed facets and therefore react faster. To measure the level of H2O2 in patients' urine, calibration curves are established in mM, μM and nM concentration ranges, which are presented in Figure 4a-c, respectively. Markedly, the βCD-AgNPs test presented with a large linear range from 5.0 nM through the whole μM range up to 17.7 mM concentration (Figure 4a) where the deviation from linearity start showing. In Figure 4d, this is represented by a refined protocol of H2O2 response in the presence of 10 mM of creatinine. The sensitivity is almost 12-folds lower, using the refined protocol with linearity maintained from 1.3 up to 37.3 nmoles of H2O2/mole of creatinine. This decrease in sensitivity is due to the encapsulation creatinine with β-CD shell of the nanoparticles. β-CD is well known to form 1:1 complexes with neutral guest species, in solution, the nonpolar cavity of β-CD is occupied with water molecules which is not favorable energetically. Water molecules are substituted with appropriate guest molecules such as creatinine that are less polar than water molecule. Limit of H2O2 detection using βCD-AgNPs is determined in the nM calibration curve applying the equation LOD = 3σ/S, where σ is the standard deviation of the response of three urine samples replicates at wavelength of 384 nm. The limit of detection is found to be 1.47 nM when Absorbance of βCD-AgNPs is used directly to assess H2O2, while 0.041 nM is found when ΔAbs is used instead. The detection limit is very low compared to other H2O2 sensors reported in Table 1. The limit of quantitation (LOQ) is found from the formula: LOQ = 10(Sy/S), where Sy and S are the slope and standard deviation of the response obtained from the calibration of the test. A LOQ of 3.76 nM is found for the βCD-AgNPs test when Abs is used and 0.14 nM when ΔAbs is used, indicating that ΔAbs always yield lower LOD and LOQ compared to Abs.  This decrease in sensitivity is due to the encapsulation creatinine with β-CD shell of the nanoparticles. β-CD is well known to form 1:1 complexes with neutral guest species, in solution, the nonpolar cavity of β-CD is occupied with water molecules which is not favorable energetically. Water molecules are substituted with appropriate guest molecules such as creatinine that are less polar than water molecule. Limit of H 2 O 2 detection using βCD-AgNPs is determined in the nM calibration curve applying the equation LOD = 3σ/S, where σ is the standard deviation of the response of three urine samples replicates at wavelength of 384 nm. The limit of detection is found to be 1.47 nM when Absorbance of βCD-AgNPs is used directly to assess H 2 O 2 , while 0.041 nM is found when ∆Abs is used instead. The detection limit is very low compared to other H 2 O 2 sensors reported in Table 1. The limit of quantitation (LOQ) is found from the formula: LOQ = 10(S y /S), where S y and S are the slope and standard deviation of the response obtained from the calibration of the test. A LOQ of 3.76 nM is found for the βCD-AgNPs test when Abs is used and 0.14 nM when ∆Abs is used, indicating that ∆Abs always yield lower LOD and LOQ compared to Abs.  In Figure 5a,b the calibration curves used to assess the levels of H2O2 in freshly voided midstream urine of the 7 healthy men aged between 20 and 40 years are presented, using the assay kit and the LSPR of βCD-AgNPs, respectively. Linear responses are obtained for both tests. For the assay kit a linear response was in the range of 1.0 nmoles to 10.0 nmoles (10-100 μM) with a slope of 0.0785 and a regression coefficient of 0.995, while a linear response 1.33 to 37.3 nmoles of H2O2/moles of creatinine with a slope of 0.0075 and regression coefficient of 0.9955 when the βCD-AgNPs test is used in the presence of 10.0 mM of creatinine. The concentration of H2O2 is dependent on the dilution for all participants, except KU-22, all participants showed lower concentration of H2O2 when samples are diluted 10 times and the assay kit is used (Figure 5c). The average concentration of H2O2 in urine using the assay kit is found to vary between 2.11 ± 0.048 nmoles (21.1 ± 0.48 μM) and 3.22 ± 0.046 nmoles (32.2 ± 0.46 μM) when the matrix is not diluted, between 1.38 ± 0.042 nmoles (13.8 ± 0.42 μM) and 2.52 ± 0.019 nmoles (25.2 ± 0.19 μM) when the matrix is diluted twice and between 0.60 ± 0.051 nmoles (6.0 ± 0.51 μM) and 1.12 ± 0.050 nmoles (12.1 ± 0.50 μM) when the matrix is diluted 10 times. In this current study, the H2O2 total concentration in freshly voided urine in the 7 healthy subjects using the assay kit are comparable to values reported by Long et al. [52] who found values between 11 and 173 μM when analyzing freshly secreted urine of subjects aged from 19 to 38 years old using oxoglutarate decarboxylation assay. H2O2 levels of 17.28 μM are also reported in normal participants  (Figure 5c). The average concentration of H 2 O 2 in urine using the assay kit is found to vary between 2.11 ± 0.048 nmoles (21.1 ± 0.48 µM) and 3.22 ± 0.046 nmoles (32.2 ± 0.46 µM) when the matrix is not diluted, between 1.38 ± 0.042 nmoles (13.8 ± 0.42 µM) and 2.52 ± 0.019 nmoles (25.2 ± 0.19 µM) when the matrix is diluted twice and between 0.60 ± 0.051 nmoles (6.0 ± 0.51 µM) and 1.12 ± 0.050 nmoles (12.1 ± 0.50 µM) when the matrix is diluted 10 times. In this current study, the H 2 O 2 total concentration in freshly voided urine in the 7 healthy subjects using the assay kit are comparable to values reported by Long et al. [52] who found values between 11 and 173 µM when analyzing freshly secreted urine of subjects aged from 19 to 38 years old using oxoglutarate decarboxylation assay. H 2 O 2 levels of 17.28 µM are also reported in normal participants not suffering from any disease [53]. However, very high concentrations of H 2 O 2 were noticed in ill subjects, 89.10 ± 8.73, 85.37 ± 15.45, 91.35 ± 17.79, and 68.02 ± 7.23 µM are reported in subjects suffering from helminth + protozoa, protozoa only, helminth and intestinal parasitic, respectively. Lower levels of H 2 O 2 in the order of 0.84 to 5.71 µM are also reported in fasting subjects [54].
available in the literature about creatinine corrected H2O2 concentration. Yuen et al. [56], for instance, reported values between 90-1164 μmoles H2O2/mole creatinine using a refined protocol taking into account the presence of creatinine in the H2O2 matrix that increases the concentration of H2O2. These values are significantly higher than values found for βCD-AgNPs test and the variations are mainly due to the stability and biological variations of the sample which limit the use of hydrogen peroxide in urine as a potential biomarker of whole-body oxidative stress.

Conclusions
βCD-AgNPs have been successfully synthesized, and the synthesized NPs are characterized by UV−visible, DLS, EDX, XRD, and HRTEM analysis. The assay using βCD acted as a shell to cap and stabilize silver nanoparticles. Furthermore, a simple and instant colorimetric assay for H2O2 detection in the commercial oxidant solution is established via synthesized βCD-AgNPs. The H2O2 detection optimum conditions were studied in detail. An increase in the volume of nanoparticles is faced with a decrease in the LSPR absorbance of the suspended nanoparticles mixed with 40.0 mM H2O2 concentration. The optimum pH is found to be equal to 10.0. The kinetic of the reaction is found to be fast, and equilibrium is established between the nanoparticles and H2O2 in the first 0.5 min. The thermodynamics of the equilibrium show that the process is exothermic, and the reaction is favored at 37 °C compared to 25 °C. The β-cyclodextrin organic shell of the synthesized  . These values are significantly higher than values found for βCD-AgNPs test and the variations are mainly due to the stability and biological variations of the sample which limit the use of hydrogen peroxide in urine as a potential biomarker of whole-body oxidative stress.

Conclusions
βCD-AgNPs have been successfully synthesized, and the synthesized NPs are characterized by UV−visible, DLS, EDX, XRD, and HRTEM analysis. The assay using βCD acted as a shell to cap and stabilize silver nanoparticles. Furthermore, a simple and instant colorimetric assay for H 2 O 2 detection in the commercial oxidant solution is established via synthesized βCD-AgNPs. The H 2 O 2 detection optimum conditions were studied in detail. An increase in the volume of nanoparticles is faced with a decrease in the LSPR absorbance of the suspended nanoparticles mixed with 40.0 mM H 2 O 2 concentration. The optimum pH is found to be equal to 10.0. The kinetic of the reaction is found to be fast, and equilibrium is established between the nanoparticles and H 2 O 2 in the first 0.5 min. The thermodynamics of the equilibrium show that the process is exothermic, and the reaction is favored at 37 • C compared to 25 • C. The β-cyclodextrin organic shell of the synthesized silver nanoparticles (βCD-AgNPs) are found to enhance the detection of hydrogen peroxide in urine through encapsulation mediated process of creatinine when compared to the commercial assay kit. Surely the assay kit gives reproducible results of total H 2 O 2 in urine, values varying between 0.60 ± 0.051 nmoles (6.0 ± 0.51 µM) and 3.22 ± 0.046 nmoles (32.2 ± 0.46 µM) are found at different dilution rates, suggesting that the test is affected by the matrix complexity. βCD-AgNPs test provided an optical creatinine corrected test with high selectivity and sensitivity for H 2 O 2 , with values ranging from 24.82 ± 1.50 nmoles of H 2 O 2 /mole of creatinine and 70.0 ± 1.57 nmoles of H 2 O 2 /mole of creatinine. The test is also affected by the complexity of the matrix. These values are lower than creatinine corrected H 2 O 2 reported in the literature (90-1164 µmoles H 2 O 2 /mole creatinine), and the variations may be due to the stability of the samples and limit the use of hydrogen peroxide in urine as a potential biomarker of whole-body oxidative stress.