Colorimetric Based Analysis Using Clustered Superparamagnetic Iron Oxide Nanoparticles for Enhanced Glucose Detection

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Introduction
Superparamagnetic iron oxide nanoparticles (SPIONs) are used in the innumerable biomedical fields such as target delivery with external sources, T2-MRI contrast agents, and hyperthermia treatment for cancers [1][2][3][4][5][6][7]. In a few decades, the SPIONs, including any other metallic particles, have been reported to exhibit an artificial peroxidase activity (i.e. nanozyme), and those have been widely studied at the earliest time.
SPIONs mediated nanozyme activity has been regarded as high stable in various temperatures and pH, also can be synthesized in bulk quantities at the comparative cost compared to the naturally expressed peroxidase [8,9]. For these advantages, SPIONs have been focused on substituting peroxidases in diverse fields, especially detecting real glucose. The glucose can be oxidated by glucose oxidase (GOX), succeeding in the release of hydrogen peroxides (H2O2) in an aqueous solution. The SPIONs can catalyze a peroxidase sensitive chromogenic substrate (i.e. 3,3′,5,5′tetramethylbenzidine (TMB) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS)), leading to the change of color in the presence of H2O2.
Several studies of the glucose detection based on the colorimetric change were 4 reported [9][10][11][12] and this procedure can be performed in a simple, easy, low-cost, and observed by our eyes intuitionally [13]. Gao et al. firstly demonstrated that Fe 2+ ions in the SPIONs can play the significant role of the nanozymes, presenting this activity were originated from instinct SPIONs property [8]. YU, Faquan, et al. investigated the effect of six differently coated SPIONs on glucose detection and shown the results were very different depending on the used substrate and core coating materials [9]. They discussed that improving electrostatic interaction between the chromogenic substrate and SPIONs is essential to yielding higher catalytic activity and sensitivity. However, few kinds of research of engineered SPIONs, in which the particles were not modified in the whole structure or surface themselves, have existed. The modification of nanocrystals characters must be done in synthesized stages and is not easy to be changed once these were made. One of the most used in the synthesis of high qualities SPIONs, the thermal decomposition method, a process that allows the generation of particles with excessive and extremely uniform size, is very important to expose the reaction mixture at a specific temperature and time but also to add a ligand to get specific characters [14]. Even if it is not a modification of the core of SPIONs, in reporting the utilization of engineered particles for nanozymes, several strategies to enhance the sensitivity and limit of detection (LOD) to glucose were tried by grafting ligands to the structure.
Focusing on the fact that the major role of SPIONs for nanozyme is Fe ions, we imaged a bulk enzyme design that can easily be made without further surface modification and promote the oxidation of the chromogenic substrate widely used in peroxidase studies. In this study, we synthesized spherical-shaped clustered SPIONs (CSPIONs). Because SPIONs consists of numerous Fe3O4 and CSPIONs also consists of many primary SPIONs, we hypothesize that CSPIONs are more sensitive toward H2O2 and glucose. For that, the glucose levels were set in conditions of real diabetes contained in physiology fluids [15,16]. We evaluated the possibility of CSPIONs nanozyme ability to H2O2 and also H2O2 produced from the oxidation of glucose by GOX to measure the glucose level in the colorimetric method. The sensitivity of CSPIONs to glucose and LOD were explored and compared to the SPIONs reported before [9][10][11].

Nanoparticles (CSPIONs)
We used the oil-in-water (O/W) method to cluster the iron oxide nanoparticles (SPIONs), and it was shown in Figure 1a. The mixture contained distilled water, SPIONs, and PLGA were emulsified with vortexing and sonication. PLGA is an amphiphilic polymer, and it was used for clustering the SPIONs that are highly hydrophobic materials in the water-abundant environment. The characterization of CSPIONs was shown in Figure 2. The CSPIONs were synthesized in highly monodisperse (PDI=0.124), and the average size and zeta-potential (ζ-potential) were measured as 120.1 nm and -61.7 mV, respectively (n=3, Fig. 2a). The CSPIONs were shown bigger size than used SPIONs whose average size is 10 ± 1 nm (provided by the manufacturer). The transmission electron microscopy (TEM) image shows that the synthesized particles have a spherical shape and successfully clustered within the PLGA (Fig. 2b).
Because the catalysis process depends on the Fe ions on the SPIONs surface [8], it is crucial to figure out how many ions were existed in the particles. In order to quantify the Fe in CSPIONs, the ferene-s assay was performed. This assay is widely used for quantification of SPIONs in nanostructure fastly and accurately [17][18][19]. The 6 representative standard curve was regressed in y=0.4671x+0.0649 (R 2 =0.996), and the back-calculated quantities of SPIONs were found in 100.73μg/100μl, indicating that the ratio of mass and volume was about 1:1 (Fig. 2c). About 20% of SPIONs were clustered into the nanostructure (500 μg SPIONs were used in this synthesis) and others were stuck in the surface of the vial due to the instinct hydrophobic property of SPIONs. The ferene-s assay was regularly performed for the quantification of SPIONs when the CSPIONs were synthesized.

The Artificial Peroxidase Activity of CSPIONs
Given that CSPIONs have responsibilities of a nanozyme, it can oxidate the substrate through a Fenton reaction. The Fenton reaction is a catalytic process, leading to converting hydrogen peroxide into a hydroxyl free radical as described in (1) and The results of the oxidated substrate ABTS in the presence of H2O2 were shown in In similar to this work, the different shape of nanocrystals (clustered sphere, octahedra, and triangular) was investigated in the study of peroxidase-like activities.  [20]. Zhou et al. reported that the catalytic mechanism can roughly be divided into two groups; structure-insensitive and structure-sensitive. They suggested that decreasing the fewer crystals planes and increasing the more reactive planes were required to enhance the catalysis effect [21]. Although further studies for the precise mechanism of the enhanced nanozyme activity of cluster shape are required, based on the previous reports, either clustering in nucleation and growth stage or structure modification of nanoparticles can increase the activity toward the chromogenic substrate. Besides, it may be related to their preferential exposure of catalytically active iron atoms or crystals planes.

The Artificial Peroxidase Activity About H2O2 Derived from Glucose
In order to investigate the performance of the CSPIONs for glucose detection, the degree of oxidated substrate ABTS in the presence of different glucose concentrations was shown in Figure 4. There is no chemical mechanism that SPIONs detect the glucose, so we measured the H2O2 originated from glucose oxidase (GOX) and glucose indirectly. It was also shown as substrate-enzyme affinity, presenting the absorbance was increased with glucose level increased. The results were highly accruable (standard deviation of all groups was less than 0.03) and demonstrated that the glucose was detected by using CSPIONs.

Comparison with Reported SPIONs
The sensitivity and LOD are presented in Figure 5. The absorbance and glucose concentration were correlated with each other linearly. The regressed line of the slope 8 represents the sensitivity of CSPIONs to glucose. The sensitivity was calculated in 1.50.
Multiplying the y-intercepts of the regression line by 3 and dividing by the slope of the line can be considered as the LOD of CSPIONs to glucose. The LOD was presented as 5.44 μM.
In order to compare the synthesized CSPIONs with others reported SPIONs, the artificial peroxidase ability of modified in nanocrystals' structure or surface was summarized in Table 1. The CSPIONs were shown more sensitive to glucose compared to the case SPIONs used. The LOD was also lower than that of SPIONs, indicating that even small amounts of CSPIONs can oxidize the chromogenic substrate and successfully detect the degree of oxidation in the colorimetric based system.

Conclusion
The CSPIONs were synthesized via the engineered bottom-up method. The particles kept the peroxidase ability after SPIONs are clustered in the amphiphilic PLGA polymer.
The various range of glucose test (below 5 mM) demonstrated the CSPIONs detect the H2O2 from the reaction between glucose and GOX in high repeatable (Standard Deviation < 0.03). The comparison with the reported SPIONs showed CSPIONs were more sensitive to glucose and had lower LOD despite using a substrate that is not proper to negatively charged particles. The results show that CSPIONs may serve as a glucose sensor. For future works, non-invasive and color-based glucose measurements using CSPIONs will be investigated.

Chemicals and reagents
All materials were purchased and used without further modification and treatment. Iron (North Carolina, USA).

The Synthesis of Clustered Superparamagnetic Iron Oxide Nanoparticles (CSPIONs)
The

The Quantification of SPIONs in CSPIONs
A ferene-s assay was performed. This assay can be used for accurate SPIONs quantification, and also regarded as reasonable compared to inductively coupled plasma mass spectrometer (ICP-MS) [17][18][19].

The Characterization of CSPIONs
The study of the size, zeta-potential (ξ-potential), and shape of CSPIONs were done to characterize the CSPIONs. The sample was prepared into 1 ml volume (100:

Assessment of Artificial Peroxidase Ability of CSPIONs
To assess the artificial peroxidase ability of CPSIONs, we utilized colorimetric based 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) assay in the presence of H2O2. Briefly, 24 μl of 60 mM ABTS, 185 μl of 0.2 M acetate buffer (pH ≈ 4), and different amounts of CSPIONs (10 μg, 20 μg, and 40 μg) were used in this assessment. The H2O2 was half-diluted from 100 mM to 0.7813 mM (i.e. 781.3 μM) using PBS and the mixture was reacted at 45 ℃ for 10 min. After centrifuging the mixture at 13,500 RPM to remove precipitated CPSIONs, the supernatant of the solution was divided into 300 μl per well to determine average absorbance. Control groups were conducted using only PBS (no glucose in the sample).

Measurement of Glucose Using CSPIONs
Similar to the procedure of the assessment of artificial peroxidase, it was also based on the chromogenic substrate's oxidation. 40 μl of GOX (10 mg/ml), 360 μl of 4 mM ABTS, and 50 μg of CSPIONs were added into various concentrations of β-Dglucose. The β-D-glucose was diluted from 5 mM to 0.078125 mM (i.e. 78.125 μM) in PBS and the blend was incubated at 45 ℃ for 45 min, following centrifuging the resulting solvent at 13,500 RPM to remove thrown CPSIONs. The supernatant was analyzed the same as the above paragraph.

The assessment of the sensitivity and limit of detection (LOD) to the glucose of CSPIONs
The sensitivity and LOD were identified below 40 μM glucose of concentration. The glucose level was half-diluted from 40 μM to 1.25 μM and analyzed ODs at 417 nm.
The sensitivity and LOD were defined as (2) and (3), respectively.