Colorimetric Nanobiosensor Design for Determining Oxidase Enzyme Substrates in Food and Biological Samples

Biological enzymes have high catalytic activity and unique substrate selectivity; their immobilization may provide cost reduction and reusability. Using magnetic nanoparticles (MNPs) as support materials for enzymes ensures easy separation from reaction media by an external magnetic field. Thus, MNPs were prepared by the coprecipitation method, coated with silanol groups, then −NH2-functionalized, and finally activated with glutaraldehyde. Finally, three different oxidase enzymes, i.e., uricase, glucose oxidase, and choline oxidase, were separately immobilized on their surfaces by covalent attachment. Hence, colorimetric nanobiosensors for the determination of three biologically important substrates, i.e., uric acid (UA), glucose (Glu), and choline (Ch), were developed. Hydrogen peroxide liberated from enzyme–substrate reactions was determined by the cupric ion reducing antioxidant capacity (CUPRAC) reagent, bis-neocuproine copper(II) chelate. In addition, UA-free total antioxidant capacity could also be measured via the developed system. Kinetic investigations were carried out for these nanobiosensors to enable the calculation of their Michaelis constants (Km), revealing no affinity loss for the substrate as a result of immobilization. Enzyme-immobilized MNPs could be reused at least five times. The linear concentration ranges were 5.43–65.22 μM for UA, 11.1–111.1 μM for Glu, and 2.78–44.4 μM for Ch, and the limit of detection (LOD) values with the same order were 0.34, 0.59, and 0.20 μM, respectively. Besides phenolic and thiol-type antioxidants, UA could be determined with an error range of 0.18–4.87%. The method is based on a clear redox reaction with a definite stoichiometry for the enzymatically generated H2O2 (which minimizes chemical deviations from Beer’s law of optical absorbances), and it was successfully applied to the determination of Glu and UA in fetal bovine serum and Ch in infant formula as real samples.


S.I.2. Examination of the effects of experimental conditions on the immobilized enzymes
As is known, although biological enzymes have unique selectivity against their substrates, they are not resistant to harsh experimental conditions. For this purpose, the effects of temperature, pH and organic solvents on the immobilized enzymes were investigated. During the experiments, the proposed methods for UA, Glu and ChCl determinations in the presence of oxidase enzymes (uricase, glucose oxidase and choline oxidase, respectively) immobilized on

. Calibration graphs for Glu and ChCl
To construct the Glu and ChCl calibration graphs, the procedure described in Section 2.4. was applied for the volumes taken from the related solutions separately. For this purpose, to obtain Glu standard calibration graph, different volumes varying between 50 μL and 0.5 mL were taken from the standard Glu solution at 1.0×10 -3 M concentration.
On the other hand, for drawing the ChCl calibration graph, different volumes between 25 μL and 0.4 mL were taken from a standard ChCl solution at a concentration of 5.0×10 -4 M. After measurement of CUPRAC absorbances, the calibration graphs were drawn between final concentrations of the standard substrate solutions and measured absorbances. Then the equation of the calibration curve was calculated with the determination coefficient.

S.I.3.2. Calibration graph for UA
For this purpose, different volumes varying between 50 μL and 0.6 mL were taken from the UA solution at a concentration of 5.0×10 -4 M, and the procedure described in Section 2.4.3.
was applied in the presence and absence of UOx@MNPs.

S.I.4. Determination of UA in the AOx mixture
To prepare a synthetic AOx mixture solution, different volumes taken from 5.0×10 -4 M UA; 1.0×10 -3 M CFA, QR, GA, CAT, FA, NAC, CYS, GSH and 1.0×10 -2 M TR were mixed. Firstly, standard CUPRAC procedure was applied to the mixture, 1 then the method described in Section 2.4.3 was applied to another identical AOx mixture. The UA concentration was calculated by using the difference between the two absorbance values measured.
Additionally, another mixture, containing UA in the presence of certain plasma AOxs, namely Bil, AA and GSH were also investigated. For this purpose, 0.1 mL of 5×10 -4 M UA was mixed with 0.1 mL of 1.0×10 -3 M GSH, 0.2 mL of 4.0×10 -4 M AA and 0.2 mL of 1,0×10 -4 M Bil separately. Here, the CUPRAC method was applied directly to the mixtures, and then the mixtures were treated with UOx@MNPs, followed by re-application of the CUPRAC method.

S.I.5. Chromatographic UA determination
To compared the results, get by the proposed method a reverse phase HPLC method was used as the standard verification method. For testing the amount of UA in the AOx mixtures, the method described earlier by George et al. 3 was applied with a few modifications. A C18 (250 mm  4.6 mm  5 µm) column was used as the stationary phase and the mobile phase was 10 mM KH 2 PO 4 at pH 4.7. The column temperature was kept constant at 25ºC during the 15minute isocratic elution at a flow rate of 0.8 mL min -1 . Analyses were performed using a photodiode array (PDA) detector within the wavelength range of 200-800 nm, and 280 nm was used as the detection wavelength. Finally, the injection volume was 20 µL.
To obtain UA calibration graph, UA standards at different concentrations between 1.0×10 -6 and 1.0×10 -4 M were injected into the HPLC system at the conditions stated above. Then the calibration graph for HPLC method between UA concentration and peak areas was drawn.

S.I.6.3. Determination of UA and UA-free TAC in FBS by the proposed UOx@MNPs method
For the determination of UA with the developed method, the FBS sample was used undiluted; 0.5 mL was taken, and the method was applied as described in Section 2.4.3.
The CUPRAC method was applied to the serum sample before and after it was treated with UOx@MNPs. The UA concentration in FBS was calculated from the difference in absorbance.

S.I.6.4. Determination of UA in the FBS by HPLC method
For this purpose, FBS was directly injected to the HPLC system first of all and then re-injected after treatment with UOx@MNPs.

S6
In addition, standard addition experiments were performed. For this purpose, 10.9 µM and 21.7 µM UA standards were added to 0.5 mL of FBS. The spiked samples were analyzed by HPLC and the proposed method.

S.I.7.1. Determination of enzyme kinetics for GOx and ChOx
To investigate enzyme kinetics, Michaelis constant (K m ) and maximum velocity of an enzymatically catalyzed reaction (V max ) values were calculated for free and immobilized enzymes. In this part, the frequently used spectrophotometric methods to investigate enzyme kinetics were preferred.

For this purpose, H 2 O 2 generated by the reaction between related enzymes (GOx and ChOx)
and their substrates (glucose (Glu) and choline chloride (ChCl) respectively) a common spectrophotometric method was used. According to the method proposed by Trinder 5 , a red quinone-imine dye was formed at the end of the reaction given below. The absorbance of the final product was measured at 506 nm. temperature, and the absorbance due to generated quinone-imine was read at 506 nm.

S7
For ChOx, the same method was applied with a few differences. Instead of 0.5 mL of PBS, 0.2 M NH 3 /NH 4 Cl buffer solution (at pH 9.0) at the same volume was used. Additionally, Glu was replaced with ChCl and similarly ChOx (4.8 U mL -1 ) was used instead of GOx.

S.II.1. The effects of pH, temperature and different solvents on the immobilized enzymes
The results of the temperature dependency experiment were given in Figure S2. When the graph was examined, it was observed that the activity of immobilized ChOx started to decrease after 37 °C. For temperatures higher than 50 °C, a significant decrease in the activity of all three immobilized enzymes (UOx, GOx and ChOx) was observed.
The results of the pH dependency experiment were given in the Figure S3. The results obtained as 'solvent free' and in the presence of different organic solvents were given in Figure S4 as a bar graph.   Table S2. As described in Section S.I.3, UA determination in the presence of some serum AOxs and experimental UA concentrations were calculated as stated above. The obtained results were collected in Table S3.  Figure S7. The calibration graph between UA concentration and HPLC peak areas.

S.II.6. Application of the proposed GOx@MNPs method to real samples
The standard addition test results were also given in Table S4.  Table S5. To compare the obtained results, the UA determination in FBS directly and in the spiked samples were analyzed by HPLC method. Figure S8. Chromatograms for FBS sample (a) obtained directly and (b) after UOx@MNPs treatment As can be seen from Figure S8, UA was completely decomposed after UOx@MNPs treatment.
The large peak seen in chromatogram (a) with an approximate retention time of 8.5 min was non-existent in chromatogram (b).

S.II.8. Investigation of enzyme kinetics
The results obtained by the classical UA monitoring method depending on the absorbance measurement at 290 nm for determination of the K m and V max values were given in Figure S10 and S11.

S.II.9. Investigation of stability and reusability of MNPs-attached enzymes
The results obtained by the experiments explained in the Section S.I.11 were given in the below for reusability (Table S7) and stability during the days ( Figure S12)