Colorimetric detection of hydrogen peroxide and cholesterol using Fe₃O₄-brominated graphene nanocomposite

Fe₃O₄-brominated graphene (Fe₃O₄-GBR) nanocomposites were synthesized via an in situ method using the precursors FeSO₄.7H₂O and GBR in different (1:1, 1:2, 2:1, 1:5, 1:10, 1:20, and 5:1) weight ratios at pH 11.5. The Fe₃O₄-GBR (1:5) nanocomposite in combination with H₂O₂ and 3,3^',5,5^'-tetramethylbenzidine (TMB) showed swift and superior intrinsic peroxidase mimetic enzyme activity compared with the other Fe₃O₄-GBR composites, GBR and Fe₃O₄, as observed by colorimetry. It was characterized using high-resolution scanning electron microscopy (HRSEM), energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), and thermogravimetric analysis (TGA). Its catalytic activity was optimized by varying different parameters, and the optimum conditions for peroxidase mimetic activity were observed using 100 μL Fe₃O₄-GBR (1 mg/mL), 50 μL TMB (1 mg/mL), and 200 μL H₂O₂(1 mM) in 400 μL of acetate buffer of pH 2.3 at 30 °C temperature. Kinetic analysis has revealed the Michaelis-Menten kinetic behavior of peroxidase activity with Michaelis-Menten constants (K_m) and maximum initial velocities (V_max) of 0.082 mM and 14.1 nMs^-1 respectively, for H₂O₂ and 0.086 mM and 5.1 nMs^-1, respectively for TMB. The limit of detection and linear range were found to be 49.6 μM and 100-880 μM, respectively, for H₂O₂ and 41.9 μM and 47.6-952.3 μM, respectively, for cholesterol. On this basis, a simple, swift, sensitive, selective, and reproducible colorimetric assay to detect cholesterol levels in blood serum samples using Fe₃O₄-GBR nanocomposite has been developed. Thus, Fe₃O₄-GBR composite as compared to Fe₃O₄ and GBR has shown better peroxidase mimicking activity for biosensing.