The absorption plots demonstrated a smooth decline in the characteristic absorbance peak at ~270 nm with the increasing amount of analyte, NH4 + , which suggests the binding of ammonium ions to few-layer graphene. In order to further investigate the UV absorption behaviour of few-layer graphene in the presence of NH4 + ions, the absorbance with increasing analyte amount ( 0.5 mM up to 3.75 mM) was plotted for different graphene amount (G-I to G-V) (Fig. and)). Here, I to V define the increasing concentration of few-layer graphene, i.e., from 0.06% up to 0.15% (v/v). The change in the absorption was quantified and plotted with respect to NH4 + ions concentration for elucidating the binding affinity and saturation conditions. The most significant reduction in the absorption was found to be for the graphene concentration of 0.1% (v/v) which suggested the most effective binding and ammonium absorption at the particular concentration. In Fig. (e), G-III showed a linear decline in the absorbance with NH4 + concentration, which was fitted to establish a linear relationship between absorbance changes and ammonium concentration. The calibration curve was further explored to estimate the limit of detection (LoD) and the limit of quantification (LoQ). The detection limit indicated the lowest concentration of ammonium ions which was detected by few-layer graphene and was found to be ~20 ppm, while the quantification limit was estimated on the basis of repeatability and precision and was calculated to be 1.8 mM. Further, the association between the analytes and detector i.e. NH4 + ions and graphene respectively was studied using the Benesi-Hildebrand equation, Fig. , which estimated the binding constant (Kb) to be ~ 0.6 ´ 10 3 M -1 . The low value of the binding constant suggested the moderate binding as reflected by the estimated LoD and LoQ values. The affinity between the prepared few-layer graphene and analyte, ammonium ions, is ordinarily governed by a plethora of factors i.e. the number of layers in the graphene, the presence of surface groups, etc. The comparative plot was drawn to collate the ammonium ions detection carried out by graphene sheets. Here, the biologically prepared few-layer graphene demonstrated a detection limit of 20 ppm which was comparable to that observed in previous reports, Fig. (g). In order to gain further insights into the sensing mechanism, control and ammonium treated graphene samples were examined for the changes in their surface chemical state and lattice structure using FTIR and Raman spectroscopy (Fig. and). Here, the reproducibility of the system was checked by heating the ammonium treated graphene and examining the changes using spectroscopic techniques. The FTIR peaks revealed the changes in the surface chemical composition of the graphene sheets upon NH4Cl application. From the plot, Fig. (h), there are significant changes observed in the NH4 + treated graphene, in comparison to control and heat treated graphene-NH4 + . The peaks at ~3100 and ~1400 cm -1 , observed for ammonium treated graphene, correspond to N-H stretching and bending respectively. Further, the band at ~1090 cm -1 corroborates to the symmetric NH3 signifying the coordination of NH3 to Lewis acid sites on the carbon surface (graphene layers). The peak at ~820 cm -1 , assigned to the deformation vibration for C-H groups in carbohydrates, i.e., glucose here, was observed to be absent for NH4 + treated graphene suspension. The absence of peak can be owed to the glucosamine formation, where hydroxyl group at the anomeric carbon can be replaced with an amine group.