Herein, composite electrospun nanofibers (NFs) based on polyacrylonitrile (PAN) and smectite clay, montmorillonite (Mt) were employed as matrices for the development of amperometric glucose biosensors. Clays are established as suitable immobilization platforms for biocatalysts and allow functionalization through intercalation with organic molecules. The present study was focused on utilization of functional Mt in biosensing by incorporation in nanocomposite NFs and cross-linking of model enzyme, glucose oxidase (GOx). The initial clay mineral utilized was industrial type Mt (K10) and a comparison between simple PAN and composite nanofibers PAN/Mt K10 as biosensing matrices was performed. Further on, the study focused on modification of Mt with dioctadecyl dimethyl ammonium chloride (DDAC), an ammonium salt with extended alkyl chain and methylene blue (MB) as a redox mediator. The functionalization of Mt with each molecule was assessed by infrared spectroscopy (FTIR), correlated with the morphology of the nanocomposite NFs, visualized by scanning electron microscopy (SEM) and with the efficiency in amperometric glucose detection. Ultimately, both incorporation and functionalization of Mt improved the performance of the constructed biosensors, increasing the linearity, which extended up to 15 10(-3) M within two linear ranges (1.0 10(-5) - 2.45 10(-3) M and 2.45 10(-3) -15 10(-3) M) and improving the sensitivity up to 52 and 25 mAM(-1)cm(-2), respectively. The detection limits were also lowered from 4.2 to 2.4 mu M and the operational stability was enhanced up to 85% as a result of incorporation of DDAC-Mt. (c) 2020 Elsevier Ltd. All rights reserved.