Development of an Effective and Economic Biosensor for Diabetic Blood Monitoring Based on MWCNTs, Artificial Redox Mediator Ferrocene, Nafion Polymer and a Local Extracted and Purified Glucose Oxidase Enzyme from Penicillium Notatum F-158 Fungus

Abstract

Enhancement of the properties of glucose oxidase (GOx) enzyme is still receiving attention due to its extensive applications. Eight different fungal strains were isolated from soil and orange fruit samples for inexpensive GOx production. Penicillium notatum F-158 (P. notatum) strain produced a remarkable amount of GOx. Its culture condition was optimized for optimum GOx production. GOx was purified and its activity, stability and kinetic parameters were studied. An effective biosensor {GCE/[MWCNTs–Fc–GOx(FAD)–NF]} based on layer by layer immbolization of MWCNTs, aritificial ferrocene (Fc) redox mediator, extracted P. notatum GOx enzyme and nafion polymer (NF) on glassy carbon electrode (GCE) surface was developed for glucose determination. Fc acts as an electron relay between enzyme and MWCNTs/GCE. The synergy of MWCNTs and Fc enhances the electrocatalytic action of Fc to the enzymatic oxidation of glucose. Low potential (+0.3V) of Fc applied in amperometric measurements avoids interference of the main electroactive substances present in the real plasma samples. This biosensor showed broad linear ranges {2.80×10-4 to 14.99×10-3 M} and low detection limit (8.68×10-6 M) for glucose determination. The achieved glucose concentrations in six plasma samples are consistent with normal values in human blood indicating that such biosensor could be used clinically.

A Mediated Enzymatic Electrochemical Sensor Using Paper-Based Laser-Induced Graphene

“…Firstly, aminoferrocene (AFc) was drop-cast on the PaperLIG electrode as the mediator layer, and glucose oxidase was then drop-cast on the PaperLIG/AFc to selectively react with the glucose molecules in the sample [ 1 , 28 , 48 ]. Finally, a Nafion thin film was applied to avoid the loss of enzyme from the PaperLIG electrode and the possible interference from other redox-reactive species [ 28 , 29 , 48 , 49 ]. As a reference, the CV of the PaperLIG electrode in the PBS solution containing 1 mg/mL AFc is shown in Figure S4b .…”

“…It was noted that magnetic stirring generated the fluctuation of the current signal, but no current increment was observed, indicating that magnetic stirring did not cause the current response of the biosensor. As Figure 4 a shows, the current signal increases after adding glucose into the solution, and this applied potential (−90 mV) is much lower than that of most developed LIG-based glucose biosensors ( Table S2 ), which can avoid the oxidation of possible redox-active interfering substances in the saliva sample [ 29 ]. The Nafion concentration for this first biosensor was 0.25%, and the biosensor showed a linear range between 0.1 and 3 mM for glucose levels in the PBS with good linearity (R 2 = 0.995) ( Figure 4 b).…”

“…The interference experiment for the biosensor was carried out in artificial saliva using a PaperLIG-based glucose biosensor containing 0.5% Nafion and the results are shown in Figure 4 d. 20 µM AA and 200 µM UA (the basal concentrations of these compounds in saliva [ 51 ]) did not cause any detectable current change in the artificial saliva with a 500 µM glucose concentration, and a further increase in the glucose concentration caused a rapid response ( Figure 4 d). The low applied potential (−90 mV) for the measurement and the application of the Nafion thin film prevents possible interference from redox-reactive species in saliva [ 29 ].…”

“…The glucose level can be detected by measuring the concentration change of the synthetic mediator under the applied potential [ 1 , 4 ]. As an example, ferrocene and its derivatives are commonly used mediators in glucose biosensors due to their fast reaction with glucose oxidase, low overpotential for redox reactions, and reversible electron transfer kinetics [ 1 , 28 , 29 ].…”

“…Finally, PaperLIG was applied in the development of a glucose biosensor by drop-casting aminoferrocene (AFc), glucose oxidase (GOx), and Nafion on its surface ( Figure 1 b). As a perfluorinated sulfonated cation exchanger, Nafion coating can improve the stability of enzymatic biosensors and prevent possible interference from the other redox species in the saliva sample such as uric acid or ascorbic acid [ 29 ]. The influence of the concentration of the Nafion solution used to cast the film on the performance of the developed glucose biosensor was also investigated.…”