Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 µA mM-1cm-2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

Tethered Lipid Membranes as a Nanoscale Arrangement towards Non-Invasive Analysis of Acute Pancreatitis.

Extracellular heat shock proteins (HSPs) mediate immunological functions and are involved in pathologies such as infection, stress, and cancer. Here, we demonstrated the dependence of an amount of HSP70 and HSP90 in serum vs. severity of acute pancreatitis (AP) on a cohort of 49 patients. Tethered bilayer lipid membranes (tBLMs) have been developed to investigate HSPs' interactions with tBLMs that can be probed by electrochemical impedance spectroscopy (EIS). The results revealed that HSP70 and HSP90 interact via different mechanisms. HSP70 shows the damage of the membrane, while HSP90 increases the insulation properties of tBLM. These findings provide evidence that EIS offers a novel approach for the study of the changes in membrane integrity induced by HSPs proteins. Herein, we present an alternative electrochemical technique, without any immunoprobes, that allows for the monitoring of HSPs on nanoscaled tBLM arrangement in biologics samples such us human urine. This study demonstrates the great potential of tBLM to be used as a membrane based biosensor for novel, simple, and non-invasive label-free analytical system for the prediction of AP severity.

The Synergy of Thermally Reduced Graphene Oxide in Amperometric Urea Biosensor: Application for Medical Technologies.

Thermally reduced graphene oxide (TRGO) is a graphene-based nanomaterial that has been identified as promising for the development of amperometric biosensors. Urease, in combination with TRGO, allowed us to create a mediator-free amperometric biosensor with the intention of precise detection of urea in clinical trials. Beyond simplicity of the technology, the biosensor exhibited high sensitivity (2.3 ± 0.1 µA cm-2 mM-1), great operational and storage stabilities (up to seven months), and appropriate reproducibility (relative standard deviation (RSD) about 2%). The analytical recovery of the TRGO-based biosensor in urine of 101 ÷ 104% with RSD of 1.2 ÷ 1.7% and in blood of 92.7 ÷ 96.4%, RSD of 1.0 ÷ 2.5%, confirmed that the biosensor is acceptable and reliable. These properties allowed us to apply the biosensor in the monitoring of urea levels in samples of urine, blood, and spent dialysate collected during hemodialysis. Accuracy of the biosensor was validated by good correlation (R = 0.9898 and R = 0.9982) for dialysate and blood, utilizing approved methods. The advantages of the proposed biosensing technology could benefit the development of point-of-care and non-invasive medical instruments.

Thermally reduced graphene oxide: The study and use for reagentless amperometric D-fructose biosensors.

Aiming to create reagentless amperometric D-fructose biosensor, graphene based electrode materials have been synthesized by newly proposed thermal reduction of graphene oxide. The method allowed to separate and collect different fractions of thermally reduced graphene oxide (TRGO) with different physicochemical properties. The structural characteristics and surface morphologies of TRGO fractions were evaluated using SEM, XRD, TGA analysis, Raman spectroscopy and BET measurements. Three different fractions of TRGO were tested as electrode materials for D-fructose amperometric biosensors. The direct electron transfer (DET) from the active site of D-fructose dehydrogenase (FDH) to the electrode was achieved with all TRGO fractions. High values of the sensitivity (up to 14.5 µA mM(-1) cm(-2)) are of the same order as these for other D-fructose sensors based on the synergistic mediated processes. The relationships between the structure of TRGO fractions and the molecular processes determining the effect of DET in bioelectrocatalysis by FDH have been studied. Stability of the D-fructose biosensors was also assessed. The best results were achieved when immobilization of FDH was performed using a crosslinking with glutaraldehyde. For the best group, after a period of 5 days the sensitivity of the biosensor for D-fructose determination decreased by less than 20%.