L-glutamate is one the most important excitatory neurotransmitter at the central nervous system level and it is implicated in several pathologies. So, it is very important to monitor its variations, in real time in animal models’ brain. The present study aimed to develop and characterize a new amperometric glutamate biosensor design that exploits the selectivity of Glutamate Oxidase (GluOx) for l-glutamate, and the capability of a small molecule as propylene glycol (PG), never used before, to influence and extend the stability and the activity of enzyme. Different designs were evaluated by modifying the main components in their concentrations to find the most suitable design. Moreover, enzyme concentrations from 100 U/ml up to 200 U/ml were verified and different PG concentrations (1%, 0.1% and 0.05%) were tested. The most suitable selected design was Ptc/PPD/PEI(1%)2/GlutOx5/PG(0.1%) and it was compared to the same already described design loading PEDGE, instead of PG, in terms of over-time performances. The PG has proved to be capable of determining an over-time stability of the glutamate biosensor in particular in terms of linear region slope (LRS) up to 21 days.

Molecular biomarkers are very important in biology, biotechnology and even in medicine, but it is quite hard to convert biology-related signals into measurable data. For this purpose, amperometric biosensors have proven to be particularly suitable because of their specificity and sensitivity. The operation and shelf stability of the biosensor are quite important features, and storage procedures therefore play an important role in preserving the performance of the biosensors. In the present study two different designs for both glucose and lactate biosensor, differing only in regards to the containment net, represented by polyurethane or glutharaldehyde, were studied under different storage conditions (+4, −20 and −80 °C) and monitored over a period of 120 days, in order to evaluate the variations of kinetic parameters, as VMAX and KM, and LRS as the analytical parameter. Surprisingly, the storage at −80 °C yielded the best results because of an unexpected and, most of all, long-lasting increase of VMAX and LRS, denoting an interesting improvement in enzyme performances and stability over time. The present study aimed to also evaluate the impact of a short-period storage in dry ice on biosensor performances, in order to simulate a hypothetical preparation-conservation-shipment condition.

The use of amperometric biosensors has attracted particular attention in recent years, both from researchers and from companies, as they have proven to be low-cost, reliable, and very sensitive devices, with a wide range of uses in different matrices. The continuous development of amperometric biosensors, since their use involves an enzyme, is specifically aimed at keeping and increasing the catalytic properties of the loaded protein, so as to be able to use the same device over time. The present study aimed to investigate the impact of glycerol and polysaccharides, in the presence of polycationic substances to constitute a hydrogel, in enhancing the enzymatic and analytic performance of a glucose biosensor. Initially, it was possible to verify how the deposition of the starch-based hydrogel, in addition to allowing the electropolymerization of the poly(p-phenylenediamine) polymer and the maintenance of its ability to shield the ascorbic acid, did not substantially limit the permeability towards hydrogen peroxide. Moreover, different biosensor designs, loading a mixture containing all the components (alone or in combination) and the enzyme, were tested in order to evaluate the changes of the apparent enzyme kinetic parameters, such as VMAX and KM, and analytical response in terms of Linear Region Slope, highlighting how the presence of all components (starch, glycerol, and polyethyleneimine) were able to substantially enhance the performance of the biosensors. The surface analysis of the biosensors was performed by scanning electron microscope (SEM). More, it was shown that the same performances were kept unchanged for seven days, proving the suitability of this biosensor design for short- and mid-term use.

Glutamate is a major excitatory neurotransmitter in the brain. It is involved in many normal physiological brain activities, but also neurological disorders and excitotoxicity. Hence, glutamate measurement is important both in clinical and pre-clinical studies. Pre-clinical studies often use amperometric biosensors due to their low invasiveness and the relatively small size of the devices. These devices also provide fast, real-time measurements because of their high sensitivity. In the present study, diethylene glycol (DEG), neopentyl glycol (NPG), triethylene glycol (TEG), and glycerol (GLY) were used to increase the long-term stability of glutamate biosensors. The evaluation was made by measuring variations of the main enzymatic (VMAX and KM) and analytical (Linear Region Slope (LRS)) parameters. Of the glycols tested, TEG was the most promising stabilizer, showing about twice as high VMAX maintained over a greater duration than with other stabilizers tested. It is also yielded the most stable linear region slope (LRS) values over the study duration. Moreover, we highlighted the ability of glycols to interact with enzyme molecules to form a containment network, able to maintain all the layered components of the biosensor adhering to the transducer.

Analyte quantification in first generation electrochemical biosensors is threatened by electro‐active molecules, such as ascorbic acid (AA). Electrochemical deposition of ortho‐phenylendiamine (oPD) on transducer considerably reduces AA interfering. These properties of poly‐oPD (PPD) are influenced by electro‐polymerization conditions. Chronoamperometry (CA) is proposed as an alternative technique for oPD electro‐deposition over Pt. Pt/PPD sensors are evaluated through functional parameters related to AA rejection and analytical performances. N2, O2, and air bubbled supporting electrolyte and several step duration times are used. Best performing CA‐PPD sensor is obtained in N2‐purged phosphate solution containing 300 mM oPD by means of a 1 s step duration/120 steps CA. High performing PPD is achieved in shorter time compared to commonly used constant potential amperometry. Aging of polymeric features and scanning electron microscopy investigations are performed and optimized CA electro‐polymerization conditions are used to build up an efficient interference blocking layer in a glucose oxidase biosensor.