Biosensors & enzymatic fuel cells based on direct electron transfer of dehydrogenases: characterization and applications

Il lavoro svolto durante i tre anni di dottorato è stato indirizzato verso lo sviluppo di nuovi metodi di sintesi ed elettrosintesi di nanomateriali metallici o carboniosi per il miglioramento del trasferimento elettronico diretto tra l’enzima e l’elettrodo. Questo miglioramento si traduce in un notevole incremento della sensibilità, stabilità e selettività dei biosensori sviluppati nonché della potenza generata da una pila enzimatica a biocombustibile, (Biofuel Cell). La prima parte della tesi riguarda lo studio e l’ottimizzazione del trasferimento elettronico diretto della cellobiosio deidrogenasi (CDH), un enzima appartenente alle flavoemeossidoreduttasi, costituito da due subunità dotate rispettivamente di cofattore FAD (subunità I) e heme b (subunità II). In questa parte abbiamo sintetizzato nanoparticelle di oro e di argento con un nuovo metodo “green”, che impiega come agente riducente la quercetina, un noto flavonoide presente in numerosi alimenti e bevande (es. tè, capperi, mirtilli, etc.). La reazione è stata condotta a temperatura ambiente e a pressione atmosferica senza ulteriore purificazione in quanto la quercetina è nota avere un comportamento stabilizzante delle sospensioni colloidali. Le suddette nanoparticelle sono state impiegate nella costruzione di biosensori per la determinazione del lattosio e di una pila a biocombustibile glucosio/ossigeno. Successivamente, abbiamo sviluppato un nuovo metodo per l’elettrodeposizione di nanoparticelle di oro in modo da ottenere una superficie nanostrutturata ordinata che ha portato allo sviluppo di un biosensore per la determinazione del glucosio nella saliva. La seconda parte della tesi riguarda lo studio del meccanismo del trasferimento elettronico diretto della fruttosio deidrogenasi (FDH), con particolare attenzione rivolta all’influenza dei cationi monovalenti e bivalenti, all’influenza della forma delle nanoparticelle sulla catalisi enzimatica, all’individuazione dei siti “heme” coinvolti nel trasferimento elettronico diretto attraverso l’accesso ad una porzione idrofobica dell’enzima, ed infine allo sviluppo di un biosensore per la determinazione del fruttosio realizzato immobilizzando la FDH su elettrodi di oro altamente poroso.The aim of this thesis is the study and the enhancement of the direct electron transfer of two different dehydrogenases, by means of a proper nanostructuration of the electrodes, for biosensors and enzymatic fuel cells (EFCs) development. Cellobiose dehydrogenase (CDH) is an extracellular enzyme belonging to the oxidoreductase group. CDH contains two subunits: (a) subunit I is the dehydrogenase domain (DHCDH), similar to the domain of other oxidoreductases, which belongs to the glucose-methanol-choline (GMC) oxidoreductase superfamily with a flavin adenine dinucleotide (FAD) co-factor covalently bound to the enzyme structure; (b) subunit II is the cytochrome domain (CYTCDH), which contains a heme b and acts as a built-in mediator by shuttling the electrons to a modified electrode. Both subunits are connected through a flexible linker responsible of the modulation of the internal electron transfer (IET) rate by varying the experimental conditions, such as changes of pH and divalent cations the concentration. Fructose dehydrogenase (FDH) is a membrane-bound flavocytochrome oxidoreductase which also belongs to the hemoflavoproteins family. FDH is a heterotrimeric membrane-bound enzyme complex with a molecular mass of 146.4 kDa, consisting of three subunits: (a) subunit I (DHFDH) is the catalytic domain with a covalently bound flavin adenine dinucleotide (FAD) cofactor, where D-(-)-fructose is involved in a 2H+/2e- oxidation to 5-dehydro-D-(-)-fructose; (b) subunit II (CYTFDH) acts as a built-in electron acceptor with three heme c moieties covalently bound to the enzyme scaffold and two of them involved in the electron transfer pathway; (c) subunit III is not involved in the electron transfer but plays a key role for the enzyme complex stability. The central target of the present thesis is the possibility to improve the electron transfer through the electrode nanostructuration, which can be realized by exploiting new nanomaterials as well as new nanostructuration methods (e.g. “green” synthesized metal nanoparticles, electrodeposition etc.). In the thesis much attention has been paid also to the understanding of the electron transfer pathway of FDH, which would be of fundamental interest in the near future for the development of highly sensitive biosensors and efficient EFCs. The biosensors realized and optimized in this thesis are prototypes of devices that, hopefully, will be commercially available on the market in the next future

Reconfigurable Implication and Inhibition Boolean logic gates based on NAD+-dependent enzymes: Application to signal-controlled biofuel cells and molecule release

AbstractThe Implication and Inhibition Boolean logic gates were realized using NAD+/NADH‐dependent dehydrogenases combined with hexokinase competing for biomolecule input signals. Both logic gates operated with the same enzyme composition and their reconfiguration was achieved simply by redefining the input signals. The output signals produced by the logic gates were analyzed optically and electrochemically, particularly using enzyme‐modified electrodes. The logically processed input signals were used to switch operation of a biofuel cell and activate a molecule release process

Metal oxide nanoparticle based electrochemical sensor for total antioxidant capacity (TAC) detection in wine samples

A single-use electrochemical screen-printed electrode is reported based on biomimetic properties of nanoceria particles (CeNPs). The developed tool showed an easy approach compared to the classical spectrophotometric methods reported in literature in terms of ease of use, cost, portability, and unnecessary secondary reagents. The sensor allowed the detection of the total antioxidant capacity (TAC) in wine samples. The sensor has been optimized and characterized electrochemically and then tested with antioxidant compounds occurred in wine samples. The electrochemical CeNPs modified sensor has been used for detection of TAC in white and red commercial wines and the data compared to the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)-based spectrophotometric method. Finally, the obtained results have demonstrated that the proposed sensor was suitable for the simple and quick evaluation of TAC in beverage samples

Catalase-based modified graphite electrode for hydrogen peroxide detection in different beverages

A catalase-based (NAF/MWCNTs) nanocomposite film modified glassy carbon electrode for hydrogen peroxide (H2O2) detection was developed. The developed biosensor was characterized in terms of its bioelectrochemical properties. Cyclic voltammetry (CV) technique was employed to study the redox features of the enzyme in the absence and in the presence of nanomaterials dispersed in Nafion polymeric solution. The electron transfer coefficient, , and the electron transfer rate constant, , were found to be 0.42 and 1.71 s−1, at pH 7.0, respectively. Subsequently, the same modification steps were applied to mesoporous graphite screenprinted electrodes. Also, these electrodes were characterized in terms of their main electrochemical and kinetic parameters. The biosensor performances improved considerably after modification with nanomaterials. Moreover, the association of Nafion with carbon nanotubes retained the biological activity of the redox protein. The enzyme electrode response was linear in the range 2.5– 1150 mol L−1, with LOD of 0.83 mol L−1. From the experimental data, we can assess the possibility of using the modified biosensor as a useful tool for H2O2 determination in packaged beverages

Inhibition-based first-generation electrochemical biosensors: theoretical aspects and application to 2,4-dichlorophenoxy acetic acid detection

In this work, several theoretical aspects involved in the first-generation inhibition-based electrochemical biosensor measurements have been discussed. In particular, we have developed a theoretical-methodological approach for the characterization of the kinetic interaction between alkaline phosphatase (AlP) and 2,4- dichlorophenoxy acetic acid (2,4-D) as representative inhibitor studied by means of cyclic voltammetry and amperometry. Based on these findings, a biosensor for the fast, simple, and inexpensive determination of 2,4-D has been developed. The enzyme has been immobilized on screen-printed electrodes (SPEs). To optimize the biosensor performances, several carbon-based SPEs, namely graphite (G), graphene (GP), and multiwalled carbon nanotubes (MWCNTs), have been evaluated. AlP was immobilized on the electrode surface by means of polyvinyl alcohol with styryl-pyridinium groups (PVA-SbQ) as cross-linking agent. In the presence of ascorbate 2-phosphate (A2P) as substrate, the herbicide has been determined, thanks to its inhibition activity towards the enzyme catalyzing the oxidation of A2P to ascorbic acid (AA). Under optimum experimental conditions, the best performance in terms of catalytic efficiency has been demonstrated by MWCNTs SPE-based biosensor. The inhibition biosensor shows a linearity range towards 2,4-D within 2.1–110 ppb, a LOD of 1 ppb, and acceptable repeatability and stability. This analysis method was applied to fortified lake water samples with recoveries above 90 %. The low cost of this device and its good analytical performances suggest its application for the screening and monitoring of 2,4-D in real matrices

The influence of the shape of Au nanoparticles on the catalytic current of fructose dehydrogenase

Graphite electrodes were modified with triangular (AuNTrs) or spherical (AuNPs) nanoparticles and further modified with fructose dehydrogenase (FDH). The present study reports the effect of the shape of these nanoparticles (NPs) on the catalytic current of immobilized FDH pointing out the different contributions on the mass transfer–limited and kinetically limited currents. The influence of the shape of the NPs on the mass transfer–limited and the kinetically limited current has been proved by using two different methods: a rotating disk electrode (RDE) and an electrode mounted in a wall jet flow-through electrochemical cell attached to a flow system. The advantages of using the wall jet flow system compared with the RDE system for kinetic investigations are as follows: no need to account for substrate consumption, especially in the case of desorption of enzyme, and studies of product-inhibited enzymes. The comparison reveals that virtually identical results can be obtained using either of the two techniques. The heterogeneous electron transfer (ET) rate constants (kS) were found to be 3.8 ± 0.3 s−1 and 0.9 ± 0.1 s−1, for triangular and spherical NPs, respectively. The improvement observed for the electrode modified with AuNTrs suggests a more effective enzyme-NP interaction, which can allocate a higher number of enzyme molecules on the electrode surface

Nanoparticles modified screen printed electrode for electrochemical determination of COD

The Chemical Oxygen Demand (COD) is a parameter widely used to determine organic pollutants in water and is defined as the number of oxygen equivalents necessary to oxidize the organic compounds. The standard method for COD measurement (the dichromate titration) suffers from several inherent drawbacks such as the long time of the process and the consumption of toxic chemicals. Hence, interest is growing towards those methods employing electrochemical oxidation of organic compounds, as they allow to dispense with toxic reagents and above all to perform a continuous determination. In this work a new electrochemical method for COD measurement has been developed based on direct oxidation of organic molecules on suitably modified electrodic surfaces. In particular, we have developed various sensors based on modified working electrode surfaces obtained by electrodepositing copper and/or nickel oxide nanoparticles onto several commercial screen printed electrodes. Glucose was used as the standard compound for COD measurements: C6H12O6 + 6O2 → 6CO2 + 6H2O The metallic nanoparticles catalyze the oxidation of the glucose, as well as of different organic pollutants, and make the detection possible at relatively low potential, also in presence of chloride as interferent. The analytical parameters were optimized and the results obtained highlight how the electrodeposition of different metallic nanoparticles onto several screen printed electrode surfaces can influence the selectivity and sensitivity towards the COD detection in real matrices, via electrochemical method. The results were compared with those obtained by the standard method and showed a good agreement. These findings provide an interesting strategy to obtain a simple, cheap, portable and eventually continuous sensor for COD measurement

Evaluation of new cholinium-amino acids based room temperature ionic liquids (RTILs) as immobilization matrix for electrochemical biosensor development: proof-of-concept with trametes versicolor laccase

In this work, we present new cholinium-amino acids room temperature ionic liquids (ChAARTILs) that can be used as an efficient immobilization matrix for electrochemical biosensor development. The ideal immobilization strategy should be able to ensure the highest enzyme loading and a tight enzymatic immobilization, preserving its native structure and biological activity. In this regard, ChAARTILs present different side chains on the amino acids giving rise to van der Waals, π-π stacking and hydrogen bonding interactions. All these interactions can affect the nanomaterial organization onto the electrode surface. To this aim, we have evaluated the main electrochemical parameters, namely electroactive area (AEA) and the heterogeneous electron transfer rate constant (k0), showing how both cations and anions of room temperature ionic liquids (RTILs) can independently affect multi-walled carbon nanotubes (MWCNTs) organization. In particular, [Ch][Phe] showed the best performance in terms of AEA (3.432 cm2) and k0 (4.71·10−3 cm s−1) with a homogeneous distribution of MWCNTs bundles onto the electrodes and a faster electron transfer rate

A Comparative Study between Hydrogen Peroxide Amperometric Biosensors Based on Different Peroxidases Wired by Os-Polymer: Applications in Water, Milk and Human Urine

In the last few years, hydrogen peroxide [...

Electrochemical and X-ray Photoelectron Spectroscopy Surface Characterization of Interchain-Driven Self-Assembled Monolayer (SAM) Reorganization

Herein, we report a combined strategy encompassing electrochemical and X-ray photoelectron spectroscopy (XPS) experiments to investigate self-assembled monolayer (SAM) conformational reorganization onto an electrode surface due to the application of an electrical field. In particular, 3-mercaptopriopionic acid SAM (3MPA SAM) modified gold electrodes are activated with a 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHSS) (EDC-NHSS) mixture by shortening the activation time, from 2 h to 15/20 min, labelled as Protocol-A, -B and -C, respectively. This step, later followed by a deactivation process with ethanolamine (EA), plays a key role in the reaction yields (formation of N-(2-hydroxyethyl)-3-mercaptopropanamide, NMPA) but also in the conformational rearrangement observed during the application of the electrical field. This study aims at explaining the high performance (i.e., single-molecule detection at a large electrode interface) of bioelectronic devices, where the 3MPA-based SAM structure is pivotal in achieving extremely high sensing performance levels due to its interchain interaction. Cyclic voltammetry (CV) experiments performed in K4Fe(CN)6:K3Fe(CN)6 for 3MPA SAMs that are activated/deactivated show similar trends of anodic peak current (IA) over time, mainly related to the presence of interchain hydrogen bonds, driving the conformational rearrangements (tightening of SAMs structure) while applying an electrical field. In addition, XPS analysis allows correlation of the deactivation yield with electrochemical data (conformational rearrangements), identifying the best protocol in terms of high reaction yield, mainly related to the shorter reaction time, and not triggering any side reactions. Finally, Protocol-C’s SAM surface coverage, determined by CV in H2SO4 and differential pulse voltammetry (DPV) in NaOH, was 1.29 * 1013 molecules cm2, being similar to the bioreceptor surface coverage in single-molecule detection at a large electrode interface