Electrochemical Protein-based Bioanalytical Devices for Drug Analysis.

Proteins are vital components of living cells and the loss of their native functions has been associated with a wide variety of medical conditions. From this point of view, investigation of the protein microenvironment is crucial to support the development of therapeutic approaches capable of ensuring cellular functions. Therefore, analytical assays for the detection, quantification, and characterization of proteins, drugs, and protein-drug complexes play an essential role in fundamental research and clinical applications. Electrochemistry arises as an alternative methodology for fast assessment of proteins and drugs and is attractive due to the adaptability to miniaturization and scalability of electroanalytical devices, which then can be further employed as strategies towards personalized medical care. Thus, this review summarizes electrochemical investigations in the past 10 years on protein-based analytical devices and biosensors. A general overview of electrochemical assays that integrate proteins with nanostructured materials and conductive polymers is presented. Applications of electrochemical assays and biosensors were divided into four categories. First, those designed for drug screening strategies that focus on targeting specific intracellular, extracellular, or membrane protein subdomains to modulate their functions, aggregation/misfolding of proteins, and protein degradation pathways. Then, drug metabolism assays that involve mimicking natural metabolic pathways to identify potential safety and efficacy issues related to a drug or its metabolites. The third was dedicated to electrochemical drug delivery systems with anchored drugs in the form of bioconjugates, while the fourth was dedicated to electroanalytical methodologies for quantitative drug assays, where the electroactivity of the target species is often used to correlate the electrochemical signal to their concentration.

Carbon Inks-Based Screen-Printed Electrodes for Qualitative Analysis of Amino Acids.

Due to the great significance of amino acids, a substantial number of research studies has been directed toward the development of effective and reliable platforms for their evaluation, detection, and identification. In order to support these studies, a new electrochemical platform based on PANI/ZnO nanowires' modified carbon inks screen-printed electrodes was developed for qualitative analysis of electroactive amino acids, with emphasis on tyrosine (Tyr) and tryptophan (Trp). A comparative investigation of the carbon ink before and after modification with the PANI/ZnO was performed by scanning electron microscopy and by Raman spectroscopy, confirming the presence of PANI and ZnO nanowires. Electrochemical investigations by cyclic voltammetry and electrochemical impedance spectroscopy have shown a higher charge-transfer rate constant, which is reflected into lower charge-transfer resistance and higher capacitance values for the PANI/ZnO modified ink when compared to the simple carbon screen-printed electrode. In order to demonstrate the electrochemical performances of the PANI/ZnO nanowires' modified carbon inks screen-printed electrodes for amino acids analysis, differential pulse voltammograms were obtained in individual and mixed solutions of electroactive amino acids. It has been shown that the PANI/ZnO nanowires' modified carbon inks screen-printed electrodes allowed for tyrosine and tryptophan a peak separation of more than 100 mV, enabling their screening and identification in mixed solutions, which is essential for the electrochemical analysis of proteins within the proteomics research field.

An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 µA cm-2 mM-1 and a LOD of 0.4 µM. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.

Immobilized Antibodies on Mercaptophenylboronic Acid Monolayers for Dual-Strategy Detection of 20S Proteasome.

A dual strategy for the electrochemical detection for 20S proteasome (20S) is proposed, based on the oriented immobilization of a capture monoclonal antibody (Abß) on a self-assembled monolayer of 4-mercaptophenylboronic acid (4-MPBA) on gold electrodes, which led to the Au/4-MPBA/Abß immunosensor. The methodology comprises the correlation of 20S concentration with (i) its proteolytic activity toward the Z-LLE-AMC substrate, using the Au/4-MPBA/Abß/20S, and (ii) the enzymatic activity of an alkaline phosphatase (AlkP) from the AlkP-labeled secondary antibody (Abcore-AlkP), which involves the conversion of aminophenylphosphate to the electroactive aminophenol using Au/4-MPBA/Abß/20S/Abcore-AlkP. The step-by-step construction of the immunosensor and the interactions at its surface were evaluated by surface plasmon resonance and gravimetric analysis with quartz crystal microbalance, showing a high affinity between both antibodies and 20S. Morphological analysis by scanning electron microscopy demonstrated a pattern of parallel lines upon immobilization of Abß on 4-MPBA and morphological changes to a well-organized granular structure upon binding of 20S. A voltametric and impedimetric characterization was performed after each step in the immunosensor construction. The two detection strategies were evaluated. It was shown that the immunosensor responds linearly with 20S concentration in the range between 5 and 100 µg mL-1, which corresponds to proteasome levels in serum in the case of diverse pathological situations, and LoD values of 1.4 and 0.2 µg mL-1 were calculated for the detection strategies. The immunosensor was applied to the detection of 20S in serum samples with recovery values ranging from 101 to 103%.

Undoped SnO2 as a Support for Ni Species to Boost Oxygen Generation through Alkaline Water Electrolysis.

In this study, the synergistic behavior of Ni species and bimodal mesoporous undoped SnO2 is investigated in oxygen evolution reactions (OERs) under alkaline conditions without any other modification of the compositional phases or using noble metals. An efficient and environmentally friendly hydrothermal method to prepare bimodal mesoporous undoped SnO2 with a very high surface area (>130 m2 g-1) and a general deposition-precipitation method for the synthesis of well-dispersed Ni species on undoped SnO2 are reported. The powders were characterized by adsorption-desorption isotherms, TG-DTA, XRD, SEM, TEM, Raman, TPR-H2, and XPS. The best NiSn composite generates, under certain experimental conditions, a very high TOF value of 1.14 s-1 and a mass activity higher than 370 A g-1, which are remarkable results considering the low amount of Ni deposited on the electrode (3.78 ng). Moreover, in 1 M NaOH electrolyte, this material produces more than 24 mA cm-2 at an overpotential value of approximately +0.33 V, with only 5 wt % Ni species. This performance stems from the dual role of undoped SnO2, on the one hand, as a support for active and well-dispersed Ni species and on the other hand as an active player through the oxygen vacancies generated upon Ni deposition.

Electrochemical Immunosensing Platform for the Determination of the 20S Proteasome Using an Aminophenylboronic/Poly-indole-6-carboxylic Acid-Modified Electrode.

The first electrochemical immunosensor for the determination of the 20S proteasome (P20S) was developed, entailing the immobilization of an antibody on an aminophenylboronic/poly-indole-6-carboxylic acid-modified electrode. The proposed electrochemical bioplatform is a simple and feasible analytical tool applicable for the determination of P20S in human plasma, considering its high clinical and biological relevance. Cyclic voltammetry, electrochemical impedance spectroscopy, and square wave voltammetry (SWV) were used to determine the optimal step-by-step process to obtain the electrochemical immunosensor. The interaction of P20S with the recognition layer of the immobilized antibody on the nanostructured surface took place by incubating the electrode in a P20S solution at 20 °C for 2 h. Using SWV as an electro-analytical technique, this immunosensor can quantify P20S. The current was linear with the P20S concentration within two dynamic concentration ranges from 20.0 to 80.0 and 80.0 to 200.0 ng·mL-1 (r2 = 0.992 and 0.98, respectively) with a limit of detection and quantification of 6 and 18 ng·mL-1, respectively. Moreover, the immunosensor showed considerable repeatability and reproducibility, when the determination was done in human serum, which confirms that it is a promising alternative for direct detection of P20S in biological fluids with minimal interference.

Palladium/palladium oxide coated electrospun fibers for wearable sweat pH-sensors.

The work describes the development of a flexible, hydrogel embedded pH-sensor that can be integrated in inexpensive wearable and non-invasive devices at epidermal level for electrochemical quantification of H+ ions in sweat. Such a device can be useful for swift, real time diagnosis and for monitoring specific conditions. The sensors' working electrodes are flexible poly(methyl methacrylate) electrospun fibers coated with a thin gold layer and electrochemically functionalized with nanostructured palladium/palladium oxide. The response to H+ ions is investigated by cyclic voltammetry and electrochemical impedance spectroscopy while open circuit potential measurements show a sensitivity of aprox. -59 mV per pH unit. The modification of the sensing interface upon basic and acid treatment is characterized by scanning and transmission electron microscopy and the chemical composition by X-ray photoelectron spectroscopy. In order to demonstrate the functionality of the pH-sensor at epidermal level, as a wearable device, the palladium/palladium oxide working electrode and silver/silver chloride reference electrode are embedded within a pad of polyacrylamide hydrogel and measurements in artificial sweat over a broad pH range were performed. Sensitivity up to -28 mV/pH unit, response time below 30 s, temperature dependence of approx. 1 mV/°C as well as the minimum volume to which the sensor responses of 250 nanoliters were obtained for this device. The proposed configuration represents a viable alternative making use of low-cost and fast fabrication processes and materials.

Direct Immobilization of Biomolecules through Magnetic Forces on Ni Electrodes via Ni Nanoparticles: Applications in Electrochemical Biosensors.

The present work describes a new simple procedure for the direct immobilization of biomolecules on Ni electrodes using magnetic Ni nanoparticles (NiNPs) as biomolecule carriers. Ni electrodes were fabricated by electroplating, and NiNPs were chemically synthesized. The chemical composition, crystallinity, and granular size of Ni electrodes, NiNP, and NiNP-modified Ni electrodes (NiNP/Ni) were determined by X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy (XPS). The electrochemical characterization of Ni electrodes by cyclic voltammetry and electrochemical impedance spectroscopy confirmed the existence of nickel oxides, hydroxides, and oxohydroxide films at the surface of Ni. Magnetic characterization and micromagnetic simulations were performed in order to prove that the magnetic force is responsible for the immobilization process. Further, Ni electrodes were employed as amperometric sensors for the detection of hydrogen peroxide because it is an important performance indicator for a material to be applied in biosensing. The working principle for magnetic immobilization of the enzyme-functionalized NiNP, without the use of external magnetic sources, was demonstrated for glucose oxidase (GOx) as a model enzyme. XPS results enabled to identify the presence of GOx attached to the NiNP (GOx-NiNP) on Ni electrodes. Finally, glucose detection and quantification were evaluated with the newly developed GOx-NiNP/Ni biosensor by amperometry at different potentials, and control experiments at different electrode materials in the presence and absence of NiNP demonstrated their importance in the biosensor architecture.

Flexible Delivery Patch Systems based on Thermoresponsive Hydrogels and Submicronic Fiber Heaters.

This paper proposes a novel, flexible, low cost administration patch which could be used as a non-invasive, controlled transdermal drug delivery system. The fabricated device consists in a flexible microfiber architecture heater covered with a thermoresponsive hydrogel, namely poly(N-isopropylacrylamide), as a matrix for the incorporation of active molecules. The manufacturing process consists of two main steps. First, the electrospun poly(methyl methacrylate) fiber networks are sputter coated with a thin gold layer and attached to flexible poly(ethylene terephthalate) substrates to obtain the heating platforms. Second, the heaters are encapsulated in poly(ethylene terephthalate) foils and covered with poly(N-isopropylacrylamide) hydrogel sheets. In order to illustrate the functionality of the fabricated patch, the hydrogel layer is loaded with methylene blue aqueous solution and is afterwards heated via Joule effect, by applying a voltage on the metalized fibers. The methylene blue releasing profiles of the heated patch are compared with those of the unheated one and the influence of parameters such as hydrogel composition and morphology, as well as the applied voltage values for microheating are investigated. The results indicate that the fabricated patch can be used as a drug administration instrument, while its performance can be tuned depending on the targeted application.

Polypyrrole Actuator Based on Electrospun Microribbons.

The development of soft actuators by using inexpensive raw materials and straightforward fabrication techniques, aiming at creating and developing muscle like micromanipulators, represents an important challenge nowadays. Providing such devices with biomimetic qualities, for example, sensing different external stimuli, adds even more complexity to the task. We developed electroactive polymer-coated microribbons that undergo conformational changes in response to external physical and chemical parameters. These were prepared following three simple steps. During the first step nylon-6/6 microribbons were fabricated by electrospinning. In a second step the microribbons were one side coated with a metallic layer. Finally, a conducting layer of polypyrrole was added by means of electrochemical deposition. Strips of polypyrrole-coated aligned microribbon meshes were tested as actuators responding to current, pH, and temperature. The electrochemical activity of the microstructured actuators was investigated by recording cyclic voltammograms. Chronopontentiograms for specific current, pH, and temperature values were obtained in electrolytes with different compositions. It was shown that, upon variation of the external stimulus, the actuator undergoes conformational changes due to the reduction processes of the polypyrrole layer. The ability of the actuator to hold and release thin wires, and to collect polystyrene microspheres from the bottom of the electrochemical cell, was also investigated.

In situ dsDNA-bevacizumab anticancer monoclonal antibody interaction electrochemical evaluation.

The interaction of the anticancer monoclonal antibody bevacizumab (BEVA) with double-stranded DNA (dsDNA) was studied by voltammetry and gel-electrophoresis in incubated samples and using the dsDNA-electrochemical biosensor. The voltammetric results revealed a decrease and disappearance of the dsDNA oxidation peaks with increasing incubation time, showing that BEVA binds to the dsDNA but no DNA oxidative damage was detected electrochemically. Non denaturing agarose gel-electrophoresis experiments were in agreement with the voltammetric results showing the formation of compact BEVA-dsDNA adduct. The dsDNA-electrochemical biosensor in incubated solutions showed that BEVA also undergoes structural modification upon binding dsDNA, and BEVA electroactive amino acid residues oxidation peaks were detected.

Electrochemical evaluation of Abelson tyrosine-protein kinase 1 activity and inhibition by imatinib mesylate and danusertib.

Abelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide. The phosphorylation reactions were carried out by incubating ABL1, ATP and the substrate abltide. Adsorption at the glassy carbon electrode surface in either reaction mixtures or control solutions, followed by differential pulse voltammetry in buffer allowed detection of the variation of abltide tyrosine residue oxidation peak reflecting the occurrence of the phosphorylation reaction. The effect of abltide, ATP and ABL1 concentrations as well as the time course of the phosphorylation reaction were studied. The influence of co-adsorption of ABL1, ATP and phosphorylated abltide was evaluated and the conditions for the electrochemical detection of ABL1-catalysed phosphorylation optimised. The Michaelis-Menten constant for abltide binding KM∼4.5 µM, turnover number kcat∼11 s(-1) and enzyme efficiency kcat/KM∼2.3 s(-1) µM(-1) were calculated. The inhibition of ABL1 by imatinib mesylate and danusertib was also electrochemically investigated and IC50 values of 0.53 and 0.08 µM determined.

In situ evaluation of gemcitabine-DNA interaction using a DNA-electrochemical biosensor.

The electrochemical behaviour of the cytosine nucleoside analogue and anti-cancer drug gemcitabine (GEM) was investigated at glassy carbon electrode, using cyclic, differential pulse and square wave voltammetry, in different pH supporting electrolytes, and no electrochemical redox process was observed. The evaluation of the interaction between GEM and DNA in incubated solutions and using the DNA-electrochemical biosensor was studied. The DNA structural modifications and damage were electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues and the occurrence of the free guanine residues electrochemical signal. The DNA-GEM interaction mechanism occurred in two sequential steps. The initial process was independent of the DNA sequence and led to the condensation/aggregation of the DNA strands, producing rigid structures, which favoured a second step, in which the guanine hydrogen atoms, participating in the C-G base pair, interacted with the GEM ribose moiety fluorine atoms.

Microcystin-LR and chemically degraded microcystin-LR electrochemical oxidation.

Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by certain strains of freshwater cyanobacteria toxic for humans and animals. The electrochemical behaviour of microcystin-LR (MC-LR) at a glassy carbon electrode (GCE) was investigated using cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). The oxidation of MC-LR is a diffusion-controlled irreversible and pH-independent process that occurs with the transfer of only one electron and does not involve the formation of any electroactive oxidation product. Upon incubation in different pH electrolytes, homogeneous degradation of MC-LR in solution was electrochemically detected by the appearance of a new oxidation peak at a lower potential. The electrochemical behaviour of chemically degraded MC-LR is an irreversible, pH-dependent process, and involves the formation of two redox products that undergo reversible oxidation. The formation of degradation products of MC-LR was confirmed by HPLC with UV detection at room temperature. Experiments were also carried out in solutions containing constituent MC-LR amino acids, which enabled the understanding of the MC-LR electron transfer reaction and degradation. An oxidation mechanism for MC-LR is proposed.

Nucleoside analogue electrochemical behaviour and in situ evaluation of DNA-clofarabine interaction.

Nucleoside analogues have had a substantial impact on the treatment of cancer, especially haematological malignancies. The electrochemical behaviour of the nucleoside analogue clofarabine (CLF) was investigated at a glassy carbon electrode using cyclic, differential pulse and square wave voltammetry in different pH supporting electrolytes. The oxidation process of CLF is irreversible and pH-dependent with transfer of two protons and two electrons, following a diffusion controlled mechanism. The oxidation mechanism of CLF involves deprotonation and leads to the formation of a hydroxylated species that undergoes reversible redox reactions. The interaction of DNA and the antileukemia drug CLF was investigated using a dsDNA-electrochemical biosensor in incubated solutions by differential pulse voltammetry. The CLF-DNA interaction leads to changes in the DNA morphological structure, confirmed using the purinic homo-polynucleotide single stranded sequences of guanosine and adenosine, poly[G] and poly[A]-electrochemical biosensors.

Electrochemical reduction mechanism of camptothecin at glassy carbon electrode.

Camptothecin (CPT) is a cytotoxic quinoline alkaloid endowed with the inhibition of topoisomerase I, an essential enzyme for the normal functioning of DNA. The redox behaviour of CPT was investigated at a glassy carbon electrode using cyclic, differential pulse and square wave voltammetry. It was shown that CPT can undergo reduction in a pH-dependent mechanism. In acid media only one irreversible charge transfer reaction was observed whereas by increasing the pH of the supporting electrolyte, two reduction peaks occurred. The diffusion coefficient of CPT was calculated in pH 4.5 0.1M acetate buffer using cyclic voltammetry to be D(CPT)=5.77x10(-6)cm(2)s(-1). Differential pulse voltammetry measurements were carried out over a wide pH range and allowed the determination of the number of electrons and protons transferred during each step in the CPT reduction mechanism, one electron and one proton. The use of square wave voltammetry proved the quasi-reversibility of CPT reduction as a function of the pH of the supporting electrolyte. Based upon the results obtained a reduction mechanism was proposed and the observed waves were attributed to the hydroxylation of the lactone ring of CPT to a lactol ring.

Electrochemical behaviour of dimethyl-2-oxoglutarate on glassy carbon electrode.

The electrochemical behaviour of dimethyl-2-oxoglutarate (MOG), a key intermediate in the Krebs cycle and an important nitrogen transporter in the metabolic pathways in biological processes, was investigated by cyclic voltammetry, square wave voltammetry and differential pulse voltammetry using a glassy carbon electrode. The reduction of MOG is an irreversible diffusion-controlled process that occurs in a cascade mechanism. For electrolytes with pH <3.0 and pH >7.0 one peak occurred and for 3.0

Interaction of imatinib with liposomes: voltammetric and AFM characterization.

The interaction of imatinib with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 2-oleoyl-1-stearoyl-sn-glycero-3-phosphocholine (OSPC) liposomes and the adsorption of DPPC and OSPC were studied using atomic force microscopy (AFM) at highly oriented pyrolytic graphite (HOPG) and differential pulse voltammetry at glassy carbon electrode (GCE). The HOPG induces the rupture of the liposomes and allows the lipids to adsorb along one of the three axes of symmetry of the HOPG basal planes, forming well-ordered lamellar structures. After interaction, both DPPC monolayers and DPPC-imatinib complexes are adsorbed onto HOPG. The OSPC-imatinib complexes self-organize only into ordered but larger domains of parallel stripes that maintain the threefold symmetry of the HOPG, due to an easier imatinib penetration into the unsaturated OSPC liposome bilayers. The voltammetric results show that upon interaction, the electrochemical active moiety of imatinib is incorporated into the lipid bilayer becoming unavailable to the GCE surface for oxidation, leading to local structural modifications of the lipid bilayer which were also electrochemically detected. A model is proposed for the liposome-imatinib interaction considering that imatinib interacts primarily by van der Waals and hydrogen bonds with the phosphatidylcholine headgroups, leading to defects in the liposome bilayer and allowing further incorporation of imatinib into the liposome lamellae.

Electrochemical behaviour of 2,8-dihydroxyadenine at a glassy carbon electrode.

The electrochemical behaviour of 2,8-dihydroxyadenine (2,8-DHA)- the main adenine oxidation product- has been investigated over a wide pH range at a glassy carbon electrode (GCE) using cyclic, differential pulse and square wave voltammetry. The oxidation of 2,8-DHA is a quasi-reversible process, pH dependent and occurs with the formation of a main oxidation product, P(2,8-DHA), that strongly adsorbs on the electrode surface. The reduction of 2,8-DHA also occurs and is a reversible process in the absence of molecular oxygen. In electrolytes with pH between 4 and 9 two consecutive reversible charge transfer reactions were identified. However, it was observed that O(2) interfered with the reductive electron transfer process of 2,8-DHA and that, in the presence of oxygen, the reduction of 2,8-DHA occurs at less negative potentials than in the absence of oxygen.

Electrochemical behaviour of isatin at a glassy carbon electrode.

The electrochemical behavior of isatin--a molecule with a broad range of applications in synthetic, biological and clinical activity--has been investigated over a wide pH range at a glassy carbon electrode (GCE) using cyclic, square wave and differential pulse voltammetry. The oxidation of isatin is an irreversible process, pH dependent and occurs with the formation of a main oxidation product that strongly adsorbs on the electrode surface. The reduction of isatin is also a pH dependent irreversible process. Cyclic voltammograms show two consecutive charge transfer reactions. The diffusion coefficient of isatin was calculated in pH 7.0 phosphate buffer to be D(0)=4.9 x 10(-7) cm2 s(-1). The limit of detection obtained in a solution of pH 7.0 phosphate buffer was LOD=0.194 microM, based on three times the noise level.