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.

Novel cells integrated biosensor based on superoxide dismutase on electrospun fiber scaffolds for the electrochemical screening of cellular stress.

A novel electrochemical biosensor was developed to monitor fibroblast cells stress levels for the first time in situ under external stimuli based on the recognition of superoxide anion released upon cell damage. The biosensor comprised metallized polycaprolactone electrospun fibers covered with zinc oxide for improved cell adhesion and signal transduction, whilst stable bioconjugates of mercaptobenzoic acid-functionalized gold nanoparticles/superoxide dismutase were employed as recognition bioelements. Biosensors were first tested and optimized for in situ generated superoxide detection by fixed potential amperometry at +0.3 V, with minimal interferences from electroactive species in cell culture media. L929 fibroblast cells were then implanted on the optimized biosensor surface and the biosensor morphologically characterized by scanning electron microscopy (SEM) and fluorescence microscopy, which illustrated the network-type pattern of fibroblasts adjacent to the fiber scaffold. Fibroblast stress was induced by zymosan and monitored at the cells integrated biosensor using fixed potential amperometry (CA) with a sensitivity of 26 nA cm-2 µg mL-1 zymosan and electrochemical impedance spectroscopy (EIS), with similar sensitivity of the biosensor considering the Rs and Z' parameters of around 0.13 Ω cm2 µg-1 mL and high correlation factors R2 of 0.9994. The obtained results underline the applicability of the here developed biosensor for the electrochemical screening of the fibroblast cells stress. The concept in using low-cost biocompatible polymeric fibers as versatile scaffolds for both enzyme immobilization and cell adhesion, opens a new path in developing biosensors for the in-situ investigation of a variety of cellular events.

PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications.

PC-12 cells have been widely used as a neuronal line study model in many biosensing devices, mainly due to the neurogenic characteristics acquired after differentiation, such as high level of secreted neurotransmitter, neuron morphology characterized by neurite outgrowth, and expression of ion and neurotransmitter receptors. For understanding the pathophysiology processes involved in brain disorders, PC-12 cell line is extensively assessed in neuroscience research, including studies on neurotoxicity, neuroprotection, or neurosecretion. Various analytical technologies have been developed to investigate physicochemical processes and the biosensors based on optical and electrochemical techniques, among others, have been at the forefront of this development. This article summarizes the application of different biosensors in PC-12 cell cultures and presents the modern approaches employed in neuronal networks biosensing.

Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring.

The use of gold nanoparticles/superoxide dismutase (AuNP/SOD) bioconjugates is described as building blocks in SOD biosensor development for the quantification of superoxide in cell culture media. AuNP functionalization with 11-mercaptoundecanoic acid (MUA) and 4-mercaptobenzoic acid (MBA) (AuNPMUA and AuNPMBA) was used to improve SOD immobilization through EDC/NHS coupling using their -COOH terminus, leading to the formation of more stable bioconjugates. AuNP and AuNP/SOD bioconjugates were characterized by SEM to determine their size and morphology, UV-Vis for optical properties, FT-IR, and Raman spectroscopies for chemical functional group analysis and EDX for elemental analysis. Electrochemical methods were used to characterize the Au/AuNP-modified electrodes. For the optimization of the biosensor architecture, different AuNP/enzyme bioconjugates were prepared by varying the amount of both enzyme and AuNP, as well as their incubation time. Finally, the biosensors incorporating the bioconjugates were characterized by fixed potential amperometry and voltammetric analysis in order to establish the enzymatic mechanism and to elucidate the best biosensor architecture for monitoring superoxide in cell culture media. The best sensitivity value for superoxide detection corresponded to 41.2 nA µM cm-2, achieved by a biosensor based on AuNPMBA/SOD bioconjugates monitored through fixed potential amperometry at 0.3 V vs. Ag/AgCl, with a limit of detection of 1.0 µM, and overall very good operational stability, maintaining 91% of the initial sensitivity after 30 days. Finally, the optimized biosensor was employed for the quantification of successive additions of superoxide in cell culture media, with excellent recovery values.

Disposable superoxide dismutase biosensors based on gold covered polycaprolactone fibers for the detection of superoxide in cell culture media.

A novel and disposable biosensor based on superoxide dismutase (SOD) immobilized on gold metallized polycaprolactone electrospun polymeric fibers (PCl/Au) has been developed for the determination of superoxide (O2•-) in cell culture media. SOD biosensors were constructed employing three immobilization methods: cross-linking with EDC/NHS at a cysteine self-assembled monolayer (PCl/Au/SODCYS), biopolymer encapsulation with chitosan (PCl/Au/SODCHI) and cross-linking with glutaraldehyde (PCl/Au/SODGA). Scanning electron microscopy was performed at the three different biosensors to evaluate their surface morphologies. Biosensors were employed for the electrochemical detection of superoxide by fixed potential amperometry at different applied potentials, with two distinct enzymatic mechanisms being proposed: i) the reduction of the enzymatically generated peroxide, at -0.3 V, for which the PCl/Au/SODCHI biosensor presented the highest value of sensitivity of 40.1 µA mM-1 cm-2, and ii) the regeneration of the enzyme catalytic copper centre, at +0.3 V, for which the PCl/Au/SODCYS biosensor had the highest sensitivity value of 16.1 µA mM-1 cm-2. The proposed recognition mechanisms were further confirmed by cyclic voltammetric measurements, which enabled also to determine the amount of immobilized electroactive SOD, with highest value corresponding to the PCl/Au/SODCYS biosensor. The biosensors with best analytical performance, PCl/Au/SODCYS and PCl/Au/SODCHI, were further investigated for stability and selectivity, with best results for the PCl/Au/SODCYS, chosen for superoxide monitoring in cell culture media. The study is promising for future application of PCl/Au/SODCYS for the on-line superoxide monitoring of superoxide in cell cultures, grown directly on the biosensor itself.

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%.