Publications

Abstract Over the years, literature reported inumerous applications of electrochemical and surface plasmon resonance (SPR) immunoassays for biosensing but, so far, the combination of the two methods in the same sensing spot for analytical purposes is much less explored and discussed. The aim of this Review is to highlight the great potential of electrochemistry combined-SPR (eSPR) as analytical tool for screening chemically and biologically relevant (bio)molecules by combining the unique features of SPR integrated with electrochemical readout.In the first part of the Review, we describe the urgent need of innovative methods for screening clinical biological markers (General Introduction), briefly discuss general concepts of SPR and electrochemical sensing (Concepts behind eSPR biosensors) and highlight the hyphenation of two methods to developed combined biosensing systems (Set-up configuration and eSPR principles). Firstly, we briefly give an overview of the setup for implementation of eSPR technique and discuss some relevant experimental conditions to perform the combined optical and electrochemical measurements. Then, the principles and fundamentals of eSPR biosensors are presented and described. We also present representative examples of eSPR biosensors in the literature (Applications of eSPR biosensors).In the second part, we review studies on how combined electrical and plasmonic detection contributed to the biosensing field, in particular, for the successful screening of clinically relevant biomolecules, namely proteins (Detection of proteins), nucleic acids (Detection of nucleic acids), small size chemical species (Detection of small molecules) and cells (Living-cell Analysis).Finally, we discuss the current limitations of eSPR biosensors performance and suggest possible ways to overcome these limitations (Limitations and optimization) and then we explore aspects about the development of the method and its applications and discuss areas of likely future growth (Conclusions and perspectives). (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abstract <jats:p>The design and synthesis of artificial receptors based on molecular imprinting (MI) technology for the development of a new MIP-based biosensor for detection of the stress biomarker α-amylase in human saliva in point-of-care (PoC) applications is described in this work. The portable electrochemical devices for monitoring α-amylase consists of cost-effective and disposable gold screen-printed electrodes (AuSPEs). To build the electrochemical device, the template biomolecule was firstly immobilized directly over the working area of the gold chip previously activated with a self-assembled monolayer (SAM) of cysteamine (CA). Then, pyrrole (Py) monomer was selected as building block of a polymeric network prepared by CV electropolymerization. After the electropolymerization process, the enzyme was removed from the polymer film in order to build the specific recognition sites for the target enzyme. The MIP biosensor showed a very wide linear concentration range (between 3.0 × 10−4 to 0.60 mg mL−1 in buffer solution and between 3.0 × 10−4 to 3.0 × 10−2 mg mL−1 in human saliva) and low detection levels were achieved (LOD &lt; 3.0 × 10−4 mg mL−1) using square wave voltammetry (SWV) as the electroanalytical technique.</jats:p>

Abstract This work reports the development of a simple and rapid electrochemical immunosensor for the determination of breast cancer biomarker Cancer Antigen 15-3 (CA 15-3). Disposable and cost-effective chips, consisting of gold screen-printed electrodes (AuSPEs), were used to develop the portable electrochemical devices for monitoring the biomarker in point-of-care (PoC), under clinical context. The biosensor preparation consisted of two simple steps. First, a self-assembled monolayer (SAM) of mercaptosuccinic acid (MSA) was formed at the AuSPE surface. Then, the CA 15-3 antibody was covalently bound to the carboxylic groups standing at the electrode surface using EDC/NHS chemistry. The performance of the developed immunosensor was evaluated by assessing the sensor sensitivity, linear response interval, selectivity and detection limit (LOD). The developed immunosensor provided a wide linear concentration range (from 1.0 to 1000 U mL(-1)) and low detection levels were achieved (LOD of 0.95 U mL(-1)), enabling the sensitive detection of the cancer biomarker at clinically relevant levels, using square wave voltammetry (SWV) as electroanalytical technique. Moreover, selectivity studies performed against other cancer biomarkers (CA 125 and CA 19-9) revealed that the antibody has high selectivity for CA 15-3 antigen. The immunosensor was applied to the quantification of CA 15-3 in artificial serum samples with satisfactory results.

Abstract In this work, we describe an innovative methodology based on combined surface plasmon resonance (SPR) and electrochemical responses (eSPR) in the same immunoassay for screening CA 15-3 cancer biomarker with high sensitivity (and selectivity), in a very simple, label-free, accurate, and fully automated manner. Detection was achieved by performing two simple steps. In the first step, direct SPR was used to monitor CA 15-3 interaction with surface immobilized antibody. Two linear response ranges were obtained and the detection limit achieved is poor (LOD of 21 U mL(-1)). However, in the second detection step, electrochemical measurements at the SPR gold surface were performed to measure the decrease of redox probe peak current upon antigen-antibody interaction, providing a suitable amplification strategy to lower detection levels of CA 15-3 (LOD of 0.0998 U mL(-1)), without the need of additional complex and/or expensive amplification steps to enhance the sensitivity. Moreover, selectivity studies were performed against other common cancer biomarkers and the results showed that the eSPR immunosensor is selective for the CA 15-3 protein. Finally, the clinical applicability of the developed eSPR biosensing methodology was successfully applied to detect CA 15-3 in human serum samples at clinically relevant levels due to the high sensitivity of electrochemical readout. The same concept may be further extended to other proteins of interest.

Abstract In this work, we combined electrochemical techniques with SPR (eSPR) for the label-free detection of cancer biomarker miRNA-145. Detection was performed in a simple two-step assay. In the first step, the gold sensor surface, previously functionalized with a self-assembled monolayer (SAM) of thiolated RNA probes is incubated with the sample containing the target RNA biomarker. In this step, hybridization of RNA fragments with complementary immobilized probes was monitored in real-time by SPR. In the second step, eSPR measurements were performed to improve the sensitivity of the hybridization assay. Potential-induced deposition of a redox probe at the sensor surface resulted in enhanced SPR response promoted by the electrochemical process, thereby allowing the detection of miRNA-145 at femtomolar level (LOD = 0.56 fM), without sample derivatization or post-hybridization treatment for signal amplification. Good linearity was achieved (R-2 = 0.984) over the concentration range from 1.0 fM and 10 nM. Furthermore, the developed eSPR biosensor showed high selectivity towards single-base and two-base mismatch sequences and detection of target miRNA-145 in synthetic human serum was successful achieved.

Abstract In this work, three universally used redox probes in amperometric biosensing devices, [Fe(CN)(6)](3-)/[Fe(CN)(6)](4-), Ru[(NH3)(6)](3+), and ferrocenedimethanol (FDM), were selected to evaluate the stability of electrochemical signals provide by the reporting systems. Studies were carried out at disposable gold screen-printed electrode (AuSPE) biosensing platforms, commonly used for screening chemical and biological relevant biomolecules. Firstly, electrochemical combined-surface plasmon resonance (eSPR) studies were performed to evaluated adsorption reversibility and/or formation of redox probe complexes at the bare gold surface when routinely used electrochemical techniques, namely cyclic voltammetry (CV) and square-wave voltammetry (SWV), are recorded. Then, the results obtained were compared with those obtained at the AuSPE under the same electrochemical conditions. Based on our findings, best experimental conditions, including the type of electrochemical technique used, are speculated for each reporting system in order to improve the analytical signal stability. Finally, a methodology based on SWV technique was applied to modified electrodes to provide a simple and easy tool to ensure diffusion controlled permeability of probes thorough the films to electrode surface.

Abstract In this work, electrically-conducting poly(Toludine Blue) was employed for the first time as synthetic receptor film, prepared by Molecular Imprinting strategies and using electrochemical methods, for the specific screening of breast cancer biomarker Carbohydrate Antigen 15.3 (CA 15-3). The protein imprinted poly(Toluidine Blue) film was grown in a pre-formed Toluidine Blue (TB) tailed SAM at the AuSPE surface, which greatly enhanced the stability against degradation of the Molecular Imprinted Polymer (MIP) film at the electrode surface. The MIP receptor film recognition ability towards the protein was investigated by fitting data to Freundlich isotherm. The binding affinity (K-p) obtained for the MIP system was significantly higher (similar to 12-fold) to that obtained for the NIP system, demonstrating the success of the approach in creating imprinted materials that specifically respond to CA 15-3 protein. The incubation of the MIP modified electrode with increasing concentration of protein (from 0.10 U mL(-1) to 1000 U mL(-1)) resulted in a decrease of the ferro/ferricyanide redox current. The device displayed linear response from 0.10 U mL(-1) to 100 U mL(-1) and LODs below 0.10 U mL(-1) were obtained from calibration curves built in neutral buffer and diluted artificial serum, using DPV technique, enabling the detection of the protein biomarker at clinically relevant levels. The developed MIP biosensor was applied to the determination of CA 15-3 in spiked serum samples with satisfactory results. The developed device provides a new strategy for sensitive, rapid, simple and cost-effective screening of CA 15-3 biomarker. Importantly, the overall approach seems suitable for point-of-care (PoC) use in clinical context.

Abstract In this work, we report for the first time the accumulation activity by energized rat heart mitochondria and the ionic transfer process at a liquid liquid interface of a novel mitochondria-targeted antioxidant, named as AntiOxCIN(4), which is structurally based on a hydroxycinnamic acid. Lipophilicity studies conducted at the water/1,6-dichlorohexane (DCH) interface allowed the building up of an ionic partition diagram of AntiOxCIN(4) in accordance with the electrochemical data obtained. The partition coefficients of both positively charged (-2.3) and zwitterionic (0.2) forms of the antioxidant were determined. This study contributed to gaining an insight about the ability of the synthesized antioxidants to cross biomembrane barriers by using an interface between two immiscible electrolyte solutions (ITIES) as a model system.

Abstract An electrochemical biosensor was developed by merging the features of Molecular Imprinting technique and Screen-Printed Electrode (SPE) for the simple and fast screening of cardiac biomarker myoglobin (Myo) in point-of-care (POC). The MIP artificial receptor for Myo was prepared by electrooxidative polymerization of phenol (Ph) on a AuSPE in the presence of Myo as template molecule. The choice of the most effective protein extraction procedure from the various extraction methods tested (mildly acidic/basic solutions, pure/mixed organic solvents, solutions containing surfactants and enzymatic digestion methods), and the optimization of the thickness of the polymer film was carefully undertaken in order to improve binding characteristics of Myo to the imprinted polymer receptor and increase the sensitivity of the MIP biosensor. The film thickness was optimized by adjusting scan rate and the number of cycles during cyclic voltammetric electropolymerization of Ph. The thickness of the polyphenol nanocoating of only few nanometres (similar to 4.4 nm), and similar to the protein diameter, brought in significant improvements in terms of sensor sensitivity. The binding affinity of MIP receptor film was estimated by fitting the experimental data to Freundlich isotherm and a similar to 8 fold increase in the binding affinity of Myo to the imprinted polymer (K-F = 0.119 +/- 0.002 ng(-1) mL) when compared to the non- imprinted polymer (K-F = 0.015 +/- 0.002 ng(-1) mL) which demonstrated excellent (re) binding affinity for the imprinted protein. The incubation of the Myo MIP receptor modified electrode with increasing concentration of protein (from 0.001 ng mL(-1) to 100 mu g mL(-1)) resulted in a decrease of the ferro/ferricyanide redox current. LODs of 2.1 and 14 pg mL(-1) were obtained from calibration curves built in neutral buffer and diluted artificial serum, respectively, using SWV technique, enabling the detection of the protein biomarker at clinically relevant levels. The prepared MIP biosensor was applied to the determination of Myo spiked serum samples with satisfactory results.

Abstract Targeting mitochondrial oxidative stress is an effective therapeutic strategy. In this context, a rational design of mitochondriotropic antioxidants (compounds 22-27) based on a dietary antioxidant (caffeic acid) was performed. Jointly named as AntiOxCINs, these molecules take advantage of the known ability of the triphenylphosphonium cation to target active molecules to mitochondria. The study was guided by structure-activity-toxicity-property relationships, and we demonstrate in this work that the novel AntiOxCINs act as mitochondriotropic antioxidants. In general, AntiOxCINs derivatives prevented lipid peroxidation and acted as inhibitors of the mitochondrial permeability transition pore. AntiOxCINs toxicity profile was found to be dependent on the structural modifications performed on the dietary antioxidant. On the basis of mitochondrial and cytotoxicity/antioxidant cellular data, compound 25 emerged as a potential candidate for the development of a drug candidate with therapeutic application in mitochondrial oxidative stress-related diseases. Compound 25 increased GSH intracellular levels and showed no toxicity on mitochondrial morphology and function.