Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates

The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation

Amperometric Immunosensing Scaffolds for Rapid, Simple, Non-Invasive and Accurate Determination of Protein Biomarkers of Well-Accepted and Emerging Clinical Importance

New amperometric immune-scaffolds for the rapid, sensitive and reliable determination of [...

Schematic representation of the amperometric sensor for FGFR4 determination using magnetic immunocarriers and a sandwich format.

<p>Schematic representation of the amperometric sensor for FGFR4 determination using magnetic immunocarriers and a sandwich format.</p

Dependence of the amperometric responses obtained with the developed immunosensor with the BDAb concentration (a), and the number of steps carried out to perform the immunoassay (b).

<p>Results are shown in the absence (white bars) or in the presence of 5,000 pg mL^-1 FGFR4 (grey bars) together with the corresponding S/B ratio (). 2(A), two sequential steps involving 30 min incubation of the CAb-MBs in a mixture solution containing FGFR4 and BDAb, and 30 min incubation in the Strep-HRP solution; 2(B) two steps involving 30 min incubation of the Cab-MBs with FGFR4 solution, followed by a 30 min incubation step in a mixture solution containing BDAb and Strep-HRP. Error bars estimated as triple of the standard deviation (n = 3).</p

Evaluation of the selectivity of the developed immunosensor for FGFR4.

<p>Amperometric signals measured in the absence (white bars) and in the presence of 2,500 pg mL^-1 FGFR4 (grey bars) and the corresponding S/B ratio () in the absence (1) and in the presence of 10 ng mL^-1 TNFα (2), 200 ng mL^-1 human p53 (3), 5 mg mL^-1 BSA (4), 5 mg mL^-1 hemoglobin (5) and 1 mg mL^-1 human IgG (6). Error bars estimated as triple of the standard deviation (n = 3).</p

Optimization of the experimental variables tested, according to the measured S/B ratio in the absence and in the presence of 5,000 pg mL^-1 FGFR4 standard solutions, for the preparation of the amperometric immunosensor for FGFR4.

<p>Optimization of the experimental variables tested, according to the measured S/B ratio in the absence and in the presence of 5,000 pg mL^-1 FGFR4 standard solutions, for the preparation of the amperometric immunosensor for FGFR4.</p

Dependence of the amperometric response measured with the developed magnetic immunocarriers-based sensor with the concentration of FGFR4 standards.

<p>Error bars estimated as triple of the standard deviation (n = 3).</p