Label-free piezoelectric biosensor for prognosis and diagnosis of Systemic Lupus Erythematosus

[EN] An autoantigen piezoelectric sensor to quantify specific circulating autoantibodies in human serum is developed. The sensor consisted on a quartz crystal microbalance with dissipation monitoring (QCM-D) where TRIM21 and TROVE2 autoantigens were covalently immobilized, allowing the selective determination of autoantibodies for diagnosis and prognosis of Systemic Lupus Erythematosus (SLE). The sensitivity of the biosensor, measured as IC50 value, was 1.51 U/mL and 0.32 U/mL, for anti-TRIM21 and anti-TROVE2 circulating autoantibodies, respectively. The sensor is also able to establish a structural interaction fingerprint pattern or profile of circulating autoantibodies, what allows scorin…

Elucidation of Carbohydrate Molecular Interaction Mechanism of Recombinant and Native ArtinM

[EN] The quartz crystal microbalance (QCM) technique has been applied for monitoring the biorecognition of ArtinM lectins at low horseradish peroxidase glycoprotein (HRP) concentrations, using a simple kinetic model based on Langmuir isotherm in previous work.(18) The latter approach was consistent with the data at dilute conditions but it fails to explain the small differences existing in the jArtinM and rArtinM due to ligand binding concentration limit. Here we extend this analysis to differentiate sugar-binding event of recombinant (rArtinM) and native (jArtinM) ArtinM lectins beyond dilute conditions. Equivalently, functionalized quartz crystal microbalance with dissipation monitoring (…

Determination of titratable acidity and ascorbic acid in fruit juices in continuous-flow systems

Two continuous-flow systems for the determination of titratable acidity and ascrobic acid in fruit juice samples are described. The assemblies permit on-line dialysis of analytes prior to the reaction step, thus improving selectivity and performing sample dilution. Flow systems are built with a channel carrying the donor phase (sample in both determinations) and another channel carrying an acceptor phase, both of them entering the dialyser. The outcoming stream transporting the dialysed sample fills the valve loop, permitting its injection into a carrier stream which continuously passes through the spectrophotometric detector. For the titratable acidity, acceptor phase and carrier are disti…

Antibody-Capped Mesoporous Nanoscopic Materials: Design of a Probe for the Selective Chromo-Fluorogenic Detection of Finasteride

[EN] The synthesis of capped mesoporous silica nanoparticles (MSN) conjugated with an antibody (AB) as a gatekeeper has been carried out in order to obtain a delivery system able to release an entrapped cargo (dye) in the presence of a target molecule (antigen) to which the conjugated antibody binds selectively. In particular, MSN loaded with rhodamine B and functionalized on the external surface with a suitable derivative of N-(t-butyl)- 3-oxo-(5a,17b)-4-aza-androst-1-ene-17-carboxamide (finasteride) have been prepared (S1). The addition of polyclonal antibodies against finasteride induced capping of the pores due to the interaction with the anchored hapten-like finasteride derivative to g…

BIO Bragg gratings on microfibers for label-free biosensing

[EN] Discovering nanoscale phenomena to sense biorecognition events introduces new perspectives to exploit nano science and nanotechnology for bioanalytical purposes. Here we present Bio Bragg Gratings (BBGs), a novel biosensing approach that consists of diffractive structures of protein bioreceptors patterned on the surface of optical waveguides, and tailored to transduce the magnitude of biorecognition assays into the intensity of single peaks in the reflection spectrum. This work addresses the design, fabrication, and optimization of this system by both theoretical and experimental studies to explore the fundamental physicochemical parameters involved. Functional biomolecular gratings ar…

Dual-Polarization Interferometry: A Novel Technique To Light up the Nanomolecular World

[EN] The challenging lecture given in 1959 by physicist and Nobel Prize awarded R. P. Feynman: “There's plenty of room at the bottom” is considered to be the starting point for nanotechnology. With this peculiar title, Feynman encouraged researchers to explore beyond the atomic level and predicted exciting new phenomena that might revolutionize science and technology. Among these pioneering researchers are Eric Betzig, Stefan W. Hell and William E. Moerner, who have been awarded with the Nobel Prize in Chemistry 2014 for developing the super-resolved fluorescence microscopy. However, it is important to remark that the exploration of this amazing nanomolecular world began in the early 1980s …

Automation of the determination of hydrogen peroxide, dichromate, formaldehyde and bicarbonate in milk by flow injection analysis

Automatic flow injection methods for the determination of hydrogen peroxide, dichromate, formaldehyde and bicarbonate in dairy products are proposed. They are based on reaction with vanadium pentoxide, diphenyl-carbazide, fuchsine-sulphur dioxide and alizarin, respectively. Sample pre-treatment (paper and membrane filtration, dialysis) is widely discussed for on-line incorporation. The usefulness of these methods was tested by applying them to different commercial samples, satisfactory results being obtained in all instances.

BIO-Bragg gratings: structured molecular networks for on-fiber bioanalysis

The research on photonic biosensors is a scientific hot topic at the moment, with a significant potential impact on industry and medicine. Label-free, miniaturized, inexpensive and low-loss biosensors are developed based on optical fiber technology. Our approach is based on a Bio-Bragg-Grating (BBG) patterned on the surface of a microfiber. We present the design, fabrication and proof of concept of our device, as well as its multiplexing and tunability perspectives [1] .

Modeling of the role of conformational dynamics in kinetics of the antigen-antibody interaction in heterogeneous phase.

[EN] A novel approach that may potentially be used to study biomolecular interactions including the simultaneous determination of structural and kinetic binding parameters is described in this Article for the first time. It allows a rigid distinction between the possible reaction mechanisms of biomolecular recognition, induced fit and conformational selection. The relative importance of the two pathways is determined not by comparing rate constants but the structural aspects of the interaction instead. So the exact location of antigen molecules with respect to the capture antibody is depicted experimentally, avoiding the use of X-ray crystallography. The proposed pattern is applied to study…

Fast determination of prominent carotenoids in tomato fruits by CEC using methacrylate ester-based monolithic columns.

In this study, the major carotenoids (beta-carotene and lycopene) present in tomato fruits were analyzed by CEC with a methacrylate ester-based monolithic column. The effects of the porogenic solvent ratio, and the hydrophobicity of bulk monomer employed were examined on carotenoids separations. A fast separation of these analytes was achieved in less than 5.0 min in a mobile phase containing 35% THF, 30% ACN, 30% methanol, and 5% of a 5 mM Tris aqueous buffer, pH 8, with lauryl methacrylate-based monoliths. The CEC method was evaluated in terms of detection limit and reproducibility (retention time, area, and column preparation) with values below 1.6 microg/mL and 7.2%, respectively. The p…

Experimental Study of the Oriented Immobilization of Antibodies on Photonic Sensing Structures by Using Protein A as an Intermediate Layer

[EN] A proper antibody immobilization on a biosensor is a crucial step in order to obtain a high sensitivity to be able to detect low target analyte concentrations. In this paper, we present an experimental study of the immobilization process of antibodies as bioreceptors on a photonic ring resonator sensor. A protein A intermediate layer was created on the sensor surface in order to obtain an oriented immobilization of the antibodies, which enhances the interaction with the target antigens to be detected. The anti-bovine serum albumin (antiBSA)-bovine serum albumin (BSA) pair was used as a model for our study. An opto-fluidic setup was developed in order to flow the different reagents and,…

Denaturing for Nanoarchitectonics: Local and Periodic UV-Laser Photodeactivation of Protein Biolayers to Create Functional Patterns for Biosensing

[EN] The nanostructuration of biolayers has become a paradigm for exploiting nanoscopic light-matter phenomena for biosensing, among other biomedical purposes. In this work, we present a photopatterning method to create periodic structures of biomacromolecules based on a local and periodic mild denaturation of protein biolayers mediated by UV-laser irradiation. These nanostructures are constituted by a periodic modulation of the protein activity, so they are free of topographic and compositional changes along the pattern. Herein, we introduce the approach, explore the patterning parameters, characterize the resulting structures, and assess their overall homogeneity. This UV-based patterning…

Simultaneous enzymatic determination of l(−) malic acid and l(+) lactic acid in wine by flow injection analysis

Abstract A simultaneous determination of two organic acid species in wine by a flow injection analysis method using enzyme-immobilized open tubular reactors is described. The design of the optimal geometry of the FIA system is especially important to avoid the matrix fluorescence. Reproducible results were obtained in the determination of l (−) malic acid and l (+) lactic acid using l -MDH and l -LDH, respectively. The assay reactions utilize the fluorimetric determination of a common product, NADH. The sample was dialyzed on line and injected into the FIA-system. The system responds linearly (log-log calibration graphs) to the injected sample (80 μl) in concentrations ranging from 0·1 m m …

Enzyme Inhibition Microassays on Blu-Ray Disks for Drug Discovery

[EN] An enzyme inhibition-based assay for drug discovery developed by microarraing on Blu-ray disks is presented. As a proof-of-concept, the system screens a selected molecule library of potential chemical inhibitors against the glycoenzyme peroxidase, identifying the promising lead compounds with high selectivity using standard Blu-ray disks and drives. In order to face the first drug discovery stages, we establish the bases for a high-throughput screening assay and a methodology based on hypersurfaces suitable to manage a high number of data as well.

Controlled delivery using oligonucleotide-capped mesoporous silica nanoparticles.

Mapping molecular binding by means of conformational dynamics measurements

[EN] Protein-protein interactions are key in virtually all biological processes. The study of these interactions and the interfaces that mediate them play a key role in the understanding of biological function. In particular, the observation of protein¿protein interactions in their dynamic environment is technically difficult. Here two surface analysis techniques, dual polarization interferometry and quartz crystal microbalance with dissipation monitoring, were paired for real-time mapping of the conformational dynamics of protein¿ protein interactions. Our approach monitors this dynamics in real time and in situ, which is a great advancement within technological platforms for drug discover…

Identification of high-affinity phage-displayed VH fragments by use of a quartz crystal microbalance with dissipation monitoring

10 Pág. Departamento de Tecnología de Alimentos​​

Determination of -Ascorbic Acid in Lycopersicon Fruits by Capillary Zone Electrophoresis

This study shows an improved method for the determination of L-ascorbic acid (l-AA) in fruits of Lycopersicon by capillary zone electrophoresis (CZE). Two backgrounds electrolytes (BGEs) have been tested: (i) 400 mM borate at pH 8.0 and 1 x 10(-2)% hexadimethrine bromide, for the separation of Eulycopersicon subgenus species; and (ii) as in BGE(i) but supplemented with 20% (v/v) acetonitrile, for the separation of species of the Eriopersicon subgenus. The present procedures were compared with two routine methods-enzymatic assay and potentiometric titration with 2,6-dichlorophenol-indophenol. While these routine methods presented some difficulties in quantifying l-AA in several Lycopersicon …

Simultaneous quantification of the main organic acids and carbohydrates involved in tomato flavour using capillary zone electrophoresis

A capillary zone electrophoresis (CZE) procedure for the simultaneous determination of the major organic acids (oxalate, malate and citrate) and carbohydrates (fructose, glucose and sucrose) in Lycopersicon fruits is reported. Comparison of this method with routine HPLC methods indicates that the CZE method offers several attractive features (speed, resolution, sensitivity and cost) which significantly improve the determination of these compounds. Detection limits were better than 1.6 µg ml−1 for organic acids and from 13 to 24 µg ml−1 for carbohydrates; repeatabilities were better than 2.1% for migration times and between 1.4 and 7.3% for peak areas. The proposed protocol is very useful to…