Patterned Biolayers of Protein Antigens for Label-Free Biosensing in Cow Milk Allergy.

This paper focuses on creating one-dimensional diffractive grooved structures of antigen proteins on glass substrates for the label-free detection of antibodies to dairy allergens. In particular, the fabrication of protein structures is carried out by combining microcontact printing with physisorption, imines coupling, and thiol-ene click chemistry. The work first sets up these patterning methods and discusses and compares the main aspects involved in them (structure, biolayer thickness, functionality, stability).

Large-Scale Nanogrooved Photonic Crystals for Label-Free Biosensing by Guided-Mode Resonance.

We have developed large-scale one-dimensional photonic crystals from standard recordable Blu-ray disks, tailored to sense unlabeled biorecognition events on their surface. These materials rely on coating, with layers of 80 nm of titanium oxide, nanogrooved polycarbonate plates obtained from regular disks. As a result, they present guided-mode resonances that we have demonstrated that can be exploited to quantify biorecognition events by means of the bandgap positions in the transmission spectra.

Phosphorylcholine-based hydrogel for immobilization of biomolecules. Application to fluorometric microarrays for use in hybridization assays and immunoassays, and nanophotonic biosensing.

An approach is presented for covalent immobilization of biomolecules on an acrylate phosphorylcholine hydrogel. The immobilization and the hydrogel formation take place simultaneously by a thiol-acrylate coupling reaction, induced by UV-light (254 nm). The hydrogel is prepared on two polymeric surfaces (the HardCoat protective layer of Blu-Ray discs, and SU-8) and applied to fluorescence microarray and label-free interferometric detection.

New structural insights into the role of TROVE2 complexes in the on-set and pathogenesis of systemic lupus erythematosus determined by a combination of QCM-D and DPI.

The mechanism of self-recognition of the autoantigen TROVE2, a common biomarker in autoimmune diseases, has been studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarization interferometry (DPI). The complementarity and remarkable analytical features of both techniques has allowed new insights into the onset of systemic lupus erythematosus (SLE) to be achieved at the molecular level. The in vitro study for SLE patients and healthy subjects suggests that anti-TROVE2 autoantibodies may undergo an antibody bipolar bridging.

Indirect Microcontact Printing to Create Functional Patterns of Physisorbed Antibodies.

Microcontact printing (µCP) is a practical and versatile approach to create nanostructured patterns of biomolecular probes, but it involves conformational changes on the patterned bioreceptors that often lead to a loss on the biological activity of the resulting structures. Herein we introduce indirect µCP to create functional patterns of bioreceptors on solid substrates. This is a simple strategy that relies on physisorbing biomolecular probes of interest in the nanostructured gaps that result after patterning backfilling agents by standard µCP.

Fluor-thiol Photocoupling Reaction for Developing High Performance Nucleic Acid (NA) Microarrays.

Spatially controlled anchoring of NA probes onto microscope glass slides by a novel fluor-thiol coupling reaction is performed. By this UV-initiated reaction, covalent immobilization in very short times (30 s at 254 nm) is achieved with probe densities of up to 39.6 pmol/cm.

A label-free diffraction-based sensing displacement immunosensor to quantify low molecular weight organic compounds.

Herein we present a diffractometric immunosensor to quantify low molecular weight organic compounds in a label-free, simple, and sensitive fashion. The approach is based on patterning analyte analogues (haptens) on solid surfaces according to a diffractive structure, and then loading specific antibodies on them to be subsequently displaced by free analytes in solution. This displacement generates a measurable change in the diffractive response that enables to quantify the analyte concentration.

Modulating receptor-ligand binding in biorecognition by setting surface wettability.

Modulation of support wettability used for microarray format biosensing has led to an improvement of results. Hydrophobicity of glass chips was set by derivatizing with single vinyl organosilanes of different chain length and silane mixtures. Thiol-ene photochemical linking has been used as effective chemistry for covalent anchoring of thiolated probes.

Diffractive Protein Gratings as Optically Active Transducers for High-Throughput Label-free Immunosensing.

A novel label-free biosensing approach based on bioreceptor networks patterned as diffractive gratings (biogratings) has been developed. Nanogrooved structures were used as optically active scaffolds for producing arrays of functional BSA biogratings on low energy surfaces by a water-assisted variant of microcontact printing. An analytical scanner, comprising a LightScribe compact disk drive, was developed to measure the diffraction patterns of these biogratings, thus allowing biointeractions to be quantitatively sensed in a multiplex and label-free fashion by means of diffraction efficiency changes.

Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. pruni in symptomatic field samples.

Xanthomonas arboricola pv. pruni is a quarantine pathogen and the causal agent of the bacterial spot disease of stone fruits and almond, a major threat to Prunus species. Rapid and specific detection methods are essential to improve disease management, and therefore a prototype of a lateral flow immunoassay (LFIA) was designed for the detection of X.

Primer design for SNP genotyping based on allele-specific amplification-Application to organ transplantation pharmacogenomics.

Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the real adoption of personalized medicine. Allele specific amplification in a homogeneous format or combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization.

Improved Performance of DNA Microarray Multiplex Hybridization Using Probes Anchored at Several Points by Thiol-Ene or Thiol-Yne Coupling Chemistry.

Nucleic acid microarray-based assay technology has shown lacks in reproducibility, reliability, and analytical sensitivity. Here, a new strategy of probe attachment modes for silicon-based materials is built up. Thus, hybridization ability is enhanced by combining thiol-ene or thiol-yne click chemistry reactions with a multipoint attachment of polythiolated probes.

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

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 IC value, was 1.

Disc-based microarrays: principles and analytical applications.

The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices.

Massive immuno multiresidue screening of water pollutants.

An immuno multiresidue screening assay in microarray format for the determination of complex chemical mixtures at the microgram per liter level, using antibody-functionalized gold nanoparticles, is presented. The analytical method relies on the use of a cocktail of nanogold-labeled specific antibodies, acting as recognition and detection species. The concept of multireside screening is proved by developing a multiplex assay on a compact disk support for the determination of 2-(2,4,5-trichlorophenoxy)propionic acid, 3-phenoxybenozic acid, 4-nitrophenol, alachlor, atrazine, azoxystrobin, chlorpyrifos, diazinon, diuron, endosulfan, fenthion, forchlorfenuron, imidacloprid, malathion, pentachlorophenol, pyraclostrobin, sulfasalazine, and triclosan, achieving detection limits of 0.

Aluminum Nanoholes for Optical Biosensing.

Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that make them highly suitable for (bio)sensing applications. Either as efficient light scattering centers for surface plasmon excitation or metal-clad optical waveguides, they are able to form strongly localized optical fields that can effectively interact with biomolecules and/or nanoparticles on the nanoscale. As the metal of choice, aluminum exhibits good optical and electrical properties, is easy to manufacture and process and, unlike gold and silver, its low cost makes it very promising for commercial applications.

Site-specific immobilization of DNA on silicon surfaces by using the thiol-yne reaction.

Covalent immobilization of ssDNA fragments onto silicon-based materials was performed using the thiol-yne reaction. Chemical functionalization provided alkyne groups on the surface where the thiol-modified oligonucleotide probes can be easily photoattached as microarrays, reaching an immobilization density around 30 pmol cm. The developed method presents the advantages of spatially controlled probe anchoring (by using a photomask), direct attachment without using cross-linkers, and short irradiation times (20 min).

Isothermal DNA amplification strategies for duplex microorganism detection.

A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp.

Total analysis systems with Thermochromic Etching Discs technology.

A new analytical system based on Thermochromic Etching Discs (TED) technology is presented. TED comprises a number of attractive features such as track independency, selective irradiation, a high power laser, and the capability to create useful assay platforms. The analytical versatility of this tool opens up a wide range of possibilities to design new compact disc-based total analysis systems applicable in chemistry and life sciences.

Direct covalent attachment of DNA microarrays by rapid thiol-ene "click" chemistry.

A rapid strategy for the covalent immobilization of DNA onto silicon-based materials using the UV-initiated radical thiol-ene reaction is presented in this study. Following this approach, thiol- and alkene-modified oligonucleotide probes were covalently attached in microarray format, reaching immobilization densities around 6 pmol·cm(-2). The developed methodology presents the advantages of spatially controlled probe anchoring (using a photomask), direct attachment without using cross-linkers (one-pot fashion), and short irradiation times (20 min).

Photoattachment of thiolated DNA probes on SU-8 spin-coated Blu-ray disk surfaces for biosensing.

A methodology to modify Blu-ray disk (BD) surfaces by spin-coating an SU-8 epoxy photoresist is evaluated, to create a hybrid material with new functionalities. The optical performance of the new film surface disk is analyzed. The light-mediated activation of epoxy-to-thiol chemistry is applied to the site-specific and covalent binding of thiol-ended oligonucleotide probes to the SU-8 disk surface.

INSEL: an in silico method for optimizing and exploring biorecognition assays.

A practical in silico method for optimizing and exploring biointeraction-based events is developed.

High density microarrays on Blu-ray discs for massive screening.

The potential and the capabilities of Blu-ray technology (discs and drives) for massive screening applications are presented. High density microarrays are fabricated onto a Blu-ray disc and applied for the determination of microcystin residues and pathogenic microorganisms. Specific probes were physisorbed onto the BD surface and the biorecognition event was displayed using labeled secondary antibody solution and subsequent signal amplification.