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. An epitope-paratope-specific binding initially occurs to activate a hidden Fc receptor in the TROVE2 tertiary structure. This bipolar mechanism may contribute to the pathogenic accumulation of anti-TROVE2 autoantibody immune complex in autoimmune disease. Furthermore, the specific calcium-dependent protein-protein bridges point out at how the TRIM21/TROVE2 association might occur, suggesting that the TROVE2 protein could stimulate the intracellular immune signaling via the TRIM21 PRY-SPRY domain. These findings may help to better understand the origins of the specificity and affinity of TROVE2 interactions, which might play a key role in the SLE pathogenesis. This manuscript gives one of the first practical applications of two novel functions (-df/dD and Δh/molec) for the analysis of the data provided by QCM-D and DPI. In addition, it is the first time that QCM-D has been used for mapping hidden Fc receptors as well as linear epitopes in a protein tertiary structure. Graphical abstract ᅟ.

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. For the first, Cy5 labeled analytes are used (λem 635 nm) and, for the second, a periodic array of high-aspect ratio nanopillars detects unlabeled analytes by interferometry. Bioavailability of the immobilized probes is demonstrated in labeled assays; for the case of oligonucleotides by discriminating single nucleotide polymorphisms, and, for the case of antibodies, by BSA immunorecognition. The raw hydrogel is employed to detect human C-reactive protein, in both labeled and non-labeled assay formats, with sensitivities of 30 ng·mL-1 and 2 pg·mL-1, respectively. Graphical abstract Schematic presentation of the phosphorylcholine (MPC) hydrogel preparation onto BluRay disc and SU-8 nanopillars to perform fluorescence and label-free interferometric detection, respectively. It selectively detects C-reactive protein (CRP), but it can covalently immobilize antibodies or nucleid acid probes to detect other analytes.

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/cm2. Modulating the surface hydrophobicity by combining a hydrophobic silane and a hydrophilic silane allows the fabrication of tuned surfaces where the analyte approaches only the anchored probe, which notably reduces nonspecific adsorption and the background. The generated substrates have proven clear advantages for discriminating single-base-pair mismatches, and for detecting bacterial PCR products. The hybridization sensitivity achieved by these high-performance surfaces is about 1.7 pM. Finally, this anchoring reaction is demonstrated using two additional surfaces: polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes. This provides a very interesting pathway for anchoring thiolated biomolecules onto surfaces with C-F motifs via a quick clean UV reaction.

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. Lowest unspecific binding and highest signal intensity and SNR were obtained with large hydrocarbon chain (C22) silanes or a shorter one (C10) containing fluorine atoms. SNR resulting values are improved, reaching levels higher than 1500 in some cases, when using vinyl silanes modified with 1% C10 alkyl fluorinated one, because mild hydrophobicity was achieved (water contact angle ca. 110°) for all silanes, including the short C2 and C3, thus giving rise to smaller and better defined array spots. In addition, unspecific binding of reagents and targets was totally withdrawn. Hence, good-performing surfaces for biosensing applications can be built using appropriate organosilane reagent selection, including fluorinated ones. Graphical abstract ᅟ.

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. This study presents the approach, assesses bovine serum albumin as backfilling agent for indirect µCP on different materials, reports the limitations of standard µCP on the functionality of patterned antibodies, and demonstrates the capabilities of indirect µCP to solve this issue. Bioreceptors were herein structured as diffractive gratings and used to measure biorecognition events in label-free conditions. Besides, as a preliminary approach towards sensing biomarkers, this work also reports the implementation of indirect µCP in an immunoassay to detect human immunoglobulin E.

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. The approach was demonstrated by immunoassaying IgGs, reaching well-correlated responses with quantification and detection limits of 1.3 and 5.2 nM, respectively. These results provide appealing insights into cost-effective, portable, and scalable alternatives for designing new analytical technologies based on diffractive gratings of bioreceptors.

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. The viability and performance of this approach was demonstrated by specifically determining Salmonella PCR products up to a 20 pM sensitivity level.

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. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays.

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 0.07, 0.24, 10.9, 0.21, 0.14, 0.11, 0.11, 102, 0.36, 1.8, 1.7, 0.06, 0.08, 5.8, 1.0, 0.39, 0.003, and 12 µg/L, respectively. Due to the selectivity of the antibody-functionalized nanoparticles, the developed screening methodology allows the simultaneous determination of mixtures of water pollutants in a 10-plex configuration. The analytical performances were compared with those of reference chromatographic methods by the analysis of spiked water samples, the sensitivity and recovery results being in good agreement. The presented screening approach directly quantifies the concentration of complex chemical mixtures without sample treatment or preconcentration steps in a total time of 35 min.

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. In this paper, TED analytical implementation is described and discussed, and their analytical potential is supported by several applications. Microarray immunoassay, immunofiltration assay, solution measurement, and cell culture approaches are herein addressed in order to demonstrate the practical capacity of this system. The analytical usefulness of TED technology is herein demonstrated, describing how to exploit this tool for developing truly integrated analytical systems that provide solutions within the point of care framework.

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). Using the described strategy, hybridization efficiencies up to 65% with full complementary strands were reached. The approach was evaluated by scoring single-base pair mismatches with discrimination ratios around 15. Moreover, the efficacy of the proposed DNA detection scheme is further demonstrated through the assay on a genomic target of bacterial Escherichia coli.

Gold, carbon, and aluminum low-reflectivity compact discs as microassaying platforms.

Compact disc (CD) surface modification with different reflective materials offers new working perspectives as bioanalytical platforms. The potential of gold, carbon, and aluminum low-reflectivity compact discs as a substrate for microassaying is presented. CD polycarbonate bases were coated with these reflecting materials, maintaining optical properties. Probes immobilization onto reflective layers was studied by both passive adsorption and covalent linking. Different chemical modifications were made on substrates to provide functional groups capable of anchoring probes with primary amines covalently. Thus, self-assembled monolayers were performed by chemisorption of 11-mercaptoundecanoic acid on gold surfaces, and silanization with N-(trimethoxysilylpropyl)ethylenediamine triacetic acid on aluminum to provide a carboxylic acid functional group. Carbon oxidation with oxygen plasma afforded similar functionalization on the discs coated with this material. Performance of the studied materials as reflective layers was evaluated, and as proof of concept, a microimmunoassay for a neurotoxic compound was studied on the three surfaces. The results show the possibility of doing assays on this new supports with good analytical performances while maintaining discs' optical and mechanical properties to be read by a CD player.

Dispersive solid-phase extraction and immunoassay with internal reference calibration using fatty acid-coated inorganic fluorescent nanoparticles.

Dispersive solid-phase extraction (dSPE) using fatty acid-coated Eu2O3 nanoparticles (NPs) was developed, and a direct immunoassay was carried out employing these NPs as support. Secondary antibodies labeled with fluorophore groups were used as reporters, and the intrinsic optical properties of the Eu2O3 NPs were employed as an internal calibration of the detection system. The methodology was optimized for both dSPE-NP amount, sample volume, extraction time, ionic strength, and pH-and immunoassay-immunoreagent concentrations, ionic strength, and incubation time. As proof of concept, the methodology was applied to the bovine serum albumin (BSA)/anti-BSA system, and precision of the method was between 5% and 17% with an IC50 of 100 nM. Then, water samples with high saline content (sea water) were assayed to observe the matrix effect, and average recoveries (n = 3) between 78% and 108% were obtained, demonstrating the reliability of the developed analytical method. Finally, the simultaneous dSPE-immunoassay methodology was applied to other compounds with very different chemical characteristics such as an oligonucleotide, the antibiotic sulfamerazine, and the pesticide chlorpyriphos. Although the IC50 values for sulfamerazine were approximately 2400 nM, satisfactory standard curves were obtained. However, poor reproducibility and sensitivity results were obtained for the oligonucleotide and chlorpyriphos preliminary assays.

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). Homogeneous periodic submicron structures of proteins are created and characterized by diffractive measurements, AFM, FESEM, and fluorescence scanning. Then, this patterning method is applied to proteins involved in cow milk allergy, and the resulting structures are implemented as optical transducers to sense specific immunoglobulins G. In particular, gratings of bovine serum albumin, casein, and ß-lactoglobulin are created and assessed, reaching limits of detection in the range of 30-45 ng·mL-1 of unlabeled antibodies by diffractive biosensing.

Aluminum oxide nanoparticles as carriers and adjuvants for eliciting antibodies from non-immunogenic haptens.

Functionalized nanomaterials have applications yet to be discovered, especially in the biological field and, in particular, the in vivo production of antibodies. In this paper, we show that aluminum oxide nanoparticles that are covalently coupled to haptens (small molecular mass compounds) activate the immune system, eliciting antibodies (polyclonal and monoclonal) specific for the herbicide atrazine, the antibiotic sulfasalazine, and the vitamin biotin in mice and rabbits. The particles play the role of carrier and adjuvant, with the immune response being dependent on size and crystallinity. The affinity constants of the antibodies are similar to those reached with traditional immunization strategies based on the use of carrier proteins. This approach has not been previously described, being of scientific and practical interest, as it can lead to immunogens safer than the conventional ones, potentially applicable to human vaccination. In addition, the useful advantages of this technique include the stability of the metallic particles, the synthesis of immunogens in organic media, the versatility of particle derivatization, the ease of purification, the full chemical characterization of immunogens, the lack of a requirement for an adjuvant, the reduction of the cross reactivity, and the low cost of materials. Also, the analytical performances of the immunoassays developed using these antibodies are comparable to those obtained by the standard protocols. Analytical applications of the developed ELISAs were fully demonstrated. Characterization (IgG nature, affinity constants, etc.) of the immunoreagents were also fully assessed.

Development of oligonucleotide microarrays onto Si-based surfaces via thioether linkage mediated by UV irradiation.

Selective covalent immobilization of thiolated oligonucleotides onto an epoxy-functionalized silicon-substrate can be achieved via light radiation (365 nm). Following this approach, thiol-modified oligonucleotide probes were covalently attached as microarrays, reaching an immobilization density of 2.5 pmol·cm(-2), with a yield of 53%. The developed method presents the advantages of spatially controlled probe anchoring (by means of using a photomask), direct attachment without using cross-linkers, and short irradiation times (10 min). Hybridization efficiencies up to 65%, with full complementary strands, were reached. The approach was evaluated by scoring single nucleotide polymorphisms with a discrimination ratio around 15. Moreover, sensitive and selective detection of bacterial Escherichia coli was demonstrated.

Chemical silicon surface modification and bioreceptor attachment to develop competitive integrated photonic biosensors.

Methodology for the functionalization of silicon-based materials employed for the development of photonic label-free nanobiosensors is reported. The studied functionalization based on organosilane chemistry allowed the direct attachment of biomolecules in a single step, maintaining their bioavailability. Using this immobilization approach in probe microarrays, successful specific detection of bacterial DNA is achieved, reaching hybridization sensitivities of 10 pM. The utility of the immobilization approach for the functionalization of label-free nanobiosensors based on photonic crystals and ring resonators was demonstrated using bovine serum albumin (BSA)/anti-BSA as a model system.

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. These photonic crystals have displayed well-correlated dose-response curves in immunoassays to quantify IgGs, C-reactive protein, and lactate dehydrogenase. The detection limit reached is 16 ng/mL, 2µg/mL, and 18 ng/mL, respectively. Herein we describe the experimental procedures and methods to fabricate and functionalize these photonic crystals, perform immunoassays on them, set up an optical system to measure their response, and process the resulting data to perform bioanalytical determinations in label-free format.

Oxygen plasma treated interactive polycarbonate DNA microarraying platform.

A novel DNA microarrying platform based on oxygen plasma activation of polycarbonate surface of compact disks (DVD) is presented. Carboxylic acid groups are generated in few seconds on polycarbonate in an efficient, fast, and clean way. Following this surface activation strategy, amino-modified oligonucleotide probes were covalently attached, reaching an immobilization density of 2 pmol cm(-2). Atomic force microscopy imaging revealed the nondestructive character of this treatment when applied for short times, allowing for disk scanning in standard DVD drives. DNA assays performed on oxygen plasma treated disks resulted very efficient with maximum hybridization yield of 93% and reaching a low limit of detection (200 pM) for perfect match synthetic oligonucleotide targets when reading the disk with a standard drive as detector. The approach was also evaluated by scoring single nucleotide polymorphisms with a discrimination ratio of 12.8. As proof of concept, the oxygen plasma treated interactive polycarbonate DNA microarraying platform was applied to the detection of PCR products of Salmonella spp., reaching a detection limit of 2 nM that corresponds to a DNA concentration of only 1 c.f.u./mL. The results confirm the suitability of the microarray platform for analysis of biological samples with high sensitivity.