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In the last years, magnetoresistive biosensors arrays have drawn a great interest due to their high sensitivity and integrability in lab-on-chip platforms. In such devices, the selective functionalization of the sensor active area is a major issue, in order to achieve high sensitivity and quantification capability. Here, we present a straightforward photolithographic procedure to create patterns of bio-reactive polymer regions on the sensor's surface, with micrometric resolution. The effectiveness of the procedure in providing high specificity and improved sensor performance is demonstrated in the case of magnetoresistive biosensors based on magnetic tunneling junctions (MTJs). On-chip biomolecular recognition assays demonstrate an enhanced sensitivity in selectively functionalized sensors with respect to non-patterned sensors, eventually leading to a limit of detection below the pM range, without target pre-concentration or chemical amplification.

The nature of protein microarray platforms is favorable for multiplexing, leading to the development of tools for personalised medicine and highly precise diagnostics. However, to date, only a limited number of protein microarrays are available in the in vitro diagnostics (IVD) market. This review article will focus on the following operational challenges that are crucial for the use of microarrays in clinical settings: (1) probe printing and quality control; (2) procurement of bio-reagents and antibody cross-reactivity; (3) mass transport limitations and assay automation; (4) calibration and quantification. A selection of microarray assays applicable to IVD and a summary of the diagnostic products currently available on the market are provided. © 2014 The Royal Society of Chemistry.

This review focuses on the fluorescent protein microarrays applied to neurodegenerative disorders, a major health problem in our aging society. Biomarker discovery studies and work on new diagnostic tests are both included. Three platforms are described: antibody planar microarrays, comprising an array of well-defined antibodies for the simultaneous capture of biomarkers; antigen planar microarrays in which proteins, peptides or small molecules are immobilized and probed with biological samples to chase specific antibodies; and bead-based array platforms, which are flow cytometry-based methods in which capture molecules are coupled to fluorescent microspheres, facilitating the simultaneous quantification of several analytes.

The interaction of carbohydrates with a variety of biological targets, including antibodies, proteins, viruses, and cells are of utmost importance in many aspects of biology. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins. In this study, a novel microarray support is reported for the fabrication of glycan arrays that combines the higher sensitivity of a layered Si-SiO

The interaction of carbohydrates with a variety of biological targets, including antibodies, proteins, viruses, and cells are of utmost importance in many aspects of biology. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins. In this study, a novel microarray support is reported for the fabrication of glycan arrays that combines the higher sensitivity of a layered Si-SiO2 surface with a novel polymeric coating easily modifiable by subsequent click reaction. The alkyne-containing copolymer, adsorbed from an aqueous solution, produces a coating by a single step procedure and serves as a soft, tridimensional support for the oriented immobilization of carbohydrates via azide/alkyne Cu (I) catalyzed "click" reaction. The advantages of a functional 3D polymer coating making use of a click chemistry immobilization are combined with the high fluorescence sensitivity and superior signal-to-noise ratio of a Si-SiO2 substrate. The proposed approach enables the attachment of complex sugars on a silicon oxide surface by a method that does not require skilled personnel and chemistry laboratories.

The generation of robust analytical data using microarray platforms strictly relies on optimal ligand-target interaction at the sensor surface, which, in turn, is inherently bound to the correct immobilization scheme of the interrogated bioprobes. In the present work, we performed a rigorous comparative analysis of the impact of peptide ligands immobilization strategy in the screening of Burkholderia cepacia complex (BCC) infections in patients affected by cystic fibrosis (CF). We generated arrays of previously validated Burkholderia derived peptide probes that were selectively oriented on polymeric coatings by means of different click-type reactions including thiol maleimide, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and strain-promoted azide-alkyne cycloaddition (SPAAC). We compared immobilization efficiency among the different chemoselective reactions, and we evaluated diagnostic performances at a statistically significant level, also in contrast to random immobilization strategies. Our findings clearly support the favorable role of correct bioprobe orientation in discriminating seronegative from infected individuals and, in the last analysis, in generating more-reliable and more-reproducible data. Spacing biomolecules from the sensor surface by means of small hydrophilic linkers also positively affects the analytical performance and leads to increased statistical significance of data. Overall, all of the click immobilization strategies that were considered displayed a good efficiency. Interestingly, SPAAC-mediated conjugation using DBCO cyclooctyne for some peptides resulted in sequence-dependent autofluorescence in the Cy5 emission range wavelength, which could be circumvented by using a different fluorescence detection channel. On the basis of our results, we critically discuss the immobilization parameters that need to be carefully considered for peptide ligand immobilization purposes.

Efficient diagnosis of emerging and novel bacterial infections is fundamental to guide decisions on therapeutic treatments. Here, we engineered a novel rational strategy to design peptide microarray platforms, which combines structural and genomic analyses to predict the binding interfaces between diverse protein antigens and antibodies against Burkholderia cepacia complex infections present in the sera of Cystic Fibrosis (CF) patients. The predicted binding interfaces on the antigens are synthesized in the form of isolated peptides and chemically optimized for controlled orientation on the surface. Our platform displays multiple Burkholderia-related epitopes and is shown to diagnose infected individuals even in presence of superinfections caused by other prevalent CF pathogens, with limited cost and time requirements. Moreover, our data point out that the specific patterns determined by combined probe responses might provide a characterization of Burkholderia infections even at the subtype level (genomovars). The method is general and immediately applicable to other bacteria.

Capillary electrophoresis (CE) is one of the most powerful techniques for the separation of biomolecules. However, the separation efficiency of proteins in CE is often compromised by their tendency to interact with the silanol groups on the surface of the inner capillary and by an uncontrolled electroosmotic flow. Herein, we report on the use of novel hydrophilic polymeric coatings that can modulate the properties of the capillary walls. The novelty of these poly(N,N-dimethylacrylamide)-based copolymers relies on the simultaneous presence of chemically reactive groups (N-acryloyloxysuccinimide and glycidyl methacrylate) and silane groups in the backbone, which results in highly stable films due to the covalent reaction between the polymer and the glass silanols. A careful optimization of monomer concentration confers anti-fouling properties to the polymer coatings, and thus allows for highly efficient acidic and alkaline protein separations. Furthermore, the presence of these monomers makes it possible to modulate the electroosmotic flow from negligible to reduced values, depending on the desired application.

A new methodology for the fabrication of an high-performance peptide microarray is reported, combining the higher sensitivity of a layered Si-SiO2 substrate with the oriented immobilization of peptides using a N,N-dimethylacrylamide-based polymeric coating that contains alkyne monomers as functional groups. This clickable polymer allows the oriented attachment of azido-modified peptides via a copper-mediated azide/alkyne cycloaddition. A similar coating that does not contain the alkyne functionality has been used as comparison, to demonstrate the importance of a proper orientation for facilitating the probe recognition and interaction with the target antibody.

The interest towards extracellular vesicles (EVs) has grown exponentially over the last few years; being involved in intercellular communication and serving as reservoirs for biomarkers for tumors, they have a great potential for liquid biopsy development, possibly replacing many costly and invasive tissue biopsies. Here we propose, for the first time, the use of a Si/SiO2 interferometric, microarray platform for multiparametric intact EVs analysis combining label-free EVs mass quantitation and high sensitivity fluorescence based phenotyping. Label free interferometric measurement allows to quantify the amount of vesicles captured by printed antibodies while, on the same chip, EVs are also detected by fluorescence in a sandwich immunoassay. The proposed method simultaneously detects, quantify and phenotype intact EVs in a microarray format.

The fabrication of high density microarrays requires a precise control of surface physical and chemical properties. In fact, spot morphology has a deep impact on signal level and might affect seriously the reliability and reproducibility of the assay. As a consequence, a proper biomolecule deposition and immobilization increases the accuracy of data analysis. Here we propose a new polymeric coating with intermediate hydrophilic/hydrophobic characteristics able to control the spreading of spots onto the surface as well as their size, thus enabling the reduction of the pitch between spots. The new coating exemplifies the versatility of a copolymer system based on the simultaneous presence of N,N-Dimethylacrylamide (DMA), 3-(trimethoxylsilyl)propyl methacrylate (MAPS) and fluorinated monomers, which impart to the surface a more hydrophobic behavior, reducing the drops spreading and merging. The controlled hydrophobicity allows to control also the size of the spots, thus the possibility of reducing their pitch to 140 μm and to obtain arrays with smaller dimentions, a very useful characteristics in those applications which imply the use of reduced areas, for example in miniaturized biosensors or microfluidic devices. The obtained copolymer is easily adsorbed onto glass or silicon oxide from a diluted aqueous solution of the polymer and the result is a microarray surface with intermediate hydrophobic/hydrophilic properties (average water contact angle is 50° and surface energy values are reduced from 114 mN/m to 50 mN/m), which allows decrease of the spot distance without merging of the droplets. To further improve the spot size and morphology, we have also investigated the use of several detergents added to the spotting buffer: in particular, the use of sucrose monolaurate, together with the hydrophobic surface, permitted the fabrication of surfaces for dense DNA microarray with very high fluorescence signals and low background noise. In fact, as an example of application in DNA microarray, the fluorinated coating has been used for the genotyping of KRAS G12D mutation, a common variant in the KRAS gene implicated in the colorectal cancer.

Capillary electrophoresis (CE) is one of the most powerful techniques for the separation of biomolecules. However, the separation efficiency of proteins in CE is often compromised by their tendency to interact with the silanol groups on the surface of the inner capillary and by an uncontrolled electroosmotic flow. Herein, we report on the use of novel hydrophilic polymeric coatings that can modulate the properties of the capillary walls. The novelty of these poly(N,N-dimethylacrylamide)-based copolymers relies on the simultaneous presence of chemically reactive groups (N-acryloyloxysuccinimide and glycidyl methacrylate) and silane groups in the backbone, which results in highly stable films due to the covalent reaction between the polymer and the glass silanols. A careful optimization of monomer concentration confers anti-fouling properties to the polymer coatings, and thus allows for highly efficient acidic and alkaline protein separations. Furthermore, the presence of these monomers makes it possible to modulate the electroosmotic flow from negligible to reduced values, depending on the desired application.

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.

We present a new multidisciplinary strategy integrating computational biology with high-throughput microarray analysis aimed to translate molecular understanding of protein-antibody recognition into the design of efficient and selective protein-based analytical and diagnostic tools. The structures of two proteins with different folds and secondary structure contents, namely, the beta-barrel FABP and the α-helical S100B, were used as the basis for the prediction and design of potential antibody-binding epitopes using the recently developed MLCE computational method. Starting from the idea that the structure, dynamics, and stability of a protein-antigen play a key role in the interaction with antibodies, MLCE integrates the analysis of the dynamical and energetic properties of proteins to identify nonoptimized, low-intensity energetic interaction-networks on the surface of the isolated antigens, which correspond to substructures that can aptly be recognized by a binding partner. The identified epitopes were next synthesized as free peptides and used to elicit specific antibodies in rabbits. Importantly, the resulting antibodies were proven to specifically and selectively recognize the original, full-length proteins in microarray-based tests. Competition experiments further demonstrated the specificity of the molecular recognition between the target immobilized proteins and the generated antibodies. Our integrated computational and microarray-based results demonstrate the possibility to rationally discover and design synthetic epitopes able to elicit antibodies specific for full-length proteins starting only from three-dimensional structural information on the target. We discuss implications for diagnosis and vaccine development purposes.

A dual detection system for protein arrays is presented that combines label-free detection by optical interference with chemiluminescence. A planar protein array that targets hepatitis B surface antigen is developed. Surface densities for individual antibody spots are quantitated using optical interference prior to use. Target binding (10 ng/ml) is detected label-free. Target binding (1 ng/ml) is detected by both optical interference and chemiluminescence with the inclusion of secondary antibodies. Binding results using both methods are found to be directly proportion to the capture probe density measured initially. The dual detection system provides the analytical utility of optical interference detection with the established clinical utility of chemiluminescence detection. (C) 2011 Elsevier B.V. All rights reserved.

By applying integrated-waveguide laser excitation to an optofluidic chip, fluorescently labeled DNA molecules of 12 or 17 different sizes are separated by CE with high operating speed and low sample consumption of approximately 600 pL. When detecting the fluorescence signals of migrating DNA molecules with a PMT, the LOD is as low as 2.1 pM. In the diagnostically relevant size range (approximately 150-1000 base-pairs) the molecules are separated with reproducibly high sizing accuracy (99%) and the plug broadening follows Poissonian statistics. Variation of the power dependence of migration time on base-pair size--probably with temperature and condition of the sieving gel matrix--indicates that the capillary migration cannot be described by a simple physical law. Integrated-waveguide excitation of a 12-microm narrow microfluidic segment provides a spatio-temporal resolution that would, in principle, allow for a 20-fold better accuracy than the currently supported by state-of-the-art electrophoretic separation in microchips, thereby demonstrating the potential of this integrated optical approach to fulfill the resolution demands of future electrophoretic microchips.

We report on the modification of a nitrocellulose film with copoly(DMA-NAS-MAPS), a tercopolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS). The chains of this polymer, interacting with nitrocellulose fibers, introduce active ester functionalities that promote the covalent binding of short oligonucleotide fragments to the nitrocellulose thin film. Using colorimetric detection, naked eye visible DNA microarrays are developed for easy identification of foodborne pathogens. The fast and robust procedure of nitrocellulose functionalization opens the opportunity to implement this material in disposable analytical microdevices that do not require sophisticated readout systems.

A novel microchip electrophoresis instrument based on single-photon avalanche diodes was used for the molecular characterization of mutations in disease genes. The identification of the main mutation causing cystic fibrosis, named DeltaF508, by the Amplification Refractory Mutation System was used to validate the technology. In our implemented protocol the wild-type and mutant allele-specific primers are labeled with Cy5 and Cy5.5, respectively. The protocol enables the amplification of the DNA sample in a single PCR. The genotype was deduced from the fluorescence of the amplicons run in the CE microchip. Validation on 15 DNA samples from either homozygous wild-type or heterozygous and homozygous mutated control subjects proved the complete reliability of the system, thus confirming its high diagnostic potential.

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90 degree angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques.

A robust method for coating polydimethylsiloxane (PDMS) allowing covalent binding of biomolecules is reported. The coating is based on adsorption of a copolymer, copoly(DMA-NAS-MAPS), bearing functional groups. The polymer is synthesized by free radical polymerization; it self-adsorbs onto the PDMS very rapidly, typically in 30 min, even without any PDMS surface pre-treatment. PDMS slides coated by copoly(DMA-NAS-MAPS) covalently bind amino-modified DNA fragments and have been tested in bacterial genotyping experiments. The usefulness of this functional coating on PDMS structures in lab-on-chip devices powered by microfluidics has been demonstrated. The copoly(DMA-NAS-MAPS) coated PDMS slides were used as flexible components of a microcell in semi-automated DNA microarray experiments for rapid food pathogen identification.

In the few last years, the need of reliable, fast and inexpensive methods for selective analysis of specific substances in complex mixtures has grown exponentially. In particular, the detection of biomolecules, such as oligonucleotides, proteins, peptides and carbohydrates is of outstanding importance in gene expression, drug design and medicine studies. To these purposes, molecular recognition on microarray-configured devices is one of the most promising tools. This technology uses a number of different substrates such as glass, silicon, alumina or gold-coated slides. The use of polymers is a very effective way to tailor surface properties introducing functional groups able to bind biomolecules and prevent denaturation and non-specific binding. Furthermore, advanced polymers, thanks to their particular physico-chemical properties, can be used to improve selectivity and sensitivity during assays. This review will provide very recent examples of polymer-mediated molecular recognition between guest molecules in solution and host molecules located at the solid phase.

In this work a high-throughput method based on the single-nucleotide extension (SNE) reaction and multicolour detection in a DNA sequencer was developed to screen for eight mutations in the human beta-globin gene: IVSI.110, cd39, IVSI.1, IVSI.6, IVSII.745, HbC, HbS and cd6. The method has been validated on a large number of samples for the two most common mutations causing beta-thalassemia in the Mediterranean area (IVSI.110 and cd39). The development of a high-throughput, fast and reliable method to assay beta-thalassemia mutations represents a significant improvement in molecular diagnosis of this disease. The multicolour detection and the use of multiple injections further enhances the throughput of mutational screening by the DNA sequencer and facilitates automated genotyping for routine molecular diagnostics.

A new methodology for the fabrication of an high-performance peptide microarray is reported, combining the higher sensitivity of a layered Si-SiO2 substrate with the oriented immobilization of peptides using a N,N-dimethylacrylamide-based polymeric coating that contains alkyne monomers as functional groups. This clickable polymer allows the oriented attachment of azido-modified peptides via a copper-mediated azide/alkyne cycloaddition. A similar coating that does not contain the alkyne functionality has been used as comparison, to demonstrate the importance of a proper orientation for facilitating the probe recognition and interaction with the target antibody.

Single-molecule detection and counting is the new frontier in biomarker analysis. Here, we report on recent techniques for the digital detection of biomolecules for clinical application. First we highlight methods based on the immunocapture of proteins onto microparticles, followed by isolation of individual particles in microenvironments so that a sufficient signal is acquired for each. binding event to make a binary decision, thus dramatically enhancing the signal:noise ratio. Various approaches are categorized based on the method used for particle confinement in an isolated microenvironment. We go on to describe methods for the detection of individual biological nanoparticles as well as the digital detection of proteins by artificial nanoparticle labels. The discussion of the methods emphasizes the practical considerations and their clinical applicability.

Nanofluidic sensing devices promise high performance by overcoming issues of mass transport of analyte molecules to the sensing surface, whilst micro-porous membranes promise high sensitivity due to a large surface for their capture. Anodic alumina (AAO) filter membranes allow the flow through of samples, and could be used as a convenient and readily available fluidic platform for the targeted delivering of analytes to bioreceptors immobilized on the pore walls. The relatively small pore dimensions, compared to fluidic diffusion lengths, promise highly efficient capture of analytes from the whole sample volume, enabling relatively fast sensing response times and the use of small sample volumes (

Microarraying peptides is a powerful proteomics technique for studying molecular recognition events. Since peptides have small molecular mass, they are not easily accessible when adsorbed onto solid supports. Moreover, peptides can lack a well-defined three-dimensional structure, and therefore a correct orientation is essential to promote the interaction with their target. In this work, we investigated the suitability as a peptide array substrate of a glass slide coated with a copolymer of N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and [3-(methacryloyl-oxy)propyl]trimethoxysilyl. This polymeric surface was used as substrate for peptides in the characterization of linear antigenic sites of human chromogranin A, a useful tissue and serum marker for neuroendocrine tumors and a precursor of many biologically active peptides. The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which linear epitopes are freely exposed despite peptide random orientation. The results reported in this article are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.

In this research, reproducible detection of fluorescently labelled DNA fragments is reported. Separation was carried out on a new microchip device composed of an array of 40 parallel microchannels (length 63 mm, width 300 μm, depth 500 μm). A comparison between coated and uncoated chips is presented, demonstrating the importance of surface coating in microchip devices for DNA separation. After a 6 min run, 10 DNA fragments were clearly observed in the coated chip with excellent reproducibility for intra-channel separation in the array of parallel capillaries. On the other hand, the efficiency of DNA separation in an uncoated microchip was adversely affected by band broadening and irreproducibility band position between channels.

Adsorbed polymers are widely used to suppress electroosmotic flow (EOF) in capillary electrophoresis (CE). Polymeric coatings, physisorbed onto the surface of the capillary wall, are often unstable under harsh conditions. This can be attributed to the reversible nature of the coating which becomes apparent when the adsorbed layer competes with a second species in the electrophoresis buffer solution for attachment/interaction with the capillary surface. In an effort to overcome the problem of coating instability, trimethoxysilane-modified polydimethylacrylamide was synthesized. This copolymer rapidly adsorbs on the wall from ultradilute aqueous solutions. After incubation at a temperature of 60 degrees C silyl groups, which extend from the polymer backbone, form condensation bonds with the silanols on the capillary surface. This enables subsequent formation of strong covalent bonds between the copolymer and the capillary wall. In this research, we establish that physisorption of polymer chains to the surface is essential for close alignment of surface and polymer silane groups which facilitates the formation of covalent bonds.

We are investigating the molecular biophysics of the early-stage etiology of sporadic Alzheimer’s Disease (AD). What might cause initial accumulation of Aβ peptide-rich fibrils and plaques in the AD brain? What is Aβ’s physiological function? We focus on Aβ’s interactions with the human cathelicidin peptide, LL-37, an antibacterial and antiviral innate immune system effector and modulator ubiquitous in tissues, expressed by myriad cell types, yet unique in the proteome. We present experimental evidence and discuss a hypothesis that LL-37 is a binding partner of Aβ1–42 can inhibit the formation of AD fibrils and plaques. We demonstrate binding between LL-37 and Aβ1–42 by capillary electrophoresis, ELISA, Transmission Electron Microscopy (TEM), and circular dichroism (CD) spectroscopy. TEM shows that LL-37 inhibits the fibrillization of Aβ1–42, especially the formation of long, straight fibrils characteristic of AD, while CD spectroscopy reveals that LL-37 binding prevents Aβ1–42 from adopting β-type secondary structure.

Here we combine the use of fluorescence-enhancing silicon substrates coated by copoly(DMA-NAS-MAPS), a ter-copolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), with an efficient dynamic incubation to overcome mass transport limitations and obtain femtomolar limits of detection. The high sensitivity was obtained with a conventional microarray scanner without the use of any sophisticated detection strategy or protocol. When the method was applied, an improvement of the analytical sensitivity of approximately three orders of magnitude was achieved for antibody detection when compared with the same assay performed on regular glass slides and static conditions. Moreover, limits of detection of 45 and 54 pg/ml were obtained for hepatitis B superficial antigen and HIV p24 antigen, respectively. (C) 2011 Elsevier Inc. All rights reserved.

To date, photonic biosensing with porous membranes has produced slow responses and long sensing times, due to the narrow (less than 100 nm) closed end pores of the membranes used. Recently, polarimetry was used to demonstrate analyte flow through, and real time biosensing in, free-standing porous alumina membranes. Here, we demonstrate how an improved functionalization technology, has for the first time enabled a real-time immunoassay within a porous membrane with a total assay time below one hour. With the new approach, we show a noise floor for individual biosensing measurements of 3.7 ng/ml (25 pM), and a bulk refractive index detection limit of 5×10-6 RIU, with a standard deviation of less than 5%. The membranes, with their 200 nm pore diameter enabling targeted delivering of analytes to bioreceptors immobilized on the pore walls, therefore provide a route towards rapid and low cost real-time opto-fluidic biosensors for small sample volumes. QC 20150203 Positive

Through the presentation of analytical data from bioassay experiments, measured by polarimetry, we demonstrate for the first time a real time immunoassay within a free standing macroporous alumina membrane. The 200 nm nominal pore diameter of the membrane enables flow-through, thereby providing an ideal fluidic platform for the targeted delivery of analytes to bioreceptors immobilized on the pore walls, enabling fast sensing response times and the use of small sample volumes (

Here we present a new and rapid immunofiltration assay for simultaneous detection of HIV p24 and hepatitis B virus antigens. The assay platform is composed of a 13 mm nitrocellulose filter spotted with capturing bioprobes and inserted in a Swinnex ® syringe filter holder. Samples and reagents are flown through the nitrocellulose filter by manual pressure on the syringe. A colorimetric detection allows for naked eye results interpretation. The assay provides sensitivity in the picomolar range in just 5 min, even using low volumes of sample in complex matrix. Probe deposition by spotting allows for flexible combinations of different capturing agents and multiple diagnoses; furthermore, the very simple and inexpensive set-up makes the syringe-based immunoassay on paper microarray a suitable diagnostic system for resource-limited settings.

The demand for high sensitivity in microarray technology has stimulated the research of new labeling strategies, new substrates with increased loading capacity and new approaches to amplify the fluorescence signals. Although these methods improve the sensitivity, they are based on non-routine procedures which limit their widespread usage. A simple way to achieve fluorescence enhancement is the optical interference (OI) coating technology based on the use of substrates with films of well-defined thickness that maximize photoabsorption of the dye molecules in the vicinity of the surface and reflect the emitted light toward the detector. Here we introduce a new substrate for fluorescence enhancement composed of a thin aluminum mirror and a single quarter wave silicon oxide as a dielectric layer. The use of such material allows the development of a substrate suited for parallel production in conventional IC (Integrated Circuit's) technology, which produces a 20-fold enhancement of fluorescence compared to glass, which is the most common material used in microarrays. The new substrate was coated with a copolymer of N , N -dimethylacrylamide, N -acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate, copoly(DMA-NAS-MAPS), which forms, by a simple and robust procedure, a functional nanometric film that covalently binds bio-probes on the surface and efficiently suppresses non-specific adsorption. The performance of the new fluorescence-enhancing substrate in microarray technology was demonstrated for the detection of a panel of inflammation biomarkers pushing detection limits into the femtomolar range.

The quality of separation in capillaries electrophoresis is strongly affected by the magnitude of the electroosmotic flow (EOF). The EOF can be efficiently suppressed by coating the capillary wall with hydrophilic polymers. In this paper, experimental data are presented to show the effect of coating thickness and charge on mass transport. Simulations performed with the lattice Boltzmann technique quantitatively reproduce the EOF with and without the coating and with either a neutral or charged coating layer. Experimental, simulation and theoretical analyses converge toward the interpretation that EO suppression arises from the frictional forces acting on the ionic currents and that a detailed representation of the polymeric coating is not needed in order to capture the phenomenon. © 2014 Springer-Verlag Berlin Heidelberg.

A fast and simple method for the internal coating of capillaries in capillary zone electrophoresis (CZE) is that with epoxy-poly(dimethylacrylamide) (EPDMA). Duration of coating by that method is 30 min, compared with that of 24 h when using uncross-linked polyacrylamide (PA) under otherwise identical conditions. Under the conditions used for the CZE of proteins (pH 9.0, 2% polyethylene glycol), the capillary coating with EPDMA is stable for at least 50 consecutive runs as judged by the constancy of low electroosmotic flow, equalling the stability of coating achieved by PA. Protein mobilities and protein peak asymmetry (suggestive of reversible interaction with the capillary wall) are also found to be the same in EPDMA and PA coated capillaries. Differences between EPDMA and PA coating also exist: The former is unstable upon lowering the ionic strength of the buffer to 0.003, upon the addition of sodium dodecyl sulfate (SDS) to the buffer and in application to the hydrophobic analyte, polystyrene carboxylate.

We have synthesized two new sugar monomers, allylamine of gluconic and lactobionic acid, by opening the corresponding lactone ring with allylamine. These monomers were copolymerized with acrylamide leading to formation of copolymers with a relative molecular mass of 288000 and 180000 Da, respectively. Double-stranded DNA fragments were separated in entangled solutions of these linear polymers in capillary electrophoresis. Resolution, peak spacing and peak width were the parameters taken into account to evaluate the quality of the separation achieved with the new polymers. This work indicates that the copolymers of acrylamide and allyl gluconic acid have a high sieving capacity and provide a performance similar to that of hydroxyethylcellulose (HEC) of comparable viscosity. Unlike HEC, this copolymer selfcoats onto the capillary wall, allowing DNA fragments to be efficiently separated in an uncoated capillary.

A novel, positively charged, copolymer of allylamine and 2-hydroxy-3-methacryloyl-β-cyclodextrin was synthesized to be used as a chiral selector in capillary electrophoresis. In the copolymer, cyclodextrin molecules are spaced from the backbone though a spacer arm which prevents sterical hindrance of the CD cavity. The self-mobility of the CD polymer in its charged form, opposite to the analytes, is the cause for the enhanced separation factor provided by this selector. Moreover, the positive charged polymer induces a reversal of electroosmotic flow which is beneficial in enantioseparations of acidic compounds as it reduces analysis time and increases peak efficiency. The ability of this copolymer to act as a CE chiral selector in the separation of 2,4-dinitrophenylamino acid enantiomers was investigated in coated and uncoated capillaries and its performance was much better then that of native β-cyclodextrin.

A fully automated coating procedure was devised based on adsorption of a new polymer, poly(dimethylacrylamide-co-allyl glycidyl ether) onto the capillary surface. The whole procedure takes less than 30 min and does not require the use of organic solvents, viscous solutions, or elevated temperature. The coating is stable even under harsh conditions such as alkaline pH, elevated temperature, and denaturant conditions that destroy most other presently available adsorbed coatings. This new adsorbed coating is highly stable and easy to produce in quantity, making it quite unique, and further making it possible to operate with any DNA sieving matrix. Finally, the above-mentioned properties facilitate coating regeneration by a simple wash with a strongly alkaline solution, thus extending the lifetime of the capillary, a highly desirable property for any coating used in biopolymer separations.

The synthesis and characterization of a novel polymer consisting of an alkyl backbone and pendant β-cyclodextrin units, obtained by radical copolymerization of vinylpyrrolidone and methacryloyl-β-cyclodextrin (PVP-β-CD), was reported. The ability of this copolymer to act as a capillary electrophoresis (CE) chiral selector was investigated in the separation of a mixture of basic drugs. The influence of polymeric cyclodextrin concentration, temperature, and pH on the separation of the test analytes was assessed and the advantage of using the polymeric selector over native β-cyclodextrin was demonstrated.

Polymeric coatings, usually referred as tridimensional chemistries, provide homogenous surface derivatization methods presenting a high reactive group concentration and resulting in an increased binding capacity of targets. Furthermore, they act as linkers distributing the bound probe also in the axial position, thus causing a faster reaction with the target involved in biomolecular recognition and can be engineered to custom tailor their properties for specific applications. Most approaches which aim at attaching polymers to a surface use a system where the polymer carries an "anchor" group either as an end group or in a side chain. This anchor group can reacts with appropriate sites at the substrate surface, thus yielding surface-attached monolayers of polymer molecules (termed "grafting to"). Another technique is to carry out a polymerization reaction in the presence of a substrate onto which monomers had been attached leading to the so called "grafting from" approach. In this chapter, protocols to functionalize glass and silicon surfaces by "grafting to" as well as by "grafting-from" approach are shown using copolymers made of N,N-dimethylacrylamide (DMA) or Glycidyl methacrylate (GMA) as the polymer backbone, N-acryloyloxysuccinimide (NAS) as reactive group, and 3-(trimethoxysilyl)propyl methacrylate (MAPS) or 3-mercaptopropyl trimethoxy silane (MPS) as anchoring groups.

We report the first demonstration of real-time biosensing in free standing macroporous alumina membranes. The membranes with their 200 nm diameter pores are ideal candidates for biosensing applications where fast response times for small sample volumes are needed as they allow analytes to flow through the pores close to the bioreceptors immobilized on the pores walls. A bulk refractive index sensitivity of 5.2x10 -6 refractive index units was obtained from signal responses to different concentrations of NaCl solutions flowing through the pores. Finally, after functionalizing the alumina pore surfaces with an epoxysilane and then spotting it with β-Lactoglobulin protein, the interactions between the β-lactoglobulin and rabbit anti-β-lactoglobulin, as well as the interaction between the rabbit anti-β-lactoglobulin and a secondary antibody anti-rabbit Immunoglobulin G were monitored in real-time.

The production of biosensors for point of care diagnostics usually requires the immobilisation and storage of protein (for example, antigen or antibody) on a sensor surface, in a manner that retains a high degree of activity and low levels of non-specific binding. These characteristics have been assessed for polymer immobilised antigens (allergens) using an IgG binding assay and demonstrated further by assay with serum containing reactive IgEs. The activity of allergens immobilised on sensor chips using copoly(DMA–NAS–MAPS) and a spotting technique, as well as the specificity of their binding interactions with cognate immunoglobulins was assessed using Dual Polarisation Interferometry (DPI). The data obtained indicate that the allergens studied remain stable over long periods of time (at least 114 days). This performance compared favourably with other immobilisation methods. Allergen coated chips were tested in an anti-casein IgE assay using human serum from allergic and non-allergic donors. Detection of both total Ig and specific IgE was demonstrated using a secondary anti-IgE antibody. Furthermore, optical signal enhancement with streptavidin conjugated quantum dots was shown to yield responses for samples below 0.84 ng/mL (0.35 KU/L) of IgE, which overlap with the industrial quasi-standard ImmunoCAP ® and is the clinically relevant threshold used to classify serum samples from allergic individuals.

In this work, we present a highly sensitive immunoassay for the detection of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aβ42) based on a label/label-free microarray platform that utilises silicon/silicon oxide (Si/SiO2) substrates. Due to constructive interference, Si/SiO2 layered slides allow enhancement of the fluorescence intensity on the surface with significant improvements in sensitivity of detection. The same substrate allows the label-free multiplexed detection of targets using the Interferometric Reflectance Imaging Sensor (IRIS), a platform amenable to high-throughput detection of mass changes on microarray substrates. Silicon chips are coated with copoly(DMA-NAS-MAPS), a ter-copolymer made from dimethylacrylamide (DMA), 3-(trimethoxysilyl)propyl methacrylate (MAPS) and N-Acryloyloxy succinimide (NAS). Aβ42 aggregation was studied by circular dichroism (CD), and an optimal antibody pair was selected based on specificity of recognition, binding yield and spot morphology of the capture antibody on the coated silicon surface as analysed by IRIS. Finally, incubation conditions were optimised, and an unprecedented Aβ42 detection sensitivity of 73pg/mL was achieved using an artificial cerebrospinal fluid (CSF) sample. Because of their multiplexing capability, low volume sample consumption and efficient sample-to-result time for population-wide screening, microarrays are ideal tools for the identification of individuals with preclinical AD who are still cognitively healthy. The high sensitivity of this assay format, potentially coupled to a pre-concentration step or signal-enhancing modifications, could lead to a non-invasive, inexpensive diagnostic tool for population-wide screening of AD biomarkers in biological fluids other than CSF, such as serum or plasma.

The accuracy of antibody-based microarrays depends on eliminating or at least minimizing the effect of cross-reactive components. In this work, a method to create a versatile, compartmentalized storage system for antibodies and soluble microarray reagents is described. These containers are made of hydrogel plugs immobilized on a slide facing the corresponding sub-array on a printed surface. The hydrogels were polymerized using a 4% w/v N,N -dimethylacrylamide and 3% w/v bisacrylamide solution, thus generating large pore sizes to allow the facile transfer of intact, functional biomolecules. The hydrogel microenvironments can be desiccated and rehydrated with a desired solution, and they can store reagents in a dry form. These hydrogels were shown to provide limits of detection similar to those obtained by conventional incubation conditions in an assay for HIV p24 antigen. Moreover, they prevent cross-reactivity issues in the detection of Staphylococcus aureus enterotoxins A and B and promote the accurate quantification of interleukin 10 by a microarray-integrated calibration curve.

A nanomechanical biosensor based on microcantilevers was implemented to test low molecular weight (small) compounds for their ability to stabilize beta(2)-microglobulin (beta(2)-m) in its native conformation. beta(2)-m was immobilized on the top face of silicon microcantilevers and it was demonstrated that pH induced unfolding of the immobilized beta(2)-m drives a specific microcantilever bending. This beta(2)-m microcantilever assay was then implemented to probe the effect of a pilot set small ligands on beta(2)-m conformational stability. Among the tested ligands, congo red was the only one able to protect beta(2)-m from unfolding, that is known to be the primary trigger of its self-polymerization into fibrils and in turn of the onset of amyloidosis. These findings disclose the high potentiality of nanomechanical sensors in the field of protein conformation related diseases, as they bring the unique advantage of directly screening compounds for their specific pharmacological activity rather than for generic binding preferences, effectively shortcutting the identification of the active ones. (C) 2012 Elsevier B. V. All rights reserved.

In this work, calculations and preliminary experimental data for determining the optimal condition for the selective bio-functionalization of magnetic tunneling junction (MTJ)-based biosensors are presented. Results on the detection of biomolecular recognition events employing MTJ-based sensor and magnetic beads are presented and interpreted through calculations, taking into account the dependence of the signal on the distribution of the beads with respect to the sensor. Furthermore, it is demonstrated by calculations that a significant increase in the sensor sensitivity and quantification capability can be achieved by selectively bio-functionalizing an area which corresponds to the sensor active area.

Protein and DNA microarrays hold the promise to revolutionize the field of molecular diagnostics. Traditional microarray applications employ labeled detection strategies based on the use of fluorescent and chemiluminescent secondary antibodies. However, the development of high throughput, sensitive, label-free detection techniques is attracting attention as they do not require labeled reactants and provide quantitative information on binding kinetics. In this article, we will provide an overview of the recent author's work in label and label-free sensing platforms employing silicon/silicon oxide (Si/SiO(2)) substrates for interferometric and/or fluorescence detection of microarrays. The review will focus on applications of Si/SiO(2) with controlled oxide layers to (i) enhance the fluorescence intensity by optical interferences, (ii) quantify with sub-nanometer accuracy the axial locations of fluorophore-labeled probes tethered to the surface, and (iii) detect protein-protein interactions label free. Different methods of biofunctionalization of the sensing surface will be discussed. In particular, organosilanization reactions for monodimensional coatings and polymeric coatings will be extensively reviewed. Finally, the importance of calibration of protein microarrays through the dual use of labeled and label-free detection schemes on the same chip will be illustrated.