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Articles by Marcella Chiari in JoVE
JoVE Bioengineering
Biyomoleküler Algılama istihdam İnterferometrik Yansıtma Görüntüleme Sensörü (IRIS)
1Department of Electrical and Computer Engineering, Boston University, 2Department of Biomedical Engineering, Boston University, 3Center for Advanced Genomics Technology, Boston University, 4Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 5Department of Microbiology, Boston University School of Medicine, 6CNR (National Research Council), Istituto di Chimica del Riconoscimento Molecolare
SiO Kantitatif, yüksek verimli, gerçek zamanlı ve etiket ücretsiz biyomoleküler algılama (DNA, protein, vb.)
Other articles by Marcella Chiari on PubMed
Separation of DNA Fragments in Hydroxylated Poly(dimethylacrylamide) Copolymers
The novel polymer matrices reported here are low-viscosity sieving media for DNA capillary electrophoresis. This new family of matrices comprises copolymers of N,N-dimethylacrylamide with different monomers which increase polymer hydrophilicity. All these new copolymers self-coat on fused-silica capillaries. Resolution, peak spacing and peak width were the parameters taken into account to assess the influence of polymer structure on separation selectivity and efficiency. This work demonstrates that the performance of polydimethylacrylamide (PDMA) can be improved through copolymerization with hydrophilic monomers. The improvement is related to the efficiency parameter. The new copolymers, due to their low viscosity high sieving capacity and ability to suppress EOF, represent a better alternative to PDMA and are suitable replaceable matrices for capillary and microchip electrophoresis.
Use of Peptide Nucleic Acid Probes for Detecting DNA Single-base Mutations by Capillary Electrophoresis
Peptide nucleic acid (PNA) oligomers can be used as probes in pre-gel hybridization experiments, as an alternative to Southern hybridization. In this technique, the PNA probe is hybridized to a cyanine-5 labeled DNA sample denatured at low ionic strength, and the mixture is directly injected for size separation into a capillary electrophoresis (CE) system equipped with laser-induced fluorescence (LIF) detector. The neutral backbone of PNA allows hybridization to occur at low ionic strength and assures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. We have used as a model system the cystic fibrosis R553X and R1162X single-base mutations and we have assessed the influence of various factors, such as temperature and denaturants concentration on DNA/PNA hybrid stability in order to achieve the high specificity required for a single base pair discrimination.
Multimodal Open-tubular Capillary Electrochromatographic Analysis of Amines and Peptides
This study describes a comparison of different modes of open-tubular electrochromatography (OTCEC) in bare and etched capillaries. To carry out the investigation, the separation of impurities of two synthetic peptides and the separation of a mixture of five heterocyclic aromatic amines were studied. Three different types of stationary phase were evaluated: (i) fluorosurfactants (anionic and zwitterionic) adsorbed in the inner wall of the capillary (electrochromatography with dynamically modified stationary phases (DMS)CEC); (ii) physically adsorbed polymers (DMA-SO(3-) and DMA-N(+)(CH(3))(3)) and (iii) chemically modified capillaries (C(18), cholesteryl 10-undecanoate and diol). The results confirm that electrochromatography can be a viable alternative to capillary electrophoresis (CE) and liquid chromatography, more established separation techniques. It is possible to differentiate some minor species for the synthetic peptides that cannot be resolved by CE or high-performance liquid chromatography (HPLC). Moreover the separation of the amine mixture depends strongly on the stationary phase used.
Decreased Protein Peak Asymmetry and Width Due to Static Capillary Coating with Hydrophilic Derivatives of Polydimethylacrylamide
We have recently described [1] a fast and simple method for the "adsorbed static" coating of capillaries in capillary zone electrophoresis (CZE) with epoxy-poly(dimethylacrylamide) (EPDMA). Protein CZE peaks in the EPDMA-coated capillaries exhibited a peak asymmetry similar to that obtained in capillaries with "covalent static" coating of polyacrylamide, suggesting a similar degree of adsorption of the protein onto the coating [2]. Instability of such coating at very low ionic strength and its stripping from the capillary in the presence of sodium dodecyl sulfate (SDS) also indicated a hydrophilic bonding of EPDMA to the silanol surface of the capillary, while its stripping in the CZE of "carboxylate-modified" polystyrene suggested a competition between carboxylate and EPDMA for the hydrophilic bonds to silanol. To test those propositions, a number of EPDMA-derived coating agents with increased hydrophilicity were synthesized. Of a number of the hydrophilic coating agents tested (Table 1) only two, 2% hydrolyzed EPDMA (HPDMA) hydrolyzed in sulfuric acid to effect the conversion of the epoxy groups to diols (Table 1, No. 38), and 20% EPDMA (Table 1, No. 44) exhibited for representative proteins a decreased peak asymmetry and width while the stability of the suppression of electroosmotic flow (EOF), and the stability of mobility in consecutive CZE runs was reduced relative to EPDMA. Coating agents which were more highly hydrophilic than those two (Table 1, No. 49) or less hydrophilic than 2% EPDMA (Table 1, Nos. 57, 53, 46) provided no stable static coating.
Separation of Proteins in a Multicompartment Electrolyzer with Chambers Defined by a Bed of Gel Beads
Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.
Nonconventional Synthesis and Characterization of Ultrahigh-molar-mass Polyacrylamides
In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.
Use of High-molecular-mass Polyacrylamides As Matrices for Microchip Electrophoresis of DNA Fragments
DNA fragment analysis requires the use of polymer solutions as sieving matrices. Generally, such matrices are constituted of high-molar-weight polymers employed at a concentration higher than their entanglement threshold concentration. These polymer solutions are highly viscous and difficult to use in the narrow channels of a microchip. Ultralarge polyacrylamides synthesized via a nonconventional method, being the low-temperature plasma-induced polymerization (PIP), were used as DNA sieving matrices for microchip electrophoresis. The distinctive features of these polymers (ultralarge molecular mass and linearity) allow their use at a dilute concentration. Dilute PIP polyacrylamides revealed a constant value of resolution in a broad range of DNA fragment sizes (123 bp-1353 bp), thus proving to be effective in common genotyping applications. Moreover, the low viscosity of the dilute solutions enable it to be easier and faster in filling the channel between runs, thus enhancing the throughput of the microchip devices.
Characterization of a Polymeric Adsorbed Coating for DNA Microarray Glass Slides
A new method was developed to covalently attach target molecules onto the surface of glass substrates such as microwell plates, beads, tubes, and microscope slides, for hybridization assays with fluorescent targets. The innovative concept introduced by this work is to physically adsorb onto underivatized glass surfaces a functional copolymer, able to graft amino-modified DNA molecules. The polymer, obtained by radical copolymerization of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate, copoly(DMA-NAS-MAPS), self-adsorbs onto the glass surface very quickly, typically in 5-30 min. The film, formed on the surface, bears active esters, which react with amino-modified DNA targets. The surface layer is stable in an aqueous buffer containing various additives (SDS, urea, salt), even at boiling temperature. It should be emphasized that the coating is formed by the immersion of glass slides in a diluted aqueous solution of the polymer. Therefore, the procedure is fast, inexpensive, robust, and reliable, and it does not require time-consuming glass pretreatments. Slides, coated with copoly(DMA-NAS-MAPS), were profitably used as substrates for the preparation of low-density DNA microarrays. The density and the thickness of the films were evaluated by X-ray reflectivity measurements whereas the extent of reaction of functional groups with DNA molecules was determined by a functional test. The experiments indicate that half of the active groups present on the surface reacts with oligonucleotide probes.
A New Polymeric Coating for Protein Microarrays
Despite the increasing interest in arraying proteins in a high-density format, several technical issues still impede the development of protein microarray technology. One of the major problems is the availability of substrates that are able to bind native proteins with high density. In this study, we investigated the suitability of a novel surface as a support for protein microarrays. A polymeric glass coating is obtained by physical adsorption of a N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS), and [3-(methacryloyl-oxy)propyl]trimethoxysilyl (MAPS) copolymer. The coating procedure provides a fast and inexpensive method of producing hydrophilic functional surfaces. The slide performance was investigated in a protein-protein interaction experiment and in the assessment of rheumatoid factor (RF) in human serum samples. The results demonstrate that the ligands immobilized on the polymeric surface maintain an active conformation and are easily accessible, providing a detection limit of 54amol/spot. Moreover, in the RF assay, after hybridization with the sera, the slides have a low background, leading to a detection limit of 900amol/spot.
Electroosmotic Flow Suppression in Capillary Electrophoresis: Chemisorption of Trimethoxy Silane-modified Polydimethylacrylamide
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.
Acrylamide-agarose Copolymers: Improved Resolution of High Molecular Mass Proteins in Two-dimensional Gel Electrophoresis
A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.
Peptide Microarrays for the Characterization of Antigenic Regions of Human Chromogranin A
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.
Detection of the R553X DNA Single Point Mutation Related to Cystic Fibrosis by a "chiral Box" D-lysine-peptide Nucleic Acid Probe by Capillary Electrophoresis
In order to recognize the presence of the R553X point mutation of the cystic fibrosis (CF) gene in the human genome, a peptide nucleic acid (PNA) complementary to the mutated gene tract and bearing three adjacent chiral monomers based on D-lysine (chiral box) was synthesized and used as a probe in CE. Binding specificity was preliminarily studied with complementary and mismatched oligonucleotides by UV spectroscopy, electrospray MS, and electrophoresis, indicating a very high sequence selectivity. The chiral PNA probe was then hybridized to cyanine-5-labeled DNA samples (186 bp), obtained by PCR amplification, respectively, from: (a) normal homozygous subjects (wtDNA), (b) CF-affected homozygous subjects (mutDNA), (c) heterozygous subjects (healthy carriers) and denatured at low ionic strength. The PNA-DNA mixture was directly analyzed by CE with LIF detection: a new signal corresponding to the PNA-mutDNA duplex was observed, in the case of CF-affected homozygous subjects, whereas for the sample containing the mismatched sequence (normal homozygous wtDNA) only the signal corresponding to ssDNA (ss, single strand) was detected. In the case of heterozygous DNA, both PNA-mutDNA duplex and ssDNA were detected. With this simple assay, it was possible to discriminate in an easy way among the three cases (mutated homozygous, normal homozygous, and heterozygous subjects) with a total specificity, thus allowing a decisive advance for the diagnosis of CF.
Protein and Peptide Arrays: Recent Trends and New Directions
Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.
Microarray Glass Slides Coated with Block Copolymer Brushes Obtained by Reversible Addition Chain-transfer Polymerization
The reversible addition-fragmentation chain-transfer polymerization was used to prepare microarray slides grafted with polymer brushes for DNA-based applications. Block copolymer brushes of N,N-dimethylacrylamide (DMA) and glycidyl methacrylate (GMA), poly(DMA-b-GMA) were prepared by extending living poly(dimethylacrylamide) chains. The functional surface was used as a substrate for oligonucleotide hybridization experiments. The results were compared to those provided by glass slides coated by a self-assembled monolayer made of (3-glycidyloxypropyl)trimethoxysilane. Surfaces coated with block polymer brushes bearing oxirane groups are more efficient as substrates for oligonucleotide hybridization than surfaces coated with nonpolymeric self-assembled monolayers containing the same functional group. The high probe grafting density and hybridization efficiency achieved with this polymeric coating reveal the importance of the block architecture to ensure good accessibility of the immobilized probe. The new surface was characterized by static angle measurements and diffuse reflectance FT-IR spectroscopy on a silica model system.
Surface Behavior and Molecular Recognition in DNA Microarrays from N,N-dimethylacrylamide Terpolymers with Activated Esters As Linking Groups
A series of terpolymers made of DMA, NAS and MAPS were synthesized by free radical copolymerization and used as functional coatings for the fabrication of glass slide DNA microarrays. The surface properties of coated glass slides were investigated through contact angle measurements, ellipsometry and atomic force microscopy. The terpolymer molecular weight showed a moderate effect on surface tension (gamma(s) = 56-62 mN x m(-1)), but no clear effect on polymeric layer thickness (5-8 nm) and roughness. Hybridization experiments with amine-functionalized oligonucleotides gave the best fluorescence intensity results for microarrays coated with intermediate-molecular-weight terpolymers. Finally, an accelerated ageing test of the microarray in a humidity chamber showed a nice relationship between decay curves of contact angle against water and fluorescence intensity.
Microchips and Single-photon Avalanche Diodes for DNA Separation with High Sensitivity
Modern techniques for DNA and protein analysis and separation rely on measurements of LIF and face a trend toward employing progressively smaller samples. The currently employed detectors that provide the required ultrahigh sensitivity, e.g. photomultiplier tubes (PMTs), are bulky and/or costly and delicate, whereas a key issue for the development of compact and economical instruments is the availability of miniaturized, inexpensive, and ultrasensitive photodetectors. The planar epitaxial silicon single-photon avalanche diodes (SPADs) combine the typical advantages of microelectronics (miniaturization, ruggedness, low voltage, low power, low cost, etc.) with high sensitivity, even better than that of PMTs. The suitability of such SPADs to microchip CE has been here ascertained by developing a new apparatus with dual-wavelength LIF detection. The apparatus has been experimented in studies on the EOF suppression and on the coating stability and tested in rapid sizing of DNA fragments. The experimental results obtained in the separation of Cy5-labeled oligonucleotide demonstrate sensitivity better than 3 pM, which corresponds to less than 100 fluorescent molecules in the 50 pL illuminated volume.
Different Approaches for Noninvasive Prenatal Diagnosis of Genetic Diseases Based on PNA-mediated Enriched PCR
The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.
High-throughput Mutational Screening for Beta-thalassemia by Single-nucleotide Extension
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.
Advances in Parallel Screening of Drug Candidates
In the hit to lead process, a drug candidate is selected from a set of potential leads by screening its binding with potential targets. This review focuses on the lead identification assays that employ a bio-chemical or bio-physical test to detect molecular recognition events between proteins and small molecules in a parallel format. These tests require either the lead or the target immobilization followed by incubation with the set of potential interaction partners and detection of a signal related to the target-ligand binding. In the first part of the review the different detection strategies amenable for drug screening are discussed. In the second part, a review of immobilization approaches for leads or targets, allowing the parallel screening of arrays of molecules, is presented.
Genotyping Beta-globin Gene Mutations on Copolymer-coated Glass Slides with the Ligation Detection Reaction
Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity.
Dual-color Microchip Electrophoresis with Single-photon Avalanche Diodes: Application to Mutation Detection
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.
A Biofunctional Polymeric Coating for Microcantilever Molecular Recognition
An innovative route to activate silicon microcantilevers (MCs) for label free molecular recognition is presented. The method consists in coating the underivatized MCs with a functional ter-polymer based on N,N-dimethylacrylamide (DMA) bearing N-acryloyloxysuccinimide (NAS) and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), two functional monomers that confer to the polymer the ability to react with nucleophilic species on biomolecules and with glass silanols, respectively. The polymer was deposited onto MCs by dip coating. Polymer coated MCs were tested in both static and dynamic modes of actuation, featuring detection of DNA hybridization as well as protein/protein interaction. In the dynamic experiments, focused on protein detection, the MCs showed an average mass responsivity of 0.4 Hz/pg for the first resonant mode and of 2.5 Hz/pg for the second resonant mode. The results of the static experiments, dedicated to DNA hybridization detection, allowed for direct estimation of the DNA duplex formation energetics, which resulted fully consistent with the nominal expected values. These results, together with easiness and cheapness, high versatility, and excellent stability of the recognition signal, make the presented route a reliable alternative to standard SAM functionalization (for microcantilevers (MCs) and for micro-electro-mechanical systems (MEMS) in general).
Development of New Substrates for High-sensitive Genotyping of Minority Mutated Alleles
An unsurpassed level of sensitivity was reached in the detection of minority mutated alleles. A low-density microarray was printed on a substrate specifically designed to provide an interference effect which amplifies the collection of the light emitted on the support and reinforces the intensity of excitation light. Optimal performance of the array was obtained by maximizing the probe density and the binding efficiency to the target through a polymeric coating made by the adsorption of a copolymer of N,N-dimethylacrylamide (97% of moles), N,N-acryloyloxysuccinimide (2%) and 3-(trimethoxysilyl)propyl methacrylate (1%) synthesized by free radical copolymerization. The new substrate was used in the identification of fetal mutations in the maternal plasma DNA. Amino-modified amplicons from genomic DNA corresponding to the locus of eight beta-thalassemia mutations were immobilized and interrogated with dual-color oligonucleotide targets. Compared with the conventional glass substrates, the new substrate showed a great enhancement of fluorescence signals thanks to the combination of the optics with the highly efficient polymeric coating, allowing specific detection of all mutations. The high sensitivity and selectivity obtained made it possible to develop assays for the identification of paternally inherited mutations on fetal DNA in the maternal plasma in couples at risk for beta-thalassemia.
Direct Observation of Conformation of a Polymeric Coating with Implications in Microarray Applications
The conformation of a three-dimensional polymeric coating (copoly(DMA-NAS-MAPS)) and immobilization and hybridization of DNA strands on the polymer coated surface are investigated. A conformational change, specifically the swelling of the surface adsorbed polymer upon hydration, is quantified in conjunction with the application of this polymer coating for DNA microarray applications. Fluorescently labeled short DNA strands (23mers) covalently linked to the functional groups on the adsorbed polymer are used as probes to measure the swelling of the polymer. A fluorescence microscopy technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), is utilized to directly measure the change in axial position of fluorophores due to swelling with subnanometer accuracy. Additionally, immobilization characteristics of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) probes, as well as hybridization of ssDNA with target strands have been studied. The results show that ssDNA further away from the surface is hybridized more efficiently, which strengthens the earlier analysis of this polymeric coating as a simple but highly efficient and robust DNA microarray surface chemistry.
Detection of Allergen Specific Immunoglobulins by Microarrays Coupled to Microfluidics
Allergen microarrays are under development for a component-resolved diagnosis of Type I (IgE-mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA-co-NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software-controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion-limited to a kinetic-limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.
High Sensitivity Protein Assays on Microarray Silicon Slides
In this work, we report on the improvement of microarray sensitivity provided by a crystalline silicon substrate coated with thermal silicon oxide functionalized by a polymeric coating. The improvement is intended for experimental procedures and instrumentations typically involved in microarray technology, such as fluorescence labeling and a confocal laser scanning apparatus. The optimized layer of thermally grown silicon oxide (SiO(2)) of a highly reproducible thickness, low roughness, and fluorescence background provides fluorescence intensification due to the constructive interference between the incident and reflected waves of the fluorescence radiation. The oxide surface is coated by 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. The polymeric coating with a thickness that does not appreciably alter the optical properties of the silicon oxide confers to the slides optimal binding specificity leading to a high signal-to-noise ratio. The present work aims to demonstrate the great potential that exists by combining an optimized reflective substrate with a high performance surface chemistry. Moreover, the techniques chosen for both the substrate and surface chemistry are simple, inexpensive, and amenable to mass production. The present application highlights their potential use for diagnostic applications of real clinical relevance. The coated silicon slides, tested in protein and peptide microarrays for detection of specific antibodies, lead to a 5-10-fold enhancement of the fluorescence signals in comparison to glass slides.
High-sensitive Microarray Substrates Specifically Designed to Improve Sensitivity for the Identification of Fetal Paternally Inherited Sequences in Maternal Plasma
The identification of very low-levels of minority sequences has interesting clinical and diagnostic applications. Among these, non-invasive prenatal diagnosis of genetic diseases on fetal DNA circulating in maternal plasma is an emerging field of application.
Quantification of DNA and Protein Adsorption by Optical Phase Shift
A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA. Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to approximately 1 ng/mm(2) surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.
Epitope Mapping of Human Chromogranin A by Peptide Microarrays
In this chapter we report on the characterization of linear antigenic sites of human chromogranin A (CgA), a useful tissue and serum marker for neuroendocrine tumours and a precursor of many biologically active peptides. The epitope mapping of CgA has been carried out by peptide microarrays on glass slides coated by a copolymer of N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS) and [3-(methacryloyl-oxy) propyl] trimethoxysilyl (MAPS). 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, despite peptide random orientation, linear epitopes are freely exposed. The results reported are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.
Optical Sensing in Microfluidic Lab-on-a-chip by Femtosecond-laser-written Waveguides
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.
Coating of Nitrocellulose for Colorimetric DNA Microarrays
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.
Label-free Microarray Imaging for Direct Detection of DNA Hybridization and Single-nucleotide Mismatches
A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by high-throughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single-nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.
High-resolution Electrophoretic Separation and Integrated-waveguide Excitation of Fluorescent DNA Molecules in a Lab on a Chip
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.
Allergen Microarrays on High-sensitivity Silicon Slides
We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.
Peptide Microarrays on Coated Silicon Slides for Highly Sensitive Antibody Detection
Peptides, with their well-established chemistry and fully automated synthesis, provide an invaluable tool for the screening of protein ligands, for epitope mapping, and for antibody diagnostics on the microarray format.The method described in this chapter shows that the sensitivity of a peptide-based microimmunoassay is greatly improved by using a new, specifically developed substrate made of silicon coated by an optimized layer of silicon oxide. A set of six peptides corresponding to the sequences of human and rat acetylcholine receptor subunits was immobilized on glass and silicon slides coated by a copolymer of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl) propyl methacrylate, copoly(DMA-NAS-MAPS). The spotted probes were incubated with rabbit anti-sera and with purified antibodies raised against the corresponding peptides. The coated silicon slides, in comparison against the glass substrates, showed a five- to tenfold enhancement of the fluorescence signals, leading to the specific detection of the full set of antibodies down to a concentration of 0.5-1 ng/mL in serum. The sensitivity provided by the test allows its use for the diagnosis of antibodies in clinical samples.
Silicon Biochips for Dual Label-free and Fluorescence Detection: Application to Protein Microarray Development
A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500nm SiO(2) layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100nm SiO(2) providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fluorescence detection of the molecular interaction counterparts. Such a combined chip is of high practical utility during assay development process to image arrays, check consistency and quality of the protein array, quantify the amount of immobilized probes and finally detect fluorescence of bioassays.
Overcoming Mass Transport Limitations to Achieve Femtomolar Detection Limits on Silicon Protein Microarrays
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.
Multiplexed Method to Calibrate and Quantitate Fluorescence Signal for Allergen-specific IgE
Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody-antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.
A Nanoelectromechanical Biosensor Based on Precise Quantification and Control of DNA Orientation
We utilize spectral self-interference fluorescent microscopy (SSFM) to measure fluorophore height with sub-nm precision to precisely quantify DNA orientation. A novel polymeric 3D scaffold is used to functionalize the sensor surface and to control orientation of the surface anchored DNA.
