Marcella Chiari

New adsorbed coatings for capillary electrophoresis.

New acrylic polymers bearing oxirane groups were synthesized to be used in the production of coated capillaries. A fully automated coating procedure was devised based on the use of diluted water solutions of these polymers. The whole procedure required less than 30 min. The new polymers rapidly adsorbed from water onto the capillary wall, thus suppressing electroosmotic flow (EOF) to a negligible value. The adsorbed coatings were stable for hundreds of hours at high pH, temperature, and in the presence of 8 M urea. Efficient separations of acidic and basic proteins were achieved in the new phases.

Capillary electrophoretic separation of proteins using stable, hydrophilic poly(acryloylaminoethoxyethanol)-coated columns

Abstract Capillary electrophoretic separations of high efficiency and resolution were obtained using poly(acryloylaminoethoxyethanol)-coated capillaries. The polymer was covalently attached to a silica surface previously modified with a α-methacryloxlypropyl functionality. The latter was realized by catalytic hydrosilylation of allyl methacrylate on an SiH-modified fused-silica capillary. The lifetime of the new type of coating used at pH 8.5 was more than twice that of conventional polyacrylamide.

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.

Movement of DNA fragments during capillary zone electrophoresis in liquid polyacrylamide

Abstract Migration of DNA fragments in the 51-23 130 base pair (bp) size range was investigated by capillary zone electrophoresis in solutions of linear polyacrylamide of concentration from 4 to 10%. Plots of log (mobility) vs . log [size (in bp)] clearly indicate three different migration regimes: according to Ogston ( i.e ., as spherical globules) up to 200 bp, reptation-without-stretching up to 3000–4000 bp and reptation with partial stretching for larger fragments. Guidelines on what percentage of the polymer should be used for optimum resolution can be obtained from plots of peak spacing (in seconds per base pair) versus percentage of polymer in solution and from standard plots of peak resolution versus percentage of polymer. An optimum linear polyacrylamide concentration, allowing for good resolution of most fragments, from small to large, is found at a level of 6% polymer. It is hypothesized that in situ polymerization allows for the formation of a large distribution of polymer sizes (centred at M r ≈ 100 000), thus facilitating simultaneous separation of short and long DNA fragments based on the principle that shorter polyacrylamide chains will sieve shorter DNA fragments and vice versa .

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.

Capillary zone electrophoresis in organic solvents: separation of anions in methanolic buffer solutions

Abstract A tris(hydroxymethyl)aminomethane-acetate buffer system with methanol as solvent has been used at an apparent pH of 8.5 for the separation of six aromatic and aliphatic acids. Compared to pure aqueous buffer systems with various pH values (4.4, 5.5, 6.2 and 8.0) improved separation was obtained due to increased selectivity. This improvement is related to a specific shift in the p K a values of the solutes in the organic solvent, and is interpreted by the concept of the transfer activity coefficient and the medium effect.

Determination of total vitamin C in fruits by capillary zone electrophoresis

A simple capillary zone electrophoretic (CZE) method is described for the rapid determination of ascorbic acid and dehydroascorbic acid, the physiologically active forms of vitamin C, in fruits. The electrophoretic run was accomplished in 9 min on a coated capillary column using 20 mM phosphate buffer (pH 7.0). Total ascorbic acid was determined by first reducing the dehydroascorbic acid to ascorbic acid by treatment with DL-homocysteine. This reaction was complete in 15 min and total ascorbic acid determination was performed immediately. The data obtained by CZE were in good agreement with HPLC data.

A fast and simple label-free immunoassay based on a smartphone.

Despite the continuous advancements in bio-molecular detection and fluidic systems integration, the realization of portable and high performance devices for diagnostic applications still presents major difficulties, mostly because of the need to combine adequate sensitivity with low cost of production and operational simplicity and speed. In this context, we propose a compact device composed of a smartphone and a custom-designed cradle, containing only a disposable sensing cartridge, a tiny magnetic stirrer and a few passive optical components. The detection principle is the previously proposed Reflective Phantom Interface that is based on measuring the intensity of light reflected by the surface of an amorphous fluoropolymer substrate, which has a refractive index very close to that of the aqueous sample solution and hosts various antibodies immobilized within spots. The reflectivity of dozens of spots is monitored in real time by the phone׳s camera using the embedded flash LED as the illumination source. We test the performance of the combined device targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV. Target concentrations as low as a few ng/ml can be rapidly and robustly determined by comparing the rate of increase of the signal after the addition of the sample with that measured after the subsequent addition of a standard solution with known concentration. The features of the proposed system enable the realization of novel handheld biosensing devices suitable for those applications where multiple targets have to be rapidly detected even without the presence of trained personnel.

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.

A new absorbed coating for DNA fragment analysis by capillary electrophoresis.

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.