Immobilization of biotinylated antibodies through streptavidin binding aptamer.

Liquid biopsy approaches are powerful strategies that potentially allow the diagnosis and prognosis of a number of diseases. The field is continuously and rapidly growing, encouraging the discovery of novel predictory biomarkers. Antibodies are usually exploited in sensors to validate biomarker candidates. Unfortunately, the immobilization of antibodies on the surface of sensors represents a challenging task. Immobilization strategies need to be optimized for each antibody, representing a huge obstacle to overcome in the discovery of new biomarkers. Herein we propose a novel strategy for the immobilization of antibodies, based on the use of a streptavidin-binding aptamer. Using this approach it is possible to immobilize antibodies on the surface of sensors with no need for optimization, with the only requirement for antibody to be biotinylated. The proposed strategy potentially paves the way towards a straightforward immobilization of antibodies on biosensors, making their use in biomarker validation more accessible.

Label-free detection of DNA single-base mismatches using a simple reflectance-based optical technique.

Rapid and quantitative detection of the binding of nucleic acids to surface-immobilized probes remains a challenge in many biomedical applications. We investigated the hybridization of a set of fully complementary and defected 12-base long DNA oligomers by using the Reflective Phantom Interface (RPI), a recently developed multiplexed label-free detection technique. Based on the simple measurement of reflected light intensity, this technology enables to quantify the hybridization directly as it occurs on the surface with a sensitivity of 10 pg mm(-2). We found a strong effect of single-base mismatches and of their location on hybridization kinetics and equilibrium binding. In line with previous studies, we found that DNA-DNA binding is weaker on a surface than in the bulk. Our data indicate that this effect is a consequence of weak nonspecific binding of the probes to the surface.

Conditions for efficient on-chip magnetic bead detection via magnetoresistive sensors.

A commonly used figure of merit of magnetoresistive sensors employed to detect magnetic beads labeling biomolecules in lab-on-chip applications is the sensor sensitivity (S0) to external magnetic fields in the linear region of the sensor. In this paper we show that, in case of lock-in detection and bead excitation by a small AC magnetic field, S0 is not the good figure of merit to optimize. Indeed, the highest sensitivity to the magnetic beads is achieved biasing the sensor in the region of its characteristics where the product between the DC bias field and the second derivative of the resistance with respect to the magnetic field is maximum. The validity of this criterion, derived from a phenomenological model of bead detection, is proved in case of magnetic tunneling junction sensors detecting magnetic beads with 250nm diameter. This work paves the way to the development of a new generation of sensors properly designed to maximize the bead sensitivity.

Use of a fluorosurfactant in micellar electrokinetic capillary chromatography.

A fluorosurfactant, the anionic N-ethyl-N-[(heptadecafluorooctyl)sulfonyl]glycine potassium salt, trade name FC-129 [CAS 2991-51-7] was investigated for possible application in micellar electrokinetic capillary chromatography (MEKC). The surfactant was characterized with conductometric titration and test sample mixtures were investigated in MEKC systems, and compared with sodium dodecylsulphate. An increased efficiency and interesting selectivity differences were observed.

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.

Separation of oligonucleotides and DNA fragments by capillary electrophoresis in dynamically and permanently coated capillaries, using a copolymer of acrylamide and beta-D-glucopyranoside as a new low viscosity matrix with high sieving capacity.

New copolymers of acrylamide and beta-D-glucopyranoside were synthesized and characterized. The different reactivity of the two monomers towards radical polymerization meant we could control the growth of the polymer chains whose length was inversely related to the number of glucose residues incorporated in the copolymers. The properties of these polymers were investigated in the separation of oligonucleotides and double-stranded DNA by capillary electrophoresis (CE) in coated and uncoated capillaries. The new copolymers were a suitable matrix for CE due to their high-resolving capacity and low viscosity. We also looked into the advantages of a new method of dynamic suppression of electroosmotic flow based on the addition of small amounts (0.03-0.05%) of dimethylacrylamide to the sieving and to the running buffer. A complete test was run on the reproducibility and efficiency of separations carried out in a permanently and dynamically coated capillary, and the advantages and disadvantages of the two methods were compared.