Imaging and spectroscopic comparison of multi-step methods to form DNA arrays based on the biotin–streptavidin system

Abstract

Three multi-step multi-molecular approaches using the biotin-streptavidin system to contact-print DNA arrays on SiO2 surfaces modified with (3-glycidoxypropyl)trimethoxysilane are examined after each deposition/reaction step by atomic force microscopy, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry. Surface modification involves the spotting of preformed conjugates of biotinylated oligonucleotides with streptavidin onto surfaces coated with biotinylated bovine serum albumin b-BSA (approach I) or the spotting of biotinylated oligonucleotides onto a streptavidin coating, the latter prepared through a reaction with immobilized b-BSA (approach II) or direct adsorption (approach III). AFM micrographs, quantified by autocorrelation and height histogram parameters (e.g. roughness), reveal uniform coverage after each modification step with distinct nanostructures after the reaction of biotinylated BSA with streptavidin or of a streptavidin conjugate with biotinylated oligonucleotides. XPS relates the immobilization of biomolecules with covalent binding to the epoxy-silanized surface. Protein coverage, estimated from photoelectron attenuation, shows that regarding streptavidin the highest and the lowest immobilization efficiency is achieved by following approaches I and III, respectively, as confirmed by TOF-SIMS microanalysis. The size of the DNA spot reflects the contact radius of the printed droplet and increases with protein coverage (and roughness) prior to the spotting, as epoxy-silanized surfaces are hardly hydrophilic. Representative TOF-SIMS images show sub-millimeter spots: uniform for approach I, doughnut-like (with a small non-zero minimum) for approach II, both with coffee-rings or peak-shaped for approach III. Spot features, originating from pinned contact lines and DNA surface binding and revealed by complementary molecular distributions (all material, DNA, streptavidin, BSA, epoxy, SiO2), indicate two modes of droplet evaporation depending on the details of each applied approach.

Fluorescence Imaging Study of Film Coating Structure and Composition Effects on DNA Hybridization

“…[ 14,15 ] Among them, the patterning into microarrays has become one of the most popular and efficient fabrication approaches for sensing platforms since it evolved in the 1990s, [ 16 ] thus broadly used in DNA hybridization, [ 17 ] protein detection, [ 18 ] and other biosensing systems. [ 19–22 ] Normally, spot uniformity, density of immobilized DNA probes, and repeatability are known as primary factors for detecting probe microarray sensitivity and reliability, and these factors are strongly influenced by the physical and chemical properties of the sensor platform interface. [ 23–25 ] Furthermore, steric issues between the DNA probe and the interface, as well as steric hindrance between adjacent probes and electrostatic forces, all affect the hybridization efficiency and capacity in surface hybridization.…”

Silica microbeads capture fetal nucleated red blood cells for noninvasive prenatal testing of fetal ABO genotype

“…As Fig. shows, silica microbeads (SiO 2 MBs) of 30 µm in diameter and 2 g/mL in density were first wholly modified with streptavidin and then conjugated with biotinylated anti‐CD147 antibody to specifically capture fnRBCs from maternal blood. After cell capture, Percoll separation solution (1.13 g/mL) was utilized as a density‐selective sedimentation to purify the cell‐attached MBs (>1.13 g/mL) from white blood cells (WBCs, 1.07–1.09 g/mL).…”

Orientation of Biotin-Binding Sites in Streptavidin Adsorbed onto the Surface of Polythiophene Films

“…The streptavidin–biotin binding system has unique properties, enabling formation of bridges between the surface and biotin-labeled biomolecules, − rendering them more accessible for reaction with their counterpart molecules in the reaction mixture. Thus, it has been employed to immobilize biotinylated oligonucleotide probes onto surfaces, leading not only to immobilization of a high oligonucleotide amount but also to excellent spot repeatability of size and shape. , Biotin–streptavidin linkage has been also used to immobilize antibodies in a controlled manner, enhancing the analytical sensitivity of the immunoassay developed using these antibodies as compared to that developed employing randomly biotinylated ones . Additionally, Magliulo et al presented a biosensor based on OFET enabling detection of biotin at the low part per trillion concentration level .…”

“…Thus, it has been employed to immobilize biotinylated oligonucleotide probes onto surfaces, 25 leading not only to immobilization of a high oligonucleotide amount but also to excellent spot repeatability of size and shape. 23,24 Biotin−streptavidin linkage has been also used to immobilize antibodies in a controlled manner, 26 enhancing the analytical sensitivity of the immunoassay developed using these antibodies as compared to that developed employing randomly biotinylated ones. 27 Additionally, Magliulo et al presented a biosensor based on OFET enabling detection of biotin at the low part per trillion concentration level.…”