ABSTRACT
Spatially controlled anchoring of NA probes onto microscope glass slides by a novel fluor-thiol coupling reaction is performed. By this UV-initiated reaction, covalent immobilization in very short times (30 s at 254 nm) is achieved with probe densities of up to 39.6 pmol/cm2. Modulating the surface hydrophobicity by combining a hydrophobic silane and a hydrophilic silane allows the fabrication of tuned surfaces where the analyte approaches only the anchored probe, which notably reduces nonspecific adsorption and the background. The generated substrates have proven clear advantages for discriminating single-base-pair mismatches, and for detecting bacterial PCR products. The hybridization sensitivity achieved by these high-performance surfaces is about 1.7 pM. Finally, this anchoring reaction is demonstrated using two additional surfaces: polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes. This provides a very interesting pathway for anchoring thiolated biomolecules onto surfaces with C-F motifs via a quick clean UV reaction.
Subject(s)
Oligonucleotide Array Sequence Analysis/methods , Photochemical Processes , Sulfhydryl Compounds/chemistry , Hydrophobic and Hydrophilic Interactions , Nucleic Acid Hybridization , Optical Imaging , Polytetrafluoroethylene/chemistry , Polyvinyls/chemistry , Silanes/chemistryABSTRACT
A UV light-induced thiol-ene coupling reaction (TEC) between half-antibodies (hIgG) and vinyl functionalized glass surfaces was run for biosensing in the microarray format. The accomplished performance improved that obtained with whole antibodies.
Subject(s)
Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Sulfhydryl Compounds/chemistry , Vinyl Compounds/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Biosensing Techniques/methods , C-Reactive Protein/analysis , C-Reactive Protein/immunology , Carbocyanines/chemistry , Cattle , Click Chemistry/methods , Fluorescent Dyes/chemistry , Humans , Immunoassay/instrumentation , Limit of Detection , Microarray Analysis/instrumentation , Oxidation-Reduction , Serum Albumin, Bovine , Sulfhydryl Compounds/radiation effects , Troponin I/blood , Troponin I/immunology , Ultraviolet Rays , Vinyl Compounds/radiation effectsABSTRACT
Nucleic acid microarray-based assay technology has shown lacks in reproducibility, reliability, and analytical sensitivity. Here, a new strategy of probe attachment modes for silicon-based materials is built up. Thus, hybridization ability is enhanced by combining thiol-ene or thiol-yne click chemistry reactions with a multipoint attachment of polythiolated probes. The viability and performance of this approach was demonstrated by specifically determining Salmonella PCR products up to a 20 pM sensitivity level.
