ABSTRACT
Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.
Subject(s)
Antigens/isolation & purification , Biomarkers/metabolism , Chemistry Techniques, Analytical/methods , Plastics/chemistry , Water/chemistry , Antibodies/metabolism , Antigens/metabolism , HIV Infections/diagnosis , Hepatitis B/diagnosis , Humans , Immunoassay , Light , Optical Phenomena , Protein Array AnalysisABSTRACT
An elastic light-scattering-based method that makes use of phantom nanoparticles as a substrate organizer (see illustration) allowed the quantitative evaluation of the molecular recognition events between a series of inactivated mutants of phospholipase D and lysophosphatidylcholine. The results highlight the remarkable effects on binding capability caused by single amino acid substitutions.
Subject(s)
Light , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Phospholipase D/genetics , Phospholipase D/metabolism , Scattering, Radiation , Animals , Cattle , Mutant Proteins/chemistry , Nanoparticles/chemistry , Phospholipase D/chemistry , Protein Binding , Streptomyces/enzymology , Substrate SpecificityABSTRACT
After a broad scouting based on quantum chemical calculations, optical absorption measurements in the vacuum ultraviolet (VUV) wavelength region between 140 and 190 nm were performed on a narrower series of commercial and experimental liquids. By elimination of sources of external contamination, mainly due to atmospheric gases, the analysis of the contributions to the absorption related to the backbone structure and to the chain end composition allowed the synthesis of a novel family of linear perfluoropolyethers (PFPEs) with optical absorbance at 157 nm between 0.3 and 0.6 cm(-1) in a broad range of compositions and molecular weights. The dependence of the optical threshold on the PFPE composition demonstrates that -OCF2- is the most transparent segmental unit in the VUV region.