ABSTRACT
A new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidin molecules. Amplified detection of avidin-biotin binding is obtained on supported membranes composed of biotin layers on quartz surface, which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to the octadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin and biotin-avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was able to detect less than 10.3 ng/cm2 avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry and reflectometry techniques. Further improvement is underway using highly refractive beads together with appropriate surface chemistry. This newly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand-analyte affinity binding.
Subject(s)
Avidin/analysis , Avidin/chemistry , Biosensing Techniques/instrumentation , Biotin/analysis , Biotin/chemistry , Microwaves , Protein Interaction Mapping/instrumentation , Affinity Labels , Biosensing Techniques/methods , Coated Materials, Biocompatible/chemistry , Equipment Design , Equipment Failure Analysis , Protein Interaction Mapping/methodsABSTRACT
Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. Using dynamic light scattering and circular dichroism, solutions of purified allophycocyanin were shown to consist of homogeneous trimers (alpha3beta3) with a nonspherical shape over a very wide range of protein concentrations at pH 6.0 and 20 degrees C. Deconvolutions of the visible circular dichroism spectrum of the trimer were carried out for the first determination of the individual spectra of all six-component chromophores. The chromophores were shown to be in different microenvironments that helped determine the spectrum of the trimer. Monomers (alpha beta) that were formed in either the presence of 0.50M NaSCN or at 45 degrees C were shown to be completely reversible to trimers. However, subunits (alpha and beta) that were formed in either the presence of 8M urea or at 60 degrees C, using spectroscopy and gel-filtration column chromatography, were observed to only partially reconstitute trimers. Homodimers (alpha2 and/or beta2) formed during the regeneration of trimers. The homodimer, which was detected for the first time when both subunits were present, was shown to be in equilibrium with its subunits. Unlike the trimer situation, subunits were found to fully reconstitute monomers in the presence of 0.50M NaSCN. These results suggest a route to trimer assembly from subunits with monomers serving as intermediaries and the homodimers forming in a nonproductive step that did not interfere with the overall assembly scheme.
Subject(s)
Phycocyanin/chemistry , Chromatography, Gel , Circular Dichroism , Cyanobacteria/metabolism , Dimerization , Hydrogen-Ion Concentration , Light , Protein Conformation , Protein Structure, Secondary , Scattering, Radiation , Temperature , Time FactorsABSTRACT
We report the first use of differential terahertz time-domain spectroscopy for bioaffinity sensing. Binding is observed by measuring the transmission of a thin layer of biotin bound to the sensor protein avidin. We demonstrate the THz wave transmission of a sub-micron-thick film and sensitivity to 0.1 microg cm(-2) of biotin. These results point the way for a host of biosensor applications using T-rays, or pulsed far-infrared (FIR) radiation.
