Quantitative aspects of SPR spectroscopy and SPR microscopy: Applications in protein binding to immobilized vesicles anddsDNA arrays.

摘要

Surface plasmon resonance (SPR) spectroscopy is used to measure biomolecular interactions in real time with high sensitivity and without labels. SPR microscopy provides the same advantages as SPR spectroscopy with the added feature of monitoring adsorption across a large area of the sensor surface with good spatial resolution (∼4 μm).; This dissertation describes the development of quantitative techniques based on SPR spectroscopy and SPR microscopy to study protein adsorption to functionalized sensor surfaces. First, SPR spectroscopy is combined with an enzyme assay to measure the binding constant of phospholipase A₂ to surface-immobilized vesicles. We show how a mixed self-assembled monolayer containing a few biotin headgroups may be used to form a streptavidin monolayer that becomes a linker layer for attachment of intact, biotinylated lipid vesicles.; Next, the development of SPR microscopy for parallel, high-throughput array-based analysis is described. The design and construction of an SPR microscope is presented. We show how a single, high-contrast angle selected from a linear region of an SPR curve allows real-time reflectivity changes to be converted to effective refractive index changes by extending SPR spectroscopy data quantitation methods to SPR microscopy. Detection limits based on the system noise are shown to be ∼2 × 10−5 refractive index units, corresponding to 1.2 ng/cm2 of protein with a time resolution of 1 data point per second. For longer time (∼20 minutes) measurements, the detection limits are shown to improve slightly due to an increase in data averaging and the use of a “reference” to account for system drift.; We demonstrate the ability to measure adsorption and desorption curves for a large number (>100) of regions of a sensor surface simultaneously by measuring adsorption of a DNA-binding protein to its DNA binding site immobilized in an array on a streptavidin linker layer. We describe the array fabrication process and show how elements containing DNA without the protein binding site can be used as “reference” regions to correct for non-specific adsorption and common refractive index changes. The real-time detection limit for fast time resolution measurements is shown to be less than ∼8 × 106 proteins per 200-μm spot.