A simple but quantitative mathematical formalism for interpretation of surface plasmon resonance (SPR) signals from adsorbed films of a wide variety of structures is presented. It can be used to estimate adsorbed film thicknesses, surface coverages, or surface concentrations from the SPR response over the entire range of film thicknesses without relying on calibration curves of response versus known thicknesses or surface concentrations. This formalism is compared to more complex optical simulations. It is further tested by (1) calibrating the response of two SPR spectrometers to changes in bulk index of refraction, (2) using these calibrations with this formalism to predict responses to several well-characterized adlayer structures (alkanethiolates and serum albumin on gold, propylamine on COOH-functionalized gold), and then (3) comparing these predictions to measured SPR responses. Methods for estimating the refractive index of the adlayer material are also discussed. Detection limits in both bulk and adsorption-based analyses are discussed. The planar system used here has a detection limit of ∼0.003 nm in average film thickness for adsorbates whose refractive index differs from that of the solvent by only 0.1. The temperature sensitivities of these two SPR spectrometers are characterized and discussed in terms of detection limits.

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We report the construction and characterization of a new compact surface plasmon resonance imaging instrument. Surface plasmon resonance imaging is a versatile technique for detection, quantification, and visualization of bio-molecular binding events which have spatial structure. The imager uses a folded light path, wide-field optics and a tilted detector to implement a high performance optical system in a volume 7″×4″×2″. A bright diode light source and an image detector with fast frame rate and integrated digital signal processor enable real-time averaging of multiple images for improved signal-to-noise ratio. Operating angle of the imager is adjusted by linear translation of the light source. Imager performance is illustrated using resolution test targets, refractive index test solutions, and competition assays for the anti-epileptic drug phenytoin. Microfluidic flowcells are used to enable simultaneous assay of three sample streams. Noise level of refractive index measurements was found to decrease proportional to the square root of the number of pixels averaged, reaching approximately 5 × 10 −7 refractive index units root-mean-square for 160×120 pixel image regions imaged for one second. The simple, compact construction and high performance of the imager will allow the device to be readily applied to a wide range of applications.