Theoretical and experimental analysis of arrayed imaging reflectometry as a sensitive proteomics technique.
Arrayed imaging reflectometry (AIR) is a newly developed label-free optical biosensing technique based on the creation and perturbation of a condition of zero reflectance on a silicon substrate. The antireflective coating is formed by covalently immobilizing arrayed probes on a silicon dioxide film. Probe-target complex formation causes a localized increase in optical thickness and a measurable reflectance change. To evaluate the performance of AIR, we have employed two proteins, intimin and tir, from enteropathogenic E. coli that are critical to the bacterium's mechanism of host infection. Using substrates functionalized with the intimin-binding domain of tir, we demonstrate detection of the extracellular domain of intimin at concentrations as low as 10 pM. Through the use of a diffusion-limited model for the intimin-tir binding interaction at this concentration, we estimate the detected intimin surface concentration to be 0.33 pg/mm2.
Mace
Charles R
CR
Department of Biochemistry and Biophysics, The Center for Future Health, University of Rochester, Rochester, New York 14642, USA.
Striemer
Christopher C
CC
Miller
Benjamin L
BL
D018829:Adhesins, Bacterial / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D029968:Escherichia coli Proteins / Q000032:analysis*; D007368:Interferometry / Q000295:instrumentation / Q000379:methods*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D011956:Receptors, Cell Surface / Q000032:analysis*; D012680:Sensitivity and Specificity
D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; C073819:eaeA protein, E coli