Label-free quantitative detection of protein using macroporous silicon photonic bandgap biosensors.
A label-free biosensor was demonstrated using macroporous silicon (pore size >100 nm) one-dimensional photonic band gap structures that are very sensitive to refractive index changes. In this study, we employed Tir-IBD (translocated Intimin receptor-Intimin binding domain) and Intimin-ECD (extracellular domain of Intimin) as the probe and target, respectively. These two recombinant proteins comprise the extracellular domains of two key proteins responsible for the pathogenicity of enteropathogenic Escherichia coli (EPEC). The optical response of the sensor was characterized so that the capture of Intimin-ECD could be quantitatively determined. Our result shows that the concentration sensitivity limit of the sensor is currently 4 microM of Intimin-ECD. This corresponds to a detection limit of approximately 130 fmol of Intimin-ECD. We have also investigated the dependence of the sensor performance on the Tir-IBD probe molecule concentration and the effect of immobilization on the Tir-IBD/Intimin-ECD equilibrium dissociation constant. A calibration curve generated from purified Intimin-ECD solutions was used to quantify the concentration of Intimin-ECD in an E. coli BL21 bacterial cell lysate, and results were validated using gel electrophoresis. This work demonstrates for the first time that a macroporous silicon microcavity sensor can be used to selectively and quantitatively detect a specific target protein with micromolar dissociation constant (Kd) in a milieu of bacterial proteins with minimal sample preparation.
Ouyang
Huimin
H
Center for Future Health, University of Rochester, Rochester, New York 14642, USA. ouyang@ece.rochester.edu
Delouise
Lisa A
LA
Miller
Benjamin L
BL
Fauchet
Philippe M
PM
D018829:Adhesins, Bacterial / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis*; D010316:Particle Size; D017785:Photons; D016062:Porosity; D011956:Receptors, Cell Surface / Q000032:analysis*; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry* / Q000528:radiation effects
D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; C073819:eaeA protein, E coli; D012825:Silicon