Analytical Chemistry on the Micro- and Nanometer Scale
소속 :
연사 : Prof. Hans-Heiner Gorris(University of Regensburg)
일시 : 2013-10-10 16:30 ~
장소 : 500동 목암홀
일 시 : 2013년 10월 10일, 오후 4:30
장 소: 500동 목암홀
-Abstract-
Large arrays of femtoliter-sized chambers etched into the surface of fused silica slides are an ideal platform for isolating and monitoring the catalytic activity of hundreds of individual enzyme molecules by fluorescence microscopy. Individual molecules of ß-galactosidase, ß-glucuronidase, and horseradish peroxidase exhibit long-lived substrate turnover rates that are broadly distributed that can be attributed to different protein conformations. By using the slow-binding inhibitor Dgalactal to single b-galactosidase molecules, we were able to observe repeated inhibitor binding and release events. The rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics. I will demonstrate how femtoliter arrays can be used for implementing a so-called single molecule ELISA to reach the ultimate detection limit of bioassays. Photon-upconverting nanoparticles (UCNPs) are a relatively new class of luminescent materials that provide many advantages for optical labeling and imaging compared to fluorescent dyes and quantum dots. For example, UCNPs display multiple emission lines of visible light under near-infrared excitation (anti-Stokes emission), which strongly reduces autofluorescence and light scattering in biological samples and enables deep tissue imaging. We have prepared protein reactive labels by conjugating maleimide groups to the surface of UCNPs. This labeling scheme is well amenable to multiplexing because the narrow and multiple emission lines UCNPs can be adjusted individually to generate large sets of codes. The multiple emission lines of UCNPs have also been used for ratiometric temperature sensing. We are further optimizing the readout of UCNPs to devise essentially background-free immunoassays with a very wide dynamic range.
장 소: 500동 목암홀
-Abstract-
Large arrays of femtoliter-sized chambers etched into the surface of fused silica slides are an ideal platform for isolating and monitoring the catalytic activity of hundreds of individual enzyme molecules by fluorescence microscopy. Individual molecules of ß-galactosidase, ß-glucuronidase, and horseradish peroxidase exhibit long-lived substrate turnover rates that are broadly distributed that can be attributed to different protein conformations. By using the slow-binding inhibitor Dgalactal to single b-galactosidase molecules, we were able to observe repeated inhibitor binding and release events. The rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics. I will demonstrate how femtoliter arrays can be used for implementing a so-called single molecule ELISA to reach the ultimate detection limit of bioassays. Photon-upconverting nanoparticles (UCNPs) are a relatively new class of luminescent materials that provide many advantages for optical labeling and imaging compared to fluorescent dyes and quantum dots. For example, UCNPs display multiple emission lines of visible light under near-infrared excitation (anti-Stokes emission), which strongly reduces autofluorescence and light scattering in biological samples and enables deep tissue imaging. We have prepared protein reactive labels by conjugating maleimide groups to the surface of UCNPs. This labeling scheme is well amenable to multiplexing because the narrow and multiple emission lines UCNPs can be adjusted individually to generate large sets of codes. The multiple emission lines of UCNPs have also been used for ratiometric temperature sensing. We are further optimizing the readout of UCNPs to devise essentially background-free immunoassays with a very wide dynamic range.
