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PD Dr. Hans H. Gorris

(University of Regensburg)


Single molecule analysis enabled by photon-upconversion nanoparticles (UCNPs) and femtoliter arrays

Photon-upconversion nanoparticles (UCNPs) have found wide interest because they emit short-wavelength light under near-infrared (NIR, 980 nm) excitation (anti-Stokes emission), which avoids optical background interference from autofluorescence and light scattering. I will present new (nano-) analytical techniques based on UCNPs. Agarose gel electrophoresis enables the separation of UCNPs that can be detected directly in the gel by a 980-nm laser scanner. UCNP-protein conjugates were purified by agarose gel electrophoresis. Such well-defined nanoconjugates have been used as labels in lateral flow assays and upconversion-linked immunoassays (ULISAs). A competitive ULISA allowed for the sensitive detection of the pharmaceutical diclofenac in environmental water samples (50 pg mL-1 (170 pM). UCNPs can be detected at the single nanoparticle level using a relatively simple wide-field upconversion microscope. In this way, it is possible to implement a single molecule sandwich immunoassay for the digital readout of diagnostic markers such as prostate-specific antigen (PSA) with ten-fold lower limit of detection (1.2 pg mL-1 (42 fM)) com¬pared to conventional (analog) immunoassays. In the second part of my talk, I will present our research on single enzyme molecule kinetics. Large arrays of 62,500 femtoliter-sized chambers are etched into the surface of fused silica slides to isolate hundreds of individual enzyme molecules and observe their substrate turnover in parallel. If a fluorogenic reaction takes place in a femtoliter volume, a single en-zyme molecule generates a product concentration high enough for detection by wide-field fluorescence microscopy. I will demonstrate that individual molecules of ß-galactosidase, ß-glucuronidase, horseradish peroxidase, and phosphatase display broadly distributed catalytic activities as a consequence of different protein conformations. This single molecule approach also provides new insights into enzyme inhibition and molecular evolu

Kdy a kde:

Čtvrtek 18.10.2018 ve 10:00 hod v klubu B

(Vytvořeno 16.10.2018 z dat upravených naposled 15.10.2018)

Centrum polymerních materiálů a technologií Otty Wichterla - CPMTOW

Centrum biomedicinálních polymerů - CBMP

Centrum polymerních senzorů - CPS

Polymery pro energetiku - EnergoLab

Ústav makromolekulární chemie AV ČR v.v.i.

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