Fluoreszenzdetektion und -Imaging von Biomolekülen unter Verwendung aufkonvertierender Nanopartikel
Zusammenfassung der Projektergebnisse
It was shown that upconverting nanoparticles with an optimized size and a vectorperceptive functionalized shell can be produced with good reproducibility. Quenching of the luminescence of the UCLNP using sub-nanometer sized gold particles was not successful. Furthermore, labeling of the molecular beacons (MB) with the fluorophore TAMRA was conducted, and FRET (or a related process) between the UCLNP and TAMRA is demonstrated. However, no DNA-dependent signal change was observed. A plausible explanation for these negative results is that the particle size (40 nm) is still too big for such a sensory system. The bulk fluorescence of the UCLNPs is efficient enough to excite the TAMRA fluorophores even in the hybridized (= open) form of the MBs, where TAMRA is no longer in close proximity to the UCLNPs. Since efficient energy transfer occurs both in the closed and open forms of the MBs, we could not see any significant changes in the TAMRA-specific signal. These results suggest that a further decrease in the size of UCLNPs is necessary. Throughout the second part of the work, particles with an average diameter of 32 nm were used that contained an active luminescent core and an inactive shell. Several techniques for coating of the particles were evaluated. Eventually, a functionalization of the UCLNPs coated with poly(acrylic acid) that introduces carboxy groups at the surface was chosen. Different dyes were evaluated to screen for the best acceptor fluorophore. Chromeo 642 and Cy5 were chosen for labeling of the MB because they showed fairly good quenching of the 655 nm emission peak of the UCLNPs. The MBs labeled with the quencher was then loaded onto the particles either via streptavidin/biotin coupling or covalently immobilized using standard NHS chemistry. Unfortunately, none of the configurations showed a response to the target DNA. Furthermore, the quenching ("shielding") effect of the attached fluorophores was only minimal even at high concentrations. The molecular beacon was split in two parts – a capture strand immobilized at the surface of the particle and a target strand (of varying length) labeled with the quencher – for a proof of principle. This setup clearly showed that the basic idea of shielding the emission of the UCLNP with a quencher bound to an oligonucleotide is functional. Furthermore, it revealed that the design of the molecular beacon has to be revisited with regard to the distance of the quencher after hybridization with the target DNA.
Projektbezogene Publikationen (Auswahl)
- Probing the Activity of Matrix Metalloproteinase II Using a Sequentially Click-Labeled Silica Nanoparticle FRET Probe. ChemBioChem (2009), 10, 2316-1320
D. E. Achatz, G. Mezö, P. Kele, O. S. Wolfbeis
(Siehe online unter https://doi.org/10.1002/cbic.200900261) - Upconverting Luminescent Nanoparticles for Use in Bioconjugation and Bioimaging. Curr. Opin.. Chem. Biol. (2010), 14, 582
H. S. Mader, P. Kele, S. Saleh, O. S. Wolfbeis
(Siehe online unter https://doi.org/10.1016/j.cbpa.2010.08.014) - Luminescent Chemical Sensing, Biosensing, and Screening Using Upconverting Nanoparticles. Topics Curr. Chem. (2011), 300, 29-50
D. E. Achatz, A. Reham, O. S. Wolfbeis
(Siehe online unter https://doi.org/10.1007/128_2010_98) - DNA "Nanolamps": Clicked DNA Conjugates with Photon Upconverting Nanoparticles as Highly Emissive Biomaterials. ChemPlusChem (2012), 77, 129-134
M. Rubner, D. E. Achatz, H. S. Mader, J. A. Stolwijk, J. Wegener, G. S. Harms, O. S. Wolfbeis, H.-A. Wagenknecht
(Siehe online unter https://doi.org/10.1002/cplu.201100055)
