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Preparation of quantum dots conjugates with redox-active proteins

Krzysztof Jakub Szczepaniak

Abstract

Introduction Since their discovery in the mid 80’s of the last century, luminescent nanocrystals are of great interest of scientists. These structures, later named quantum dots (QD), have a potential to revolutionize cellular imagining. Such colloidal nanocrystals are also useful in variety of other fields due to their unique features. One of the most important characteristics of QDs is a correlation between maximum wavelength of their emission and their diameter. What is more, the nanocrystals have wide Stockes shift, what means that, unlike emission, extinction maximum in not dependent on the nanocrystal size. The conjugation of the quantum dots with enzymes, especially with redox-active proteins, is not yet widely studied. It is known that QDs may accept or donate electrons, therefore they may became a donor or acceptor in redox reactions. What is more, increased concentration of free electrons occurs in a close vicinity of nanocrystals surface. For that reason redox-active proteins attached to the surface of the quantum dots may possibly modify emission characteristics of those fluorophores. The purpose of presented work was to obtain stable conjugate of QDs and photosynthetic proteins- ferredoxin:NADP+ oxidoreductase (FNR) and ferredoxin (Fd), with a special attention put on preserving the native activity of both proteins. The process and final product of conjugate formation was characterized with spectroscopic methods. Results and discussion Hydrophilic quantum dots with strong emission and only slight maximum shift were obtained as a result of first phase of experiments. Such nanocrystals dissolved in water were stable over long period of time in neutral and basic pH. Several conjugation methods were tested, and two of them, using popular cross-linker 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide (EDC), with or without addition of N-hydroxysuccinimide gave the best results. Stable conjugates with bovine serum albumin were obtained, confirmed with fluorescence spectroscopy. The method using only EDC was found to be more effective. Both methods were further tested for FNR and Fd conjugation. Obtaining of both protein conjugates were confirmed by spectroscopic and fluorimetric techniques. Method using only EDC was again found to be more effective. Unfortunately, during conjugation procedure the active center of ferredoxin was destroyed. Taking into account all results, a method using only EDC was chosen as the best for following work. It was further found that in QD-FNR structures the protein was enzymatically active, which was confirmed by in vitro NADPH oxidation reaction . The kinetic data proved that at least 50% of the enzyme native activity was preserved. In vitro reconstruction of an active center of ferredoxin was tested, although without satisfying results. All experiments confirmed effectiveness of conjugation methods described in scientific literature and alloved to select the best conjugation technique for proteins of interest. Stable QD-FNR conjugates were obtained, with the protein retaining over 50% of its native activity.
Record ID
WUT9a84ba54b70a4aa590fd0ced8bda84d4
Diploma type
Master of Science
Author
Krzysztof Jakub Szczepaniak Krzysztof Jakub Szczepaniak,, Undefined Affiliation
Title in Polish
Utworzenie i charakterystyka in vitro i in vivo konjugatu kropki kwantowej CdSe/ZnS z wybranymi białkami modelowymi
Supervisor
Antoni Pietrzykowski (FC/DCOC) Antoni Pietrzykowski,, Department Of Catalysis And Organometallic Chemistry (FC/DCOC)Faculty of Chemistry (FC)
Certifying unit
Faculty of Chemistry (FC)
Affiliation unit
Department Of Catalysis And Organometallic Chemistry (FC/DCOC)
Study subject / specialization
, Mikrobioanalityka
Language
(pl) Polish
Status
Finished
Defense Date
29-08-2012
Issue date (year)
2012
Keywords in Polish
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Keywords in English
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Abstract in Polish
urn:pw-repo:WUT9a84ba54b70a4aa590fd0ced8bda84d4