Lanthanides and Quantum Dots - Time-Resolved Laser Spectroscopy of Biochemical F¨orster Resonance Energy Transfer (FRET) Systems by Niko Hildebrandt A Dissertation submitted to the Mathematics-Natural Science Faculty of the University of Potsdam in partial fulfillment of the requirements for the degree of Doctor of Natural Science Potsdam, December 2006 To Ella and Monique. You make my life complete. Contents List of publications III Abstract V Kurzzusammenfassung VII 1 Introduction 1 2 Theoretical and practical background 3 2.1 F¨orsterresonanceenergytransfer.......................... 3 2.1.1 Introduction.................................. 3 2.1.2 Theoreticaltreatment............................ 3 2.1.3 FRETapplications.............................. 6 2.2Luminescencequenching............................... 7 2.3Lanthanidecomplexes................................ 8 2.3.1 Lanthanideions................................ 8 2.3.2 Lanthanideioncomplexingligands..................... 15 2.3.3 Biochemical applications of lanthanide complexes . 17 2.4 Quantum dot semiconductor nanocrystals . 19 2.4.1 Introduction.................................. 19 2.4.2 The quasiparticle approach in semiconductor crystals . 19 2.4.3 Quantumconfinement............................ 20 2.4.4 Core/shell semiconductor QD . 23 2.4.5 BiocompatibleQD.............................. 25 2.5BiochemicalFRETsystems............................. 27 2.5.1 Immunoassays................................. 27 2.5.2 Biotin-(strept)avidin............................. 30 2.5.3 Conjugationmethods............................. 32 3 Experimental section 35 3.1Measuringsolutions.................................. 35 3.2 FRET donors and acceptors . 35 3.3Analyticalmethods.................................. 36 3.3.1 Stationary absorption and luminescence spectroscopy . 36 3.3.2 Time-resolved luminescence spectroscopy and fluoro immunoassays . 36 3.3.3 MALDI-TOF................................. 38 3.4Dataprocessing.................................... 38 I II 4 Results and discussion 39 4.1CharacterizationoftheFRETdonors........................ 39 4.1.1 Bioconjugation................................ 39 4.1.2 Photophysicalcharacterization........................ 42 4.2 Characterization of the FRET acceptors . 49 4.2.1 Bioconjugation................................ 50 4.2.2 Photophysicalcharacterization........................ 50 4.3FRETexperiments.................................. 53 4.3.1 Streptavidin-biotin FRET system . 53 4.3.2 HCGFRETimmunoassays......................... 62 5 Summary and outlook 67 6 Appendix 71 6.1 Fundamental constants . 71 6.2Abbreviations..................................... 71 6.3Emissionspectra................................... 73 6.4Luminescencedecaykinetics............................. 82 6.5FRETexperiments.................................. 88 6.5.1 Biot-QD655 as acceptor . 88 6.5.2 Biot-APC and Biot-DY633 as acceptors . 92 References 97 List of publications Original publications 1. Hildebrandt N, Charbonni`ereLJ, Ziessel RF, L¨ohmannsr¨oben H-G; ”Homogeneous FRET Immunoassay Based on Lanthanides to Quantum Dots Energy Transfer” In: Osinski M, Jovin TM, Yamamoto K, editors. Proceedings of SPIE Vol. 6448 - Colloidal Quantum Dots for Biomedical Applications II; 2007; 64480K (12 pages). 2. Niederkr¨uger M, Salb C, Marowsky G, Beck M, Hildebrandt N, L¨ohmannsr¨oben H-G; ”Improvement of a Fluorescence Immunoassay with a Compact Diode-Pumped Solid State Laser at 315 nm” In: Proceedings of SPIE Vol. 6380 - Smart Medical and Biomedical Sensor Technology IV; 2006; 63800M (9 pages). 3. Charbonni`ere LJ, Hildebrandt N, Ziessel RF, L¨ohmannsr¨oben H-G; ”Lanthanides to Quantum Dots Resonance Energy Transfer in Time-Resolved FluoroImmunoAssays and Lu- minescence Microscopy” Journal of the American Chemical Society 2006;128(39): 12800-12809. 4. Hildebrandt N, Charbonni`ere LJ, Ziessel RF, L¨ohmannsr¨oben H-G; ”Quantum Dots as Resonance Energy Transfer Acceptors for Monitoring Biological Interactions” In: Grzymala R, Haeberle O, editors. Proceedings of SPIE Vol. 6191 - Biophotonics and New Therapy Frontiers; 2006; 61910W (9 pages). 5. Beck M, Hildebrandt N, L¨ohmannsr¨oben H-G; ”Quantum Dots as Acceptors in FRET Assays Containing Serum” In: Grzymala R, Haeberle O, editors. Proceedings of SPIE Vol. 6191 - Biophotonics and New Therapy Frontiers; 2006; 61910X (8 pages). 6. L¨ohmannsr¨oben H-G, Beck M, Hildebrandt N, Schm¨alzlin E, van Dongen JT; ”Laser-Based Fluoroimmuno Analysis and In-Vivo Optical Oxygen Monitoring” In: Gries W, Pearsall TP, editors. Proceedings of SPIE Vol. 6157 - Workshop on Laser Applications in Europe; 2006; E1-E6. 7. Hildebrandt N, Charbonni`ere LJ, Beck M, Ziessel RF, L¨ohmannsr¨oben H-G; ”Quan- tum Dots As Efficient Energy Acceptors in a Time-Resolved Fluoroimmunoassay” Angewandte Chemie - International Edition 2005;44(46): 7612-7615. 8. Hildebrandt N, Charbonni`ere LJ, Beck M, Ziessel RF, L¨ohmannsr¨oben H-G; ”Quan- tenpunkte als effiziente Energieacceptoren in einem zeitaufgel¨ostenFluoroimmuntestsystem” Angewandte Chemie 2005;117(46): 7784-7788. Review 1. Hildebrandt N, L¨ohmannsr¨oben H-G; ”Quantum Dot Nanocrystals and Supramolecular Lan- thanide Complexes - Energy Transfer Systems for Sensitive In Vitro Diagnostics and High Throuput Screening in Chemical Biology” Current Chemical Biology 2007; accepted for pub- lication. III IV Oral presentations at conferences and meetings 1. Hildebrandt N, Charbonni`ere LJ, Ziessel RF, L¨ohmannsr¨oben H-G; ”Homogeneous FRET Immunoassay Based on Lanthanides to Quantum Dots Energy Transfer” BiOS 2007 - Biomedical Optics - Colloidal Quantum Dots for Biomedical Applications II, San Jose, USA; 2007. 2. Hildebrandt N, Charbonni`ere LJ, Beck M, Ziessel RF, L¨ohmannsr¨oben H-G; ”Lan- thanides and Quantum Dots as Efficient Donor-Acceptor Pairs for Sensitive Homogeneous F¨orster Resonance Energy Transfer Fluoroimmunoassays (FRET-FIA)” EOS Topical Meeting on Biophotonics and Biomedical Optics 2006, Paris, France; 2006. 3. Hildebrandt N, Charbonni`ere LJ, Beck M, Ziessel RF, L¨ohmannsr¨oben H-G; ”Lan- thanide Donors and Quantum Dot Acceptors for Homogeneous FRET Immunoassays” 105. Bunsentagung 2006, Erlangen, Germany; 2006. 4. Hildebrandt N, Charbonni`ere LJ, Ziessel RF, L¨ohmannsr¨oben H-G; ”Quantum Dots as Resonance Energy Transfer Acceptors for Monitoring Biological Interactions” Photonics Europe 2006 - Biophotonics and New Therapy Frontiers, Strasbourg, France; 2006. 5. Hildebrandt N, Flehr R, Bois E, L¨ohmannsr¨oben H-G; ”Optimized Homogeneous Immunoas- say Based on XeCl-Laser Excited F¨orster Resonance Energy Transfer” CLEO/EUROPE 2005 - Conferences on Lasers and Electro-Optics/Europe - Photonic Manipulation and Bio-Sensing, M¨unchen, Germany; 2005. 6. Hildebrandt N,BoisE,NutiD,L¨ohmannsr¨oben H-G; ”New lasers for better fluores- cence immunoassays (FIA)” OPTO 2003, Paris - France; 2003. Poster presentations at conferences and meetings 1. Hildebrandt N, Bois E, Nuti D, L¨ohmannsr¨oben H-G; ”Sensitivity Improvement of a Laser- Based Homogeneous Fluorescence Immunoassay (FIA) Laboratory System” EPA summer school: New Perspectives in Photochemistry, Egmond aan See - Netherlands; 2003. 2. Hildebrandt N, Bois E, Nuti D, L¨ohmannsr¨oben H-G; ”Miniaturized Laser-Based Ho- mogeneous Fluorescence Immunoassay (FIA) Reader System” MINIT 2003, Potsdam, Germany; 2003. Abstract F¨orster Resonance Energy Transfer (FRET) plays an important role for biochemical applica- tions such as DNA sequencing, intracellular protein-protein interactions, molecular binding studies, in vitro diagnostics and many others. For qualitative and quantitative analysis, FRET systems are usually assembled through molecular recognition of biomolecules conjugated with donor and acceptor luminophores. Lanthanide (Ln) complexes, as well as semiconductor quantum dot nanocrystals (QD), possess unique photophysical properties that make them especially suitable for applied FRET. In this work the possibility of using QD as very efficient FRET acceptors in combination with Ln complexes as donors in biochemical systems is demonstrated. The necessary theoretical and practical background of FRET, Ln complexes, QD and the applied biochemical models is outlined. In addition, scientific as well as commercial applications are presented. FRET can be used to measure structural changes or dynamics at distances ranging from approximately 1 to 10 nm. The very strong and well characterized binding process between streptavidin (Strep) and biotin (Biot) is used as a biomolecular model system. A FRET system is established by Strep conjugation with the Ln complexes and QD biotinylation. Three Ln complexes (one with Tb3+ and two with Eu3+ as central ion) are used as FRET donors. Besides the QD two further acceptors, the luminescent crosslinked protein allophycocyanin (APC) and a commercial fluorescence dye (DY633), are investigated for direct comparison. FRET is demonstrated for all donor-acceptor pairs by acceptor emission sensitization and a more than 1000-fold increase of the luminescence decay time in the case of QD reaching the hundred microsecond regime. Detailed photophysical characterization of donors and acceptors permits analysis of the bioconjugates and calculation of the FRET parameters. Extremely large F¨orster radii of more than 100 A˚ are achieved for QD as acceptors, considerably larger than for APC and DY633 (ca. 80 and 60 A).˚ Special attention is paid to interactions with different additives in aqueous solutions, namely borate buffer, bovine serum albumin (BSA), sodium azide