Alexander P. Savitsky A.N. Bach Institute of Biochemistry, Moscow 1 th German-Russian Symposium on Nanobiotechnology

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1 Alexander P. Savitsky A.N. Bach Institute of Biochemistry, Moscow 1 th German-Russian Symposium on Nanobiotechnology ctober 2009 Lichtenwalde (Sachsen)

2 Fluorescent detection systems - high sensitivity (up to M); - real time measurements (milli or microseconds per sample or array); - possibility of automatization; - possibility to use micro and nanovolumes; - long shelf-life of the labeled reagents; - Genetical encoding; - in vivo application, small animal whole body imaging.

3 Mode of measurement

4 Room Temperature Phosphorescence RTP Biomedical Applications xygen detection xygen imaging Immunoassay DNA biding assay Microscopy

5 Excitation and emission spectra of PtCP, PdCP and detection limit in 2% met-β-cyclodextrin A.P.Savitsky. Subpicomolar Assay of antibodies and DNA using phosphorescence labels. In Fluorescence sensors and biosensors. R.B.Thompson Ed.CRC Taylor and Francis Group, London-N.Y., 2005

6 Calibration curve for thyrotropin assay obtained by MS- PhIA on an Arcus 1230 instrument (Wallac, Finland) with 400 nm excitation and 660 nm emission filters, delay time was 200 ms, measuring time was 800 ms, signal accumulation time was 1 s.

7 Normalized absorption and fluorescence spectra of NIR metalloporphyrins at room temperature in 2% Triton X-100, ph 7.2.

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9 F 3 C N CF 3 N N N N N N N CF 3 CF 3 C 58 H N 8 F 12

10 Nonseparation competitive assay of human IgG lg[intensity] lg[bsa-dtpa-abtfa] Savitsky et.al. Advances in Fluorescence Sensing Technology II, J.R.Lakowicz, Ed. Proc.SPIE Bellingham, Washington Vol.2388, pp , 1995 IgG, ng/ml

11 Tomography Molecular biology Cell biology Histology Physiology Medicine DNA Proteins Cells Tissue Animal Human Information level Sesitivity to malecular events Genomics Proteomics Cytomics Phenomic Clinic $$ $$ $ $$$$ $-$$ CT US CT NMR FDT no no no М М

12 Light penetration depth, mm Wavelength, nm Therapeutic windows

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14 Spectral diversity and brightness of fluorescent proteins brightness=qxε

15 Target tumor cells 50,000 cells per well of 6-wells plate in RPMI % serum Packaging cells PT67-GFP, DMEM+10% serum reaching of 70-80% confluency of monolayer polybrene 18 hours Packaging cells PT67-GFP, DMEM +5% serum 24 hours Target tumor cells + virus + polybrene filtering cellulose-acetate or polysulphonic filter 0,45 µm next slide virus-containing supernatant

16 24 hours Changing of medium to RPMI % serum 24 hours Transfected target cells selection cloning Individual clone of target cells expressing GFP method of limited dilution using of cloning cylinders Individual clone of target cells expressing GFP

17 Phase contrast Fluorescence

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19 Molecular imaging in living subjects Visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms. Each disease has molecular origin Diagnostic New therapeutic methods targeted to molecular processes.

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22 P CMV IE TagRFP GTGGSGGDEVDGTGGSGDPPVAT KFP

23 normalized intensity 1,0 TagRFP 0,8 TagRFP-23-KFP 0,6 0, time, ns τ 1 (No FRET) τ 2 (FRET) TagRFP I( t) n = A e i t τ i TagRFP- 23-KFP i= 1

24 Campthotecin induced apoptosis FLIM MicroTime 200, PicoQuant (Berlin) 1,2 1 log intensity 0,8 0,6 0,4 0,2 no with campth 0-0, Time, ns

25 fluorescence resonance energy transfer for Tb red fluorescent protein pair FRET DEVD Donor Terbium chelate Excitation max 280 nm Emission max 545 nm Acceptor DsRed2 or TagRFP Excitation max 562 nm Emission max 586 nm

26 verlapping of emission spectrum of terbium and excitation spectrum of DsRed2 or TagRFP

27 Emission of long-lived fluorescence of Tb-DEVD- TagRFP concentration of protein - 10 mkm concentration of Tb - 10 mkm 35 Intensity, a.e without Tb 10 mm HEPES ph 7 physiological conditions Wavelength, nm

28 IN VIV DETECTIN F QUANTUM DTS IN NUDE MICE. Kat+ Kat+ Kat+ - - S S S S S - Kat+ Kat+ Kat+ Kat+ CdS ZnS S S S S S Kat+ Kat+ Kat+ Kat+ Kat+ Kat+ Stomach Duodenum F/Fmax 1,2 1 0,8 0,6 0 time 3 weeks stomach 2 h after administration of QD MPA excrements 24 h after administration of QD MPA QD MPA in buffer Control 10 min 20 min Time after administration 0,4 0, Wavelength, nm 30 min 1 h 2 h Intestine

29 Conclusion Developed mouse model of the human fluorescent tumor (Mel-Kor) Life-time based FRET substrate for caspase 3 for FLIM Lanthanide red fluorescent protein FRET pare Nude mouse model for QD traffic and localization

30 Perspective Fluorescence life-time tomography Lanthanide FRET substrate for multiphoton imaging In vivo QD traffic and localization (skin, lung)

31 L.R. Arslanbaeva T.V. Ivashina V.V. Jerdeva G. D. Lapshin I.G. Meerovich A.L. Rusanov This research was supported by Federal Agency for Science and Innovations (state contract no ) Molecular and Cell Biology Program of the Russian Academy of Sciences Russian Foundation for Basic Research (grant a)