Fluorescent Oligomer Oligomer Used Used Fluorescent in a Pathogen Biosensor in a Pathogen Biosensor Presented By: Presented By: Danielle Okerblom of Danielle Okerblom of Cuesta Cuesta College College UCSB Dept of Chemistry and Biochemistry UCSB Dept of Chemistry and Biochemistry Mentor: Aidee Aidee Gonzalez Gonzalez Mentor: Advisor: Gui Gui Bazan Bazan Advisor: Funding provide by: INSET
Recent New s and Outbreaks � January 27, 2009- More Salmonella Peanut Butter Recalls Coming (5) � June 2008- Canada listeria outbreak 22 people died . Concluded that listeria is difficult to detect (6) � August 7, 2008- Years After Anthrax Attacks, Bioterrorism Threat Still Looms (3) � June 18, 2008- Investigation of Multistate Outbreak of E. coli (6)
Standard Methods of Detecting Bacteria � Coliform Bacteria in Water Samples � Lauryl tryptose broth or The Autoanalysis Colilert System. � 24hrs to detect � positive or negative results only does not identify of bacteia. Aalso marine bacteria have been shown to give false positive (1) � Food Samples Salmonella- � Grown inTrypticase soy broth � To grow and detect CDC Biohazard level 2 guidelines must be met � 30hrs to detect (2) � Mail out for Identification to an Industry Lab � $50 per sample for test � 10 business days www.water-research.net/images/ � Possible additional costs for coliformcolony.jpg meat samples. (4)
Expanding Uses of Organic Expanding Uses of Organic Polymers and Oligomers Oligomers Polymers and � Synthesizing � Synthesizing Oligomers Oligomers � � Prepare new applications Prepare new applications for Oligomers Oligomers for � Chacterize � Chacterize oligomers oligomers � Our lab has created � Our lab has created aggregates which detect aggregates which detect presence of proteins in presence of proteins in solution solution � Biospecies � Biospecies interacts with interacts with aggregates this modifies aggregates this modifies the fluorescence spectra the fluorescence spectra Huaping Li, Gui Bazan, Advance Material Journal Volume 21 No 9 March 6 th 2009
Synthesizing and Testing the Synthesizing and Testing the Limitations of Aggregate Limitations of Aggregate Interactions Interactions � � Synthesize conjugated, water Synthesize conjugated, water soluble oligomer oligomer soluble � � Oligomer and peptides form and peptides form Oligomer aggregates by electrostatic aggregates by electrostatic interactions interactions � � Test dependence of FRET Test dependence of FRET Anionic signal on solvents signal on solvents Oligomer http://www.activorcorporation.net/sitebui ldercontent/sitebuilderpictures/peptide1 0.jpg Cationic Peptide
Electro-statically Formed Aggregates � Oligomer is seen in blue � Peptide tagged with Fluorescence is seen in red � Negatively charged oligomer binds to positively charged peptide to form aggregate � Energy transfer within aggregates measured with Forester Resonance Energy Advanced Functional Materials 2008, 18, 3606- Transfer 3612
Synthesis of Monomer: Synthesis of Monomer: ptc NaOH (aq) Yield 39% 1.61g P Outline- - Outline 1) Reaction- - Dissolve Dissolve Fluorene Fluorene, , NaOH NaOH, , 1) Reaction ptc in DMSO. Add in DMSO. Add Sultone Sultone. . ptc 2) Transfer to acetone solution & 2) Transfer to acetone solution & precipitate precipitate 3) Purification 3) Purification 4) 4) Crystallization Crystallization 5) NMR Confirmation of product. 5) NMR Confirmation of product. 6) Used to create Oligomer Oligomer 6) Used to create www.bridgat.com/files/Caustoc_so da.jpg
Solvent Investigations: • The 400nm region is Milli Pore H20 HPF to Oligomer Ratio where the oligomer emissions is. • The 550nm region is R e la tiv e E m is s io n where the fluorescence emission is. 1.3 : 1.0 In te n s ity 1.5 : 1.0 • Difference charge 1.75 : 1.0 concentration which were tested. • Stability of signals over 60 minutes time 350 400 450 500 550 600 650 was measured. Wavelength (nm) • Relavence- 1 st peak compared to 2 nd peak intensity.
Further Solvent Investigations PBS HPF to Oligomer Ratio PB HPF to Oligomer Ratio Relative E m issio n In ten sity R e l a ti v e E m i s s i o n 1.1 : 1.0 1.5 : 1.0 I n te n s i ty 1.3 : 1.0 1.1 : 1.0 1.5 : 1.0 350 400 450 500 550 600 650 375 425 475 525 575 625 Wavelength (nm) Wavelength (nm)
Solvent Investigation of FRET Signal : Phosphate Buffer vs Phosphate Buffer Saline vs Milli Pore Water PB HPF to Oligomer Ratio Milli Pore H20 HPF to Oligomer Ratio Relative Emission Relative Emission 1.3 : 1.0 Intensity 1.5 : 1.0 Intensity 1.1 : 1.0 1.5 : 1.0 1.75 : 1.0 350 400 450 500 550 600 650 350 400 450 500 550 600 650 Wavelength (nm) Wavelength (nm) PBS HPF to Oligomer Ratio Fluorscence emission (550nm)/ Oligomer emission (414nm) ission Intensity 6 5 1.1 : 1.0 FRET Ratio 4 1.3 : 1.0 Conc 1.0 : 1.5 elative Em 3 1.5 : 1.0 2 1 R 0 0 1 2 3 H20 PB PBS 375 425 475 525 575 625 Wavelength (nm) FRET ratio= Fluorescence emission / Oligomer emission
Future Work: � Look at E. Coli isolated samples within the optimal peptide oligomer ratios � Measuring aggregation formation with other peptides. � Determine FRET signal using different bacteria and effect of media such soil and water.
References: (1) EPA Analytic analysis drinking water http://www.epa.gov/nerlcwww/online.htm (2) USDA Analysis of Meat, Poultry and Egg http://www.fsis.usda.gov/PDF/MLG_4_04.pd (3) http://www.pbs.org/newshour/bb/terrorism/july- dec08/bioterrorprep_08-07.html (4) MRPK Industry lab 1(866)888-6653 nbc33tv.com (5) http://www.cdc.gov/salmonella/
What is FRET What is FRET � � FRET- - Forester Resonance Energy Forester Resonance Energy FRET Transfer Transfer � � describes energy transfer between describes energy transfer between two chromophores two chromophores located less than located less than 10nm from each other 10nm from each other � � Excited electron from donor Excited electron from donor chromophore are transferred to lower are transferred to lower chromophore levels of energy at acceptor levels of energy at acceptor chromophore. . chromophore *Chromophores Chromophores almost always arise in almost always arise in * one of two forms: conjugated pi conjugated pi one of two forms: systems. . systems
Isolated Oligomer Fractions Isolated FPFC4S03 Fract 1-3 0.5 Methanol blank 0.4 A b s o r b a n c e ( n m ) Frac 3 0.3 H20 blank 0.2 *Fraction 2 is Frac 2 0.1 FPFC4SO3. Frac 1 0 200 250 300 350 400 *We use long UV -0.1 waves lights to see Wavelength the fluorescent glow
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