Fluorescent Oligomer Oligomer Used Used Fluorescent in a Pathogen - - PowerPoint PPT Presentation

Fluorescent Fluorescent Oligomer Oligomer Used Used in a Pathogen Biosensor in a Pathogen Biosensor

Presented By: Presented By:

Danielle Okerblom Danielle Okerblom of

• f Cuesta

Cuesta College College

UCSB Dept of Chemistry and Biochemistry UCSB Dept of Chemistry and Biochemistry Mentor: Mentor: Aidee Aidee Gonzalez Gonzalez Advisor: Advisor: Gui Gui Bazan Bazan

Funding provide by: INSET

Recent New s and Outbreaks

• January 27, 2009- More

Salmonella Peanut Butter Recalls Coming (5)

• June 2008- Canada listeria
• utbreak 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
• f 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
• nly 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
• Possible additional costs for

meat samples. (4) www.water-research.net/images/ coliformcolony.jpg

Expanding Uses of Organic Expanding Uses of Organic Polymers and Polymers and Oligomers Oligomers

• Synthesizing

Synthesizing Oligomers Oligomers

• Prepare new applications

Prepare new applications for for Oligomers Oligomers

• Chacterize

Chacterize oligomers

• ligomers
• 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 6th 2009

Synthesizing and Testing the Synthesizing and Testing the Limitations of Aggregate Limitations of Aggregate Interactions Interactions

• Synthesize conjugated, water

Synthesize conjugated, water soluble soluble oligomer

• ligomer
• Oligomer

Oligomer and peptides form and peptides form aggregates by electrostatic aggregates by electrostatic interactions interactions

• Test dependence of FRET

Test dependence of FRET signal on solvents signal on solvents

http://www.activorcorporation.net/sitebui ldercontent/sitebuilderpictures/peptide1 0.jpg

Cationic Peptide Anionic Oligomer

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 Transfer

Advanced Functional Materials 2008, 18, 3606- 3612

Synthesis of Monomer: Synthesis of Monomer:

Outline Outline-

• 1)

1) Reaction Reaction-

• Dissolve

Dissolve Fluorene Fluorene, , NaOH NaOH, , ptc ptc in DMSO. Add in DMSO. Add Sultone Sultone. . 2) 2) Transfer to acetone solution & Transfer to acetone solution & precipitate precipitate 3) 3) Purification Purification 4) 4) Crystallization Crystallization 5) 5) NMR Confirmation of product. NMR Confirmation of product. 6) 6) Used to create Used to create Oligomer Oligomer

NaOH (aq)

www.bridgat.com/files/Caustoc_so da.jpg

P

ptc

Yield 39% 1.61g

Solvent Investigations:

Milli Pore H20 HPF to Oligomer Ratio

350 400 450 500 550 600 650 Wavelength (nm) R e la tiv e E m is s io n In te n s ity

1.3 : 1.0 1.5 : 1.0 1.75 : 1.0

• The 400nm region is

where the oligomer emissions is.

• The 550nm region is

where the fluorescence emission is.

• Difference charge

concentration which were tested.

• Stability of signals
• ver 60 minutes time

was measured.

• Relavence- 1st peak

compared to 2nd peak intensity.

Further Solvent Investigations

PBS HPF to Oligomer Ratio

375 425 475 525 575 625 Wavelength (nm) Relative E m issio n In ten sity 1.1 : 1.0 1.3 : 1.0 1.5 : 1.0

PB HPF to Oligomer Ratio

350 400 450 500 550 600 650 Wavelength (nm) R e l a ti v e E m i s s i o n I n te n s i ty

1.5 : 1.0 1.1 : 1.0

Solvent Investigation of FRET Signal : Phosphate Buffer vs

Phosphate Buffer Saline vs Milli Pore Water

Milli Pore H20 HPF to Oligomer Ratio

350 400 450 500 550 600 650 Wavelength (nm) Relative Emission Intensity

1.3 : 1.0 1.5 : 1.0 1.75 : 1.0

PB HPF to Oligomer Ratio

350 400 450 500 550 600 650 Wavelength (nm) Relative Emission Intensity

1.5 : 1.0 1.1 : 1.0

PBS HPF to Oligomer Ratio

375 425 475 525 575 625 Wavelength (nm) R elative Em ission Intensity 1.1 : 1.0 1.3 : 1.0 1.5 : 1.0

FRET ratio= Fluorescence emission / Oligomer emission

Fluorscence emission (550nm)/ Oligomer emission (414nm)

1 2 3 4 5 6 1 2 3 H20 PB PBS FRET Ratio Conc 1.0 : 1.5

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

FRET-

• Forester Resonance Energy

Forester Resonance Energy Transfer Transfer

• describes energy transfer between

describes energy transfer between two two chromophores chromophores located less than located less than 10nm from each other 10nm from each other

• Excited electron from donor

Excited electron from donor chromophore chromophore are transferred to lower are transferred to lower levels of energy at acceptor levels of energy at acceptor chromophore chromophore. . * *Chromophores Chromophores almost always arise in almost always arise in

• ne of two forms:
• ne of two forms: conjugated pi

conjugated pi systems systems. .

Isolated Oligomer Fractions

Isolated FPFC4S03 Fract 1-3

• 0.1

0.1 0.2 0.3 0.4 0.5 200 250 300 350 400 Wavelength A b s o r b a n c e ( n m ) Methanol blank Frac 3 H20 blank Frac 2 Frac 1

*Fraction 2 is FPFC4SO3. *We use long UV waves lights to see the fluorescent glow