Design of a Fluorescent Capillary Flow Immunosensor with Increased Sensitivity

1
Design of a Fluorescent Capillary Flow
Immunosensor with Increased Sensitivity

• Katrina Schweiker
• PI Greg Zimmerli
• Biosciences and Technology Branch
• 15 July 2005

2
Overview

• Background
• Objectives
• Results?
• Future Directions
• The Big Picture
• Other Interesting Information
• Acknowledgements

3
Background Definitions

• Gram Negative Bacteria
• E. coli

• Gram Positive Bacteria
• S. aureus

Both images from http//pathmicro.med.sc.edu/f
ox/cell_envelope.htm
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Background Definitions

• Antigen
• Epitope

Epitope
http//pathmicro.med.sc.edu/fox/cell_envelope.htm
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Background Definitions

• Antibody
• How does it differ from a protein?
• Polyclonal vs. Monoclonal

http//www.accessexcellence.org/RC/VL/GG/antiBD_mo
l.html
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Background Basic Technique

• In the 1990s Ligler, et al. developed the
  capillary flow immunosensor
• Based on ELISA
• Presence of antigen detected by fluorescence
  microscopy

http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/E/Elisa.html
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Project Objectives

• Decrease the size of the sensor to 1 cm
• Need to reduce the length dependence of the
  sensitivity of the biosensor
• Increase the sensitivity
• CPG increases surface to volume ratio
• Quantum dots
• Colloidal gold

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Work Completed

• Design of apparatus
• Wire stoppers
• Cleaned Equipment
• Capillary Tube
• CPG
• Sialinization

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Work Completed

• Design of stage for microscope
• Redesign of microscope stage

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Three Types of Experiments

• CPG in a Capillary Tube
• Patterned CPG with anti- E. coli capture
  antibody
• Control was silanized CPG
• Put CPG into capillary tubes
• Ran assay
• Antigen concentration 0.1 mg/mL LPS in PBS
• Tried a Regeneration Procedure
• 0.1 M HCl for 15 minutes at Room Temp Rinse with
  DI H2O
• Plain Capillary Tube
• Patterned Capillary Glass with anti-E. coli
  capture antibodies
• Ran same assay
• Hoped to reduce the noise
• Madtech Dish
• Painstakingly put one CPG bead in dish
• Performed same assay, but left time for diffusion
  to occur (4-12 hr incubations)
• All steps done under a microscope so as not to
  lose the bead

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CPG/Capillary Experiment
Ab CPG before assay
Ab CPG after assay
Conrol CPG before assay
Control CPG after assay
Flow ? ? ?
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Regeneration with 0.1 M HCl Reuse
Ab CPG Regeneration
Control After Regen
Control CPG Regeneration
Flow ? ? ?
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Capillaries with no CPG
Ab coated capillary tube
Control capillary tube
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Madtech Dish Experiment
Ab Ag Regeneration
Ab Ag
Ab Buffer Regeneration
Ab Buffer
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Madtec Dish Experiment
Control Ag Regeneration
Control Ag
Control Buffer Regeneration
Control Buffer
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Future Work

• CPG works in dish
• Understand why CPG in capillaries traps
  fluorophore
• Get access to a pump
• might help wash unbound antibody away in
  capillaries
• Figure out a good image analysis protocol to
  extract data from the pictures
• Look into colloidal gold or quantum dots as an
  alternative method

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The Big Picture
http//www.nasa.gov/centers/glenn/events/vcjan05_p
lanetnews.html
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The Big Picture
Original Credit The Mars Society taken from
http//www.astronomie.de/bibliothek/interview/tart
er/
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The Big Picture
Original Credit NASA JSC taken from
www.bu.edu/bridge/archive/ 2004/12-03/fraser.html
http//ares.jsc.nasa.gov/HumanExplore/Exploration/
EXLibrary/images/Pics/MarsScenario1/08Ascent.gif
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The Big Picture
www.imagineeringezine.com/ e-zine/mars.html
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Fun Facts

• Fluorescence Microscopy
• Other sorts of microscopy

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Summary

• Building biosensors is tricky business
• A lot more work to be done
• It would be good to look into other methods
• Colloidal gold or quantum dot conjugated
  antibodies still rely on the ELISA technique
  there might be a better way

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Acknowledgements

• My Family
• George Makhatadze
• Ann Marie Daniel
• Greg Zimmerli
• Nicole, Mike, and Marius
• NASA Academy
• PA Space Grant

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Questions?