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SPR for Aptamer-Based Molecular Interactions in Programmable Materials
- 1. SPR for Aptamer-Based Molecular Interactions in Programmable Materials Reichert Technologies Webinar September 22, 2015 Erin Gaddes The Wang Lab: Biomolecular & Biomimetic Materials The Pennsylvania State University University Park, PA 16802 SPR for Aptamer-Molecule Interactions
- 2. Outline SPR for Aptamer-Molecule Interactions • Introduction to surface plasmon resonance (SPR) • What is it? How does it work? • Samples and detection strategies • Data analysis • SPR for analysis of oligo-biomolecule interactions • Cell Capture and Release • Growth factor loading and release • Signal amplification
- 3. Surface Plasmon Resonance SPR for Aptamer-Molecule Interactions Technique used to examine molecular interactions in real time • Sensor chip contains immobilized ligand • Microfluidic system delivers analyte • Optical measurement system: changes in local refractive index changes in mass at sensor chip-solution Daghestani & Day. Sensors 2010, 10, 9630-9646. Wang lab SPR setup: Reichert SR7500DC dual channel system
- 4. Optical Detection SPR for Aptamer-Molecule Interactions • Light entering the prism above the critical angle is totally internally reflected • These photons produce an evanescent wave at surface interface • Mobile electrons of metal surface treated as plasma • Surface plasmons, from density fluctuations at the interface, n1 > n2 ϴSPR > ϴCritical n2 n1 ϴSPR
- 5. Optical Detection SPR for Aptamer-Molecule Interactions • When momentum of photons matches that of surface plasmons, resonance occurs, based on: • Light angle • Wavelength • Refractive indices of materials • Photons excite the plasmons reduction in detected light • This reduction occurs as resonance angle is approached
- 6. Optical detection SPR for Aptamer-Molecule Interactions • Light illuminated on surface at range of angles • Output determines angle of minimum reflectivity • Mass changes at interface alter local refractive index alter resonance angle http://www.reichertspr.co m/
- 7. Sensor Chip SPR for Aptamer-Molecule Interactions • Sensor chip consists of glass coated with a thin metal layer • Metal functionalized for immobilization of ligand • Amine coupling (EDC/NHS) • Streptavidin/neutravidin- biotin • Gold-thiol • Polymer matrix for balance between binding sites andhttp://www.reichertspr.co N.J. de Mol, M.J.E. Fischer (eds.), Surface Plasmon Resonance, Methods in Molecular
- 8. Flow Cell SPR for Aptamer-Molecule Interactions • Two channels • Immobilization of ligand on sample channel • Reference channel with no ligand • Both channels treated with analyte for binding analysis http://www.reichertspr.co m/ Jahanshahi et al. Scientific Reports 2014, 4, 3851.
- 9. Data Analysis SPR for Aptamer-Molecule Interactions • Kinetic analysis software (TraceDrawer) • Alignment for start time, response • Blank subtractions • Kinetic model fitting • 1:1 • 2:1, 1:2 • Mass transport depletion considerations N.J. de Mol, M.J.E. Fischer (eds.), Surface Plasmon Resonance, Methods in Molecular Biology, 2010. http://www.reichertspr.co m/
- 10. SPR Data SPR for Aptamer-Molecule Interactions • Binding kinetics • Equilibrium analysis • Binding specificity • Molecular interactions • Protein • Small molecules • Cells • Oligonucleotides Le et al. Analytica Chimica Acta 2013, 761, 143-148. Stephenson-Brown et al. Analyst 2013, 138, 7140- 7145.
- 11. Aptamers SPR for Aptamer-Molecule Interactions • Merits • Robust • High throughput chemical synthesis • Little batch-to-batch variation • No significant immunogenicity http://www.amsbio.com/ 2013 • Nucleic acid aptamers: • Short, single-stranded oligonucleotides • Selected from randomized libraries • Interact with biomolecules (e.g. surface receptors)
- 12. SPR for Oligonucleotide Interactions SPR for Aptamer-Molecule Interactions • Biosensors • Sequence specificity • Hybridization • DNA polymers • Competitive displacement • Aptamer affinity • VEGF • Thrombin • PTK7 receptors Hasegawa et al. Sensors 2008, 8, 1090-1098. Chen et al. Biosensors and Bioelectronics 2014, 61, 83-87.
- 13. Sequence Specificity SPR for Aptamer-Molecule Interactions 0 50 100 150 200 250 300 350 0 500 1000 1500 2000 ∆µRIU Time [s] Sequence A immobilize d on chip Sequence B Sequence C + A B C Scrambled B B + Scrambled CB + C (20 base pairs) + + + B + C (25 base pairs) Zhang et al. JACS 2012, 134, 15716-15719. C displaces B from A by forming 25 base pairs B binds A with 20 base pairs
- 14. Application: Tumor Cell Capture and Release SPR for Aptamer-Molecule Interactions Zhang et al. JACS 2012, 134, 15716-15719. Cell Release Complementary Sequence Release Functional Scrambled Cell Catch Hydrogel regeneration Aptamer Display
- 15. Aptamer Length vs Affinity SPR for Aptamer-Molecule Interactions Zhang et al. Chemical Communications 2013, 49, 9600- 0 30 60 90 120 150 180 0 200 400 600 ΔμRIU Injection time [s] 10mer 9mer 8mer 7mer 6mer 0 30 60 90 120 150 180 0 200 400 600 ΔμRIU Injection time [s] 10mer + trigger 10mer + control
- 16. Aptamer Affinity Evaluation via SPR SPR for Aptamer-Molecule Interactions Battig et al. Biomaterials 2014, 35, 8040-8048. 0 100 200 300 0 250 500 Response [µRIU] Time [s] 100 nM 50 nM 25 nM 0 50 100 150 200 250 0 250 500 Response[µRIU] Time [s] 0 100 200 0 250 500 Response [µRIU] Time [s] 100 nM 50 nM 25 nM 12.5 nM 6.25 nM 3.125 nM 0 50 100 150 200 250 0 250 500 Response [µRIU] Time [s] 100 nM 50 nM 25 nM 12.5 nM 6.25 nM 3.125 nM High Affinity Aptamer Moderate Affinity Aptamer Low Affinity Aptamer High Moderat e Low Comparison of Anti-PDGF BB Aptamers at 100 nM
- 17. Application: Loading and Release of Growth Factors SPR for Aptamer-Molecule Interactions Battig et al. Biomaterials 2014, 35, 8040-8048.
- 18. SPR Examination of DNA Polymerization SPR for Aptamer-Molecule Interactions 0 200 400 600 800 1000 1200 1400 1600 0 1000 2000 3000 4000 ΔμRIU Time [s] DI DM1 + DM2 DNA Polymer DM1 DM2 DM1 + DM2 Chen et al. Small 2013, 23, 3944-3949. DI is immobilized on sensor chip
- 19. Application: DNA Polymer Nanoparticles for Signal Amplification SPR for Aptamer-Molecule Interactions Chen et al. Small 2013, 23, 3944-3949.
- 20. Formation of Polyvalent Aptamers SPR for Aptamer-Molecule Interactions Richards et al. Biomacromolecules 2014, 15, 4561-
- 21. Application: Polyvalent Aptamers for Tumor Cell Capture SPR for Aptamer-Molecule Interactions Richards et al. Biomacromolecules 2014, 15, 4561-
- 22. Polyvalent Aptamer Triggered Depolymerization SPR for Aptamer-Molecule Interactions Gaddes et al. Biomacromolecules 2015, 16, 1382-
- 23. Summary SPR for Aptamer-Molecule Interactions • SPR is a technique to detect molecular interactions in real time via alterations in local refractive index • Used to determine binding specificity, kinetics, and molecular interactions between proteins, nucleic acids, cells, and other small molecules • Nucleic acid aptamers have ability to bind targets with tunable affinity • SPR utilized to evaluate aptamer specificity, DNA hybridization, and triggered dissociation
- 24. Resources SPR for Aptamer-Molecule Interactions • Mol, Nico J. de, and Marcel J. E. Fischer, eds. Surface Plasmon Resonance Methods and Protocols. New York, NY: Humana Press, 2010. • Wong, Chi Lok, and Malini Olivo. “Surface Plasmon Resonance Imaging Sensors: A Review.” Plasmonics 9.4 (2014): 809–824. • Reichert Technologies: http://www.reichertspr.com/ • TraceDrawer: http://www.ridgeview.eu/software/tracedrawer/ • Scrubber: http://www.biologic.com.au/
- 25. Acknowledgements SPR for Aptamer-Molecule Interactions Funding: • National Science Foundation (DMR 1322332) • Pennsylvania State College of Engineering • Pennsylvania State Materials Research Institute • Dr. Yong Wang • The Wang Lab • Dr. Mark R. Battig • Dr. Niancao Chen • Shihui Li http://www.mri.psu.edu/about/millennium-science- complex.asp • Reichert Technologies