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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

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

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 interface

Daghestani & Day. Sensors 2010, 10, 9630-9646. Wang lab SPR setup: Reichert SR7500DC dual channel system

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, propagate along the interface

n1 > n2 ϴSPR > ϴCritical

n2 n1

ϴSPR

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

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/

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 and signal strength

http://www.reichertspr.co m/

N.J. de Mol, M.J.E. Fischer (eds.), Surface Plasmon Resonance, Methods in Molecular Biology, 2010.

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.

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/

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.

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
• ligonucleotides
• Selected from randomized libraries
• Interact with biomolecules (e.g.

surface receptors)

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.

Sequence Specificity

SPR for Aptamer-Molecule Interactions

50 100 150 200 250 300 350 500 1000 1500 2000

∆µRIU Time [s]

Sequence A immobilize d on chip Sequence B Sequence C

+ A B C

Scrambled B B + Scrambled C B + 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

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

Aptamer Length vs Affinity

SPR for Aptamer-Molecule Interactions

Zhang et al. Chemical Communications 2013, 49, 9600- 9602. 30 60 90 120 150 180 200 400 600

ΔμRIU Injection time [s]

10mer 9mer 8mer 7mer 6mer 30 60 90 120 150 180 200 400 600

ΔμRIU Injection time [s]

10mer + trigger 10mer + control

Aptamer Affinity Evaluation via SPR

SPR for Aptamer-Molecule Interactions

Battig et al. Biomaterials 2014, 35, 8040-8048. 100 200 300 250 500 Response [µRIU] Time [s]

100 nM 50 nM 25 nM

50 100 150 200 250 250 500

Response [µRIU] Time [s]

100 200 250 500 Response [µRIU] Time [s]

100 nM 50 nM 25 nM 12.5 nM 6.25 nM 3.125 nM

50 100 150 200 250 250 500 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

Application: Loading and Release of Growth Factors

SPR for Aptamer-Molecule Interactions

Battig et al. Biomaterials 2014, 35, 8040-8048.

SPR Examination of DNA Polymerization

SPR for Aptamer-Molecule Interactions

200 400 600 800 1000 1200 1400 1600 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

Application: DNA Polymer Nanoparticles for Signal Amplification

SPR for Aptamer-Molecule Interactions

Chen et al. Small 2013, 23, 3944-3949.

Formation of Polyvalent Aptamers

SPR for Aptamer-Molecule Interactions

Richards et al. Biomacromolecules 2014, 15, 4561- 4569.

Application: Polyvalent Aptamers for Tumor Cell Capture

SPR for Aptamer-Molecule Interactions

Richards et al. Biomacromolecules 2014, 15, 4561- 4569.

Polyvalent Aptamer Triggered Depolymerization

SPR for Aptamer-Molecule Interactions

Gaddes et al. Biomacromolecules 2015, 16, 1382- 1389.

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

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/

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