SPR for Aptamer-Based Molecular Interactions in Programmable Materials

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

Outline


• 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


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 Æ 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


• 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 surfacetreated as plasma

• Surface plasmons, from density fluctuations at the interface, propagate along the interface

n1 > n2

ϴSPR > ϴCritical

n2

n1

ϴSPR

Optical Detection


• When momentum of photons matches that of surface plasmons, resonance occurs, based on:

• Light angle• Wavelength• Refractive indices of materials

• Photons excite the plasmonsÆreduction in detected light

• This reduction occurs as resonance angle is approached

Optical detection


• Light illuminated on surface at range of angles

• Output determines angle of minimum reflectivity

• Mass changes at interface alter local refractive index Æalter resonance angle

http://www.reichertspr.com/

Sensor Chip


• 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


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

Flow Cell


• Two channels• Immobilization of ligand on

sample channel• Reference channel with no

ligand

• Both channels treated with analyte for binding analysis


Jahanshahi et al. Scientific Reports 2014, 4, 3851.

Data Analysis


• Kinetic analysis software (TraceDrawer)

• Alignment for start time, response

• Blank subtractions• Kinetic model fitting

• 1:1• 2:1, 1:2• Mass transport

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


SPR Data


• Binding kinetics• Equilibrium analysis• Binding specificity• Molecular interactions

• Protein• Small molecules• Cells• Oligonucleotides

Le et al. Analytica ChimicaActa 2013, 761, 143-148.

Stephenson-Brown et al. Analyst 2013, 138, 7140-7145.

Aptamers


• 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)

SPR for Oligonucleotide 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


0

50

100

150

200

250

300

350

0 500 1000 1500 2000

∆µR

IUTime [s]

Sequence A

immobilized on chip

Sequence B

Sequence C

+

A

BC

Scrambled B

B + Scrambled CB + C (20 base pairs)

+

+

+

B + C (25 base pairs)

Zhang et al. JACS 2012, 134, 15716-15719.

C displaces Bfrom A by forming 25 base pairs

B binds A with 20

base pairs

Application: Tumor Cell Capture and Release


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


Zhang et al. Chemical Communications 2013, 49, 9600-9602.

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

Aptamer Affinity Evaluation via SPR


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

0

100

200

300

0 250 500

Res

pon

se

[µR

IU]

Time [s]

100 nM

50 nM

25 nM

0

50

100

150

200

250

0 250 500

Res

pon

se [

µRIU

]

Time [s]

0

100

200

0 250 500

Res

pon

se

[µR

IU]

Time [s]

100 nM50 nM25 nM12.5 nM6.25 nM3.125 nM

050

100150200250

0 250 500

Res

pon

se

[µR

IU]

Time [s]

100 nM50 nM25 nM12.5 nM6.25 nM3.125 nM

High Affinity Aptamer

Moderate Affinity Aptamer

Low Affinity Aptamer

High

Moderate

Low

Comparison of Anti-PDGF BB Aptamers at 100 nM

Application: Loading and Release of Growth Factors


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

SPR Examination of DNA Polymerization


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

Application: DNA Polymer Nanoparticles for Signal Amplification


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

Formation of Polyvalent Aptamers


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

Application: Polyvalent Aptamers for Tumor Cell Capture


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

Polyvalent Aptamer Triggered Depolymerization


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

Summary


• 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


• 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/


http://www.biologic.com.au/

Acknowledgements


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

SPR for Aptamer-Based Molecular Interactions in Programmable Materials

Science

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