Stimuli-responsive reagents enable rapid biomarker capture and separation
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1 Stimuli-responsive reagents enable rapid biomarker capture and separation Barrett J. Nehilla, PhD Nexgenia, Inc 1
2 Evolution of Immunoassay Supports Where are the magnetic nanoparticles? VS VS 96-Well Plates Current Magnetic Microbeads (2 µm) Magnetic Nanoparticles (.2 µm) Surface Area/Volume Lowest Lower 1X Higher Diffusion None Slower 1X faster Magnetic Separation None Rapid Slow or N/A Assay Time Hours-days ~Hour Minutes Nanoparticles offer advantages if separation issues can be addressed 2
3 Stimuli-responsive, Smart Polymers 2 Polymer Smart Polymer Behavior 1.5 Polymer+NaCl Insoluble polymer Absorbance (55nm) 1.5 Soluble polymer Lower Critical Solution Temperature (LCST) Celsius * Sharp and significant response to small stimulus * 3
4 RAFT Polymerization monomer 1 polymer 1 = macrocta RAFT monomers polymer 3 = endfunctionalized polymer Roy, et al. ACS Macro Lett available for conjugation polymer 2 = diblock copolymer available for conjugation 4
5 RAFT Polymerization 4.E-5 3.E-5 3.E-5 Gel permeation chromatography of RAFT-produced, stimuli-responsive polymers 5kDa 1kDa 2kDa dri 2.E-5 2.E-5 1.E-5 5.E-6.E+ 36kDa ml * Excellent and reproducible molecular weight control and very low PDI * 5
6 + A Possible Solution: Stimuli-Responsive Binary Reagent System + Stimuli-responsive mnp Stimuli-responsive biomolecule conjugate Sample (antigen) Stimulus: ph, T, Ι + Stimuli-responsive, biomoleculelabeled mnp Sample (antigen) Co-aggregate of stimuli-responsive mnps, polymer-ab conjugates and captured antigens * Large enough for magnetic separation * Magnetic-based system = little/no instrument modification 6
7 Magnetic Nanoparticles Transmission Electron Micrograph (TEM) Normalized Absorbance Aggregation Kinetics T-mNP ph-mnp Seconds Distribution Dynamic Light Scattering Batch 1 Batch 2 Batch 3 % Separation Separation Kinetics ph-mnp T-mNP Hydrodynamic Diameter (nm) Seconds 7
8 Stimuli-responsive polymer-biomolecule conjugates or + Na2CO3/NaH 2 CO3 ph ~ 9.5 <1% DMSO 2 16 pnipam IgG Conjugate Aqueous GPC UV Signal Retention (ml) 8
9 Stimuli-Responsive Conjugates % Antigen Bound Antigen Binding and Specificity anti-p24 IgG Conjugate Conjugate + PTH Normalized ABS Polymer Conjugate Ab:Ag LCST Behavior Capture (%) mnp and Conjugate Co-Aggregation Celsius Conjugate/mNP Conjugate/no mnp IgG/mNP 9
10 Binary Reagents Outperform Conventional Microbeads % p24 Bound time= does not include 2 minutes at 37 C and 2 minutes of magnetic separation at 37 C Binding Time (Minutes) NXG Binary Reagent Dynabeads The stimuli-responsive binary reagent system captured HIV p24 antigen more rapidly than magnetic microbeads 1
11 Other Targets 1 Oligo Separation Annealed and Biotinylated Oligos Polymer- Streptavidin Conjugate mnp % Depletion fmol pmol SA-pNIPAAM conjugate BSA Separation Biotinylated Protein Streptavidin Biotinylated Polymer mnp * Other captured targets: PfHRP2 (malaria antigen, parathyroid hormone) * % BSA Depletion pmol pmol Streptavidin 11
12 Conclusions Reagent system consists of stimuli-responsive biomolecules and magnetic nanoparticles Many chemistries available to make stimuliresponsive polymer-biomolecule reagents Technology amenable to diverse targets/antigens Magnetic nanoparticles and polymer conjugates respond to stimuli in seconds for complete magnetic separation of targets in < 2 minutes 12
13 Thank You Co-authors and collaborators Thomas H. Schulte, Nexgenia Debashish Roy, Yen-chi Chen, James J. Lai, Patrick S. Stayton, University of Washington, Seattle, WA Funding NIH, LSDF, UW C4C, Coulter Foundation For more information Poster presentation this evening #749 13
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