Characteriza*on of Protein G B1 Immobilized Gold Nanopar*cles Using
ToF-‐SIMS and XPS
Yung-‐Chen Wang and David G. Castner University of Washington
• Nanopar;cles’ applica;on in biotechnology – Diagnos;c – Therapeu;cs – Sensing
• Nanopar;cles have unique proper;es – Small size – Op;cal proper;es
• Nanopar;cle behavior in biological systems is highly dependent on their surface proper;es – Important to characterize nanopar/cle surfaces
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Background and motivation
Grainger & Castner, Advanced Materials 20 (2008) 867.
Nanoparticle
Antigen
IgG antibody
http://www.biology.arizona.edu/immunology/tutorials/antibody/structure.html
Controlling IgG orientation using Protein G
• Protein G can control the orienta;on of Immunoglobulin G (IgG) – Binding to the Fc domain of IgG
• IgG binding domain of protein G: – Protein G B1
• Protein G B1 were immobilized onto:
– Gold nanopar;cles
Protein G
IgG
Fab Domain
Protein G B1
Fc Domain
hNp://www.biology.arizona.edu/immunology/tutorials/an;body/structure.html
• EDC/NHS chemistry - (OEG)-COOH thiol
hNps://ja.wikipedia.org/wiki/N-‐%E3%83%92%E3%83%89%E3%83%AD%E3%82%AD%E3%82%B7%E3%82%B3%E3%83%8F%E3%82%AF%E9%85%B8%E3%82%A4%E3%83%9F%E3%83%89
Covalent protein immobilization
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• Maleimide - Cysteine coupling – Maleimide thiol + Protein G B1
cysteine mutant
hNps://www.thermofisher.com/us/en/home/life-‐science/protein-‐biology/protein-‐biology-‐learning-‐center/protein-‐biology-‐resource-‐library/pierce-‐protein-‐methods/sul]ydryl-‐reac;ve-‐crosslinker-‐chemistry.html
Covalent protein immobilization
Protein G B1 orientation control using cysteine mutant
Baugh et al. Langmuir 2010, 26(21), 16434–16441
Approach: characterization methods
• Transmission Electron Microscopy (TEM) – Shape and size distribu/on of nanopar/cles
• X-‐ray Photoelectron Spectroscopy (XPS) – Elemental composi/on and chemical state
• Time of Flight-‐Secondary Ion Mass Spectrometry (ToF-‐SIMS) – Highly sensi/ve molecular informa/on
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x-‐rays
ions
Synthesized gold nanoparticle are monodispersed and spherical
10 Major-‐axis
Minor-‐axis
XPS result of EDC/NHS chemistry
Successful protein G B1 immobilization using EDC/NHS coupling
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XPS result of Maleimide chemistry
Successful protein G B1 immobilization using Maleimide-Cysteine coupling
ToF-SIMS characterization of Protein G B1 orientation
Protein G B1 orientation control using cysteine mutant
Baugh et al. Langmuir 2010, 26(21), 16434–16441
V21 Cysteine > T11 Cysteine
V21 Cysteine (N-terminus)
Σ(C-terminus amino acid peaks) : Σ(N-terminus amino acid peaks)
Comparison of C-terminus and N-terminus secondary ion peaks
Asparagine (Asn), C4H4NO2+ Methionine (Met), C5H9OS+
C-terminus amino acid
V21 Cysteine
V21 Cysteine T11 Cysteine
T11 Cysteine
N-terminus amino acid
Protein orientation can be determined using amino acid specific ToF-SIMS secondary ions
Orientation of immobilized protein G B1
V21 Cysteine > T11 Cysteine Σ(C-terminus amino acid peaks) : Σ(N-terminus amino acid peaks)
Conclusions
• Systematic evaluation of protein G B1 immobilized gold nanoparticles – Detailed characterization using TEM, XPS and
ToF-SIMS
• Ability to control and determine the orientation of immobilized protein – Maleimide-cysteine coupling: controlled
orientation
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NESAC/BIO Group David Castner Dan Graham Winston Ciridon Gerry Hammer
Acknowledgement
Thank you for your attention!
TEM result and particle analysis
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TEM of synthesized gold nanopar/cles 125kX magnifica/on ImageJ par/cle analysis
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• Ultra High Vacuum (UHV) • Primary ion impact causes collision cascade • >99% ejected molecules are neutral, <1% are ions
Animations from Iontof.com
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)
Baugh et al. Langmuir 2010, 26(21), 16434–16441
Unfunc/onalized nanopar/cle
Func/onalized nanopar/cle
Func/onalized nanopar/cle chemically ac/vated
Func/onalized nanopar/cle Without ac/va/on
+ Protein
Func/onalized nanopar/cle chemically ac/vated
+ Protein
Purified
Controlled conjuga/on
Uncontrolled adsorp/on
Protein AYachment
Purified
Protein conjugation strategies: .
- EDC/NHS - With (OEG)-COOH thiol
- Maleimide-Cysteine - With protein G B1 cysteine
mutant