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