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

Transcription

1 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

2 Background and motivation 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 2

3 Grainger & Castner, Advanced Materials 20 (2008) 867.

4 Antigen Nanoparticle IgG antibody

5 Controlling IgG orientation using Protein G Protein G can control the orienta;on of Immunoglobulin G (IgG) Binding to the Fc domain of IgG Fab Domain IgG Fc Domain Protein G IgG binding domain of protein G: Protein G B1 Protein G B1 were immobilized onto: Protein G B1 Gold nanopar;cles hnp://

6 Covalent protein immobilization EDC/NHS chemistry - (OEG)-COOH thiol 4 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

7 Covalent protein immobilization Maleimide - Cysteine coupling Maleimide thiol + Protein G B1 cysteine mutant hnps:// science/protein- biology/protein- biology- learning- center/protein- biology- resource- library/pierce- protein- methods/sul]ydryl- reac;ve- crosslinker- chemistry.html

8 Protein G B1 orientation control using cysteine mutant Baugh et al. Langmuir 2010, 26(21),

9 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 x- rays ions 9

10 Synthesized gold nanoparticle are monodispersed and spherical Minor- axis Major- axis 10

11 XPS result of EDC/NHS chemistry 4 Successful protein G B1 immobilization using EDC/NHS coupling

12 XPS result of Maleimide chemistry Successful protein G B1 immobilization using Maleimide-Cysteine coupling

13 ToF-SIMS characterization of Protein G B1 orientation

14 Protein G B1 orientation control using cysteine mutant V21 Cysteine (N-terminus) Σ(C-terminus amino acid peaks) : Σ(N-terminus amino acid peaks) V21 Cysteine > T11 Cysteine Baugh et al. Langmuir 2010, 26(21),

15 Comparison of C-terminus and N-terminus secondary ion peaks C-terminus amino acid N-terminus amino acid V21 Cysteine T11 Cysteine T11 Cysteine V21 Cysteine Asparagine (Asn), C 4 H 4 NO 2 + Methionine (Met), C 5 H 9 OS + Protein orientation can be determined using amino acid specific ToF-SIMS secondary ions

16 Orientation of immobilized protein G B1 Σ(C-terminus amino acid peaks) : Σ(N-terminus amino acid peaks) V21 Cysteine > T11 Cysteine

17 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 17

18 Acknowledgement NESAC/BIO Group David Castner Dan Graham Winston Ciridon Gerry Hammer

19 Thank you for your attention!

20 TEM result and particle analysis TEM of synthesized gold nanopar/cles 125kX magnifica/on ImageJ par/cle analysis 20

21 Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) Ultra High Vacuum (UHV) Primary ion impact causes collision cascade >99% ejected molecules are neutral, <1% are ions Animations from Iontof.com 21

22 Baugh et al. Langmuir 2010, 26(21),

23 Protein conjugation strategies:. - EDC/NHS - With (OEG)-COOH thiol - Maleimide-Cysteine - With protein G B1 cysteine mutant Func/onalized nanopar/cle chemically ac/vated + Protein Purified Unfunc/onalized nanopar/cle Func/onalized nanopar/cle Func/onalized nanopar/cle chemically ac/vated Controlled conjuga/on Uncontrolled adsorp/on Protein AYachment Func/onalized nanopar/cle Without ac/va/on + Protein Purified