Antibody Immobilisation on Gold and Silver Nanoparticles for the

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Antibody Immobilisation on Gold and Silver Nanoparticles for the Powered By Docstoc
					 Antibody Immobilisation on Gold and
  Silver Nanoparticles for the Use in
    Electrochemical Immunoassay
    El t    h i lI




Mateusz Szymanski, Simon Attree and Robert Porter
              Bristol 10.11.2009
Nanoparticles                   Antibodies




Unique properties              Very specific strong
               Nanoparticle – antibody
                    conjugate        binders
Applications of metal nanoparticle-
antibody conjugates:

Lateral flow tests:
Metaloimmunoassays
Biosensing
Probes in light and electron microscopy
 Mechanism of protein binding to metal NPs
                                   g
      Proteins bind to a colloidal gold or silver surface
through their amino acids that form complexes with
the metal.
    1. Hydrophobic interaction.
    2. Electrostatic i t   ti
    2 El t t ti interaction.
    3. Sharing of a lone pair of electrons by silver
       or gold and sulphur/nitrogen atoms.
Mechanism of protein binding to metal NPs
            Hydrophobic attractions:

 Via aromatic amino acids (Tyr, Trp, His and Phe)
Mechanism of protein binding to metal NPs
            Ionic attractions:

 Via carboxylate groups of the Asp, Glu and C-
                 termini group




                        - COO
            +
                +
                    +
Mechanism of protein binding to metal NPs
        Sharing of a lone pair of electrons:
      Via the sulphur atom (Cys, Met) or via
nitrogen atom in imino group of Trp and His
  t oge ato          o g oup o p a d s
         Silver Sol – Antibody Conjugate
                       lit    j   t
              poor-quality conjugates:


                                 Aggregation
Wrong orientation




        Denaturated/Inactive antibody
       Metal NP – Antibody Conjugate
                   ti i ti
                optimisation:

1. Buffer type (PBS, TBS, Borate)
2. Silver sol : antibody ratio
3. Buffer molarity and pH
         Borate, Phosphate,
Buffers: Borate Phosphate Tris
               Flocculation Test:
    Silver sol is stable in suspension as there is a balance
                          p
between electrostatic repulsion and Van der Waals attraction
among the particles. When we add ionic substance the
attracting force becomes greater than the counteraction,
leading to an aggregation.

   Coating the colloidal surfaces with protein molecules can
prevent this instability.
0   2   4   6   8                                  [µg/mL]
                    10 20 30 40 50 - - - - - - BSA [ / L]



                    + NaCl




                    Centrifugation




                    Resuspension




                                                             12
Electrochemical Measurement Process:

                     Ag+ x 106

Silver Dissolution
                                 Silver Quantification

                                   ASV




                                                         13
Electrochemical Measurement Process:



                      No Signal

            ASV




                                       14
Effect of the buffer pH




                          15
Effect of the buffer pH




                          16
Performance of 6 different silver conjugates
    Molarity of the buffer


High ionic strength




Low ionic strength


                             18
pH of the buffer


pH 14




        pI = ?




pH 1


                   19
Antibody Loading

   Gold NP + Anti-Myoglobin Antibody in 0.2 M Borate Buffer pH 6.5




                                                                 20
free antibody compete with conjugate




                                       21
Antibody Loading




                   22
Antibody Loading




     NSB




                   23
 Conclusion:



Silver + anti-Myoglobin   0 2 M H3BO3 pH 6 5 10 µg/mL
                          0.2            6.5,
Silver + anti-Troponin    0.1 M H3BO3 pH 7.5, 10 µg/mL

Gold + anti-Myoglobin     0.1 M H3BO3 pH 6.5, 5 µg/mL
Gold + anti-Troponin
               p                      p         µg/mL
                          0.1 M H3BO3 pH 7.5, 5 µg




                                                        24
Acknowledgements:
   Robert Porter
   Simon Attree
   Smita Thobhani
   James Noble
   Neelam Kumarswami


   www.npl.co.uk/biotech
   mateusz.szymanski@npl.co.uk