Peptide Mediated Nanoparticle Assembly investigated using Small Angle X-ray Scattering
Author(s)
Gryko, Piotr
Type
Thesis
Abstract
Nanoparticle systems arranged through bio-chemical self-assembly
mechanisms display novel electronic and optical characteristics applicable
to biosensing, tissue engineering, therapeutics and drug delivery.
Metallic Nanoparticles such as those composed of gold or CdSe are
expected to replace standard chemical fluorophore due to their high
optical scattering cross-section. The colloidal nature of nanoparticles
necessitates the engineering of NPs surface chemistry to control particle
stability and assembly order. The high intensity of modern synchrotron
sources, combined with the high electron density contrast of metallic
NPs, allows for fast, sensitive, in-situ synchrotron measurements. Here
Small Angle X-ray scattering is used to observe nanoscale structural
changes and relate them to changes in polypeptide mediated interaction
potential, particle surface charge and mobility within aggregates. These
results enable strategies for optimising the structure and the optical
characteristics of NP assemblies, which are vital for their performance
as colourimetric sensors. Here three NP systems are investigated and
characterised:
(1) Gold nanoparticles assembled using de novo designed polypeptides
developed by D. Aili (Chapter 3). The assembly is achieved
by the folding dependent bridging of the particles induced by the
heteroassociation of immobilised helix-loop-helix polypeptides and a
complementary non-linear polypeptide found in solution. Changes in
the optical properties of the assembly as a function of particle size and
linker concentration are observed, with SAXS used to obtain in-situ
structural information on the aggregates. The particles are found to
assemble into highly ordered close packed structures with an interparticle
spacing of 4.8±0.4nm, corresponding to the length of a folded
polypeptide. Changes in particle ordering, aggregation dynamics and
mass fractal dimension are observed as a function of linker concentration
and particle size. These transitions are related to changes in linker
mediated interaction potential, particle surface charge and mobility
within the aggregates.
(2) Interaction of BSA with AuNP aggregates (Chapter 4). Bovine
serum albumin (BSA) is a vital component of standard immunological
assays and it is often extended for use in de-novo assays based on
peptide modified metallic nanoparticles. Whilst a wide range of plasma
proteins such as BSA and HSA have been shown to associate with
nanoparticles, there is little work done on the effect of BSA on NP
assemblies, which are highly dependent on particle stability. Here the
effect of BSA on AuNP aggregates is characterised, using stability
assays, DLS, UV-VIS and SAXS. Far from being an inert component,
the introduction of BSA directly alters the structure and interaction
of AuNP aggregates. Temperature is observed to be a factor in these
interactions with a specific affinity for hydrophobic systems.
(3) A Quantum Dot - Gold Nanoparticle conjugate assay for the
detection of urokinase plasminogen activator (uPA) (Chapter 5). Proteolytic
enzymes are used as disease biomarkers for several forms of
cancer. The surface assembly of gold nanoparticles onto the surface
of quantum dots is demonstrated, with the growth in conjugate size
characterised using in-situ SAXS. Complete conjugate disassembly is
observed upon the addition of the enzyme uPA, however QDs luminescence
does not fully recover. This indicates that other effects may result
in the permanent quenching of quantum dots.
The work presented here is designed to guide in the synthesis of general
principles for assembly of NPs by biomolecular mediated bridging,
whilst contributing to the fundamental understanding of the assembly
process.
mechanisms display novel electronic and optical characteristics applicable
to biosensing, tissue engineering, therapeutics and drug delivery.
Metallic Nanoparticles such as those composed of gold or CdSe are
expected to replace standard chemical fluorophore due to their high
optical scattering cross-section. The colloidal nature of nanoparticles
necessitates the engineering of NPs surface chemistry to control particle
stability and assembly order. The high intensity of modern synchrotron
sources, combined with the high electron density contrast of metallic
NPs, allows for fast, sensitive, in-situ synchrotron measurements. Here
Small Angle X-ray scattering is used to observe nanoscale structural
changes and relate them to changes in polypeptide mediated interaction
potential, particle surface charge and mobility within aggregates. These
results enable strategies for optimising the structure and the optical
characteristics of NP assemblies, which are vital for their performance
as colourimetric sensors. Here three NP systems are investigated and
characterised:
(1) Gold nanoparticles assembled using de novo designed polypeptides
developed by D. Aili (Chapter 3). The assembly is achieved
by the folding dependent bridging of the particles induced by the
heteroassociation of immobilised helix-loop-helix polypeptides and a
complementary non-linear polypeptide found in solution. Changes in
the optical properties of the assembly as a function of particle size and
linker concentration are observed, with SAXS used to obtain in-situ
structural information on the aggregates. The particles are found to
assemble into highly ordered close packed structures with an interparticle
spacing of 4.8±0.4nm, corresponding to the length of a folded
polypeptide. Changes in particle ordering, aggregation dynamics and
mass fractal dimension are observed as a function of linker concentration
and particle size. These transitions are related to changes in linker
mediated interaction potential, particle surface charge and mobility
within the aggregates.
(2) Interaction of BSA with AuNP aggregates (Chapter 4). Bovine
serum albumin (BSA) is a vital component of standard immunological
assays and it is often extended for use in de-novo assays based on
peptide modified metallic nanoparticles. Whilst a wide range of plasma
proteins such as BSA and HSA have been shown to associate with
nanoparticles, there is little work done on the effect of BSA on NP
assemblies, which are highly dependent on particle stability. Here the
effect of BSA on AuNP aggregates is characterised, using stability
assays, DLS, UV-VIS and SAXS. Far from being an inert component,
the introduction of BSA directly alters the structure and interaction
of AuNP aggregates. Temperature is observed to be a factor in these
interactions with a specific affinity for hydrophobic systems.
(3) A Quantum Dot - Gold Nanoparticle conjugate assay for the
detection of urokinase plasminogen activator (uPA) (Chapter 5). Proteolytic
enzymes are used as disease biomarkers for several forms of
cancer. The surface assembly of gold nanoparticles onto the surface
of quantum dots is demonstrated, with the growth in conjugate size
characterised using in-situ SAXS. Complete conjugate disassembly is
observed upon the addition of the enzyme uPA, however QDs luminescence
does not fully recover. This indicates that other effects may result
in the permanent quenching of quantum dots.
The work presented here is designed to guide in the synthesis of general
principles for assembly of NPs by biomolecular mediated bridging,
whilst contributing to the fundamental understanding of the assembly
process.
Date Issued
2012-03
Date Awarded
2012-04
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Stevens, Molly
Ryan, Mary
Sponsor
Engineering and Physical Sciences Research Council ; Nihon Gakujutsu Shinkōkai
Publisher Department
Materials
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)