Publicação
Interaction between gold nanoparticles and blood proteins to define disease states
| Resumo: | Noble metal nanoparticles constitute promising biosensors due to a high and tailored affinity to biomolecules such as proteins, forming protein coronas of distinct compositions on the surface. Gold nanoparticles (AuNP) are particularly interesting for a relatively easy, quick and inexpensive synthesis, low toxicity and ease of functionalization with bifunctional molecules, which typically have thiol groups bound to the AuNP surface and bio-friendly chemical groups at the opposite end, allowing for controlled protein adsorption. Functionalized AuNP can be used as probing agents for blood samples and health states determined by the protein corona composition, which is divided into a strongly bound innermost hard corona and a looser external soft corona. The objective of this work was to further understand the behaviour of plasma proteins integrating the corona. To attain these objectives, conjugates were prepared with ca. 15 and 40 nm diameter AuNP and two important plasma proteins: serum albumin and fibrinogen. AuNP synthesis was by a modified Turkevich method, with diameter and concentration determined by UV-Vis spectroscopy. AuNP were functionalized with 11-mercaptoundecanoic acid and conjugated with bovine or human serum albumin or fibrinogen. These single protein conjugates were evaluated for colloidal stability with ionic strength and pH variation through UV-Vis, protein conformational changes through circular dichroism (CD), hydrodynamic diameter changes upon centrifugation through dynamic light scattering (DLS) and electrophoretic mobility and concentration dependent conjugation efficiency were determined through agarose gel electrophoresis (AGE). Analysis of AGE profiles was by the open source electrophoresis gel image processing software eReuss. CD confirmed α-helix loss for conjugated serum albumins. DLS showed a hydrodynamic diameter decrease for centrifuged 42 nm AuNP conjugates, with high polydispersity indexes for 13 nm ones, suggesting aggregation. AGE revealed electrophoretic mobility decreases as the protein:AuNP ratio increases, data fitted to a Langmuir adsorption model. Serum albumin undergoes conformational alterations upon conjugation with AuNP. Centrifugation affects the protein corona, despite its tendency to aggregate AuNP. Overall, CD, DLS and AGE were demonstrated as useful techniques for the characterisation of the protein corona. |
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| Autores principais: | Costa, Carlos Filipe Santos |
| Assunto: | Blood plasma proteins protein corona gold nanoparticles circular dichroism dynamic light scattering electrophoresis |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Noble metal nanoparticles constitute promising biosensors due to a high and tailored affinity to biomolecules such as proteins, forming protein coronas of distinct compositions on the surface. Gold nanoparticles (AuNP) are particularly interesting for a relatively easy, quick and inexpensive synthesis, low toxicity and ease of functionalization with bifunctional molecules, which typically have thiol groups bound to the AuNP surface and bio-friendly chemical groups at the opposite end, allowing for controlled protein adsorption. Functionalized AuNP can be used as probing agents for blood samples and health states determined by the protein corona composition, which is divided into a strongly bound innermost hard corona and a looser external soft corona. The objective of this work was to further understand the behaviour of plasma proteins integrating the corona. To attain these objectives, conjugates were prepared with ca. 15 and 40 nm diameter AuNP and two important plasma proteins: serum albumin and fibrinogen. AuNP synthesis was by a modified Turkevich method, with diameter and concentration determined by UV-Vis spectroscopy. AuNP were functionalized with 11-mercaptoundecanoic acid and conjugated with bovine or human serum albumin or fibrinogen. These single protein conjugates were evaluated for colloidal stability with ionic strength and pH variation through UV-Vis, protein conformational changes through circular dichroism (CD), hydrodynamic diameter changes upon centrifugation through dynamic light scattering (DLS) and electrophoretic mobility and concentration dependent conjugation efficiency were determined through agarose gel electrophoresis (AGE). Analysis of AGE profiles was by the open source electrophoresis gel image processing software eReuss. CD confirmed α-helix loss for conjugated serum albumins. DLS showed a hydrodynamic diameter decrease for centrifuged 42 nm AuNP conjugates, with high polydispersity indexes for 13 nm ones, suggesting aggregation. AGE revealed electrophoretic mobility decreases as the protein:AuNP ratio increases, data fitted to a Langmuir adsorption model. Serum albumin undergoes conformational alterations upon conjugation with AuNP. Centrifugation affects the protein corona, despite its tendency to aggregate AuNP. Overall, CD, DLS and AGE were demonstrated as useful techniques for the characterisation of the protein corona. |
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