Author: Andrew Oliver Jane
Jane, Andrew Oliver, 2014 New Approaches in Porous Silicon Based Optical Immunosensors, Flinders University, School of Chemical and Physical Sciences
This electronic version is made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact firstname.lastname@example.org with the details.
The rapid and reliable detection of diseases and pathogens is essential to modern healthcare systems. Development of new and more efficient sensing techniques is continuously being undertaken to meet this requirement with a large focus on immuno- and biosensing devices. The work conducted in this thesis seeks to address these needs via the development of new detection mechanisms and receptor immobilisation techniques for porous silicon (pSi) based optical immunosensors. Initial investigations focused on the fabrication of an appropriate pSi optical sensor platform via changes in the electrochemical etching parameters. Higher current density applied during the etching cycle resulted in increased pore size, porosity and etching rate of n-type pSi. Monolayer pSi containing higher levels of porosity were demonstrated to be more sensitive to changes in refractive index through interferometric reflectance spectroscopy (IRS). Optimisation of these parameters yielded a sensitive and flexible sensor platform. Development of a new absorbance based pSi optical biosensor was then undertaken. Detection of a human IgG analyte was achieved via a cascade of immunological reactions at the pore walls to form a sandwich assay. The detection strategy involved an alkaline phosphatase (AP) labelled secondary antibody and precipitation of the enzyme substrate 5-bromo-4-chloro-3-indoyl phosphate (BCIP)/nitro blue tetrazolium (NBT) within the porous matrix. The intense colour change and strong absorbance of the biocatalysed BCIP/NBT compounds at 600 nm provided a measurable response on the intensity of the reflected optical profile of the porous layer. This approach yielded a limit of detection of 2.14 ng/mL, well within the working range required for analysis of clinical samples. Following development of the new pSi sensor, a special protein based IgG affinity coating was investigated as a new method of receptor immobilisation on optical sensors. Adaptation of a previously reported genetically modified bacterial surface layer (S-layer) protein from Lysinibacillus sphaericus containing twin IgG binding domains (SbpA31-1064/ZZ) provided a route to the formation of a self-assembling protein layer capable of immobilising receptor IgG molecules with defined orientation. In vitro self-assembly of purified recombinant rSbpA31-1068/ZZ fusion protein was demonstrated by the formation of crystalline protein layers on various surface chemistries. IgG binding capacity was shown on rSbpA31-1068/ZZ coated ELISA microtiter plates via the immobilisation of IgG capture antibodies and detection of human IgG and human Interleukin-6 analytes. Integration of this coating into the previously developed pSi biosensor yielded a general improvement in sensor performance compared to covalent attachment of capture antibodies indicating that this new approach resulted in less receptor inhibition and greater numbers of viable binding sites. Finally, the development of a new pSi optical interferometric biosensor based on metallic deposition was investigated. Chemical reduction of silver and deposition within gold treated pSi was found to result in a significant decrease to the EOT of the material due to a refractive index change. This refractive index 'contrast' enhancement was demonstrated on both gold nanoparticle decorated pSi and gold plated pSi and optimised to provide maximum signal change. An enzyme mediated silver deposition system was then developed using alkaline phosphatase and a synthesised enzyme substrate, hydroquinone diphosphate. Enzyme mediated silver deposition on gold plated pSi was demonstrated and optimised. Finally, adaptation of this system to a pSi optical immunosensor was demonstrated via the detection of human IgG. The new organic and metallic enhancement immunosensors developed in this thesis demonstrate strong sensor platforms and with further investigation may be viable as future diagnostic techniques. In addition, the S-layer affinity coating has vast potential for use in a variety of immunosensors and a swath of other applications including patterned microarrays, biomimetics and drug delivery.
Keywords: biosensor,porous silicon,immunosensor,protein,s-layer,antibody,interferometry,enzyme,assay,optical sensor
Subject: Chemistry thesis
Thesis type: Doctor of Philosophy
School: School of Chemical and Physical Sciences
Supervisor: Professor Joseph Shapter