Recently, a novel diffractive-based encoded system has been developed for various multiplexed suspension biological assays. These microparticles, which are manufactured using photolithography of a commercial epoxy-based negative photoresist (SU-8), contain micrometre-sized diffractive elements and can encode millions of unique codes. In this thesis, the preparation and surface modification of different diffractive microparticles; the attachment of a range of biological molecules (e.g. proteins and peptides) onto the functionalised surfaces; and different on-bead analytical techniques for surface characterisation are described. From a thermodynamic study of a multiplexed immunoassay for immunoglobulins (Ig, MW ~150 kDa), the immobilised probe molecules exhibit high affinity (Kd = 9 ± 3 nM) and excellent specificity (S/N >36:1) for the target analytes. The suspension assay resembles solution-like reaction kinetics allowing detection of multiple target proteins in <20 min. The encoded microparticles can also be used for quantifying small proteins, such as cytokines (MW ~20 kDa). In a particle-based sandwich suspension immunoassay, human tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) are detected and compared with conventional enzyme-linked immunosorbent assays (ELISA). Correlation coefficients (R2) for the two cytokines are found to be >0.96. Intra-assay variability (%CV) is determined to be <25%. The sensitivity of the multiplexed immunoassay, expressed as Lowest Detection Limit (LDL), is found to be 379 fM and 1.47 pM for TNF-α and IL-6 respectively, and are comparable to the corresponding ELISAs as demonstrated by the suppliers
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:548245 |
| Date | January 2011 |
| Creators | She, Joseph K. |
| Contributors | Roach, Peter |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/209093/ |
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