Thesis

Fabricating protein arrays using Dip-pen Nanolithography techniques for improved sensitivity of biomarker detection

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Awarding institution
  • University of Strathclyde
Date of award
  • 2012
Thesis identifier
  • T13882
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Abstract
  • Dip-pen Nanolithography (DPN) was first established as a fabrication technique in 1999, as an alternative method for printing various small molecules and biomolecules on the low nanoscale. Thirteen years on, DPN plays a far more prominent role in nanotechnology investigations. Early studies demonstrated that a single tip can place a series of molecules on a surface, which have lead to robust, consistent printing methods that are currently applicable in electronics, biosensing and cell interactions. In the present study, DPN was used to develop a platform for the detection of the biomarker, prostate-specific antigen (PSA), using optical microscopy techniques. Investigations into creating a faster patterning method, with reduced feature sizes of antibodies are first detailed. The aim was to develop a diagnostic system that competes with current biomarker detection formats such as ELISA and microarray formats produced by alternative printing techniques. DPN methods were optimised to create PSA immunoassay arrays, which were detected by fluorescence microscopy and gave good limits of detection (LOD). Furthermore, the assay method proved adaptable, as consistent results were obtained from two different functional glass substrates. To improve the sensitivity of PSA detection, the assay was modified to render it compatible with SERS analysis. Two different approaches were investigated. It was shown that reproducible results could be achieved through a conjugate based system, giving an improved calculated LOD compared to that of the fluorescence assay. Finally, the optimised method for printing proteins was used for an alternative application, enzymatically generating a novel SE(R)RS active substrate by DPN. Arrays of streptavidin conjugated horseradish peroxidase (HRP) were fabricated, acting as enzymatic templates for the reaction of a silver crystal growth kit, namely, EnzMet™. The development of the first DPN-made SERS active substrate is discussed. Ultimately this research has optimised a high-throughput protein array printing method using the DPN technique that can be used for creating both a biomarker detection platform and SERS enhancing metal micro-structures. It highlights the importance of combining DPN with a sensitive optical detection method (SERS) for improved biomarker detection.
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DOI
Date Created
  • 2012
Former identifier
  • 1041728

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