Surface Enhanced Affinity Capture Proteins On Porous Silicon And Laser Desorption/Ionization Mass Spectrometry Read-out

After genomics, proteomics is considered the next step in the study of biological systems. The proteome encompasses all proteins that result from the genome of cells, a tissue or an organism. It is not a static parameter, as is the genome, but a dynamic collection of proteins that reflects both the intrinsic genetic of the cell and the impact of its immediate environment. Compared with the genome, the proteome provides a more realistic view of a biological status and is, therefore, expected to be more useful than gene analysis for evaluating disease presence, progression and response to treatment. The major tasks of proteomics are to separate and analyze the whole proteome and to process the massive amount of data into meaningful results using statistics and bioinformatics. Thus, the ability to visualize the full depth of the proteome in a high-throughput manner is a major goal. Methodologies, which combine higher throughput with the ability to observe differential protein expression levels, have been applied to this goal. An example of such a system is the combination of robotic sample processing to matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Within this paradigm is a modification of MALDI termed surface enhanced laser desorption/ionization (SELDI), which incorporates straightforward sample preparation with on-chip affinity capturing and detection, lessening the need for sample preparation and mass spectrometric expertise. Different SELDI chips, from chromatographic chemistries surfaces that bind many different molecules, to surfaces with a specific biomolecular affinity that bind one specific molecule or molecular class, have been developed to retain different proteins. Porous silicon has attracted much attention because of its high surface area-to-volume ratio, simple fabrication process, and compatibility with some commercialized instruments. This thesis describes the preparations for porous silicon (PSi) based protein chip surfaces used in laser desorption/ionization (LDI) MS. Silver nanoparticles (AgNPs) deposited porous silicon, Niâ…¡-NTA derivatized Ag nanoparticles/porous silicon and antibody immobilized porous silicon for LDI will be presented.Chapter 1 presents a summary of proteomics technology, protein microarray (types of chips, detection methods, substrate materials and porous silicon), MALDI-TOF MS (ionization mechanism, applications, preanalytical sample process and Integrated Selective Enrichment Target), SELDI (surface-enhanced neat desorption LDI and surface-enhanced affinity capture LDI)Chapter 2 describes an improved DIOS (desorption ionization on porous silicon) method for laser desorption/ionization mass spectrometry (LDI MS) by electroless plating of AgNPs on PSi. By addition of 4-aminothiophenol (4-ATP) into the AgNO3 plating solution, the plating speed is slowed down and 4-ATP self-assembled monolayer (SAMs) on AgNPs is formed. Both AgNPs and 4-ATP/AgNPs coated PSi substrates present much higher stability, sensitivity and reproducibility for LDI MS for small molecules than the un-treated porous silicon ones.Chapter 3 develops an affinity chip via chemical conversion of amino groups of 4-ATP/AgNPs coated PSi to Ni"-nitrilotriacetic acid (Niâ…¡-NTA) termini, to purify his-tagged proteins from the sample solution with high concentration salts and denaturants.Chapter 4 presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an Integrated Sample Enrichment Target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on the 3D-structured porous silicon surface, which allows efficient antibody immobilization by surface adsorption in 30-60 min. The hydrophilic nature of the porous silicon surface at molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples and the hydrophobic behavior of the nanostructured surface facilitates liquid confinement during the assay. The performance of the proposed immunoaffinity PSi-ISET iMALDI assay was evaluated by specific detection of Angiotensin I at 10 femtomol level in diluted plasma samples (10 uL,1 nM) and more important, direct adaption of the protocol into standard sample handling robots is possible.