Preparation Of Aptamer Based Organic-inorganic Hybrid Monolithic Column With Gold Nanoparticles As Intermediary For Enrichment Of Proteins

Aptamer is a single-stranded oligonucleotide, which can specifically bind to targets, showing advantages, such as strong affinity, good stability and ease of chemical modification. Organic-inorganic hybrid monolith is a novel monolith material with several advantages, such as ease of preparation, a wide pH stability and good mechanical strength. Gold nanoparticle is a kind of nanoparticle material, which has been widely applied in life science with various advantages, such as ease of preparation, ease of modification and good stability. Recently, gold nanoparticle attached monolith has attracted more and more attention and has been widely used in high performance liquid chromatography, capillary electrophoresis and solid phase micro-extraction, etc.In this work, a novel method for the preparation of aptamer based organic-inorganic hybrid monolithic column with gold nanoparticles as intermediary was developed. First, amino hybrid monolith was fabricated via "one-step" sol-gel condensation. Then, with gold nanoparticle as intermediary, aptamer was modified on the surface of monolith and aptamer based monolith was prepared. Finally, with the combination of aptamer based monolith and the enzymatic reaction, it was applied in the isolation, enrichment and detection of low abundance proteins.1. Preparation of aptamer based organic-inorganic hybrid monolithic column with gold nanoparticle as intermediaryAmino hybrid monolith was synthesized via "one-step" sol-gel condensation. With gold nanoparticle as intermediary, aptamer was modified on the surface of monolith and aptamer based monolith was prepared. This monolith material possessed good performances, such as good mechanical strength, good permeability and stability. This preparation method largely simplified the process of fabrication of gold nanoparticle attached monolith. The material was also characterized with scanning electron microscope (SEM), energy dispersive spectroscopy (EDS) and Fourier transform infrared spectrum (FT-IR). The results demonstrated that hybrid monolith had high quality content of nitrogen,13.3% (w/w), and gold nanoparticle was attached to the surface of monolith with high quality content of Au,74.9% (w/w), and the coverage density of aptamer modified on monolith was 342 pmol/μL.2. Isolation, enrichment and detection of low abundance proteinsIn this work, human a-thrombin was chosen as target protein. With the combination of aptamer based monolith and the enzymatic reaction, it was applied in the isolation, enrichment and detection of thrombin. Under optimal conditions, the enrichment factor for thrombin in the affinity column was up to 5 and assay of thrombin spiked in human serum showed the recovery of 89.9%. These results indicated that a novel protocol for the isolation, enrichment and detection of low abundance proteins was successfully developed.