Glyconanoparticle as a High Affinity Probe for Target Protein Enrichment and Identification
Date Issued
2007
Date
2007
Author(s)
Chien, Yu-Yi
DOI
zh-TW
Abstract
In this thesis, six Pk encapsulated AuNPs (Pk-AuNPs) with the combination of three different sizes (4 nm, 13 nm and 20 nm) and two different lengths of linker were synthesized. The carbohydrate ligand amount on each AuNP was determined by anthrone method. For the Pk ligand with short linker, there are 60, 826 and 1498 ligands on each 4 nm, 13 nm and 20 nm AuNPs, respectively, while those for the long linker ligand are 113, 1298 and 1970, respectively.
The multivalent interaction between Pk-AuNPs and B subunit of shiga-like toxin (Slt-B) were evaluated by surface plasma resonance (SPR) competition assays. The inhibition avidity results showed that the binding affinity was affected by nanoparticle size, linker length and the ligand density on nanoparticle surface. In comparison with the binding affinity of mono Pk ligand with Slt-B, the highest affinity increment was showed by the 20-Pk-l-AuNP (20 nm size and long tether) and its binding affinity was enhanced by 4.48x108 times.
Pk-AuNP was also used as affinity probe to simultaneously enrich and isolate the target PA-IL at a femto-mole level from a protein mixture. The captured protein was directly identified by treating with protease and analyzing the hydrolytic peptide fragments. Furthermore, the binding-epitope-containing peptides were revealed by MS-MS analysis of remained peptides on AuNP after washing step.
The high affinity AuNP probe was also used as affinity probe to purify recombinant Slt-B from E. Coli cell lysat. The purity of extracted protein is higher than 95% based on the MALDI-TOF and SDS-PAGE analyses, and the purification yield is 1.085 mg of Slt-B from 100 mL of cell culture.
Combination with sliver enhancement, a Pk-AuNP based method for the detection of shiga-like toxin was developed. The monoclonal antibody for the Slt-A was immobilized on the glass slide. Thus, the toxin was captured by antibody and the carbohydrate-binding site (Slt-B) was exposed. The presence of toxin was then visualized by staining the chip with the high affinity Pk-AuNP followed by silver enhancement. Although the detection limit of current un-optimized Pk-AuNP based method is not as sensitive as ELISA, our method provides a relative cost effective, rapid and accurate platform.
Subjects
醣
金奈米粒子
綠膿桿菌
類志賀氏毒素
carbohdydrate
gold nanoparticle
Pseudomonas aeruginosa
shiga-like toxin
Type
thesis
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