| Summary: | 碩士 === 國立中正大學 === 化學暨生物化學研究所 === 99 === In this research, the Particle Plasmon Resonance（PPR）sensing system combining with the hybridization technique is used to detect methylated oligonucleotide. We design the probe and its complementary oligonucleotide from the part of sequence which is easily methylated in lung cancer. Then we conjugated the probe on the surface of gold nanoparticles and a specific biosensor is developed for this application.
The gold nanoparticles for PPR sensing were prepared by chemical reduction and were modified on substrate by sol-gel and chemisorption reactions. Finally, we modified the probe on the surface of gold nanoparticles with two different approaches and observed the hybridization reactions.
Results show that direct conjugation of a thiol-modified probe on gold nanoparticle surface can detect the complementary oligonucleotide. Moreover, we developed another way to conjugate the probe by using the N-Succinimidyl 6-maleimidohexanoate（EMCS）to be a linker. Unlike the former linker, EMCS without the activation process is able to conjugate with the probe. EMCS reacts with amino group on the gold nanoparticle surface and use carbon-sulfur bond to fix the probe on the gold nanoparticle surface. Based on this approach, the detection limit is about 10-10 M and the binding constant is about 108 M-1. The results are similar to those by method of direct conjugation of a thiol-modified probe. Hence the EMCS conjugation technology is another feasible modification approach to conjugate oligonucleotides. Preliminary show that this sensing system has the ability to detect methylation DNA.
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