臺灣博碩士論文加值系統

利用光纖全反射及逐漸消失場 ( Evanescent field ) 原理配合金
奈米特殊的光學特性，我們發展出 DNA 及 mRNA 免標記光纖定域
式電漿共振生物感測器 (Label-Free Fiber-Optic Localized Plasmon
Resonance Biosensors)。
將金奈米粒子修飾於已除去覆遞h的光纖表面，並修飾上不同長
度（19mer、21mer、23mer、25mer 及27mer）單股寡核苷酸（single strand
oligonucleotide）作為探針（primer），偵測濃度2.5×10-10M ~10-6M的
互補寡核苷酸（complement oligonucleotide, c-oligonucleotide）。推算
出最低偵測極限（limit of detection, LOD）濃度約在1.30×10-9M ~
6.06×10-11M。oligonucleotide鹼基數越多，LOD值就越低。相差兩個
鹼基的oligonucleotide間，推算出LOD值約相差1.5~3倍。
我們將此生物感測器運用在生醫疾病檢測上，與大林慈濟醫院合
作，對僵直性脊椎炎的HLA-B27 mRNA基因表現量進行感測。我們以
同步PCR (real-time PCR) 的 CT值對不同病人的檢體進行相對定
量。 CT值越負者，檢體中HLA-B27 mRNA含量越高。經由本實驗結
果顯示， CT值越負者，其相對訊號變化量亦越多。將 CT值與相對
訊號變化量作圖具有良好的線性關係。初步證實光纖生化感測器在生
醫疾病檢測上的可行性。

Using Fiber-Optic Localized Plasmon Resonance (FO-LPR) method,
we have developed a novel label-free biosensor for DNA and message
RNA (mRNA). Upon functionalization of the colloidal gold surface with
an oligonucleotide probe, the hybridization of the probe with
complementary DNA or mRNA was interrogated by the unique optical
properties of colloidal gold. The thiolated DNA was immobilized onto the
gold surface via the well-known self-assembly chemistry, where thiolated
DNA is assembled onto a gold surface followed by adsorption of a diluent
of an alkanethiol. The diluent not only helps to project the probe DNA off
the surface but also resists nonspecific adsorption onto the gold
nanoparticle surface.
Since the optical properties and hence the attenuated total reflection
spectrum of self-assembled gold colloids on the optical fiber changes
with the adsorbate induced refractive index of the environment near the
colloidal gold surface, the realization of the sensing platform is through
the measurement of intensity from the colloidal gold-modified optical
fiber. The limit of detection for DNA hybridization was
about 10-8~10-11M, depending on the number of base in the target DNA.
The FO-LPR method was further applied to detection of mRNA in
serum samples from patients. This study, we used real-time PCR to
quantify mRNA during HLA-B27 gene expression in patients with
Ankylosing Spondylitis and compared with results from FO-LPR method.
Results show that mRNA formed during HLA-B27 gene expression can
be detected and the sensor response correlate with results from real-time
PCR.

總目錄………………...………………………………………………..Ⅰ
圖表目錄……………………..………………………………………..Ⅳ
中文摘要………………………..……………………………………..Ⅷ
英文摘要……..………………………………………………………..Ⅸ
第一章、緒論………………………………………………………....1
1-1、奈米材料…………………………………………………………..1
1-1-1、奈米材料之簡介……………………………………………...1
1-1-2、金屬奈米粒子之光學特性…………………………………...2
1-1-3、金奈米粒子簡介及應用……………………………………...3
1-2、光纖生物感測器……………………...………………………….5
1-2-1、生物感測器之簡介…………………………………………...5
1-2-2、光纖特性……………………………………………………...6
1-2-3、光纖生物感測器……………………………………………...7
1-3、核酸………………………..……………………………………….9
1-3-1、去氧核醣核酸.………………………………………………11
1-3-2、核醣核酸.……………………………………………………12
1-3-3、聚合酶鏈反應.………………………………………………14
1-4、僵直性脊椎炎…………………………………………………..19
第二章、實驗方法…………………………………………………21
2-1、實驗藥品及儀器設備……………………..…………………...21
2-1-1、實驗藥品…………………………………………………….21
2-1-2、寡核苷酸（oligonucleotide）序列…………………..………..22
2-1-3、儀器設備…………………………………………………….23
2-2、實驗步驟………………………..………………………………..24
2-2-1、製備有機相圓球型金奈米粒子…………………………….24
2-2-2、金奈米粒子固定化於玻璃片表面………………………….24
2-2-3、金奈米粒子固定化於光纖表面…………………………….25
2-2-4、微流體系統………………………………………………….26
2-2-5、光纖電漿共振感測系統之儀器架構……………………….26
2-2-6、定域化電漿共振感測系統靈敏度測定…………………….27
2-2-7、修飾primer結合MCE 混合自我組裝單層………………..27
2-2-8、雜交（Hybridization）條件………………………………..28
2-2-9、再生（Regeneration）條件…………………………………28
第三章、結果與討論……………………...………………………31
3-1、定域化電漿共振感測系統的製備及鑑定…………….…...31
3-1-1、圓球形金奈米粒子之鑑定………………………………….31
3-1-2、圓球形金奈米粒子修飾於玻璃表面之鑑定……………….32
3-2、定域式電漿共振系統偵測去寡核苷酸分子………….…...34
3-2-1、修飾探針寡核苷酸測定其互補寡核苷酸之光譜分析…….35
3-2-2、修飾MCE / MCH對雜交反應的影響……………………...38
3-2-3、緩衝溶液對寡核苷酸雜交及再生反應的影響…………….43
3-3、光纖定域式電漿共振系統偵測寡核苷酸分子…...51
3-3-1、製備光纖定域化表面電漿共振感測器…………………….52
3-3-2、光纖定域化表面電將共振感測器靈敏度測定…………….53
3-3-3、光纖定域式電漿共振系統偵測寡核苷酸分子…………….55
3-3-4、光纖定域式電漿共振系統專一性測試…………………….65
3-3-5、濃度折射率對光纖定域式電漿共振系統訊號之影響…….66
3-4、光纖定域式電漿共振系統感測測僵直性脊椎炎的訊息核
醣核酸 (HLA-B27 mRNA)…………..……………………...67
3-4-1、檢體濃度折射率對光纖定域式電漿共振系統訊號之影響.68
3-4-2、不同探針oligo對DNA-mRNA 雜交的影響………………69
3-4-3、單一檢體在不同濃度下的訊號相對變化量……………….71
3-4-4、不同檢體在不同濃度下的訊號相對變化量……………….72
第四章、結論…………………………………………..……………75
參考文獻……………………………………………………………..76

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