金奈米電極核酸感測器之製造與電性量測研究

Abstract

近幾年來，有許多以電性偵測為基礎的核酸分析技術已經被開發出來並且應用於基因疾病的診斷。在那些電性偵測技術中，其偵測方法最主要特色為避免使用聚合酶連鎖反應(PCR)來放大核酸(DNA)分子的濃度與避免使用昂貴的光學設備以擷取螢光DNA的放射訊號。 本論文主要在於研究以醛類衍生的核酸分子(aldehyde-derivatIzed DNA) 來達到電性偵測。在本實驗中，我們結合電子微影技術及lift-off 技術在矽晶圓表面上製造出次奈米間隙的金奈米電極，在兩電極中所暴露出的二氧化矽層先修飾上氨基分子接著在固定上27-mer 的單股核酸分子(capture DNA)，然後加入已醛基官能化的核酸分子(target DNA)進行專一性的辨識。經由PBS緩衝溶液及去離子水的沖洗，核酸晶片以具有氧化性的多倫試劑(Tollen's reagent)處理；當target DNA 的序列與 capture DNA 互補時，銀離子將會被限定在target DNA的醛基所還原出來並且延著DNA分子的骨架沈積，因此互補的雙股核酸分子被多倫試劑金屬化，而且金奈米電極所增加的電流可以直接被分析器量測出來。

In recent years, a variety of DNA assay techniques based on electrical detection for diagnosis of genetic and pathogenic diseases had been explored. Among those skills, the major characterizations of detection methods were to avoid the use of polymerase chain reaction for amplifying the concentration of analytic DNA and expensive optical microscope equipments to obtain emitting signals from fluorescent DNA. Herein, I investigated a novel strategy for the electrical detection of DNA by aldehyde-derivatized oligonucleotide probes. In this experimental protocol, we combined the shaped electron beam lithography to fabricate sub-nanometer gap between two electrode pads on silicon wafers, and the gold electrodes were fabricated by lift-off process. 27-mer single-stranded nucleic acids called as capture DNA were immobilized on exposed SiO2 surface modified with amino groups between two electrodes. Then analytical DNA (namely targets DNA) functionalized with aldehyde groups were spotted for occurring specific recognition. After washing with PBS buffer and DI water strictly, the DNA chip was handled with Tollen's reagent acted as oxidation agent. When the sequences of the target DNA were complementary to the capture strand, silver ions would be reduced by the aldehyde groups localized on base within DNA and deposited along with the DNA skeleton. Hence, the complementary double-stranded DNA molecules were metallized by Tollen's reagent and the increased currents across gold nanoelectrodes could be measured by analyzer directly.