開發多重放大機制之光電流DNA生物感測器

Turn-on photoelectrochemical DNA biosensor accompanied with enzyme based dual cascade amplification strategy

左仁德(Jen-Te Tso)

指導教授 ： 謝有容

國立交通大學/理學院/應用化學系所/碩士(2020年)

摘要

本論文之主旨在開發高靈敏度、低成本以及方便檢測炭疽桿菌中致病基因之光電流生物感測器。實驗中透過兩種不同酵素核酸外切酶 III (exonuclease III，Exo III)、限制酶 Nb.BbvCI (nicking enzyme，NE) 來設計有多重循環放大機制與兩階段光電流改變機制的感測器。炭疽桿菌中的致病基因，稱之為目標序列 (target)。於目標序列存在的情況下，會導致本研究中所設計的兩種髮夾型去氧核醣核酸序列 (hairpin，HP) 產生結構上的變形、形成對應辨識區。先供核酸外切酶 III 進行特定的水解作用，重新釋放出目標序列進行循環反應，同時循環後將兩種 HP 水解成兩種殘基序列 (residue)；殘基序列將與修飾在電極上的信標序列 3 (beacon sequence 3，BS3) 互補並使限制酶對其水解。透過序列末端修飾的增益物 rhodamine 6G (R6G)、淬滅物 ferrocene (Fc) 影響修飾於電極上的硫化鎘量子點 (quantum dots，QDs)。以光電流 (photocurrent) 判讀訊號變化，探討炭疽病毒致病基因影響此感測器之程度，藉著雙重循環放大機制以及兩階段光電流改變機制，提高訊號改變程度，訊號隨著目標序列濃度越高而提高，本篇論文藉此檢測並成功能有效定量目標序列，達成開發新型具高靈敏度的生物感測器，盼在未來可做更加廣泛的應用。

關鍵字

致病基因 ； 量子點 ； 光電流 ； 循環放大機制

並列摘要

A photoelectrochemical (PEC) biosensor was developed with signal-on, multiple amplification strategy for the detection of pathogenicity gene. The photocurrent is obtained by CdS quantum dots (QDs) modified indium tin oxide (ITO) electrode. With appropriate excitation light, the photo-current was obtained by migration of excited electron from QDs to ITO directly due to its lower energy gap instead of emitting fluorescence. The obtained photo-current is affected by species close by ITO surface and used for target sequence quantitation. Herein, designed hairpin 1 (HP1) works as a probe, could hybridize with target sequence to form dsDNA, which could be further digested by exonuclease III (Exo III) from its 3’-blunt termini, provides a residue 1 (R1) fragment and release target sequence for further repeating processes. Subsequently, R1 could hybridize with rhodamine 6G (R6G)-tagged hairpin 2 (HP2), triggered the same digestion by Exo III, led a R6G tagged residue 2 (R2) to work as enhancer for QDs and R1 was released for more amplification cycles. After 2 amplification cycles, the produced R1 and R2 were put on ferrocene-tagged beacon sequence 3 (BS3), which used as a quencher for QDs, modified ITO electrode. Both R1 and R2 hybridized with BS3 in different regions, respectively. The R1 triggered the cleavage of related nicking enzyme, remove quenching effect of Fc and restore photocurrent. Moreover, hybridized R2 with R6G tag further enhanced the photocurrent for better performance of biosensor.
As a universal approach, it played an important role for applications in DNA sequence detection. The proposed strategy provided an ultrasensitive electrochemical detection of DNA with photocurrent biosensor. Thus, it held great potential for the development of biosensor platform for the applications in bioanalysis and clinical diagnostics.

並列關鍵字

pathogenicity genes ； CdS quantum dots ； photocurrent ； amplification strategy

參考文獻

1. Kimmel, D.W., G. LeBlanc, M.E. Meschievitz, and D.E. Cliffel, Electrochemical sensors and biosensors. Analytical chemistry, 2012. 84(2): p. 685-707.

2. Byrem, T., P. Bartlett, H. Donohue, B. Voisinet, and J. Houseman, Performance of a commercial serum ELISA for the detection of antibodies to Neospora caninum in whole and skim milk samples. Veterinary parasitology, 2012. 190(1-2): p. 249-253.

3. Kumada, Y., Y. Ohigashi, Y. Emori, K. Imamura, Y. Omura, and M. Kishimoto, Improved lectin ELISA for glycosylation analysis of biomarkers using PS-tag-fused single-chain Fv. Journal of immunological methods, 2012. 385(1-2): p. 15-22.

4. Zhou, Y., C.-Y. Li, Y.-S. Li, H.-L. Ren, S.-Y. Lu, X.-L. Tian, Y.-M. Hao, Y.-Y. Zhang, Q.-F. Shen, and Z.-S. Liu, Monoclonal antibody based inhibition ELISA as a new tool for the analysis of melamine in milk and pet food samples. Food chemistry, 2012. 135(4): p. 2681-2686.

5. Tohgi, K., K. Kohno, H. Takahashi, H. Matsuo, S. Nakayama, and E. Morita, Usability of Fag e 2 ImmunoCAP in the diagnosis of buckwheat allergy. Archives of dermatological research, 2011. 303(9): p. 635.