使用經聚對二甲苯修飾之壓電石英晶體微天平系統開發肺結核分枝桿菌核酸感測器偵測之研究

Abstract

本研究目的為發展一種新型di-para-xylene(parylene)-QCM(PQCM)系統，此系統將具有表面一致性、低介電常數、電子穩定特性之parylene與目前已廣泛應用之高靈敏度、低成本之石英晶體微量天平(Quartz Crystal Microbalance, QCM)結合而成。本研究首先測試PQCM系統之基礎特性，逐項探討該系統之靈敏度、專一性、環境安定特性、以及自組裝單層膜之可行性等條件。以兩步驟之自組裝單層膜法，於parylene表面修飾目標官能基，第一步驟先利用氣體電漿處理parylene包覆之石英晶片進行表面改質，第二步驟再使用矽烷分子在表面形成具有游離官能基之自組裝單層膜，利用此方法以電漿表面改質再經由化學連結劑作用，即可修飾出目標官能基。 本研究擬將所開發之PQCM系統應用於肺結核桿菌之檢測，根據Mycobacterium tuberculosis 上的Rv3618基因設計專一性的探針進行檢測，所設計長度為25 mer之寡核醣探針在尾端分別修飾OH與SH兩種官能基，將探討各種探針之最適固定化方法。藉由直接ㄧ步驟利用氣體電漿處理或兩步驟使用連結劑(linker)的化學修飾方法，將SH-探針與OH-探針固定於反應區上。經過探針最適固定化條件之檢討，結果顯示5’-SH-探針藉由11-Mercaptoundecanoic acid (11-MUA)作為連結劑、3’-OH-探針藉由香草醛(vanillin)作為連結劑，可以有效將探針固定在PQCM電極表面。分別以11-MUA與vanillin固定DNA探針之PQCM系統，將可針對樣品中是否存在具有互補核苷酸序列之DNA片段及其數量進行定性及定量檢測。分別探討雜合溶液、溫度、時間，測試出最適雜合條件及效率，進而偵測目標序列。結果顯示以11-MUA固定SH-探針之PQCM系統可偵測25.96– 519.2 pg/μL範圍，噪訊比(S/N ratio)為3.2305；以Vanillin及靜電吸附方法固定之OH-探針分別可偵測範圍為129.8– 519.2 pg/μL與 2.596 – 5.192 pg/μL，其噪訊比為1.925、8.6987。 PQCM系統具有便宜、可重複使用、環境安定性佳、單層膜改質安定、溫度穩定性、抗酸鹼腐蝕等優點。PQCM系統上之新型兩步驟之自組裝單層膜技術，也可適用於其他生物感測器上，作為防疫或臨床檢驗之應用。

Parylene have the property of surface regularity, low permittivity, electric stability, biocompatible, low bulk permeability, hydrophobic and resistant to damage by acid and bases. This makes them good physical, chemical, and biological barrier material. Further, a quartz crystal microbalance (QCM) sensor is a mass-sensitive sensor capable of measuring very small mass changes in nanogram levels. QCM technology has been widely used and high sensitivity and low cost. QCM are suitable transducers for chemical and biochemical sensing in general. In this study, the goal is combine parylene and QCM to developed a novel di-para-xylene(parylene)-QCM (PQCM) system. First, we test basic quality of the PQCM system, including sensitivity, specificity, and stability. All of the above, the two-step self assemble monolayer method might modify parylene to form objective group on the surface. First step, PQCM chip coated parylene processed to treat surface by plasma. Then, it reacted with silano to form amino group on the surface. Therefore, we used the PQCM system to apply for detection of Mycobacterium tuberculosis. First, we designed the specific probes (25 mer) and synthetic oligonucleotide targets (32 bp) from the M. tuberculosis RV3618 gene for the detection of M. tuberculosis. Two of oligonucleotide probe designed, one was modified thiol group at 5 terminus, and the other was modified hydroxyl group at 3 terminus. Using two methods, surface plasma modification and immobilizing with linkers, confirmed optimum immobilization methods. The result revealed the 5’-SH-probe used linker of 11-Mercaptoundecanoic acid (11-MUA) and 3’-OH-probe used linker of vanillin to immobilize probe on reaction area of QCM chip. DNA probes were immobilized on the sensor surface of a PQCM device and the hybridization between the immobilized probe and the target of complementary sequence in solution was monitored. Then, the optimum hybridization efficiency, including buffer, temperature, and time, was tested and used to detect target sequence. The result revealed that SH-Probe immobilized with 11-MUA linker detected scope from 25.96 to 519.2 pg/μL and the S/N ratio was 3.2305. Another, OH-Probe immobilized with vanillin and static electricity detected scope from 129.8 to 519.2 pg/μL and from 2.596 to 5.192 pg/μL, and the S/N ratios were 1.925 and 8.6987. The PQCM system offered many advantages including cheap, reusing, resisting electric interference, stability, measuring large sample, working in high temperature, and operates well in complex condition. It could apply other model biosensor for epidemic control and clinical detection.