開發偵測焦磷酸酶之生物感測器

Abstract

本篇論文包含兩個部分，使用具有簡單製備以及高靈敏度特性的螢光法及電化學法來進行焦磷酸酶之活性的檢測。兩個部分所使用的原理為利用銅離子來觸發反應，造成訊號發生變化。當加入焦磷酸鹽 (pyrophosphate，PPi) 時，焦磷酸鹽為雙牙基分子，對於二價銅離子具有配位性質。當存在銅離子時，會以兩個焦磷酸鹽螯合住一個銅離子，形成PPi–Cu2+–PPi之錯合物。當加入焦磷酸酶時，焦磷酸酶會催化水解焦磷酸鹽，使焦磷酸鹽分解成兩個磷酸根離子，磷酸根離子配位能力遠小於焦磷酸根，螯合住銅離子能力下降，而釋放出銅離子。本研究兩個部分均利用此原理，讓釋放後之銅離子催化特定之化學反應，藉由不同的偵測方式使訊號產生差異，用以定量焦磷酸酶之濃度。 第一部分是使用銅離子來催化 o-phenyldiamine (OPD) 聚合反應，生成2,3-diaminophenazine。其吸收光譜與所用的電化學訊號源硫化鎘相近，令其無法產生光誘導電流產生，進而抑制所偵測到之電流訊號。 第二部分是使用去氧核醣核酸酶與缺陷 DNA 形成雙股結構，並加入螢光染劑，使螢光染劑嵌入雙股結構中。當樣品中存在有焦磷酸酶時，將切斷預先加入之PPi–Cu2+–PPi錯合物並釋放出銅離子。所釋放之銅離子會進一步與去氧核醣核酸酶結合，而使互補的缺陷 DNA 斷開，斷開的缺陷 DNA 互補序列太少，無法維持雙股結構，而原本嵌入的螢光染劑則被釋放而產生螢光訊號之差異。 此兩種方法擁有高靈敏度及高選擇性來偵測焦磷酸酶可以應用於與焦磷酸酶相關的定量與疾病診斷。

Two brand new biosensors for detection of inorganic pyrophosphatase (PPase) were developed. By using specific complex between pyrophosphate (PPi) and copper ion, PPase was indirectly estimated after hydrolysis of PPi, which released the Cu2+ and triggered further reactions. In first section, released Cu2+ triggered the polymerization of o-phenylenediamine (OPD). Transparent indium tin oxide (ITO) electrode was first coated with poly(diallyldimethylammonium chloride) (PDDA) , CdS and 3,4-diaminobenzoic acid (DBA) layer by layer. Under applying 405 nm excitation light source, CdS was excited and transmitted the excited electron to ITO, led to a photo induced current and read by electrochemical analyzer. In the presence of PPase, PPi–Cu2+–PPi complex was dissembled and released Cu2+ triggered the polymerization of OPD, which covered the electrode surface and shielded the excitation light source for photo induced current. The current difference can be used for quantitation of PPase. In the second section, Cu2+ was used to trigger the reaction of DNAzyme. An artificial DNA sequence containing apurinic/apyrimidinic site was designed and used as substrate of DNAzyme. The fluorescent dye, ATMND, was introduced into the system, which will embed into the dsDNA formed by artificial DNA and DNAzyme. After Cu2+ triggered the DNAzyme and break artificial DNA, embed ATMND released due to the denaturation of dsDNA and turn on the fluorescence emission. The fluorescence intensity was corresponding to the concentration of PPase and can be used for its quantitation. Both presented methods existed several advantages as high sensitivity and selectivity for PPase detection with fast operation and low cost. These developed methods hold the potential application in the diagnosis of PPase-related diseases. A simple and highly sensitive fluorometric method and electrochemical analysis has been developed for inorganic pyrophosphatase (PPase) activity detection. Copper ions can trigger the reaction, which results in effective signal exchange. While, with the addition of pyrophosphate (PPi), the exchanged signal is effectively recovered owing to the strong interaction between PPi and Cu2+. Furthermore, under the catalytic hydrolysis of PPase, the complex of PPi–Cu2+–PPi is rapidly disassembled, and the signal is different. In the first part, copper ions can trigger the o-phenylenediamine into 2,3-diaminophenazine, and the absorption of 2,3-diaminophenazine is closed and competed with CdS, which the signal Source of electrochemical. With more and more 2,3-diaminophenazine formed, the electrochemical signal is decreased. In the second part, we used DNAzyme and apurinic/apyrimidinic site DNA to form dsDNA. We embed the fluorescent dye (ATMND) into dsDNA. Copper ions can trigger the DNAzyme to break down the apurinic/apyrimidinic site DNA. Leading the ATMND escaped, and fluorescent signal is rising. These methods are highly sensitive and selective for copper ions detection. It means the result of detect copper ions same as PPase. Thus, the proposed methods may hold a potential application in the diagnosis of PPase-related diseases the proposed methods may hold a potential application in the diagnosis of PPase-related diseases.