腎功能檢測中尿素與尿酸生化感測器之研究

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Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/3355

Title:	腎功能檢測中尿素與尿酸生化感測器之研究
Other Titles:	Urea and Uric Acid Biosensors for Renal Detection
Authors:	駱永建 Luo, Yung-Chien
Contributors:	杜景順 Do, Jing-Shan 東海大學化學工程與材料工程學系
Keywords:	尿素;尿酸;生化感測器;腎功能 Urea, Uric Acid, Biosensor, Renal
Date:	2006
Issue Date:	2011-04-09T08:31:18Z (UTC)
Abstract:	尿素與尿酸為腎功能檢測與臨床醫療之重要的指標，本論文中分別製備不同聚苯胺(polyaniline, PANI)複合膜/Au為基礎的尿素感測電極與尿酸酵素/銥修飾碳之尿酸感測電極，作為感測尿素與尿酸之感測電極，探討製備所得電極之特性及其感測特性。銨根離子為一非電化學活性物質，為尿素被尿素酵素水解後之代謝產物之一，藉由銨根離子之感測可定量尿素。文中分別以不同製備方式製備具有銨根離子選擇功能之聚苯胺複合膜，包括PANI-Nafion?、PANI-PSSMA(poly(styrenesulfonate-co-maleic acid, sodium type))(I)、PANI-PSSMA(II)、PANI-PSSMA(III)、奈米結構PANI-PSSMA(IV)、奈米結構PANI-Nafion?複合膜於Au上作為感測基礎電極，進行銨根離子感測特性之探討。發現製備所得之PANI(製備圈數=5)-Nafion?/Au與奈米結構PANI(定電流聚合時間為30分鐘)-Nafion?/Au對銨根離子感測靈敏度分別為100.4與836.39 ?A mM-1，偵測極限為0.025與0.005 mM。另一方面，在四種自制之PANI-PSSMA複合膜中，其中以奈米結構PANI-PSSMA(IV)複合膜電極對銨根離子感測靈敏度最高，在25?C環境下，當銨根離子濃度範圍為0 ~ 1.0 mM時得到感測靈敏度為126.2 ?A mM-1。分別以PANI(製備圈數=5)-Nafion?(4 ?l (2 wt%))/Au與奈米結構PANI(定電流聚合時間為30分鐘)-Nafion?(8 ?l (1 wt%))/Au為基礎電極，製備成尿素酵素感測電極。其中以澆鑄4 ?l之Nafion?溶液混合160 U尿素酵素，澆鑄於奈米結構PANI-Nafion?/Au基礎電極上，製備所得之尿素酵素感測電極對尿素的感測靈敏度為87.26 ?A mM-1，偵測極限可達到5 ?M。而利用4 ?l (2 wt%)戊二醛溶液混合320 U之尿素酵素於奈米結構之PANI-PSSMA(IV)/Au基礎電極上，對尿素感測靈敏度為52.02 ?A mM-1。在尿酸感測器之研究中，以銥(20 wt%)修飾碳/碳/Au為基礎電極，在37?C環境下，對過氧化氫之感測靈敏度為36.71 ?A mM-1，應答時間(t90)介於18.4 ~ 25秒，維他命C (AA)與葡萄糖對此種電極干擾極小。以戊二醛交聯16 U之尿酸酵素於銥(20 wt%)修飾碳/碳/Au基礎電極上，形成尿酸酵素/銥(20 wt%)修飾碳/碳/Au電極，在pH 7 PBS (phosphate buffer solution)與37?C下，對尿酸感測靈敏度為23.32 ?A mM-1，此種尿酸酵素感測電極最低酵素負載量為2 U，對尿酸感測靈敏度為14.03 ?A mM-1。直接型尿酸感測器是以銥(5 wt%)修飾碳作為電極，利用微脈衝伏安法(differential pulse voltammetry, DPV)發現AA之氧化電位為0.255 V(vs. Ag/AgCl)，高於尿酸之氧化電位，因此在0.2 V的操作電壓下，對感測電極的干擾極小。而銥(5 wt%)修飾碳電極在pH 8 PBS與37?C下，對尿酸感測靈敏度為1.08 ?A mM-1。利用銥(5 wt%)修飾碳電極在血清與血漿系統中進行感測，其結果發現此種電極可以直接用於人體血清與血漿中感測尿酸，血液中其他物質對感測電極干擾極小。 Urea and uric acid are the important indicators for the kidney diseases and clinic diagnosis. In this study, preparing urea and uric acid biosensors based on various polyaniline (PANI) conducting composite film/Au and iridium-modified carbon (Ir-C)/C/Au electrodes were carried out, respectively. And the properties of sensing electrodes and sensing characteristics were investigated.Ammonium ion (NH4+) is a non-electroactive specie. And it is the major product from catalyzing urea by enzymatic reaction. Consequently, the detection of NH4+ could be used to quantify the urea. Therefore, the preparation of the various NH4+-selective sensing electrodes based on the PANI-Nafion?, PANI-PSSMA(I), PANI-PSSMA(II), PANI-PSSMA(III), nano-structural PANI-PSSMA(IV) and nano-structural PANI-Nafion? on the Au were employed as NH4+-sensing electrodes. The sensing properties for NH4+ were also obtained in this study. The sensitivity and detecting limit of 100.4 ?A mM-1 and 0.025 mM for NH4+, respectively, were found by using the PANI(cycle no.=5)-Nafion?(casting 4 ?l (2 wt%))/Au as the working electrode. The NH4+-sensitive electrode based on nano-structural PANI-Nafion?(casting 8 ?l (1 wt%))/Au was also employed. The sensitivity of 836.39 ?A mM-1 and detecting limit of 0.005 mM were found, respectively. And there are four kinds of PANI-poly(styrenesulfonate-co-maleic acid, sodium type) (PSSMA) to be fabricated in NH4+ sensing electrodes. The highest sensitivity of 126.2 ?A mM-1 was obtained within the NH4+ concentration range of 0 ~ 1.0 mM based on nano-structural PANI-PSSMA(16 ?l (0.5 wt%))(IV)/Au electrode at 25?C.Urea biosensor based on PANI(cycle no.=5)-Nafion?(casting 8 ?l (1 wt%))/Au and nano-structural PANI(current density=80 ?A cm-2, 30 min)-Nafion?(casting 8 ?l (1 wt%))/Au were fabricated in this study. The highest sensitivity of 87.26 ?A mM-1 and detecting limit of 5 ?M were found by using Nafion? (urease, 160 U)/nano-structural PANI-Nafion?/Au as working electrode. Also, cross linked urease (320 U) onto the nano-structural PANI-PSSMA(IV) by glutaraldehyde (GA) as cross-linker was used for sensing urea. The sensitivity of 52.02 ?A mM-1 was obtained in this study.The sensitivity of 36.71 ?A mM-1 and response time (t90) between 18.4 and 25 s were found for sensing H2O2 based on Ir(20 wt%)-C/C/Au at 37?C. And the interferences caused by ascorbic acid (AA) and glucose could be minimized. Cross linked uricase (16 U) on the Ir(20 wt%)-C/C/Au formed uricase/Ir(20 wt%)-C/C/Au UA-sensing electrode. The sensitivity of 23.32 ?A mM-1 was obtained in pH 7 PBS at 37?C. And the sensitivity of 14.03 ?A mM-1 for sensing UA was obtained when the minimum enzyme loading of 2 U was cast on Ir(20 wt%)-C/C/AuThe UA sensor base on Ir(5 wt%)-C was employed in this work. The oxidative potential of 0.255 V (vs. Ag/AgCl) for AA by using differential pulse voltammetric (DPV) method was higher than the oxidative potential for UA. Therefore, the minimized interference by AA was demonstrated at the operating potential of 0.2 V. Using Ir(5 wt%)-C as UA-sensitive electrode, the sensitivity of 1.08 ?A mM-1 was found in pH 8 PBS at 37?C. Furthermore, the Ir(5 wt%)-C electrode was employed to monitor UA in the serum and plasma. The sensing results were shown the Ir(5 wt%)-C could be used to sense UA directly in human serum and plasma, and the interference cased by other species in the blood was insignificant.
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