第十三屆化學感測器科技研討會議程

中華民國九十六年五月二十六日（星期六）

時 間 會 程 主持人 08:30 - 09:30 報 到 (Registration) 09:30 - 09:40 開 幕 典 禮 (Opening Ceremony) 傅勝利 校長

劉進興 理事長09:40 - 10:20 主題演講 (Plenary Lecture)

講題：An Amperometric Nicotine Biosensor based on Molecularly Imprinted TiO2-Modified Electrodes 演講者：何國川 教授

劉進興 理事長

10:20 - 11:00 主題演講 (Plenary Lecture) 講題：Microfluidic Enabling Technologies for Sensors演講者：姚南光 組長

吳昭燕 教授

11:00 - 11:10 茶 敘 (Coffee/Tea Break)

11:10 - 12:20 論文發表口頭報告(IA, IB, IC) (10 min for each)

汪玉銘 教授 周榮泉 教授 丁信智 教授

12:20 - 13:30 午餐及壁報展示(I) (Lunch and Poster Session )

楊明長 教授 杜景順 教授 林修正 教授

13:30 - 14:10 主題演講 (Plenary Lecture) 講題：生物晶片技術 演講者：黃榮山 教授

沈季燕 教授

14:10 - 15:20 論文發表口頭報告(IIA, IIB, IIIC) (10 min for each)

汪玉銘 教授 周榮泉 教授 丁信智 教授

15:20 - 15:40 茶 敘 (Coffee/Tea Break) 15:40 - 16:20 主題演講 (Plenary Lecture)

講題：奈米技術及應用 演講者：蔡嬪嬪 教授

張憲彰 教授

16:20 - 16:50 閉幕典禮及優良壁報頒獎 (Closing Ceremony and Awards to the Best Posters)

16:50- 晚宴

=口頭報告=

IA Session 論文口頭報告（第三研討室）

編

號 時程 題 目

1 11:10- 11:20

Analysis the performance of TiO2 membrane deposited on different conductive material applied to pH sensor 鄒智元、李向晴、林智玲、覃永隆、孫台平 華梵大學電子工程／暨南大學電子工

程

2 11:20- 11:30

微波法製作鋅鎵氧化物奈米顆粒之電化學激發螢光探討

陳怡榮、林修正 長庚大學化工與材料工程研究所

3 11:30- 11:40

Development of affinity biosensor based self-assembled array micro-eletrodes

林漢鋆、黃富國、陳文章 雲林科技大學化工系

4 11:40- 11:50

Eletrochemical characteristics of SPCEs with drop-coating of fMWCNTs and its

application for glucose biosensing

徐俊旭、呂博文、陳文章、林鴻明 雲林科技大學化工系

5 11:50- 12:00

Fluorescent-signal amplification and optimization of Cytokine microarray using

rolling-circle amplification technique

葉庭秀, 吳凱智, 吳瑞璋 中原大學化工系

6 12:00- 12:10

Temperture characteristics on Hafnium Oxide/Silicon oxide gate ion-sensitive field-effect transistor devices 賴朝松、呂增富、林彥智、楊家銘、Dorota G. Pijanowska and Bohdan Jaroszewicz 長庚大學電子所／Institute of Electron Technology, Warsaw Technical University

7 12:10- 12:20

製備奈米結構之聚苯胺/Nafion®複合膜尿素生化感測器及應用於重金屬離子之感測 林凱新、太原麗子、杜景順 東海化大學工系／工研院能環所

IIA Session 論文口頭報告（第四研討室）

編

號 時程 題目

1 14:10- 14:20

以低溫燒結二氧化鈦薄膜製作延伸式閘極離子感測器

林彥名、姚品全、廖豐標 大葉大學電機工程研究所

2 14:20- 14:30

溶膠–凝膠法包覆室溫離子液體修飾玻璃碳電極的電化學行為

林正立、陳怡君、李文仁 吳鳳技術學院化學工程學系

3 14:30- 14:40

Comparison of carbon and platinum performance as TiO2 membrane substrate material applied to glucose biosensor 李向晴、鄒智元、 林智玲、覃永隆、孫台平 華梵大學電子／暨南大學電機系

4 14:40- 14:50

薄膜結構對二氧化鈦延伸式閘極離子感測器之感測特性的影響

吳嘉泰，姚品全 大葉大學電機所

5 14:50- 15:00

利用白金電化學氧化作用之電化學液態感測器測定一氧化碳濃度

蔡豐懋、林修正 長庚大學化工與材料工程研究所

6 15:00- 15:10

以聚苯胺為塗層的剪力水平表面聲波氨氣感測器之感測特性

徐正樑，劉仕苑，沈季燕 義守大學電機工程學系

7 15:10- 15:20

A planar type all-solid-state reference electrode based on a screen-printing technique

黃則衛、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子工

程系／暨南大學電子工程系

IB Session 論文口頭報告（第五研討室）

編

號 時程 題目

1 11:10- 11:20

Design a portable device for measuring ion-selective electrode

周榮泉、陳俊銘 雲林科技大學電子工程研究所

2 11:20- 11:30

The synthesis of carbon nanotubes on clay minerals, and its application to the glucose

biosensor based on a Nafion-CNTs/clay-Au-Glucose oxidase composite film

許豪麟 鄭紀民 中興大學化工學系

3 11:30- 11:40

Experimental investigation on the electrochemical reaction of histidine oxidation at

oxidized boron-doped diamond electrode

陳立家、張家欽、張憲彰 成功大學醫學工程研究所／台南大學環境與能源系

成大生醫工程

4 11:40- 11:50

The optimization of PVC membrane for reference electrode field effect transistor 賴朝松、呂承恩、呂涵薇、楊家銘、Dorota G. Pijanowska 長庚大學電子所／ Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Poland

5 11:50- 12:00

以二茂鐵修飾葡萄糖氧化酵素/聚苯胺作為感測電極之電流式葡萄糖感測器

張書豪、杜景順 東海大學化學工程研究所

6 12:00- 12:10

丙酮氣體在 PANi/Au/Cr/glass 電極上的感測特性

郭陞贏、杜景順 東海大學化學工程研究所

7 12:10- 12:20

Development of a solid-state chloride ion selective electrode

陳威廷、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電

子工程系／暨南大學電子工程系

IIB Session 論文口頭報告（第三研討室）

編

號 時程 題目

1 14:10- 14:20

Hydrogen sensing properties of a Pd/Oxide/ALGAAS (MOS) field-effect resistive device

洪慶文 蔡宗翰 陳慧英 陳梓斌 劉文超 成大電機微電子所／化工所

2 14:20- 14:30

Post N2O plasma treatment on silicon nitride membrane for pH-insensitive REFET application 王泰權、呂增富、秦啟航、呂承恩、楊家銘、賴朝松 長庚大學電子所

3 14:30- 14:40

免疫層析試紙測試法組裝最適化及對 streptavidin 蛋白質之快速偵測

吳凱智、葉庭秀、吳瑞璋 中原大學化工系

4 14:40- 14:50

Optical sensing responses of spin-coated Protoporphyrin IX ZINC (II) towards amines

K.Y.K.W. Sumpena、劉進興 台科大化工系

5 14:50- 15:00

以氧氣消耗量評估生物活性之細胞晶片的研發

蔡林彥廷、吳靖宙 中興大學生物產業機電工程研究所

6 15:00- 15:10

土壤中油分解菌之生物晶片監測鑑定應用研究

廖書賢、何宜衡、鄭宜肪、 楊穎枚、林其昌、張憲彰 成功大學醫學工程研究所

7 15:10- 15:20

發展陣列式介電泳晶片應用於捕捉及濃縮臨床上之致病菌

何宜衡、楊啟宏、廖書賢、鄭宜肪、林其昌、張憲彰 成功大學醫學工程研究所

IC Session 論文口頭報告（第四研討室）

編

號 時程 題目

1 11:10- 11:20

Novel flexible resistive-type humidity sensor

蘇平貴、王超聖 中國文化大學/化學系

2 11:20- 11:30

Characteristics analysis on EGFET of Ruthenium-doped Titanium Dioxide

周榮泉、陳正委 雲林科技大學光電所

3 11:30- 11:40

The characterization on Si3N4/SiO2/Si based light-addressable potentiometric sensor

賴朝松、戴治強、饒瑞修、楊家銘 長庚大學電子工程系／光電工程／電子研

究所

4 11:40- 11:50

Fabrication and sensing characteristics of electroless plated Pd/InGaP transistor hydrogen sensor 林金田、周彥伊、董建圻、吳忠燁、陳慧英 成功大學化工系／工研院能源與資所

5 11:50- 12:00

脫氧還原對 IrO2氣體感測性質之影響

裴紹凱、劉進興 國立台灣科技大學化學工程研究所

6 12:00- 12:10

Development of the potentiometric SnO2/ITO-based lactate biosensor 林志晟、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子工程系／暨南大學電子工程系

7 12:10- 12:20

Drift and hysteresis effects of a SnO2/carbon electrode

陳俊諺、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子工

程系／暨南大學電子工程系／中原大學電子研究所

IIC Session 論文口頭報告（第五研討室）

編

號 時程 題目

1 14:10- 14:20

具有表面增顯拉曼光譜活性的金屬奈米粒子之生物醫學應用

陳彥夫、陳志在、林其昌、張憲彰 成功大學醫學工程研究所

2 14:20- 14:30

In-situ photopolymerization of nanopowder TiO2/polypyrrole nanocomposite thin film combined

with QCM as low humidity sensors

蘇平貴、張一博、謝其華 中國文化大學化學系

3 14:30- 14:40

以 Sulfapyridine 為模板分子製備層析固定相用以分離磺胺藥物分子

洪晉頴、鄭達智、黃清江 虎尾科技大學生物科技系／明道管理學院生命科學

系

4 14:40- 14:50

Development a screen-printed miniaturized disposable ion-selective electrodes 周榮泉、蔡炎熹 雲林科技大學電子工程系

5 14:50- 15:00

利用金奈米粒子之表面電將共振現象於腸炎弧菌之檢測

張孟福、周欣穎、陳涵葳、林昭任 中正大學化工所

6 15:00- 15:10

The sensing device for chloride ion applied to the detection of fine aggregate

氯離子感測元件應用於細粒料之量測

周榮泉、周佩蘭 雲林科技大學光電研究所

7 15:10- 15:20

Potentiometric calcium ion-selective electrode based on the SnO2/ITO thin film

李易珊、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子

工程系／暨南大學電子工程系

目 錄

＝邀請演講＝

編號 題 目 頁碼

1

An Amperometric Nicotine Biosensor based on Molecularly Imprinted TiO2-Modified

Electrodes

Cheng-Tar Wu and Kuo-Chuan Ho 台灣大學化工程研究所

I-IV

2

Microfluidic enabling technologies for sensors the protable cell-based influenza micro TAS

姚南光 工業技術研究院 醫療器材科技中心

V

3

Wireless Nanomechanics-based Biosensors for Point-of-care Applications

黃榮山 Institute of Applied Mechanics, National Taiwan University

VI

4

奈米技術與應用

蔡嬪嬪 國家同步輻射研究中心

VII

＝研討會論文發表＝

＝化學及生物感測技術＝

壁報

編號 題 目 頁碼

1-P01 Analysis the performance of TiO2 membrane deposited on different conductive material applied to pH sensor 鄒智元、李向晴、林智玲、覃永隆、孫台平 華梵大學電子工程／暨南大學電子工程、

1-4

1-P02 微波法製作鋅鎵氧化物奈米顆粒之電化學激發螢光探討

陳怡榮、林修正 長庚大學化工與材料工程研究所

5-7

1-P03 Development of affinity biosensor based self-assembled array micro-eletrodes

林漢鋆、黃富國、陳文章 雲林科技大學化工系

8-11

1-P04 Eletrochemical characteristics of SPCEs with drop-coating of fMWCNTs and its

application for glucose biosensing

徐俊旭、呂博文、陳文章、林鴻明 雲林科技大學化工系

12-15

1-P05

Lactate biosensors based on Lactate Dehydrogenase-Multiwalled Carbon

nanotube-chitosan nanobiocomposite

陳筱芸、蔡毓楨 中興大學化學工程學系

16-18

1-P06 Fluorescent-signal amplification and optimization of Cytokine microarray using

rolling-circle amplification technique

葉庭秀, 吳凱智, 吳瑞璋 中原大學化工系

19-23

1-P07 Temperture characteristics on Hafnium Oxide/Silicon oxide gate ion-sensitive field-effect transistor devices 賴朝松、呂增富、林彥智、楊家銘、Dorota G. Pijanowska and Bohdan Jaroszewicz 長庚大學電子所／Institute of Electron Technology, Warsaw Technical University

24-26

1-P08 製備奈米結構之聚苯胺/Nafion®複合膜尿素生化感測器及應用於重金屬離子之感測

林凱新、太原麗子、杜景順 東海化大學工系／工研院能環所

27-30

1-P09 以低溫燒結二氧化鈦薄膜製作延伸式閘極離子感測器

林彥名、姚品全、廖豐標 大葉大學電機工程研究所

31-35

1-P10 溶膠–凝膠法包覆室溫離子液體修飾玻璃碳電極的電化學行為

林正立、陳怡君、李文仁 吳鳳技術學院化學工程學系

36-39

1-P11 Comparison of carbon and platinum performance as TiO2 membrane substrate material applied to glucose biosensor 李向晴、鄒智元、 林智玲、覃永隆、孫台平 華梵大學電子／暨南大學電機系

40-45

1-P12 薄膜結構對二氧化鈦延伸式閘極離子感測器之感測特性的影響

吳嘉泰，姚品全 大葉大學電機所

46-51

1-P13 利用白金電化學氧化作用之電化學液態感測器測定一氧化碳濃度

蔡豐懋、林修正 長庚大學化工與材料工程研究所

52-55

1-P14 以聚苯胺為塗層的剪力水平表面聲波氨氣感測器之感測特性

徐正樑，劉仕苑，沈季燕 義守大學電機工程學系

56-59

1-P15

多層壁奈米探管/全氟磺酸聚合物/酪胺酸酶生物複合材料薄膜做為酚化合物感測器之

探討 邱乾城、蔡毓楨 中興大學化學工程學系

60-62

1-P16 Hydrogen sensing properties of a Pd/Oxide/ALGAAS (MOS) field-effect resistive device

洪慶文 蔡宗翰 陳慧英 陳梓斌 劉文超 成大電機微電子所／化工所

63-68

1-P17 Post N2O plasma treatment on silicon nitride membrane for pH-insensitive REFET application 王泰權、呂增富、秦啟航、呂承恩、楊家銘、賴朝松 長庚大學電子所

69-72

1-P18 免疫層析試紙測試法組裝最適化及對 streptavidin 蛋白質之快速偵測

吳凱智、葉庭秀、吳瑞璋 中原大學化工系

73-76

1-P19 Optical sensing responses of spin-coated Protoporphyrin IX ZINC (II) towards amines

K.Y.K.W. Sumpena、劉進興 台科大化工系

77-80

1-P20 電化學式榖胺酸微感測器之研究

王詩涵、郭幸宜 義守大學化工系

81-84

＝細胞感測技術＝

3-P01 以氧氣消耗量評估生物活性之細胞晶片的研發

蔡林彥廷、吳靖宙 中興大學生物產業機電工程研究所

85-89

＝快速微生物與病毒之感測和鑑定技術＝

4-P01 土壤中油分解菌之生物晶片監測鑑定應用研究

廖書賢、何宜衡、鄭宜肪、 楊穎枚、林其昌、張憲彰 成功大學醫學工程研究所

90-93

4-P02 發展陣列式介電泳晶片應用於捕捉及濃縮臨床上之致病菌

何宜衡、楊啟宏、廖書賢、鄭宜肪、林其昌、張憲彰 成功大學醫學工程研究所

94-97

＝環境與衛生感測技術＝

5-P01 The sensing device for chloride ion applied to the detection of fine aggregate

氯離子感測元件應用於細粒料之量測

周榮泉、周佩蘭 雲林科技大學光電研究所

98-101

＝電化學及壓電感測技術＝

6-P01 Design a portable device for measuring ion-selective electrode

周榮泉、陳俊銘 雲林科技大學電子工程研究所

102-105

6-P02 The synthesis of carbon nanotubes on clay minerals, and its application to the glucose

biosensor based on a Nafion-CNTs/clay-Au-Glucose oxidase composite film

許豪麟 鄭紀民 中興大學化工學系

106-110

6-P03

Experimental investigation on the electrochemical reaction of histidine oxidation at

oxidized boron-doped diamond electrode

陳立家、張家欽、張憲彰 成功大學醫學工程研究所／台南大學環境與能源系

111-117

6-P04 The optimization of PVC membrane for reference electrode field effect transistor 賴朝松、呂承恩、呂涵薇、楊家銘、Dorota G. Pijanowska 長庚大學電子所／Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Poland

118-121

6-P05 以二茂鐵修飾葡萄糖氧化酵素/聚苯胺作為感測電極之電流式葡萄糖感測器

張書豪、杜景順 東海大學化學工程研究所

122-125

6-P06 丙酮氣體在 PANi/Au/Cr/glass 電極上的感測特性

郭陞贏、杜景順 東海大學化學工程研究所

126-129

6-P07 Development of a solid-state chloride ion selective electrode

陳威廷、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子

工程系／暨南大學電子工程系

130-133

6-P08 A planar type all-solid-state reference electrode based on a screen-printing technique

黃則衛、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子工

程系／暨南大學電子工程系

134-137

6-P09 Potentiometric calcium ion-selective electrode based on the SnO2/ITO thin film

李易珊、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子

工程系／暨南大學電子工程系

138-141

6-P10 Glucose biosensor with GODx immobilized by polyvinyl alcohol cryo-gel film

汪玉銘、周澤川 吳鳳技術學院化工系／成功大學化工系

142-145

＝光學及半導體感測技術＝

7-P01 Novel flexible resistive-type humidity sensor

蘇平貴、王超聖 中國文化大學/化學系

146-149

7-P02 Characteristics analysis on EGFET of Ruthenium-doped Titanium Dioxide

周榮泉、陳正委 雲林科技大學光電所

150-153

7-P03 The characterization on Si3N4/SiO2/Si based light-addressable potentiometric sensor

賴朝松、戴治強、饒瑞修、楊家銘 長庚大學電子工程系／光電工程／電子研究

所

154-157

7-P04 Fabrication and sensing characteristics of electroless plated Pd/InGaP transistor hydrogen sensor 林金田、周彥伊、董建圻、吳忠燁、陳慧英 成功大學化工系／工研院能源與資所

158-162

7-P05 脫氧還原對 IrO2氣體感測性質之影響

裴紹凱、劉進興 國立台灣科技大學化學工程研究所

163-166

7-P06 Development of the potentiometric SnO2/ITO-based lactate biosensor 林志晟、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子工程系／暨南大學電子工程系

167-170

7-P07 Drift and hysteresis effects of a SnO2/carbon electrode

陳俊諺、周榮泉、孫台平、熊慎幹 中原大學電子工程系／雲林科技大學電子工

程系／暨南大學電子工程系／中原大學電子研究所

171-174

＝其他相關創新前瞻檢測技術＝

8-P01 具有表面增顯拉曼光譜活性的金屬奈米粒子之生物醫學應用

陳彥夫、陳志在、林其昌、張憲彰 成功大學醫學工程研究所

175-178

8-P02

In-situ photopolymerization of nanopowder TiO2/polypyrrole nanocomposite thin film combined

with QCM as low humidity sensors

蘇平貴、張一博、謝其華 中國文化大學化學系

179-182

8-P03 以 Sulfapyridine 為模板分子製備層析固定相用以分離磺胺藥物分子

洪晉頴、鄭達智、黃清江 虎尾科技大學生物科技系／明道管理學院生命科學系

183-186

8-P04 Development a screen-printed miniaturized disposable ion-selective electrodes 周榮泉、蔡炎熹 雲林科技大學電子工程系

187-190

8-P05 利用金奈米粒子之表面電將共振現象於腸炎弧菌之檢測

張孟福、周欣穎、陳涵葳、林昭任 中正大學化工所

191-194

8-P06 製備兒茶酚胺感測電極之研究

王慶翔、許耀人、林宗榮 義守大學生物技術與化學工程研究所

195-198

I

AN AMPEROMETRIC NICOTINE BIOSENSOR BASED ON MOLECULARLY IMPRINTED TIO2-MODIFIED ELECTRODES

Cheng-Tar Wua and Kuo-Chuan Hoa,b aDepartment of Chemical Engineering and bInstitute of Polymer Science and Engineering

National Taiwan University, Taipei, Taiwan 10617 Tel:+886-2-2366-0739; Fax:+886-2-2362-3040; E-mail:kcho@ntu.edu.tw

(NSC94-2214-E-002-021) Abstract --- Amperometric detection of nicotine (NIC) was carried out on a titanium dioxide (TiO2)/

poly(3,4-ethylenedioxythiophene) (PEDOT) modified electrode by molecularly imprinted technique. In order to improve the conductivity of the substrate, PEDOT was coated on the sintered electrode by in-situ electrochemical polymerization of the monomer. The sensing potential of the NIC imprinted TiO2 electrode (ITO/TiO2[NIC]/PEDOT) in a PBS solution (pH = 7.4) containing 0.1 M KCl was determined to be 0.88 V (vs. Ag/AgCl/sat’d KCl). The linear detection range for the NIC oxidation on the ITO/TiO2[NIC]/PEDOT electrode was between 0 to 5 mM, with a sensitivity and limit of detection of 31.4 μA mM-1cm-2 and 11.1 μM respectively. The ITO/TiO2[NIC]/PEDOT electrode shows a reasonably good selectivity in distinguishing the NIC from its major interferent, (-)-cotinine (COT).

Abstract --- Amperometric, Biosensor, Nicotine, Molecularly imprinted TiO2, PEDOT

1. INTRODUCTION Nicotine (NIC) belongs to alkaloids family existing in tobacco leaves and it has been used as one of the

important drug materials for the treatment of Alzheimer’s disease (AD) [1]. The cognitive effect and clinical diagnosis of the NIC had been summarized [2]. However, the frequent intake of the NIC in the human body creates few side effects such as increase of blood pressure, heart beat, acceleration of the respiratory organs and stimulation of the central nervous system leading to disruption of arteries and cardiovascular risk factors. The toxicity of the NIC is about 60 mg, as exhibited by its fatal dose to adults [1]. To date, there are more than 1.2 billion people in the world who consumes varieties of tobacco products and this intake affects human metabolism resulting in the increase of the amount of low-density lipoprotein (LDL) and injuries to our health [3]. Hence, it is indeed necessary to develop a quick and precise sensing technique for the detection of the NIC in the human body.

A number of methods for the NIC detection in the human body or in the tobacco samples had been reported so far and this includes high performance liquid chromatography (HPLC) with different kinds of detectors such as ultraviolet (HPLC-UV), electrochemical (HPLC-ED) and high sensitivity gas chromatography-mass spectrometric (GC-MS) technique [4-6]. Molecularly imprinted polymeric (MIP) technique is becoming important in recent years for the product separation with solid phase extraction (SPE) and clinical assays where the selectivity was achieved by adjusting the monomer-template molar ratios [7].

When compared to other methods, electrochemical sensing has been considered as a simple, cheap and reliable. On this basis, amperometric detection of the NIC has been carried by molecularly imprinted technique. NIC was imprinted on a TiO2 matrix, coated on an indium tin oxide (ITO) electrode and for improving conductivity, 3,4-ethylenedioxythiophene (EDOT) was electrochemically polymerized on the electrode to get the NIC imprinted TiO2/PEDOT modified electrode (denoted as ITO/TiO2[NIC]/PEDOT). Subsequently, a non-imprinted PEDOT modified electrode (denoted as ITO/TiO2/PEDOT) was also fabricated for the NIC sensing.

Scanning electrochemical microscopy (SECM) was used to distinguish the different surface morphology of both the modified electrodes based on their redox chemistry. In this work, the SECM was employed to distinguish the surface morphology of the imprinted electrode and the non-imprinted electrode using Fe(CN)63-/ Fe(CN)64- as the redox couple.

2. EXPERIMENTAL 2.1 Preparation of the ITO/TiO2[NIC] imprinted and the ITO/TiO2 electrodes

The NIC liquid was added into the TiO2 colloids to form the 0.125 M NIC mixed solution and stirred homogenously. The paste was coated uniformly on pre-cleaned ITO electrodes by glass rod, followed by air drying for 30 min. Then the film was heated to 500 °C at a rate of 20 °C/min, and maintained for 30 min before cooling to room temperature. During this heat treatment, all NIC molecules were removed from the substrate

II

(note that the boiling point of NIC is about 247 °C) and there was no need for any extraction procedure using solvents. The removal of NIC was ensured by checking the NIC oxidation current response from the cyclic voltammogram (CV) of the imprinted electrode. Thus, the NIC imprinted TiO2 modified electrode (ITO/TiO2[NIC]) was obtained. In the same way, the non-imprinted electrode was fabricated without NIC and was used for the comparative study.

2.2 Electropolymerization of EDOT

An effective area of 0.5 × 0.5 cm2 on both imprinted and non-imprinted electrodes was obtained with the polyimide insulating tape. Both the working electrodes were dipped in anhydrous acetonitrile solution containing 0.01 M EDOT monomer and 0.1 M LiClO4. Prior to electropolymerization, the electrolyte was deoxygenated by purging with nitrogen for 5 min. A constant potential of 1.2 V (vs. Ag/Ag+) for 5 s was applied using a potentiostat. The corresponding charges passed during the electrochemical deposition of PEDOT for the ITO/TiO2[NIC]/PEDOT and ITO/TiO2/PEDOT electrodes were 6.0±0.04 and 5.4±0.03 mC, respectively. After the electropolymerization, both thin films, namely ITO/TiO2[NIC]/PEDOT and ITO/TiO2/PEDOT, were cleaned with the acetonitrile, dried with N2, and then stored in air.

2.3 Amperometric detection of the NIC

After acquiring suitable operating potential, a typical chronoamperometric i-t curve was recorded for our sensing experiment. First of all, the background current was equilibrated for 200 s and then the added NIC amount was adjusted. The concentrations of the NIC in the bulk solution was varied from 0 to 5 mM and at each concentration level, the components were mixed thoroughly by a magnetic bar with the rotation speed of 250 rpm for 20 s. In order to obtain a steady-state current, the cell was kept without any disturbance for 200 s. The net current was obtained in the same manner as mentioned previously. The calibration curves were constructed by calculating the net steady-state current densities at various NIC concentrations ranging from 0 to 5 mM, for the imprinted and the non-imprinted electrode. The sensitivity was the slope of the calibrated current vs. concentration and the detection limit (based on S/N=3) of the sensor was also obtained. Furthermore, the interference of COT was evaluated by adding 0 to 5 mM of COT to the same electrolyte solution. The interference effect with equimolar coexistence of the NIC and COT mixture were also compared.

2.4 Topographical mappings by SECM

A four-electrode system was employed in the SECM instrument and the SECM mapping topographies were obtained in a solution containing 5 mM K3Fe(CN)6 as a mediator and 0.1 M KCl as the electrolyte. In order to do topographical mapping, the tip must be kept a distance of 10 μm from both ITO/TiO2/PEDOT and ITO/TiO2[NIC]/PEDOT electrodes. The mapping area was 100 × 100 μm2. The recorded data was plotted in a three dimensional figure and in addition, the tick label of X , Y axis and Z axis are the representatives of the distance of the tip on the X and Y axis and the normalized current respectively.

3. RESULTS AND DISCUSSION 3.1 The catalytic effect of the conducting PEDOT

The CVs of 5 mM of the NIC in 0.1 M KCl solution containing PBS on (a) ITO/TiO2[NIC]/PEDOT, (b) bare ITO, and (c) ITO/TiO2[NIC] electrodes were obtained first (not shown here). From which, it is understandable that the modified ITO electrode electropolymerized with PEDOT can enhance the oxidation response of the NIC. This confirms that the PEDOT acts as a strong catalyst for the oxidation of the NIC.

3.2 Amperometric detection of the NIC and its major interferent, COT

It is necessary to choose a suitable potential for comparing the sensing performance between the imprinted and the non-imprinted electrodes. The operating potential for the amperometric detection of the NIC was determined from the LSV curve as 0.88 V. From our previous studies (section 3.1), it is noted that the PEDOT modified electrode can enhance the current response and lower the overpotential for the NIC oxidation, when compared with the oxidation process on the bare ITO electrode. Hence, the next step is to compare the electrochemical response of the molecularly imprinted (ITO/TiO2[NIC]/PEDOT) and the non-imprinted electrode (ITO/TiO2/PEDOT) on the detection of the NIC.

Fig. 1 exhibits the calibration curves obtained from the typical steady-state current-time responses for the NIC oxidation within the concentration, ranging from 0 to 5 mM. The experiment in the amperometric sensing mode was repeated for three times with a fresh electrode at each measurement and the result is shown as error bars in the figure. From Fig. 1, it is noted that the current response carried out on the imprinted electrode is statistically higher than that on the non-imprinted electrode. The limit of detection (LOD) was found to be 11.1 μM and the linear detection range (LDR) was in between 0 to 5 mM with a sensitivity value of 31.4 μA

III

mM-1cm-2 (R2=0.997) for the imprinted electrode. The sensing performance (LOD and LDR) of the non-imprinted electrode were 25.2 μA mM-1cm-2 and 13.8 μM, respectively. However, till date, there is no specific definition to distinguish the performance between MIPs and non-imprinted polymers (NMIPs). In order to quantitatively determine the attribution contributed by the template imprinting, a normalized value of imprinting efficiency (I.E.) is defined as the bounded amount of MIP divided by the bounded amount of NMIP.

While in an amperometric MIP biosensor system, it is difficult to measure the bounded amount on the MIP modified electrode. Therefore, a sensitivity enhancement (S.E.) factor has been modified from the definition of I.E. described above as the ratio of MIP sensitivity over NMIP sensitivity. Therefore, the S.E. of this work is about 1.24.

A partial list of I.E. for non-EC MIP sensors and S.E. for electrochemical (EC) MIP sensors reported in literatures has been compiled. From which, it is noted that the S.E. of this work is well comparable with the S.E. obtained from other published EC based sensors. It is noted that the value of S.E. lies between 1 and 3 for most of the EC based MIP sensors. Although the S.E. of our work is not as high as those when comparing to some of the others, this work represents a first preliminary example for an amperometric nicotine sensor imprinted on oxides of transition metal immobilized with PEDOT that shows promising results.

3.3 Interference effect

In the human body, COT has been reported as the principal metabolite of the NIC in blood and urine.12,22 Hence, the COT was considered as the key interference in this study. The corresponding calibration curve was shown in Fig. 2. Curves (a), (b) and (c) correspond to the oxidation current responses of the added NIC, equimolar coexistence of the NIC and COT mixture and the COT respectively. Curves (a) and (b) reveal that the ITO/TiO2[NIC]/PEDOT electrode exhibits a high current responses particularly for the NIC oxidation. Based on the oxidation current responses, it is noted that the ITO/TiO2[NIC]/PEDOT film shows higher selectivity for the NIC, where the NIC is adsorbed on the specific sites at the applied potential, 0.88 V.

The above investigations clearly reveal that the NIC molecules occupy specific sites on the TiO2 nanostructure during coating and leaves imprinted shapes during the thermal treatment (extraction process). These imprinted sites are selective only to the NIC molecules and the adsorption of the COT on the above specific sites is almost negligible. Hence, the ITO/TiO2[NIC]/PEDOT electrode can distinguish the NIC from the COT in terms of their sensing performance. As shown in curve (b) of Fig. 2, it appears that the oxidation current response of the equimolar coexistence of the NIC and COT is somewhat lower than the addition of the NIC alone. This may be due to the fact that the COT may slightly block a few imprinting sites on the ITO/TiO2[NIC]/PEDOT electrode. On the basis of above results, it is concluded that the imprinting sensor, ITO/TiO2[NIC]/PEDOT can be used in an environment with the coexistence of the NIC and COT.

4. CONCLUSIONS In conclusion, the concept of a molecularly imprinted TiO2 does work, which has been utilized for NIC

sensing. The imprinted (ITO/TiO2[NIC]/PEDOT) electrode shows higher response current than the non-imprinted one (ITO/TiO2/PEDOT). The sensitivity of the imprinted (31.4 μA mM-1cm-2) film is about 1.24 times higher than the non-imprinted (25.2 μA mM-1cm-2) film, i.e. the S.E. is about 1.24. The LOD was calculated as 11.1 μM. The linear detection region was from 0 to 5 mM and this offers a possible method for the NIC sensing. According to this study, the NIC-imprinted TiO2 (ITO/TiO2[NIC]/PEDOT) electrode shows a reasonably good selectivity in distinguishing the NIC from the COT. The imprinted and the non-imprinted electrodes were characterized by SECM and SEM. The FTIR spectra confirmed the formation of hydrogen bond between NIC and TiO2 in the imprinted electrode. Although the S.E. for EC MIP sensors reported in literatures is well comparable with the S.E. obtained in this work. more works are now being carried out in our laboratory for improving the S.E. up to 2 to 3.

5. REFERENCES [1] J. S. Meyer and L. F. Quenzer, Psychopharmacology: Drugs, the Brain, and Behavior, Sinauer Associates,

Sunderland, 2005. [2] A. H. Rezvani and E. D. Levin, Biol. Psychiatry, 49, pp. 258-267 (2001). [3] T. T. Denton, X. D.Zhang, and J. R. Cashman, J. of Med. Chem., 48, pp. 224-239 (2005).. [4] M. Page-Sharp, T. W. Hale, L. P. Hackett, J. H. Kristensen, and K. F. Ilett, J. Chromatogr. Biomed. Sci.

Appl., 796, pp. 173-180 (2003). [5] G. N. Mahoney and W. Al-Delaimy, J. Chromatogr., Biomed. Appl., 753, pp. 179-187 (2001). [6] H. S. Shin, J. G. Kim, Y. J. Shin, and S. H. Jee, J. Chromatogr. Biomed. Sci. Appl., 769, pp. 177-183 (2002). [7] A. Zander, P. Findlay, T. Renner, and B. Sellergren, Anal. Chem., 70, pp. 3304-3314 (1998).

IV

0 1 2 3 4 5

0

40

80

120

160

[KCl] = 0.1 MIn PBS, pH = 7.4

ITO/TiO2[NIC]/PEDOT ITO/TiO2/PEDOT Linear fit of ITO/TiO2[NIC]/PEDOT Linear fit of ITO/TiO2/PEDOT

Y = 31.35E-6 * X

R2 = 0.9971

Sensitivity = 31.35 (μA/mM⋅cm2)

Detection Potential = 0.88 V

Y = 25.21E-6 * X

R2 = 0.9968

Sensitivity = 25.21 (μA/mM⋅cm2)

Cur

rent

den

sity

(μA

/cm

2 )

Concentration of NIC (mM)

Fig. 1. Sensing performance for the ITO/TiO2/PEDOT and the ITO/TiO2[NIC]/PEDOT electrodes.

0 1 2 3 4 5

0

40

80

120

160

(c)

(b)

(a) NIC (b) NIC/COT = 1/1 (c) COT

Detection Potential = 0.88 VElectrode = ITO/TiO2[NIC]/PEDOT[KCl] = 0.1 MIn PBS, pH = 7.4

Cur

rent

den

sity

(μA

/cm

2 )

Concentration (mM)

(a)

Fig. 2. Calibration curves for (a) NIC alone, (b) equimolar coexistence of the NIC & COT mixture, and (c) COT

alone.

V

MICROFLUIDIC ENABLING TECHNOLOGIES FOR

SENSORS THE PROTABLE CELL-BASED INFLUENZA MICRO TAS

可攜式細胞級流感檢測微系統

姚南光 工業技術研究院 醫療器材科技中心

03-5913495, nkyao@itri.org.tw Abstract --- We developed a portable cell-based analytical system for influenza test by microfluidic

technology. System includes a disposable cell-culture microfluidic cartridge and a built-in EIA test strip. A wireless servo device was employed in process programming and thermal control. The protocols of influenza virus (A type, H1N1 ) identification such as virus inoculating, replicating in MDCK cells, rapid EIA testing were executed automatically by this hand-free system. We provide a portable, reliable and low cost novel tool to rapid screen the high quantity of samples for influenza surveillance.

Abstract --- 本計畫運用微流體技術 (Microfluidic Technology) 開發完成 ”可攜式 MDCK 細胞培養與流感病毒檢測微系統 (Portable influenza microTAS)”，內建細胞培養微反應器、程式化培養基導流與微域溫控裝置，可自動化執行病毒感染細胞 (Inoculation)、病毒複製 (Replication)、細胞裂解 (Cell lysis) 以及快速酵素免疫分析 (Rapid EIA test)。實驗成果顯示，利用本系統之 MDCK 細胞生物放大作用，可大幅提昇 H1N1 流感檢體之 EIA 檢測靈敏度 (0.01MOI 0.0001MOI)，對於提前檢知微量可疑檢體非常有幫助，可提供第一線防疫人員於流感疫情升溫時處理大量檢體，以便及時篩選出可疑檢體，再以活細胞保存病毒模式，優先擇送專業實驗室進行進一步診斷分析。

VI

Wireless Nanomechanics-based Biosensors for

Point-of-care Applications Long-Sun Haung (黃榮山)

Institute of Applied Mechanics, National Taiwan University

Tel: 886-2-3366-5653, E-mail: lshuang@mems.iam.ntu.edu.tw

Biomolecular recognition is nature characteristics in DNA hybridization, DNA-protein interaction, protein-protein interaction, and cell-ligand binding. In the post-genomics era, proteins and their associated unique characteristics are of great interests that drive rapid development in fusion of biotechnology, nanotechnology and microsystems. This talk will address recent work of cross-disciplines in our team, and show the technological integration of cross-disciplines to bridge the gap in which current biotechnologies fail to cohesively fill the demand in sensitive/efficient/portable diagnostics, characterization and in-depth understanding of biomolecular interaction. As for recent advances in biotechnology and clinical diagnostics as well as a growing healthcare demand on point-of-care applications, the bio-sensing tools have been rapidly moving toward miniaturization, high sensitivity, portability and wireless ubiquity.

A label-free nanomechanics-based microcantilever immunoassay has been demonstrated for detection of disease-related protein biomarkers. This study reports our recent achievement in detection of C-reactive protein (CRP), an acute-phase reactant bio-marker that is produced in the liver normally present in healthy human serum with a concentration < 1μg/mL. Various CRP levels which may rise up to 100 or even 500 times during acute infection or inflammation in blood concentration were measured in a clinical range. In addition, the detection of prostate-specific antigen (PSA) biomarker, a useful indicator of prostate cancer, was also conducted with the nanomechanics-based biosensors. Both biomarker detected signals were wirelessly transmitted to remote display. With such a technology leverage in mature semiconductor industry sectors into medical applications, this work exhibits merits of technological integration, portability, potential low-cost, and remote on-site diagnosis.

VII

奈米技術與應用

蔡嬪嬪

國家同步輻射研究中心

30076 新竹市科學園區新安路 101 號 03-5780281 轉 2206, Fax:03-5783817, pptsai@nsrrc.org.tw

Abstract --- 本報告分為二大部分：第一部分以世界上奈米技術應用與產業化情形為主，第二部分以

同步輻射光源在奈米科技研究與材料分析方面的應用為主題，並介紹國家同步輻射研究中心設施與使用情形。

Keywords: 奈米科技、產業化、同步輻射光源

第一部分以世界上奈米技術應用與產業化情形為主，包括國內奈米標章、世界上奈米指標的發展情

形，並以 Nanosys 公司為例展現奈米材料技術公司在商品化以及上市過程如何提昇產品價值。

第二部分以同步輻射光源在奈米科技研究與材料分析方面的應用為主題，並介紹國家同步輻射研究

中心設施與使用情形。「同步輻射光」是帶電荷的粒子在速度接近光速的條件下，基於愛因斯坦相對論

所產生的超強光，其光譜涵蓋微米級的紅外光、紫外光到原子尺度的 X光。目前中心已運轉多年的 15 億

電子伏特加速器光源設置完成 27 條光束線包括紅外線光束線、VUV 光束線，以及 X光成像、散射、繞射、

吸收光譜光束線，另外還有蛋白質結晶學光束線、生物膜散射光束線、微機械光束線、X光光刻光束線

等等。此光源設施的使用率年年提昇，已漸趨飽和。為了台灣先進光源設施的永續發展，國科會、經建

會業已同意建造一座最先進的 30 億電子伏特加速器光源，以建置頂尖的科學實驗站，與未來國家高科技

產業同步發展之核心設施，提升台灣擁有 21 世紀科技創新研發的優勢，可進行尖端科學如奈米探針 X光

實驗、聚焦至數微米的高亮度 X光實驗、X光生醫影像實驗等。未來「台灣光子源」的蛋白質結構核心

設施及奈米生醫影像設施，可將目前腫瘤的偵測及治療從毫米推向百分之一毫米尺度，為癌症的醫療開

拓革命性的新境界；在藥物設計方面，經由自動化 X光蛋白質結構檢測，將大幅縮短所需時間，由年變

日。其他如以 X光分析技術為基礎的半導體產業 IC 檢測技術既快速且準確。

1

Analysis the performance of TiO2 membrane deposited on different conductive material applied to pH sensor

Chih-Yuan Tsoua, Hsiang-Ching Lee b, Jyh-Ling Lin a, Yuan-Lung China, Tai-Ping Sunc aDepartment of Electronic Engineering, Huafan University, Taipei, Taiwan , R.O.C.

bDepartment of Mechatronic Engineering, Huafan University, Taipei, Taiwan , R.O.C. cDepartment of Electrical Engineering, National Chi Nan University, Nantou, Taiwan 545.

02-26632102ext4104, 02-26632102ext4102, cllin@huafan.hfu.edu.tw (NSC 95-2221-E-211-021)

Abstract---The objective of this work is the study and analysis of sol-gel TiO2 deposited on different conductive material as a pH sensor using the separated extended-gate field-effect transistor (SEGFET) structure. Conductive materials include carbon, platinum, indium tin oxide (ITO), cuprum, silver, and aluminum. Experimental results show good pH sensitivities when the conductive material adopted platinum, carbon, and indium tin oxide. The corresponding sensitivities are 48.8, 40.5 and 55.5 mV/pH, respectively at IDS= 100 μA. There is no pH recognition when the conductive materials are low work function, such as cuprum, silver, and aluminum.

Keywords：Titanium Oxide (TiO2); Separated Extended-Gate Field-Effect Transistor (SEGFET); pH meter

1. INTRODUCTION In 1970, Bergveld introduced ion-selective field-effect transistors (ISFETs), which combine the

chemical-sensitive properties of glass membranes with the impedance converting characteristics of the metal-oxide-semiconductor field-effect transistor (MOSFET). Most of the ISFET papers covered additional sensing membrane, such as Si3N4, Al2O3, Ta2O5, SnO2, presented how to fabricate an effective pH meter. In 1983, Van Der Spiegel et al proposed the framework for the extended-gate ion-sensitive field effect transistor (EGFET). So far many papers studied on separated extended-gate ion-sensitive field-effect transistor (SEGFET) [1-3]. Its structure consists of two main parts. The sensor component is separated from transistor because it has to be soaked in the testing solution. Another part is the MOSFET component. So the SEGFET has many advantages like light insensitive, easy to manufacture and package, and development disposable sensor.

There are many studies on ion-sensing membranes and various kinds of conductive materials can be used for the SEGFET electrode. But not any and every kind of conductive materials are suitable to be the sensing membrane substrate as SEGFET structure. The objective of this work is the study and analysis of sol-gel TiO2 deposited on different conductive material as a pH sensor. TiO2 is a transition-metal oxide with many applications. In this study, TiO2 thin films were prepared using the sol-gel method owing to its ability to offers such advantages as the ability to grow the films over large-area substrates, a low processing temperature, the very low cost of the production facilities.

2. EXPERIMENTAL METHODS The TiO2 film was used as the detecting membrane, which was coated on various conductive materials by

the dipping method and was prepared using the sol-gel technique supported by ONID Technology Corporation. Conductive materials include carbon, platinum, indium tin oxide (ITO), cuprum, silver, and aluminum. Carbon conductive films were patterned by screen-printing technology on a polypropylene (PP) plastic plate. Platinum films were deposited by E-gun evaporation technology. ITO materials were purchased from standard ITO glasses. The thickness of the TiO2 layer was about 0.1 μm, and the surface area of the detecting window was leaved 3×8 mm2, covered by insulating epoxy. SEGFET of TiO2/conductive-material sensing structure is shown in Fig. 1.

SEGFET corresponding IDS current of the linear region and the saturation region characteristics can be obtained through Keithley 4200 systemic detection and measurement by connecting the prepared electrode, the reference electrode, NMOS transistor (CD4007) and placing them in acidic and basic solutions. Another measurement system takes an instrumental amplifier. Figs. 2(a) and 2(b) present the SEGFET measurement structure and INA114 instrumental amplifier acid-base measurement system.

3. RESULTS AND DISCUSSION

2

Sol-gel TiO2 supported by ONID Technology Corporation, Taiwan, was coated on Pt, carbon, ITO, cuprum, silver, and aluminum substrate by dipping method. Figs. 3(a) to 3(c) show the TiO2/Pt, TiO2/carbon, TiO2/ITO SEGFET characteristic curves in various pH values for different pH values. The corresponding sensitivities are 48.8, 40.5 and 55.5 mV/pH, respectively at IDS= 100 μA.

The SEGFET turn-on voltage varies with different conductive material. Figure 4 shows IDS-VGS characteristics for TiO2/Pt, TiO2/carbon and TiO2/ITO under pH=7 solution. Obviously, TiO2/Pt had smaller turn-on voltage than others because platinum has large work function. SEGFET of TiO2/Cu, TiO2/Ag and TiO2/Al had high turn-on voltage and no pH response. Figure 5 shows the turn-on voltage versus five conductive materials under pH=7 solution. Figure 6 shows the acid-base characteristics of TiO2 on five different conductive materials using instrumentation amplifier measurement system.

ACKNOWLEDGMENT The authors would like to thank the National Science Council of the Republic of China, Taiwan, for

financially supporting this research under Contract No. NSC 95-2221-E-211-021. ONID Technology Corporation is appreciated for its scientific and technical support.

REFERENCES [1] J. C. Chou. P. K. Kwan, Z. J. Chen, “SnO2 separative structure extended gate H+- ion sensitive field

effect transistor by the sol-gel technology and the readout circuit developed by source follower,” Jpn. J. Appl. Phys. 42, pp. 6790-6794 (2003).

[2] P. D. Batista, M. Mulato, “ZnO extended-gate field effect transistors as pH sensors,” Applied Physics Letters 87, pp. 143508-143511 (2005).

[3] J. L. Lin, Y. M. Chu, S. H. Hsaio, Y. L. Chin, T. P. Sun, “Structures of anodized aluminum oxide extended-gate field-effect transistors on pH sensors,” Jpn. J. Appl. Phys. 45, pp. 7999-8004 (2006).

Fig. 1 Structure of TiO2/conductive-material

(a) (b) Fig. 2 (a) TiO2/conductive-material SEGFET measurement structure.

(b) Acid-base measurement system using INA114 instrumentation amplifier.

3

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0-50

0

50

100

150

200

250

300

350

pH2 pH3 pH4 pH5 pH6 pH7 pH8 pH9 pH10 pH11 pH12

I DS

(μA

)

VGS

(a)

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0-50

0

50

100

150

200

250

300

350

pH2 pH3 pH4 pH5 pH6 pH7 pH8 pH9 pH10 pH11 pH12

I DS

(μA

)

VGS

(b)

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0-50

0

50

100

150

200

250

300

350

pH2 pH3 pH4 pH5 pH6 pH7 pH8 pH9 pH10 pH11 pH12

I DS

(μA

)

VGS

(c)

Fig. 3 (a) IDS-VGS characteristics of TiO2/Pt SEGFET, the sensitivities is 48.8 mV/pH at IDS= 100 μA. (b) IDS-VGS characteristics of TiO2/carbon SEGFET, the sensitivities is 40.5 mV/pH at IDS= 100 μA. (c) IDS-VGS characteristics of TiO2/ITO SEGFET, the sensitivities is 55.5 mV/pH at IDS= 100 μA.

.

4

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0-50

0

50

100

150

200

250

300

350

Cur

rent

(μA

)

VGS

TiO2/Pt TiO2/C TiO2/ITO

Fig. 4 IDS-VGS characteristics for TiO2/Pt, TiO2/carbon and TiO2/ITO under pH=7 solution.

Pt C ITO Cu Al1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

V GS

(V)

Conductive material

SEGFET_pH7

Fig. 5 SEGFET turn-on voltage versus five conductive materials under pH=7 solution.

2 4 6 8 10 12

-0.5

0.0

0.5

1.0

1.5

Pt_TiO2 C_TiO2 ITO_TiO2 Cu_TiO2 Al_TiO2

Volta

ge (m

V)

pH

Fig. 6 Acid-base characteristics of TiO2 on five conductive-materials using INA114 instrumentation amplifier.

5

微波法製作鋅鎵氧化物奈米顆粒之電化學激發螢光探討

陳怡榮、林修正

長庚大學、化工與材料工程研究所

TEL:(03)211-8800, FAX:(03)211-8700, andrew@mail.cgu.edu.tw

摘要:電化學激發螢光是由電子與電洞結合而發光，亦因電子吸收能量而激發產生光的能量。 本研究是以新式的微波法製作鋅鎵氧化物奈米顆粒，而因為不同的製作方法會產生不同的物理性

質與化學性質的產物，故以SEM、XRD、PL及CV來測定，並確定微波法製作出的鋅鎵氧化物的電化

學激發螢光的反應機制。

關鍵字:電化學激發螢光、鋅鎵氧化物、奈米顆粒、直接沉析法、微波法。 電化學激發螢光（Electrochemical luminescence，ECL）是用電化學的原理提供能量，使有價電數的

離子激發產生光，亦是材料的電子吸收能量激發而產生能量，而其產生的能量以光的型式產生，因為其

發光的強度會由施加的電壓與電流的密度的關係而有所改變。此系統因其量測性:敏感度高、偵測極限

廣、線性濃度範圍大、動力工作範圍廣等，可應用於生物、化學感測器。

微波法的特性，因為它是以分子震動的方式使水產生熱能而加熱，相對的一般分子也因微波的震動使分

子產生旋轉進而使分子結構的鍵結展直，並可用較少的熱量打斷鍵結，使反應物產生反應，並可減少熱

量的消耗及反應的時間。

本研究是利用不同的製程會使ZnGa2O

4有不同的大小、發光強度、物性及化性，並且利用微波法製作

ZnGa2O

4，並從中得知相關的電化學激發螢光的機制。並且探討直接沉析法與微波法對電化學發光的影

響與差異。

前言 由於微波法是以分子震動的方式加熱可以以最少的熱量加速反應，使原本需要幾小時甚至是幾天

的反應速率可以在數小時或數分鐘使反應完成，並且製作出來的產物可能會有不同的特性。因此我們先

以直接沉降法製作ZnGa2O

4為主體，並改變加熱方式，以微波的方式加熱產生出來的產物，用XRD、SEM

和PL做測定以及測其電化學激發螢光特性，並與直接沉降法做比較。 實驗方法

直接沉析法製作ZnGa2O

4奈米顆粒電極

以ZnSO4

7H2O和Ga

2(SO

4)

3xH

2O做起始材料，溶於去離子水中，配製濃度為[Zn

2+]=2[Ga

3+]=0.1M溶

液，並加入四倍溶液體積的氨水中和酸性。並將混合液以55oC同時加熱及磁石攪拌20小時。並將混合液

置於離心管中，離心分離，取出沉澱物，並置於烘箱，以60oC烘乾。壓碎粉體，置於高溫爐中，以500

oC

加熱一小時，此為奈米ZnGa2O4。後將粉末壓成13mm的圓片，並將圓片置於爐中，並通入空氣以1400

oC

燒結10小時。再將圓片放入爐中，通入純氫氣，流量50cc/sec，並加溫至800oC維持10小時，進行還原反

應。最後用銀膠將銅線與圓片底部接合，等銀膠乾後，再塗上一層蠟固定，並將要浸入電解液中的銅線

以鐵氟籠帶纏繞。

微波法製作ZnGa2O

4奈米顆粒電極

以ZnSO4

7H2O和Ga

2(SO

4)

3xH

2O做起始材料，溶於去離子水中，配製濃度為[Zn

2+]=2[Ga

3+]=0.1M

溶液，並加入四倍溶液體積的氨水中和酸性。並將混合液放入微波裝置中以55oC微波加熱及磁石攪拌。

並將混合液置於離心管中，離心分離，取出沉澱物，並置於烘箱，以60oC烘乾。壓碎粉體，置於高溫爐

中，

6

以500

oC加熱一小時，此為奈米ZnGa

2O4。後將粉末壓成13mm的圓片，並將圓片置於爐中，並通入

空氣以1400oC燒結10小時。再將圓片放入爐中，通入純氫氣，流量50cc/sec，並加溫至800

oC維持10小時，

進行還原反應。最後用銀膠將銅線與圓片底部接合，等銀膠乾後，再塗上一層蠟固定，並將要浸入電解

液中的銅線以鐵氟籠帶纏繞。

材料結構與性質分析 我們將以氫氛還原後的ZnGa

2O

4粉末進行XRD、SEM的材料結構分析與PL發光性質分析。最後，我

們將製備完成的ZnGa2O

4電極作為本系統的工作電極；因為光譜儀內部空間狹小，所以製作鈀-氫電極作

為本實驗的參考電極。相對電極選用Pt絲；電解液為鹼性系統的2M NaOH+0.5M Na2S

2O

8，進行電分析

化學與電化學激發螢光量測。

結果與討論

XRD分析

由直接沉析法合成的奈米ZnGa2O

4的XRD圖中，在角度約33°處有一較寬的峰，並不屬於ZnGa

2O

4尖

晶石結構的特性峰。這是在製作奈米粉末過程中，部分材料尚未反應完全，此峰是屬於氧化鎵(Ga2O

3)

於32.87°的 (014)特性峰。根據Scherrer’s Eqn估計由直接沉析法合成的奈米ZnGa2O

4尺寸大小約為

9.6±0.05nm。

圖2可知經氫氛(通氫氣)還原反應後除了包含ZNGA2O

4、GA

2O

3外，另外還出現了金屬鎵(GA)及氧化鋅

(ZNO)的複合物。因此，導電度的增加，可以說部份的金屬氧化物還原成金屬所造成的。ZNGA2O

4部份

形成GA2O

3和ZNO，使得尖晶石結構中四面體格隙位置與八面體格隙位置形成缺陷，這些缺陷可讓離子

遷移，因而產生新的能階，降低能隙，促使電子躍遷，使發光強度急遽增加

圖1由直接沉析法合成的ZnGa2O

4之XRD分析 圖2 ZnGa

2O

4經氫氛還原反應後之XRD分析

SEM分析

直接沉析法合成的ZNGA2O

4型態上仍有相當程

度的聚集，難以辨別顆粒的尺寸大小，取一較清

晰之顆粒量測，顆粒直徑約為10NM，和XRD中以SCHERRER’S EQN推算之結果所述相符。

圖3由直接沉析法製得的ZnGa2O

4之SEM圖

7

PL分析

直接沉析法合成的ZnGa2O

4以波長254nm激發，會不同於一般塊材ZnGa

2O

4 PL的鐘型(bell-shape)，

其PL會產生八個峰，主峰位於468nm處，如圖4。但相較於固態反應法製成的ZnGa2O

4發光強度較低，

這是因為奈米顆粒的表面積增大，表面的非輻射緩解效應增強，與結晶性變差所導致的。

經氫氛後的波形較接近電化學發光，固推測氫氛過後結構產生變化，波形也就不同於未經氫氛的

光發光譜。氫氛過後，峰由寬變窄，並產生些微的紅位移，這是因為ZnGa2O4晶格中產生氧缺陷，使

能隙中產生新的自身活化中心，使放射波長往長波長方向偏移。兩者皆有此趨勢，如圖5。

圖4由直接沉析法製成ZnGa

2O

4的放射光譜 圖5以254nm波長激發由直接沉析法製成ZnGa

2O

4經氫氛還原後圓片的放射光譜

結論 微波法跟直接沉降法所生產的產物，因為前驅物相同及實驗方法類似，在實驗結果方面可減少反應

時間，減少熱量的損耗，相對可減少成本的損失，以相同的方式確可減少耗損量，也說明了微波法

除了可以以較低的溫度及較少的時間產出我們所要的產物。

利用XRD及SEM證明所產生的物質為ZnGa2O

4並以Scherrer’s Eqn推算直接沉降法製成的顆粒

9.6±0.5nm和SEM是相同結果，之後再以EDS佐證合成物為Zn:Ga:O約為1:2:4。

參考文獻

[1] T. Ohtake, N. Sonoyama, T. Sakata., Chem. Phys. Lett., 298 (1998) 395. [2] T. Ohtake, N. Sonoyama, T. Sakata., Chem. Phys. Lett., 318 (2000) 517. [3] T. Ohtake, N. Sonoyama, T. Sakata., Bull. Chem. Soc. Jpn., 72 (1999) 2617. [4] M. Hirano, S. Okumura, Y. Hasegawa, M. Inagaki, Int. J. Inorg. Mater., 3 (2001) 797. [5] Z. Xu, Y. Li, Z Liu, Z. Xiong, Mater. Sci. Eng. B., 110 (2004) 302. [6]林子豪，”鋅鎵氧化物奈米顆粒之電化學激發螢光探討”，長庚大學化工與材料工程研究所碩士論

文(2005)

8

Development of affinity biosensor based on self-assembled array micro-electrodes

Han-Yun Lin, Fu-Kuo Huang, Wen-Chang Chen* Department of Chemical Engineering, National Yunlin University of Science and

technology 123 University Road, Sec. 3, Douliou, Yunlin, Taiwan, R.O.C. * corresponding author, E-mail: chenwc@yuntech.edu.tw

Abstract----An affinity biosensor based on the immobilization of neutravidin onto the surface modification of interdigitated array gold electrodes (IDAEs) via self-assembled methods was investigated. The biosensor was prepared by modifying the electrode surface with cysteamine, and then cross-linked with neutravidin by glutaraldehyde. The stepwise modified procedures of the biosensor were further characterized by means of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Results show that the sensor has a good reproducibility and a linear response to biotin over the concentration range of 0-10 μg/ml.

Keywords: Affinity biosensor, Self assembly, Interdigitated array electrodes (IDAEs)

INTRODUCTION

In the last few years, many researchers have been lay stress on exploitation of alternating analytical technique in order to establish more fast and sensitive analytical method. Electrochemical affinity biosensors hold a great attraction due to their latent advantage of simple, label-free, ease of use and miniaturized device size [1]. Furthermore, they offer benefits in diminishing the cost and time of analysis compared with traditional techniques. Among the various electrochemical transduction techniques, impedance spectroscopy is an efficient method to probe the interfacial properties of modified electrodes and often used for studying electrochemical reaction kinetics and application of impedimetric biosensor. Avidin is a well-known glycoprotein, which exhibits a high affinity for biotin. The avidin-biotin system allows many biological and diagnostic applications based on their rapid and almost irreversible binding of molecule to which biotin can be linked [2]. The most universal application of avidin-biotin complex layer in biosensor is the immobilization of biomolecules due to the good compatibility with other proteins. Thus it can maintain the activity of immobilized biomolecule more successful than other immobilization methods [3]. The most important factor for affinity biosensors is the immobilization of biorecognition element. Self-assembled monolayers (SAMs) is known to be a popular, simple and reliable procedure for immobilizing enzymes and molecules on various metal and oxide surfaces, mostly due to its simplicity, versatility and the establishment of a high level of order on a molecular scale as a means of preparing modified surface [4]. The most studied self-assembled systems are thiols, sulfides and disulfides self-assembling on gold and some other metals. The binding formed between the sulfur atom and gold is very strong and the so formed SAMs are stable in air, water and ethanol at room temperature [5]. The interdigitated array electrodes (IDAEs) have been paid more attention recently because of their electrochemical advantages such as high current density, sensitivity and fast response comparing with that of conventional electrodes [6]. In this work, we choose avidin-biotin system as a model for construction of affinity impedance biosensor. Affinity biosensor based on the IDAEs was made via self-assembly methods through cysteamine monolayer. On the basis of CV and EIS investigation, it was found that neutravidin can be successfully immobilized onto the surface of IDAEs. In addition, the calibration plot for biotin detection by EIS with the proposed biosensor will be also investigated. EXPERIMENTAL Reagents

9

Biotin, cysteamine and bovine serum albumin (BSA) were purchased from Sigma. Neutravidin were purchased from Pierce. Glutaraldehyde was obtained from Fluka and hexacyanoferrate (III) (Ferri) from Merck.

Preparation of the biosensor An IDAEs was immersed in 5-ml cysteamine/ethanol solution (40 mM) for 2 h at room temperature. The resulting self-assembled electrode was thoroughly rinsed with absolute ethanol and distilled water to remove physically adsorbed cysteamine molecules. The modified electrode was subsequently immersed in 5-ml glutaraldehyde solution for 1 h. The surface was then washed with distilled water to remove any loosely bound species. The immobilization of neutravidin was then carried out by dropping 15 μl neutravidin solution onto the surface of the modified IDAEs. After kept in 4 oC for more than 4 h, the affinity biosensor was rinsed and stored at 4 oC when not in use. Instrument and measurements

Impedimetric measurements of the modified electrode were performed in the presence of a 5 mM Ferri/Ferro mixture (mole ratio = 1:1) as a redox probe by using an electrochemical analyzer (Autolab PGSTAT12, Eco chemie, B.V., The Netherlands) in the frequency range of 10-2-1×106 Hz and alternating voltage of 10 mV. CVs were performed with the same redox probe by a CHI 611A Electrochemical Workstation (CH Instrument, USA) and the potential swept from -0.6 to 0.6 V with a scan rate of 100 mV/s.

RESULTS AND DISCUSSION As shown in Fig. 1(a), an almost straight line implied the characteristic of a diffusion-limiting step of the electrochemical process on a bare gold electrode (Ret = 0.6 kΩ). Fig. 1(b) shows a lower interfacial Ret (= 90 Ω), indicating that self-assembly of cysteamine was adsorbed on the electrode surface. The electron transfer resistance of amine modified electrode decreased drastically. It might be due to electrostatic interaction between the positive charged amine group and negative charged mediator, which facilitates the electron transfer between electrolyte and electrode surface. After activation of glutaraldehyde, the neutravidin molecule was immobilized on to the modified electrode. Obviously, the Ret increased up to 5 kΩ (Fig. 1(c)). This indicated that an electron transport-blocking layer was formed on the electrode. In order to blocking the residue activated sites on the modified electrode, BSA was used as a blocking molecule. From Fig. 1(d), an increasing Ret (6.4 kΩ) can be observed due to the successful blocking with aldehyde group on the electrode. Furthermore, Fig. 2 shows the CVs of the gold electrode with different modified process at a scan rate of 100 mV/s. At the bare gold electrode, a reversible electrochemical response for Fe(CN)64-/3- can be obtained (Fig. 2(a)). After the electrode was modified by cysteamine, the peak current changed obviously to a higher extent (Fig. 2(b)), due to the promoted electron-transfer kinetics through the amine group of cysteamaine [7]. After the neutravidin was immobilized, a decreased current response can be found (Fig. 2(c)). It is well known that the reaction between amine group and aldehyde group proceeds easily in moderate condition with no contamination and significant deactivation of the protein. Based on this reaction, neutravidin was covalently attached to the aminated electrode surface. In addition, the blocking of BSA also makes the current response decrease. This may be resulted from the more densely modified protein layer (Fig. 2(d)). The CV of the Ferri/Ferro redox couple confirmed this process, and greatly diminished response indicates that an insulating layer of protein is introduced. The affinity interaction of neutravidin with biotin was investigated by EIS. Fig. 3 shows the changes in the avidin-modified electrode, and calibration plot for biotin detection with the proposed biosensor is illustrated in Fig. 4. As expected, the response signal increased with the increase of biotin concentration. The linear response range covered from 0 to 10 μg/ml of biotin with a regression equation of the form Y (kΩ) = 3.478 X (μg/ml) and correlation coefficient of 0.993. The method is thus believed to be an efficient way to quantify the biotin concentration in the sample. CONCLUSIONS In summary, the feasibility of the fabrication of an affinity biosensor by covalent attachment of neutravidin

10

to a cysteamine-monolayer modified IDAEs was investigated. The assembled cysteamine was shown to facilitate electron transfer between the redox probe and the electrode surface. Improvements of the electrode coverage by SAMs and protein immobilization are known to be very important for optimizing biosensor operation and will be further studied in the near future. REFERENCES [1] R. Pei, Z. Cheng, E. Wang and X. Yang, “Amplification of antigen-antibody interaction based on biotin labeled protein-streptavidin network complex using impedance spectroscopy”. Biosens Bioelectron, 16, pp 355-361 (2001). [2] J. I. Anzai, Y. Kobayashi, Y. Suzuki, H. Takeshita, Q. Chen, T. Osa, T. Hoshi and X. Y. Du, “Enzyme sensors prepared by layer-by-layer deposition of enzymes on a platinum electrode through avidin–biotin interaction”. Sens Actuat B, 52, pp 3-9 (1998). [3] J. W. Chung, J. M. Park, R. Bernhardt and J. C. Pyun, “Immunosensors with a controlled orientation of antibodies by using Neutravidin-protein A complex at immunoaffinity layer”, J. Biotech, 126, pp. 325-333 (2006) [4] X. Zhong, R. Yuan, Y. Chai, Y. Liu, J. Dai, D. Tang, “Glucose biosensor based on self-assembled gold nanoparticles and double-layer 2d-network (3-mercaptopropyl)-trimethoxysilane polymer onto gold sub- strate,” Sens. Actuators B, 104, pp. 191–198 (2005). [5] C. Berggren, B. Bjarnason, G. Johansson, “Capacitive biosensors,” Electroanalysis, 13, pp. 173-180 (2001) [6] W. Laureyn, D, Nelis, P. V. Gerwen, K. Baert, L, Hermans, R. Magnee, J. J. Pireaux and G. Maes, “Nanoscaled interdigitated titanium electrodes for impedimetric biosensing”, Sens. Actuators B, 68, pp. 360-370 (2000). [7] S. Zhang, N. Wang, H. Yu, Y. Niu and C. Sun, “Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor”, Bioelecrochemistry, 67, pp12-15(2005).

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Electrochemical Characteristics of SPCEs with Drop-Coating of fMWCNTs and Its Application for Glucose Biosensing

Chun-Hsu Hsu1, Bo-Wen Lu1, Wen-Chang Chen1* and Hong-Ming Lin2 1Department of Chemical Engineering, Yunlin University of Science and Technology, Douliou, Yunlin 64002, Taiwan, ROC 2Department of Materials Engineering, Tatung University, Taipei 104, Taiwan, ROC * Corresponding author, E-Mail: chenwc@yuntech.edu.tw

Abstract --- An amperometric glucose biosensor was successfully constructed by drop-coating of glucose oxidase (GOD) and ferricyanide (Ferri)-multi-wall carbon nanotubes (MWCNTs) mixture onto the screen- printed carbon electrodes (SPCEs). fMWCNTs were obtained by treating MWCNTs with concentrated HNO3/H2SO4 (1:1) mixed solution, and Ferri was used as the electron mediator. The modified processes of the SPCEs and the related electrochemical characteristics were analyzed by cyclic voltammetry (CV), chronoam- perometry (CA) and electrochemical impedance spectroscopy (EIS). Results showed that the optimum ratio of fMWCNTs and applied potential for glucose measurements were 10% (v/v) and 0.3 V, respectively. For glucose measurement, fMWCNTs-modifiedbiosensor presents a wide linear response range (25 to 600 mg/dL), a higher sensitivity (0.096 μA dL/mg), and a lower detection limit (2.76 mg/dL).

Keyword: functionalized multi-wall carbon nanotubes (fMWCNTs); amperometric glcose biosensor; screen- printed carbon electrodes (SCPEs)

INTRODUCTION Recently, carbon nanotubes (CNTs) have attracted much attention because of their unique properties.

CNTs can divide into single-wall CNTs (SWCNTs) and multi-wall CNTs (MWCNTs). They may be visualized as the results of concentric and folding graphic layers into carbon cylinders [1]. Britto et al. have first reported the application of CNTs as a modified electrode material, and an excellent electrochemical performance in the oxidation of dopamine was obtained [2]. Luo et al. used SWCNTs functionalized with carboxylic acid groups to modify a glassy carbon electrode, and favorable electrocatalytic behavior toward the oxidation of biomolecules could be observed [3]. Rubianes et al. also reported CNTs-modified electrode had better electrocatalytic activities than graphite electrode [4]. Moreover, excellent electric ability of MWCNTs could improve the transfer of electrons between the mediator in the bulk solution and active redox center of GOD [5].

In this study, we report the application of Ferri-fMWCNTs-deposited SPCEs for the fabrication of the amperometric glucose biosensor. fMWCNTs were obtained by treating MWCNTs with concentrated HNO3/ H2SO4 (1:1) mixed solution, and Ferri was used as the electron mediator. The modified processes of the SPCEs and related electrochemical characteristics were analyzed by cyclic voltammetry (CV), chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS). In addition, the optimization of the applied potential was also investigated and the biosensor demonstrated a high sensitivity and a wide linear response range to glucose. EXPERIMENT Materials: Glucose oxidase (GOD, EC 1.1.3.4 from Aspergillus niger) and β-D-glucose were purchased from Sigma (St. Louis, MO), K3Fe(CN)6 from Merck, MWCNTs with outer diameter between 20-25 nm from CNT Co., Ltd. (Korea) and all other chemicals were of analytical grade. GOD solution (0.6 U/μL) was freshly prepared with 0.1 M PBS (pH 7.0) before the fabrication of biosensors. fMWCNTs were obtained by treating MWCNTs with concentrated H2SO4/HNO3 mixed solution (1:1) and heated to boiling for 3 h. The fMWCNTs were then collected and washed with deionized water. Ferri- fMWCNTs solutions of various mixing ratios were prepared by mixing appropriate volumes of fMWCNTs suspension (in 0.1M PBS, pH 7.0) with 5 ml of 0.2 M Fe(CN)63-, and then adding PBS to the final volume of 10 ml.

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Preparation of various modified electrodes: Disposable SPCEs-test strips (gifts from Apex Biotech- nology Corp., Hsinchu, Taiwan) with a two-electrode configuration were used throughout the biosensor fabric- cation. Each SPCEs-test strip consists of two identical half-circular electrodes (with a diameter of ca. 5 mm), which were used as the working and counter electrodes, on a poly (vinyl chloride) substrate. The Ferri- fMWCNTs/GOD-deposited SPCEs were prepared by drop-coating of 10 μL Ferri-fMWCNTs and 10 μL of GOD solution, respectively, onto SPCEs, and allowed to dry at 40 oC for 15 min. The biosensor test-strips were ready for use. Measurements: CV and CA were performed using a CHI 440 electrochemical workstation (CH Instruments, USA) and EIS with an Autolab PGSTAT12 (Eco chemie, B.V., The Netherlands), controlled by the GPES 4.9 and FRA 4.9 softwares. The impedance spectra were recorded in the frequency range 0.1 Hz-100 kHz by using a sinusoidal excitation signal with amplitude of 10 mV. RESULTS AND DISCUSSION Electrochemical characterization of multi-layers modified SPCEs: From preliminary experiments, redox peak currents could not be observed with fMWCNTs-deposited SPCEs by CV from -0.6 V to +0.6 V at scan rate of 0.01 V/s. In addition, no direct electron transfer between GOD active center and electrode surface was found, either. Results suggested that fMWCNTs, prepared in this study, could not be used as an electron mediator. So, Ferri was adopted as a mediator, and the response characteristics of various glucose biosensors were determined by CV in the absence and presence of 100 mg/dL glucose, respectively (data not shown). Results suggest that fMWCNTs not only provided the necessary conduction pathways, but could act like nano-scaled electrodes in promoting the electron transfer between the analysts and the SPCEs surface [4]. The electrochemical properties of the fMWCNTs-modified SPCEs were examined by using CV with 0.1 M PBS containing 0.1 M Ferri in the potential range of -0.6 V to 0.6 V for different scan rate (Fig. 1). Signifi- cant increases of the peak current and the peak potential separation with increasing scan rate could be found. It suggests that the redox reaction on the modified SPCEs surface is not completed with the increase of scan rate [6]. At low scan rates, the redox peak currents increase linearly with the scan rate (ν), not ν1/2, indicating that the redox reaction was a surface-controlled process [7]. When the scan rate is higher then 0.05 V/s, the electrode reaction becomes irreversible (Fig. 1, inset) [3]. Effect of mixing ratio: A common way of showing the resulting data of EIS is the Nyquist plot, in which the real (Z') vs. the imaginary (Z") components of the impedance (Z) are plotted. The interface can be modeled by a simplest equivalent circuit (Fig. 2, inset), made of the electrolyte solution resistance (Rs) in series with the parallel circuit of Faradic impedance and double-layer capacitance (Cdl). Here, a constant phase element (CPE) is often introduced in the equivalent circuit instead of the Cdl for a better fitting to stem from the fact that the electrode is normally rough and the deviation or real situation from the ideal behavior. Faradic impedance is composed of Warburg impedance (Zw) and electron transfer resistance (Ret). The diameter of the semicircle in the Nyquist plot equals Ret. Various mixing ratios of fMWCNTs solution (0-20 % (v/v)) were prepared and drop-coated onto SPCEs. The interface behavior of the fMWCNTs-modified SPCEs was analyzed by EIS. Results (Fig. 2) show that Ret value decreases from 35.6 KΩ to 4.46 KΩ as the fMWCNTs mixing ratio increases. It is suggested that fMWCNTs can act as a good electron-transferring layer between the mediator and the electrode surface. In addition, since there is no significant difference in Ret between 10 % (v/v) and 20 % (v/v), the former was chosen as the optimum mixing ratio for the following experiments. Effect of applied potential: Due to a strong effect on the biosensor response, the potential dependence was examined with 200 mg/dL glucose solution to find an optimum working potential for glucose measurements. As shown in Fig. 3, the amperometric response to glucose increased significantly with an increasing of the applied potential. It reached a maximal response at around 0.3~0.4 V, and then sharply decreased. Because the choice of applied potential is very important for biosensor operation to achieve the lowest detection limit and avoid the electrochemical interfering species, a potential of 0.30 V was selected as the optimum applied potential for the amperometric glucose measurements. Measurement of glucose solution: Fig. 4 shows the calibration plots of the fMWCNTs-modified and bare SPCEs glucose biosensors, respectively. Both types of biosensor could respond linearly to glucose in the

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con- centration range of 25 to 600 mg/dL. However, the fMWCNTs-modified biosensor showed a higher sensitivity (0.096 μA dL/mg versus 0.035 μA dL/mg) and a lower detection limit (2.76 mg/dL versus 5.41 mg/dL). CONCLUSION In this study, fMWCNTs-modified glucose biosensors were successfully constructed by drop-coating fMWCNTs onto the surface of SPCEs, and GOD was then layered on. Results showed that fMWCNTs act like nano-scaled electrodes and promote the electron transfer between the analysts and the SPCEs. In addition, the optimum mixing ratio of fMWCNTs and applied potential for glucose measurements was selected as 10% (v/v) and 0.3 V. For glucose measurement, fMWCNTs-modified biosensor presents a wide linear response range (25 mg/dL to 600 mg/dL), higher sensitivity (0.096 μA dL/mg) and lower detection limit (2.76 mg/dL). REFERENCES [1] M. L. Cohen, “Nanotubes, nanoscience, and nanotechnology,” Materials Science and Engineering C,

15, pp. 1-11 (2001). [2] P. J. Britto, K. S. V. Santhanam, and P. M. Ajayan, “Carbon nanotube electrode for oxidation of

dopamine,” Bioelectrochemistry and Bioenergetics, 41, pp 121-125 (1996). [3] H. Luo, Z. Shi, N. Li, Z.Gu, and Q. Zhuang, “Investigation of the electrochemical and electrocatalytic

behavior of single-wall carbon nanotubes film on a glassy carbon electrode,” Analytical Chemistry, 73, pp 915-920 (2001).

[4] M .D. Rubianes, and G. A. Rivas, “Carbon nanotubes paste electrode, ” Electrochemistry Communications, 5, pp 689-694 (2003).

[5] W. J. Guan, Y. Li, Y. Q. Chen, X. B. Zhang, and G. Q. Hu, “Glucose biosensor based on multi-wall carbon nanotubes and screen printed carbon electrodes,” Biosensors and Bioelectronics, 21, pp 508-512 (2005).

[6] A. Erlenkötter, M. Kottbus, and G. C. Chemnitius, “Flexible amperometric transducers for biosensors based on screen print three electrode system,” Journal of Electroanalytical Chemistry, 481, pp 82-94 (2000).

[7] Y. D. Zhao, W. D. Zhang, H. Chen, and Q. M. Luo, “Direct electron transfer of glucose oxidase molecules adsorbed onto carbon nanotube power microelectrode,” Analytical Sciences, 18, pp 939-941(2002).

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16

LACTATE BIOSENSORS BASED ON LACTATE DEHYDROGENASE-MULTIWALLED CARBON

NANOTUBE-CHITOSAN NANOBIOCOMPOSITE

Siao-Yun Chen(陳筱芸)、Yu-Chen Tsai (蔡毓楨)* 中興大學化學工程學系

台中市南區國光路 250 號

04-22857257, 04-22854743, yctsai@dragon.nchu.edu.tw (NSC 95-2221-E-005-083)

Abstract --- A composite of multiwalled carbon nanotubes-chitosan (MWCNT-CHIT) was used as a matrix for entrapment of lactate dehydrogenase (LDH) on a glassy carbon electrode in order to fabricate

amperometric biosensor. The inclusion of MWCNT within MWCNT-CHIT-LDH exhibits the abilities to raise the

current responses and decrease the electrooxidation potential of NADH (β-nicotinamide adenine dinucleotide,

reduced form). The biosensor for the determination of lactate shows a sensitivity of 584 nA mM-1 and a response

time of about 3 s.

Keywords: Biosensor; Amperometry; Multiwalled carbon nanotube; Lactate dehydrogenase; Chitosan

INTRODUCTION

The determination of lactate is vital in the areas of clinical diagnostic, fermentation, and food analysis. In

general, analysis of lactate is based on spectrophotometry [1]; this method is time consuming and complicated.

In this case, amperometric biosensors fulfill these requirements. LDH catalyzes the conversion of lactate to

pyruvate in the presence of NAD+ and the NADH produced in the enzymatic reaction can be measured. Recently,

chitosan has attracted increasing attention as a biomaterial for immobilizing enzyme through the formation of

polyelectrolyte complexes between the enzymes and polysaccharide chains of the chitosan [2,3].

The electrooxidation of NADH are especially significant because they can provide high sensitivity and low

detection limit. However, the selectivity and stability of electrochemical determination of NADH have been

often insufficient because of a large overpotential for the direct electrochemical oxidation of NADH at

conventional electrodes [4]. Recent studies show that carbon nanotubes (CNTs)-based electrodes possess the

ability to significantly decrease the overpotential for electrochemical oxidation of NADH [5-7]. In this study,

chitosan is selected to suspense CNTs owing to its splendid film-forming properties, biological compatibility,

and good adhesion.

EXPERIMENTAL

The bare glassy carbon electrode (GCE) was polished with 0.3 μm alumina slurries and rinsing it with

deionizedwater. LDH and MWNTs were added to 2 ml of 0.25 wt% chitosan solution, was ultrasonicated to

form a homogeneous MWCNT–CHIT-LDH solution. The MWCNT–CHIT-LDH film was prepared by casting 6

μL aliquot of MWCNT–CHIT-LDH solution on bare GCE (3 mm). The solvent was allowed to evaporate at

room temperature in the air. In a typical measurement, 20 ml of sample was transferred to the cell.

Measurements of lactate were carried out in a 0.1 M phosphate buffer solution supporting electrolyte medium.

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RESULTS AND DISCUSSION

Figure 1(a) and 1(b) shows the AFM images of MWCNT-CHIT, MWCNT-CHIT-LDH modified glassy

carbon electrodes, respectively. AFM images clearly show that a homogeneous dispersion of MWCNT and

LDH are achieved throughout the chitosan.

Figure 2 shows the amperometric responses for 0.1 mM NADH MWCNT-CHIT modified electrode.

Different potentials (0.2, 0.4, 0.6 and 0.8 V) were applied on MWCNT-CHIT modified glassy carbon electrode.

With increasing oxidation potential, the response increases rapidly over the 0.2 – 0.6 V range and displays a

maximum value at 0.6 V. An working potential of 0.6 V was chosen for subsequent studies in order to obtain

high sensitivity and to minimize possible interferences.

The amperometric reponse of the MWCNT-CHIT-LDH modified GCE to the addition of varying

concentrations of lactate are shown in Figure 3. The sensitivity at the MWCNT-CHIT-LDH modified GCE is

584 nA mM-1 with a response time of about 3 s.

CONCLUSION

We have developed and characterized the performance of a lactate biosensor based on a novel

MWCNT-CHIT-LDH nanobiocomposite film. The biosensor was prepared by using MWCNT-CHIT as an

entrapment support of LDH. It demonstrates that the MWCNT-CHIT-LDH nanobiocomposite film combines

the advantages of efficient electrocatalytic activity of MWCNT and the excellent properties of the

nanobiocomposite. This immobilization methodology is promising for a wide range of biosensor construction

which is based on the entrapment of enzymes within a MWCNT-CHIT composite.

REFERENCES

[1] F.K. Sartain, X. Yang, C.R. Lowe, "Holographic lactate sensor", Anal. Chem., 78, 5664-5670 (2006). [2] B. Krajewska, "Application of chitin- and chitosan-based materials for enzyme immobilizations: a review",

Enzyme Microb. Technol., 35, 126-139 (2004).

[3] Y. Liu, X. Qu, H. Guo, H. Chen, B. Liu, S. Dong, "Facile preparation of amperometric laccase biosensor with multifunction based on the matrix of carbon nanotubes–chitosan composite", Biosens. Bioelectron., 21, 2195-2201 (2006).

[4] M. Zhang, W. Gorski, "Electrochemical sensing based on redox mediation at carbon nanotubes", Anal. Chem., 77, 3960-3965 (2005).

[5] M. Musameh, J. Wang, A. Merkoci, Y. Lin, "Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes", Electrochem. Commun., 4, 743-746 (2002).

[6]