利用指叉金電極阻抗感測器監測大腸桿菌之生長

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研究生：	蘇虹娜 Kalpana Settu
論文名稱：	利用指叉金電極阻抗感測器監測大腸桿菌之生長 Monitoring Escherichia coli Growth using Interdigitated Gold Microelectrode-Based Impedance Sensors
指導教授：	蔡章仁 Jang-Zern Tsai
口試委員:
學位類別：	碩士 Master
系所名稱：	資訊電機學院 - 電機工程學系 Department of Electrical Engineering
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	179
中文關鍵詞：	指叉微電極、阻抗譜、大腸桿菌
外文關鍵詞：	Escherichia coli, Impedance spectroscopy, Interdigitated microelectrode
相關次數：	點閱：12 下載：0
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摘要：
本研究以照相平版印刷術在玻璃基板製造有效面積為0.4平方毫米的指叉形金微電極感測器以利用阻抗分析法監測大腸桿菌在數種不同介質中的生長；這些介質包括去離子水、細菌生長介質、動物細胞培養基、牛的生乳、和人尿。阻抗測量是在細菌的生長過程進行，使用的儀器是IM6ex阻抗分析儀，測量的頻率範圍是1Hz至1MHz。從測量結果和等效電路分析得知大腸桿菌的生長引起電雙層電容的變化；因此，細菌生長所引起的阻抗變化宜使用低頻率（<10千赫）來監測。在去離子水裡，大腸桿菌生長時從其細胞釋放的離子造成測量到的阻抗變小。在細菌生長介質裡和在生牛乳裡，測得的阻抗下降是由於由大腸桿菌代謝造成介質中離子濃度的變化。在動物細胞培養基裡由於培養液pH值在大氣環境中迅速增加，使得大腸桿菌的生長受到抑制，因此沒有觀察到阻抗變化。在人尿中，細菌生長過程中的阻抗變化是由於感測器表面上的細菌生物膜的形成和積累。實驗的結果證實這指叉形金微電極感測器可應用於利用阻抗測量的方式在細菌生長介質、動物細胞培養基、牛的生乳、和人尿裡監測大腸桿菌的生長。

ABSTRACT:
Interdigitated gold microelectrode (IDME) sensor was designed with an active area of 0.4 mm2 and fabricated photolithographically on glass substrate for monitoring E-coli growth by impedance analysis. This in-house fabricated IDME sensor was used as impedance sensor for monitoring Escherichia coli (E.coli) growth in different medium such as DI-water, bacterial growth medium (LB Miller’s broth), animal cell culture medium (DMEM), raw cow’s milk and in human urine. Impedance measurement was carried out during bacterial growth in different mediums over a frequency range of 1 Hz to 1 MHz using IM6ex impedance analyzer. Based on equivalent circuit analysis, it was observed that double layer capacitance was responsible for the impedance change caused by the E.coli growth and hence low frequencies (<10 kHz) were suitable for the IDME impedance sensor to monitor the change in impedance during the bacterial growth. In DI-water, decrease in impedance was observed during the E-coli growth time due to the ion release from bacterial cells. In LB-broth and raw cow milk, during E-coli growth decrease in impedance was observed due to the ionic concentration change in the medium caused by E-coli metabolism. In DMEM, E-coli growth was inhibited due to the rapid increase in pH of DMEM at ambient atmosphere and hence impedance change was not observed. In urine, change in impedance during bacterial growth was due to the formation and accumulation of bacterial biofilm on the sensor surface. IDME sensor was successfully applied to monitor the growth of E-coli in LB Miller’s broth, DMEM, raw cow’s milk and in human urine using impedance measurements.

TABLE OF CONTENTS
ABSTRACT................................................................................................................................... I
ABSTRACT (Chinese).................................................................................................................. II
List of Figures..............................................................................................................................VII
List of Tables............................................................................................................................... XV
INTRODUCTION........................................................... 1
1.     Biosensors...................................................... 2
2.     Electrochemical Impedance Spectroscopy (EIS)    ...................................................................... 9
2.1.    Impedance theory..........................................  10
2.2.     Equivalent circuit analysis.............................. 12
3.    Interdigitated microelectrodes (IDME)............... 13
3.1.    Microfabrication.............................................. 14
3.2.    IDME in animal-cell analysis............................ 17
3.3.    IDME in Bacterial detection..............................19
4.    Escherichia coli O157:H7................................ 25
4.1.    Bacterial Growth Curve................................... 26
4.2.    Bacterial metabolism...................................... 28
4.3.    Bacterial biofilm............................................. 34
MOTIVATION AND OBJECTIVES................................ 38
MATERIALS AND METHODS......................................41
1.    Interdigitated microelectrode (IDME) preparation    ......................................................................41
1.1.    Interdigitated microelectrode design..................41
1.2.    Electrode material selection............................ 55
1.3.    Interdigitated microelectrode fabrication............ 55
1.4.    Interdigitated microelectrode characterization    ....................................................................  61
2.    Escherichia coli O157:H7 preparation............... 67
2.1.    Escherichia coli O157:H7 cell culture................69
2.2.    Escherichia coli O157:H7 cell counting............. 70
3.    Monitoring E-coli growth by impedance measurement................................................................. 72
RESULTS AND DISCUSSION..................................... 76
1.    Sensor characterization.................................. 76
1.1.    Microfabrication process analysis.................... 77
1.2.    Capacitance characteristics............................ 80
1.3.    Electrical characterization of sensors in different solutions........................................................................ 82
2.    E-coli O157:H7 preparation.............................. 91
3.    Monitoring E.coli growth in DI-water.................. 93
3.1.    Electrical Impedance Spectroscopy (EIS) of E.coli in DI-water    ....................................................................  93
3.2.    Effect of E-coli cells in DI-water on impedance measurement................................................................. 99
4.    Monitoring E-coli growth in LB Miller’s broth (Luria Bertani broth)............................................................... 103
4.1.    Impedance measurement of E-coli growth in LB Miller’s broth................................................................ 103
4.2.    Effect of the growth of E-coli cells in LB Miller’s broth on impedance measurement................................................................106
4.3.    E-coli concentration during growth in LB Miller’s broth............................................................................ 113
4.4.    Impedance change versus concentration of E.coli    ................................................................... 115
5.    Monitoring E-coli growth in DMEM (Dulbecco’s Modified Eagle Medium)............................................... 117
5.1.    Impedance measurement of E-coli growth in DMEM    ................................................................... 117
6.    Monitoring E-coli growth in milk...................... 120
6.1.    Impedance measurement of E-coli growth in milk    ................................................................... 120
6.2.    Effect of the growth of E-coli cells in milk on impedance measurement.............................................. 123
6.3.    E-coli growth curves in milk with different cell concentrations.............................................................. 127
6.3.     pH during E-coli growth in milk...................... 129
6.4.    Impedance change versus concentration of E.coli    ................................................................... 131
7.    Monitoring E-coli growth in urine..................... 133
7.1.    Impedance measurement of E-coli growth in urine    ................................................................... 133
7.2.    Effect of the growth of E-coli cells in urine on impedance measurement.............................................. 135
7.3.    E-coli growth curves in urine with different cell concentrations............................................................. 140
CONCLUSION.......................................................... 144
Future work.................................................................. 146
References.................................................................. 147
Appendix .....................................................................158

                                

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