在生物醫學上的醫療診斷、藥物篩選、化學分析和環境偵測等各種領域中的諸多挑戰，使新穎生物感測元件有更急迫的需求。在許多不同的感測技術當中，矽奈米線場效應電晶體感測器具有免標定、即時偵測、高靈敏度、優異的選擇性等優異特性。本論文聚焦於多條平行連結的矽奈米線場效應電晶體(Multiple-parallel-connected (MPC) SiNW-FET)之元件組裝，以及將其應用於生物相關離子濃度之感測。本研究中所使用之多條平行連結矽奈米線場效應電晶體，和傳統傳導通道(conducting channel)僅用一根或數根矽奈米線的場效應電晶體相比，擁有相當高的感測靈敏度和較佳的訊雜比。
鉀離子對於人體各樣細胞有正常功能活動扮演重要的角色，例如：人體電解質平衡、人體酸鹼平衡、細胞凋亡、神經傳遞和肌肉收縮等。為了進行鉀離子的感測，本研究於矽奈米線場效應電晶體上修飾可特異性結合鉀離子的去氧核醣核酸適體(K+-specific DNA-aptamers)，進行皮質神經元細胞鉀離子釋放的即時偵測。修飾後之矽奈米線場效應電晶體具有寬廣的線性工作範圍，可偵測鉀離子濃度從10-9 M到10-6 M，並且對鉀離子具有高靈敏度，而對其他的鹼金屬離子幾乎沒有電訊號的反應。神經細胞放置在生物感測器表面，並以去鈉/鉀離子緩衝溶液培養時，AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid，α-氨基-3-羥基-5-甲基-4-異噁唑丙酸)可以刺激神經細胞釋放鉀離子，此釋出可被AMPA的受體拮抗劑 6,7-二硝基-2,3-二酮 (6,7-dinitroquinoxaline-2,3-dione) 所抑制。此外，當神經細胞在一般生理緩衝液中培養時，分離細胞質中的鉀離子濃度下降了百分之75。這些結果說明了矽奈米線生物感測器可以專一性、高靈敏的偵測鉀離子；鉀離子在神經細胞內外的濃度則受到神經刺激的極大調控。
鋅離子對於許多生物活動扮演重要的角色，像是細胞凋亡、去氧核醣核酸合成、酶活性、基因表達、免疫系統功能和神經傳遞等。我們設計對鋅具有特異性的感測器來研究細胞外鋅離子濃度與β澱粉蛋白纖維化之關係。另外，目前於生理條件下，還無法於神經元周圍偵測到鋅離子濃度。因此，我們利用將具有鋅離子特異性螢光分子(FluoZin-3)修飾於矽奈米線場效應電晶體上，進行即時的細胞外鋅離子濃度量測。鋅離子特異性螢光分子之矽奈米線場效應電晶體在鋅離子的量測上具有約12 nM的解離常數(dissociation constant)，以及從10-11 M到10-6 M的大範圍線性工作區間，而對其他的生物相關的二價離子(例如：鐵、鈣、錳、鎂等)則沒有可偵測到的變化。本研究將培養有胚胎皮質神經元細胞的蓋玻片放置在FZ-3/SiNW-FET上，AMPA的刺激可使鋅離子濃度上升到約110 nM。AMPA受體或胞吐作用的阻斷劑可以大量抑制鋅離子濃度升高，說明了儲存於突觸微胞中的鋅離子是濃度升高的主要來源。此外，修飾有乙型類澱粉蛋白的矽奈米線元件能以約633 nM的解離常數結合鋅離子，並可量測受AMPA刺激而釋放的鋅離子。此結果顯示鋅離子濃度可達到足以結合類澱粉蛋白的濃度，提高鋅離子誘導類澱粉蛋白纖維化的可能性，為阿茲海默症發病的可能因素之一。以上這些結果表現出矽奈米線場效電晶體用於探索人體疾病中生物學基礎的無窮潛力。

The growing number of challenges in a wide variety of areas, including biomedicine (medical diagnosis, proteomics, drug screening, and toxicity), food production, chemical analysis, and environmental monitoring, has pushed the challenging demands for novel biosensors. Among different kinds of developed biosensing technologies, silicon nanowires field-effect transistor (SiNW-FET)-based biosensors stand out due to their attractive features, such as label-free and real-time detection, ultrahigh sensitivity, exquisite selectivity, multiplexing, and high integration density. The focus of this thesis is the fabrication of multiple-parallel-connected (MPC) SiNW-FETs and their potential applications for sensing biologically relevant ions. Compared with a traditional SiNW-FET, whose conducting channel is composed of only a single or a few SiNWs, an MPC SiNW-FET system possesses remarkably higher detection sensitivity (i.e., larger transconductance) and a better signal-to-noise ratio (SNR) in electrical measurements.
The homeostasis of potassium ion (K+) is essential for the proper function of various cellular activities, such as fluid-electrolyte balance, acid-base balance, apoptosis, neurotransmission, and muscle contraction. To detect K+, an MPC SiNW-FET was modified with K+-specific DNA-aptamers (aptamer/SiNW-FET) for the real-time detection of the K+ efflux from cultured cortical neurons. The aptamer/SiNW-FET showed a wide linear working range of detecting K+ from 10-9 M to 10-6 M and high sensitivity against K+ with association constant of 2.18 ± 0.44 × 106 M-1. Moreover, the aptamer/SiNW-FET showed either less or negligible response to other alkali metal ions. When neurons were placed atop the aptamer/SiNW-FET in a Na+/K+-free buffer, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA) stimulated neurons exhibited the escalated K+ efflux in a dose-dependent manner, which is greatly suppressed by 6,7-dinitroquinoxaline-2,3-dione, an AMPA receptor antagonist. In addition, when neurons were stimulated under a normal physiological buffer, the intracellular K+ concentration in the isolated cytosolic fraction was decreased by 75 %. These findings demonstrated that the aptamer/SiNW-FET is sensitive and selective for detecting K+ and the K+ concentration inside and outside of the neurons could be greatly altered to modulate the neuron excitability.
Zinc ion (Zn2+) is vital for various biological activities, such as apoptosis, DNA synthesis, enzyme activities, gene expression, immune system function, and neurotransmission. The objective for designing the Zn2+-specific sensor is to study the link between extracellular Zn2+ concentration ( ) and the amyloid-β (Aβ) fibrilization. Moreover, the exact surrounding neurons under (patho)physiological conditions is not settled. To address these concerns, a SiNW-FET was modified with the Zn2+-sensitive fluorophore, FluoZin-3 (FZ-3/SiNW-FET), to quantify the in real time. The FZ-3/SiNW-FET device has a dissociation constant of ~12 nM against Zn2+ and has a linear working range spanned from 10-11 M to 10-6 M with no appreciable conductance change for other biologially relevant divalent ions (Fe2+, Ca2+, Mn2+, or Mg2+). By placing a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, the AMPA stimulation elevated the to ~110 nM. Blockers against the AMPA receptor or exocytosis have greatly suppressed this elevation, demonstrating that the Zn2+ stored in the synaptic vesicles was the major source responsible for the elevation of . In addition, a SiNW-FET modified with Aβ could bind Zn2+ with a dissociation constant of ~633 nM and respond to the Zn2+ released from AMPA-stimulated neurons. These results showed that can reach a level high enough to bind Aβ and thus raises the possibility that Zn2+-induced Aβ fibrilization could be one of the factors for the onset of Alzheimer’s disease. These extraordinary results demonstrate the almost endless capabilities of SiNW-FET biosensors to explore the biological underpinnings of human diseases.

Abstract iii
Acknowledgements vii
Table of Contents viii
List of Figures xi
List of Tables iii
List of Abbreviation xiv

Chapter 1 Introduction–Aim and Scope 1
1.1 Introduction–Aim and Scope 1
1.2 References 4
Chapter 2 Silicon Nanowire Field-Effect Transistor–Working and Fabrication 8
2.1 Introduction–Transistor 8
2.2 Theory of MOSFET 9
2.3 From MOSFETs to ISFET to Silicon Nanowire FETs 15
2.4 SiNW Synthesis and Device Fabrication 20
2.5 References 23
Chapter 3 Biology of Metal Ions 26
3.1 A Survey of the Role of Metal lons in Biological Systems 26
3.2 Sodium and Potassium in Health and Disease 27
3.3 Zinc in Health and Disease 33
3.4 Conclusion 37
3.5 References 38
Chapter 4 Experimental Setup and Important Considerations 42
4.1 Electrical Measurments and Device Characterization 42
4.2 Sample Delivery: On-Chip Microfluidics Integration 45
4.3 Device-to-Device Reproducibility 45
4.4 Debye-Hückel Screening Effect 46
4.5 Association Constant: Langmüir Adsorption Isotherm Model 48
4.6 References 49
Chapter 5 Detection of K+ Efflux from Stimulated Cortical Neurons by
an Aptamer-Modified Silicon Nanowire Field-Effect Transistor 50
5.1 Introduction 50
5.2 Electrical Characterization 54
5.3 Functionalization of Aptamers on SiNW-FET 56
5.4 Sensitivity of Aptamer/SiNW-FET Device 58
5.5 Selectivity of Aptamer/SiNW-FET Device 61
5.6 K+ Efflux from Stimulated Neurons 64
5.7 AMPA Dose-Dependent K+ Release from Neurons 68
5.8 Estimation of K+ in Cytosolic Fraction 71
5.9 Additional Remarks 74
5.10 Conclusions 76
5.11 References 77
Chapter 6 The Extracellular Zn2+ Concentration Surrounding Excited Neurons
is High Enough to Bind Amyloid-β Revealed by a Nanowire Transistor 84
6.1 Introduction 84
6.2 Electrical Characterization of SiNW-FET Device 88
6.3 Functionalization of FluoZin-3 on SiNW-FET 90
6.4 Sensitivity and Selectivity of FZ-3/SiNW-FET Device 92
6.5 Actions of Agonists and Antagonists on Zn2+ Efflux from Stimulated Neurons 95
6.6 Non-Specific Binding of AMPA and DNQX on FZ-3/SiNW-FET 97
6.7 AMPA-induced Zn2+ release from the Cortical Neurons 97
6.8 Effect of AMPA Receptor Blockers on Zn2+ release from the Stimulated Cortical
Neurons 100
6.9 Origin of released Zn2+ 102
6.10 Interaction of Zn2+ and Aβ 105
6.11 Conclusion 108
6.12 References 109
Conclusion and Outlook 116
Appendixes
Appendix A1: Immobilization of DNA-Aptamers on an MPC SiNW-FET 119
Appendix A2: Primary Culture of Cortical Neurons 120
Appendix A3: Electrophysiology Recording 121
Appendix A4: Estimation of IC50 Values Using a Boltzmann Sigmoidal Function 122
Appendix A5: Determination of K+ from Cellular Extract 123
Appendix A6: Immobilization of FluoZin-3 on an SiNW-FET 124
Appendix A7: Ca2+ Imaging 127
Appendix A8: List of Publications 128

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