乳癌是全世界女性中最重要的癌症之一。因為乳癌易於轉移和復發，所以如果不及早診斷，則死亡率相對較高。因此，重要的是開發一種能夠快速且準確地診斷出乳癌的裝置。近來，已經報導了微核糖核酸（microRNA）例如microRNA-195和microRNA-126作為用於乳腺癌診斷的生物標記。因此本研究開發兩種生物感測器（包括電容式和場效應晶體管（FET）生物感測器）來檢測和定量microRNA，以快速，準確地診斷乳腺癌。電容式生物感測器由六個不同尺寸的叉指式電極組成，與平面電極相比，其面積增加，從而提高了固定效率。在使用延伸性結構在閘極上去避免德拜長度影響的FET生物感測器中，使用穩定的金硫醇化學方法將microRNA-195和microRNA-126特異性探針固定在金柵電極上。由於這兩個生物感測器與CMOS電路集成在一起，因此發現這些生物感測器的性能優於常規感測器。可以成功實現濃度範圍從1 fM到10 pM的microRNA-195的檢測，電容性生物感測器的檢測限為617 aM，FET生物感測器的檢測限為84 aM。頻率變化為587％，電容變化為159％。實驗結果表明，microRNA特異性探針可用於CMOS電路上的microRNA檢測。

Breast cancer is one of the most common cancers among women worldwide. Because breast cancer is prone to metastasis and relapse, if not diagnosed early, the mortality rate is relatively high. Therefore, it is important to develop a device that can diagnose breast cancer quickly and accurately. Recently, Micro ribonucleic acid (microRNAs) such as microRNA-195 and microRNA-126 have been reported as biomarkers for breast cancer diagnosis. Therefore, this study developed two kinds of biosensors including a capacitive and a field-effect transistor (FET) biosensor to quickly and accurately diagnose breast cancer. The capacitive biosensor consisted of six different-size interdigitated electrodes, which increased the area and hence enhanced the immobilization efficiency as compared with the planar electrodes. In the FET biosensor which used an extended gate structure to avoid the Debye length influence, immobilized microRNA-195 and microRNA-126 specific probes on the gold-gate electrode using stable gold-thiol chemistry was carried out. Since these two biosensors were integrated with CMOS circuits, the performance of these biosensors was found to be superior to the conventional sensors. Detection of microRNA-195 with concentrations ranging from 1 fM to 10 pM could be successfully achieved and the limits of detection were found to be 617 aM for capacitive biosensors and 84 aM for FET biosensors. The variation of the frequency was achieved to be 587% and the capacitance change was 159%. Experimental results showed that the microRNA specific probe can be used for microRNA detection by using the CMOS circuits.

Acknowledgements…………………………………………………………………...1
Abstract 3
中文摘要 5
Table of contents 6
List of tables 9
List of figures 10
Abbreviations and nomenclature 18
Chapter 1 Introduction 21
1-1 Breast cancer and cancer diagnosis methods 21
1-2 MEMS and CMOS-MEMS 24
1-3 CMOS biosensors...…………………………………………………….27
1-4 CMOS biosensors for detecting microRNA………………………………28
1-5 CMOS-based integrated microfluidic systems…………………………...30
1-6 Motivation and novelty 33
Chapter 2 Materials and methods 35
2-1 Experimental procedure of microRNA detection………………………..35
2-2 Design of the interdigitated electrodes (IDE) 40
2-3 Design of the integrated circuit and simulation 44
2-3-1 Comparator…………………………………………………………..44
2-3-2 Output driver………………………………………………………...49
2-3-3 System of sensing circuit……………………………………….........51
2-4 Influencing factors of Debye length and electric-double layer 53
2-5 Sensing mechanism of DNA field effect transistor (DNAFET) …………59
2-6 Sensing mechanism of DNAFET………...………………………………..63
2-7 Design of the microfluidic chip and integration with biosensors……….66
2-8 Immobilization of thiolated DNA probe on the gold electrode surface………………………………………………………………………….67
Chapter 3 Results and discussion 69
3-1 IDE biosensors for measurements of microRNAs……………………….69
3-1-1 Calibration of detecting microRNA-195 and microRNA-126 under different concentrations in IDE biosensors…………………………...69
3-1-2 Specificity measurements of microRNA-195 and microRNA-126 in IDE biosensors…………………………………………………………………75
3-2 DNA-FET biosensor for measurements of microRNAs…………………79
3-2-1 Calibration of detecting microRNA-195 and microRNA-126 under different concentrations in DNA-FET sensors……………………….79
3-2-2 Specificity measurements of microRNA-195 and microRNA-126 in DNA-FET biosensors…………………………………………………………83
3-2-3 Blind tests with microRNA-195 and microRNA-126 in DNA-FET sensors…………………………………………………………………………87
Chapter 4 Conclusions and future perspectives 90
4-1 Conclusions 90
4-2 Future perspectives ………………………………………………………..91
References 93

1. P.P. Rosen, S. Groshen, P.E. Saigo, D.W. Kinne, and S. Hellman, "Pathological prognostic factors in stage I (T1N0M0) and stage II (T1N1M0) breast carcinoma: a study of 644 patients with median follow-up of 18 years," J Clin Oncol, 7(9), pp. 1239-51, 1989.
2. J.W. Hadden, "The immunology and immunotherapy of breast cancer: an update," International Journal of Immunopharmacology, 21(2), pp. 79-101, 1999.
3. K. Holland, I. Sechopoulos, R.M. Mann, G.J. den Heeten, C.H. van Gils, and N. Karssemeijer, "Influence of breast compression pressure on the performance of population-based mammography screening," Breast Cancer Research, 19, pp. 126-133, 2017.
4. L. Harris, H. Fritsche, R. Mennel, L. Norton, P. Ravdin, S. Taube, M.R. Somerfield, D.F. Hayes, and R.C. Bast, "American society of clinical oncology 2007 update of recommendations for the use of tumor markers in breast cancer," Journal of Clinical Oncology, 25(33), pp. 5287-5312, 2007.
5. M.J. Duffy, A. van Dalen, C. Haglund, L. Hansson, R. Klapdor, R. Lamerz, O. Nilsson, C. Sturgeon, and O. Topolcan, "Clinical utility of biochemical markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines," European Journal of Cancer, 39(6), pp. 718-727, 2003.
6. F. Asad-Ur-Rahman and M.W. Saif, "Elevated level of serum Carcinoembryonic Antigen (CEA) and search for a malignancy: A Case Report," Cureus, 8(6), pp. 648-656, 2016.
7. S. Rontogianni, E. Synadaki, B. Li, M.C. Liefaard, E.H. Lips, J. Wesseling, W. Wu, and M. Altelaar, "Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping," Communications Biology, 2, pp. 325-338, 2019.
8. H. Wang, R. Peng, J.J. Wang, Z.L. Qin, and L.X. Xue, "Circulating microRNAs as potential cancer biomarkers: the advantage and disadvantage," Clinical Epigenetics, 10, pp. 10-18, 2018.
9. S. Shantikumar, A. Caporali, and C. Emanueli, "Role of microRNAs in diabetes and its cardiovascular complications," Cardiovascular Research, 93(4), pp. 583-593, 2012.
10. B.M. Ryan, A.I. Robles, and C.C. Harris, "Genetic variation in microRNA networks: the implications for cancer research," Nature Reviews Cancer, 10(6), pp. 389-402, 2010.
11. H.M. Heneghan, N. Miller, R. Kelly, J. Newell, and M.J. Kerin, "Systemic miRNA-195 differentiates breast cancer from other malignancies and is a potential biomarker for detecting noninvasive and early stage disease," Oncologist, 15(7), pp. 673-682, 2010.
12. M. Swellam, A. Ramadan, E.A. El-Hussieny, N.M. Bakr, N.M. Hassan, M.E. Sobeih, and L.R. EzzElArab, "Clinical significance of blood-based miRNAs as diagnostic and prognostic nucleic acid markers in breast cancer: Comparative to conventional tumor markers," Journal of Cellular Biochemistry, 120(8), pp. 12321-12330, 2019.
13. G. Cecene, S. Ak, G.G. Eskiler, E. Demirdogen, E. Erturk, S. Gokgoz, V. Polatkan, U. Egeli, B. Tunca, G. Tezcan, U. Topal, S. Tolunay, and I. Tasdelen, "Circulating miR-195 as a therapeutic biomarker in turkish breast cancer patients," Asian Pac J Cancer Prev, 17(9), pp. 4241-4246, 2016.
14. W.P. Yu, X. Liang, X.D. Li, Y. Zhang, Z.Q. Sun, Y. Liu, and J.X. Wang, "MicroRNA-195: a review of its role in cancers," Oncotargets and Therapy, 11, pp. 7109-7123, 2018.
15. J. Fluitman, "Microsystems technology: Objectives," Sensors and Actuators a-Physical, 56(1-2), pp. 151-166, 1996.
16. H.W. Qu, "CMOS MEMS fabrication technologies and devices," Micromachines, 7(1), pp. 14-35, 2016.
17. C. Stagni, C. Guiducci, L. Benini, B. Ricco, S. Carrara, B. Samori, C. Paulus, M. Schienle, M. Augustyniak, and R. Thewes, "CMOS DNA sensor array with integrated a/d conversion based on label-free capacitance measurement," IEEE Journal of Solid-State Circuits, 41(12), pp. 2956-2964, 2006.
18. J.J. Sniegowski, "Chemical-mechanical polishing: Enhancing the manufacturability of MEMS, " Micromachining and Microfabrication Process Technology Ii, 2879, pp.104-115. 1996.
19. A.N. Kozitsina, T.S. Svalova, N.N. Malysheva, A.V. Okhokhonin, M.B. Vidrevich, and K.Z. Brainina, "Sensors based on bio and biomimetic receptors in medical diagnostic, environment, and food analysis," Biosensors-Basel, 8(2), pp.34-69, 2018.
20. M.A. Morales and J.M. Halpern, "Guide to selecting a biorecognition element for biosensors," Bioconjugate Chemistry, 60(1), pp. 3231-3239, 2018.
21. S. Liebana and G.A. Drago, "Bioconjugation and stabilisation of biomolecules in biosensors," Essays in Biochemistry, 60(1), pp. 59-68, 2016.
22. P.P. Shah, L.E. Hutchinson, and S.S. Kakar, "Emerging role of microRNAs in diagnosis and treatment of various diseases including ovarian cancer," Journal of ovarian research, 2(1), pp. 11-20, 2009.
23. W. Shu, E.D. Laue, and A.A. Seshia, "Investigation of biotin-streptavidin binding interactions using microcantilever sensors," Biosens Bioelectron, 22(9-10), pp. 2003-2009, 2007.
24. N. Bhalla, P. Jolly, N. Formisano, and P. Estrela, "Introduction to biosensors," Essays in Biochemistry, 60(1), pp. 1-8, 2016.
25. M.P. Byfield and R.A. Abuknesha, " Biochemical aspects of biosensors," Biosensors and Bioelectronics, 9(4-5), pp. 373-400, 1994.
26. A. Ahmed, J.V. Rushworth, N.A. Hirst, and P.A. Millner, "Biosensors for whole-cell bacterial detection," Clinical microbiology reviews, 27(3), pp. 631-646, 2014.
27. K.-I. Chen, B.-R. Li, and Y.-T. Chen, "Silicon nanowire field-effect transistor-based biosensors for biomedical diagnosis and cellular recording investigation," Nano today, 6(2), pp. 131-154, 2011.
28. H.L. Cheng, C.Y. Fu, W.C. Kuo, Y.W. Chen, Y.S. Chen, Y.M. Lee, K.H. Li, C.C. Chen, H.P. Ma, P.C. Huang, Y.L. Wang, and G.B. Lee, "Detecting miRNA biomarkers from extracellular vesicles for cardiovascular disease with a microfluidic system," Lab on a Chip, 18(19), pp.2917-2925, 2018.
29. N. Lu, A. Gao, P. Dai, S. Song, C. Fan, Y. Wang, and T. Li, "CMOS-Compatible silicon nanowire Field-Effect Transistors for ultrasensitive and label-free micrornas sensing," Small, 10(10), pp. 2022-2028, 2014.
30. S. Damiati, U.B. Kompella, S.A. Damiati, and R. Kodzius, " Microfluidic devices for drug delivery systems and drug screening," Genes, 9(2), pp. 103-127, 2018.
31. J.C. Liou, T.Y. Su, and Ieee, "Investigation of the DNA droplet CMOS/MEMS chip microfluidic channels geometry, " IEEE International Symposium on Next-Generation Electronics. pp. 208-209, 2018.
32. K.H. Lee, J.O. Lee, M.J. Sohn, B. Lee, S.H. Choi, S.K. Kim, J.B. Yoon, and G.H. Cho, "One-chip electronic detection of DNA hybridization using precision impedance-based CMOS array sensor," Biosensors and Bioelectronics, 26(4), pp. 1373-1379, 2010.
33. Y.H. Kuo, Y.S. Chen, P.C. Huang, and G.B. Lee, "A CMOS-based capacitive biosensor for detection of a breast cancer microRNA biomarker," IEEE Open Journal of Nanotechnology, pp. 1-6, 2020.
34. J.S. Daniels and N. Pourmand, "Label-Free impedance biosensors: opportunities and challenges," Electroanalysis, 19(12), pp. 1239-1257, 2007.
35. A. Dizon and M.E. Orazem, "On the impedance response of interdigitated electrodes," Electrochimica Acta, 327, pp. 13-42, 2019.
36. J. Oberlander, Z.B. Jildeh, P. Kirchner, L. Wendeler, A. Bromm, H. Iken, P. Wagner, M. Keusgen, and M.J. Schoning, "Study of interdigitated electrode arrays using experiments and finite element models for the evaluation of sterilization processes," Sensors, 15(10), pp. 26115-26127, 2015.
37. C.R. Rodrigues, C. Muller, and D.J.M. Neto, "Hysteresis settling technique for CMOS comparators based on substrate voltage," Electronics Letters, 49(1), pp. 27-28, 2013.
38. M.S. Lu, Y.C. Chen, and P.C. Huang, "5×5 CMOS capacitive sensor array for detection of the neurotransmitter dopamine," Biosenssors and Bioelectronics, 26(3), pp. 1093-1097, 2010.
39. J.T. Villanueva, Q. Huang, N.O. Fischer, G. Arya, and D.J. Sirbuly, "Nanofiber-based total internal reflection microscopy for characterizing colloidal systems at the microscale," Journal of Physical Chemistry C, 122(38), pp. 22114-22124, 2018.
40. Y. Rosenberg-Hasson, L. Hansmann, M. Liedtke, I. Herschmann, and H.T. Maecker, "Effects of serum and plasma matrices on multiplex immunoassays," Immunologic research, 58(2-3), pp. 224-233, 2014.
41. C.G. Gray and P.J. Stiles, "Nonlinear electrostatics: the Poisson–Boltzmann equation," European Journal of Physics, 39(5), pp. 053002-053029, 2018.
42. Allen J. Bard and Larry R. Faulkner, "Electrochemical methods: fundamentals and applications, New York: Wiley, 2001, 2nd ed," Russian Journal of Electrochemistry, 38(12), pp. 1364-1365, 2002.
43. E. Gongadze, S. Petersen, U. Beck, and U. van Rienen, "Classical models of the interface between an electrode and an electrolyte," pp.1-7, 2020.
44. R.D. Silva, G.I. Wirth, and L. Brusamarello, "A novel and accurate time-domain description of MOSFET low-frequency noise based on random telegraph signals," International Journal of Modern Physics B, 24(30), pp. 5885-5894, 2010.
45. G.Y. Xu, J. Abbott, and D. Ham, " Optimization of CMOS-ISFET-based biomolecular sensing: Analysis and demonstration in DNA detection," Ieee Transactions on Electron Devices, 63(8), pp. 3249-3256, 2016.
46. P. Georgiou and C. Toumazou, "ISFET threshold voltage programming in CMOS using hot-electron injection," Electronics Letters, 45(22), pp. 1112-1113, 2009.
47. C. Chang and M.S. Lu, "CMOS Ion Sensitive Field Effect Transistors for highly sensitive detection of DNA hybridization," IEEE Sensors Journal, pp. 8930-8937, 2020.
48. Y.W. Chen, T.Y. Tai, C.P. Hsu, I. Sarangadharan, A.K. Pulikkathodi, H.L. Wang, R. Sukesan, G.Y. Lee, J.I. Chyi, C.C. Chen, G.B. Lee, and Y.L. Wang, "Direct detection of DNA using electrical double layer gated high electron mobility transistor in high ionic strength solution with high sensitivity and specificity," Sensors and Actuators B: Chemical, 271, pp. 110-117, 2018.
49. Y.W. Chen, W.C. Kuo, T.Y. Tai, C.P. Hsu, I. Sarangadharan, A.K. Pulikkathodi, S.L. Wang, R. Sukesan, H.Y. Lin, K.W. Kao, C.L. Hsu, C.C. Chen, and Y.L. Wang, "Highly sensitive and rapid MicroRNA detection for cardiovascular diseases with electrical double layer (EDL) gated AlGaN/GaN high electron mobility transistors," Sensors and Actuators B: Chemical, 262, pp. 365-370, 2018.
50. I.Y. Huang, R.S. Huang, and L.H. Lo, "Improvement of integrated Ag/AgCl thin-film electrodes by KCl-gel coating for ISFET applications," Sensors and Actuators B: Chemical, 94(1), pp. 53-64, 2003..
51. S.B. Nimse, K. Song, M.D. Sonawane, D.R. Sayyed, and T. Kim, "Immobilization techniques for microarray: challenges and applications," Sensors, 14(12), pp. 22208-22229, 2014.