黃榮山Huang, Long-Sun臺灣大學:應用力學研究所顏毅廣Yen, Yi-KuangYi-KuangYen2010-06-022018-06-292010-06-022018-06-292009U0001-2407200915594900http://ntur.lib.ntu.edu.tw//handle/246246/184844在地即時的疾病檢測已成為可攜式微型化生物感測器發展的重要目標。微型化生物感測器為了能提供使用者多樣且複雜的疾病進行診斷，需要達到針對疾病標記蛋白質與病源體之量化且準確的檢測。此外，為了因應在偏遠地區以及非醫院區域診斷的需求，生物感測器朝向具有免螢光標定、可攜式、低成本且高可靠度的特性。本研究發展一個整合式非螢光標定微型壓阻式微懸臂樑生物感測平台，用以檢測疾病相關之標記蛋白質與病源體。此平台包括壓阻微懸臂樑晶片、微流道、白金微型溫度感測器以及可拋棄式電路板之封裝。壓阻微懸臂樑係利用微機電製程技術製造，為多層材料所組成，其長度為300微米，寬度為50微米，以及1.2微米的厚度，其靈敏度為 (奈米)-1，而最低可量測之表面應力為0.065 (牛頓/米)。研究提出以單根自感式壓阻微懸臂樑為生物感測之配制，有別於一般以雙根懸臂樑於惠斯通電橋的檢測配置。此一新型配置，將可解決壓阻微懸臂樑感測器因化學效應所產生的訊號誤判的問題。此外，溫度效應所造成之感測測懸臂樑與參考懸臂樑之差異，亦無法藉由傳統惠斯通電橋配置消除。因此，本研究提出溫度控制的方法，以解決一切溫度效應所引發之雜訊，最低可將溫度控制至0.3℃以內。研究使用自行研發的整合式微型壓阻微懸臂樑生物感測器，成功地進行對心血管疾病標記蛋白質－C反應蛋白以及養殖魚類病源體－巴斯德桿菌之檢測。其中，C反應蛋白的檢測靈敏度可至1 微克/毫升，此為心臟疾病檢測的指標濃度，而其重複性之變異係數為17 %至21 %。巴斯德桿菌之初步的檢測，重複性之變異係數可低於5 %。所有的檢測都有對照組實驗，作為專一性的驗證。此微型生物感測應用，成功驗證此平台具有量化且準確的檢測能力。此外，此平台為一整合性可攜式檢測系統及可拋棄式裝置，具有可商業化的潛力。Diagnosis of complex diseases requires a quantitative and reliable detection of target proteins and pathogens. A portable, low-cost, and label-free biosensing tool is needed for diagnosis which is performed at the place far away from the examining laboratory or in the remote region. This work has developed an integrated miniaturized piezoresistive microcantilever-based biosensing platform for detections of the disease relevant protein and the pathogen. The sensing microcantilever was made up of multi-layer thin films of 1.2 μm in total thickness, of 300 μm in length and of 50 μm in width. The deflection sensitivity of the biosensing cantilever is experimentally measured to be , and the minimum detectable surface stress achieves 0.065 N/m. A disposable package was the first time being developed by integrating a sensor chip, a microfluidic device, and a platinum temperature sensor on a PCB for the convenience of a portable biosensing platform. single free-standing self-sensing piezoresistive microcantilever connected with a Wheatstone bridge electrical readout was proposed to avoid the unwanted false signals caused by the response of a reference cantilever to chemical effects during biochemical detections. Temperature effects as the noise sources also have been reduced by the temperature control system and the thermal-effect elimination method. oreover, this work also presented that the integrated miniature piezoresistive microcantilever has successfully been used to detect a clinically relevant biomarker, CRP, and a cultured fish pathogen, P. piscicida in a good reproducibility. For the assay of CRP, the analytical sensitivity achieves to diagnostic level of 1 μg/ml, and the CV % ranges from 17 % to 21%. In addition, for the preliminary study on the detection of P. piscicida, the CV% is found to be below 5%. The specificity of both these detections has been verified by processing control experiments. These biosensing applications demonstrated the reproducibility of this portable integrated biosensor which expresses its feasibility of commercialization.誌謝 I文摘要 IIbstract IVable of Contents VIist of Figures XIist of Tables XXIomenclatures XXIIhapter 1 Introduction 1.1 Motivation and innovation 3.1.1 A portable biochemical-analysis system 3.1.2 Reproducibility improvement of a label-free biosensor 4.1.3 Protein and pathogen detection 5.2 Overview of the thesis 6hapter 2 Surface-stress Based Microcantilever Biosensor 7.1 Principle of biosensor 8.2 Static mode microcantilever biosensor 10.3 Readout schemes of microcantilever deflection 15.3.1 Optical methods 16.3.2 Piezoresistive Method 17.4 Demonstrated applications 20.4.1 Protein and pathogen detection 20.4.2 DNA detection 23.4.3 Biochemical compound detection 25.5 Commercial microcantilever sensor platforms 26.6 Summary 29hapter 3 Design and Fabrication of Integrated Miniature Piezoresistive Microcantilever Biosensors 30.1 Design of piezoresistive microcantilever chip 31.1.1 Physical dimensions of piezoresistive microcantilever 31.1.2 Multilayer structure of piezoresistive microcantilever 34.1.3 Electrical considerations of piezoresistive microcantilever sensor 38.2 Fabrication of piezoresistive microcantilever chip 40.2.1 Fabrication process of chip 40.2.2 Fabrication issues during process 42.3 Microfluidic device 45.3.1 Design of microchannel 46.3.2 Fabrication process of microchannel 49.3.3 Design of channel bottom plate 50.3.4 Fabrication of channel bottom plate 51.4 Summary 53hapter 4 Package and setup of Integrated Miniature Piezoresistive Microcantilever Biosensors 54.1 Package of the sensor for disposable use 54.1.1 Package design 55.1.2 Package assembly 56.2 Measurement and readout system 58.2.1 Setup of a signal acquisition system 58.2.2 Setup of a signal acquisition system for temperature sensor 62.3 Setup of liquid handling system 63.4 Summary 64hapter 5 Characterization of Integrated Miniature Piezoresistive Microcantilever Biosensors 66.1 Actual dimensions and resistivity 66.2 Deflection sensitivity of the microcantilever 68.3 Surface stress sensitivity 72.4 Analysis of circuit noise 74.4.1 Wheatstone bridge test 74.4.2 Noise analysis of signal performance 76.5 Summary 77hapter 6 Thermal Effects on Integrated Miniature Piezoresistive Microcantilever Biosensors 79.1 Introduction 79.2 Temperature effects on piezoresistive microcantilever 80.2.1 The test of symmetrical bridge 82.2.2 The test of non-symmetrical bridge 85.3 Methods of Temperature control 87.3.1 Temperature control using a Peltier element 87.3.2 Temperature control using a Cryostat system 88.4 Method of thermal effect elimination 90.5 Summary 93hapter 7 Chemical effects on Integrated Miniature Piezoresistive Microcantilever Biosensors 95.1 Introduction 95.2 Ethanol variation in aqueous environment 96.2.1 Experiments using Au-coated cantilever with reference cantilever 96.2.2 Results of ethanol detection 97.3 Linker 8-mercaptooctanoic acid (MOA) in ethanol 100.4 pH variation in liquid environment 103.4.1 Experiments using Au-coated cantilever with reference cantilever 104.4.2 Results of using Au-coated cantilever with reference cantilever 105.4.3 Results of using a single Au-coated cantilever 108.5 Summary 110hapter 8 Biosensing Applications of Integrated Miniature Piezoresistive Microcantilever Biosensors 111.1 Introduction 111.2 C-reactive protein detection 112.2.1 Introduction of C-reactive protein 112.2.2 Surface functionalization of microcantilever 114.2.3 Immobilization of capture protein 116.2.4 Bioassay of C-reactive protein 119.3 Photobacterium damselae subsp. Piscicida detection 124.3.1 Introduction of Photobacterium damselae subsp. piscicida 124.3.2 Immobilization of capture protein 125.3.3 Detection of Photobacterium damselae subsp. Piscicida 127.4 Summary 131hapter 9 Conclusions and Future Work 133.1 Conclusions 133.2 Future work 135eferences 137application/pdf3821607 bytesapplication/pdfen-US生物感測器壓阻微懸臂樑微機電技術蛋白質檢測病源體檢測BiosensorPiezoresistive microcantileverMEMSProtein detectionPathogen detectionthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/184844/1/ntu-98-D93543001-1.pdf