【摘要】：活體細胞對外界刺激如藥物、環(huán)境因素變化和受體作用等產生響應,會引起細胞代謝等功能性變化,外在表現為生化參數的改變。利用微型化電化學生物傳感器,可以將細胞培養(yǎng)在傳感器表而,細胞內的生理變化引起胞外環(huán)境的變化,胞外的電解質溶液與傳感器之間形成電化學池,從而將這些參數的變化通過二級換能器轉換為電信號輸出,最終實現定量化監(jiān)測和細胞水平的分析。將傳感器微型化和建立良好的細胞-芯片耦合是關鍵問題所在。鑒于此,采用微機械加工技術開展的基于細胞傳感器的微電極陣列、叉指型阻抗傳感器、場效應傳感器等具有良好的生物相容性,得到了廣泛的研究,并已在藥物篩選、環(huán)境監(jiān)測等領域得到了初步應用。 本文從微型電化學生物傳感器的設計和制備等關鍵技術入手,對基于電解質/金屬-絕緣層-半導體(Electrolyte/metal-insulator-silicon, EIS/MIS)結構和和光伏檢測技術的傳感器、金微電極、碳微電極等傳感器進行了研究,實現了離體原代腎細胞在傳感器表而的良好耦合以及胞外酸化率、胞外氧化還原電位檢測、葡萄糖溶液濃度檢測,為EIS/MIS結構半導體器件拓展了新的生物學應用領域。此外,本文將碳微加工(Carbon micro-electromechanical systems, C-MEMS)工藝用于碳微電極的制備,實現了碳電極的微型化,成功應用于胰島素溶液的檢測,為細胞分泌的研究提供了一種新的可行性方法。本論文的主要內容和貢獻如下 1.提出了一種新的有效的細胞胞外氧化還原電位檢測的方法 細胞胞外氧化還原電位(Extracellular redox potential, ERP)反應了細胞外微環(huán)境的氧化還原狀態(tài),與細胞的增殖、分化、凋亡及癌細胞侵蝕都有著密切的關系。本文采用基于MIS結構的傳感器和流動注射微腔體聯用的方法,為細胞水平的氧化還原電位測試提供了一個可行的解決方案。測試無需添加其他氧化還原介質。研究表明,線粒體電子傳遞鏈對ERP有較大的影響。 2.提出了一種從細胞釋放H+和自由電子活性兩種角度研究細胞生命代謝活動的集成傳感器的設計和可行性 本文提出了同時檢測胞外酸化率和胞外氧化還原電位的一種集成傳感器的設計,該集成傳感器的性能可與單獨的EIS、MIS傳感器相媲美。此外,本文對正常生理狀態(tài)下的細胞酸化率和ERP兩種參數進行了初步的研究,獲得了與單獨測試類似的結果,驗證了該設計的可行性。該設計有望應用于揭示細胞代謝機理的研究和藥物分析測試中。 3.研制了一種新的電位型葡萄糖傳感器 本文將PPy-GOD (Polypyrrole-glucose oxidase)超薄膜修飾在MIS結構傳感器上獲得了比使用同種修飾薄膜的其他類型傳感器更高的靈敏度。MIS傳感器所采用的光伏檢測技術與流動注射微腔體聯用,有利于提高傳感器本身的靈敏度和響應時間。 4.提出了將C-MEMS工藝制備碳微電極用于胰島素的檢測 采用C-MEMS工藝將光刻膠熱解制備的碳微電極具有較好的電化學性能,在其表面電沉積納米RuOx顆粒之后實現了胰島素的電化學檢測,這是繼玻碳電極、碳糊電極、碳纖維微電極之后,首次將C-MEMS應用于胰島素測試的研究。采用C-MEMS工藝的優(yōu)勢在于,可根據實際檢測需要靈活設計電極的結構和尺(?),有利于胰島素傳感器的微型化和集成化。
[Abstract]:In vivo cells respond to external stimuli such as drugs, changes in environmental factors and receptors, which can cause functional changes in cell metabolism and external changes in biochemical parameters. An electrochemical cell is formed between the electrolyte solution and the sensor, and the changes of these parameters are converted into electrical signals by a two-stage transducer for quantitative monitoring and cell level analysis. Microelectrode arrays, interdigital impedance sensors and field effect sensors based on cell sensors have been extensively studied for their good biocompatibility, and have been preliminarily applied in drug screening and environmental monitoring.
Starting with the design and fabrication of micro-electrochemical biosensors, the electrolyte/metal-insulator-silicon (EIS/MIS) structure and photovoltaic sensors, gold microelectrode, carbon microelectrode and other sensors are studied in this paper. The in vitro primary kidney cells can be found in vivo. The good coupling of the sensor surface and the detection of extracellular acidification rate, extracellular redox potential and glucose solution concentration have opened up new biological applications for EIS/MIS semiconductor devices. In addition, the Carbon micro-electromechanical systems (C-MEMS) process has been applied to the preparation of carbon microelectrodes, which has been realized. The miniaturization of carbon electrode, which has been successfully applied to the detection of insulin solution, provides a new feasible method for the study of cell secretion.
1. a new effective method for extracellular redox potential detection is proposed.
Extracellular redox potential (ERP) reflects the redox state of the extracellular microenvironment and is closely related to cell proliferation, differentiation, apoptosis and cancer cell erosion. Potential testing provides a viable solution without adding any other redox medium. Studies have shown that mitochondrial electron transfer chains have a greater impact on ERP.
2. The design and feasibility of an integrated biosensor for the study of cell life metabolism from the point of view of H+ release and free electron activity are presented.
An integrated sensor for simultaneous detection of extracellular acidification rate and extracellular redox potential is proposed in this paper. The performance of the integrated sensor is comparable to that of the EIS and MIS sensors. In addition, the acidification rate and ERP parameters under normal physiological conditions are preliminarily studied, and the results are similar to those obtained by the single sensor. The results show that the design is feasible and is expected to be applied to reveal the mechanism of cell metabolism and drug analysis.
3. a new potential glucose sensor has been developed.
In this paper, PPy-GOD (Polypyrrole-glucose oxidase) ultrathin film was modified to obtain higher sensitivity than other sensors using the same modified film on MIS structure sensor.
4. it is proposed to prepare carbon microelectrodes for the detection of insulin by C-MEMS process.
Carbon microelectrode prepared by pyrolysis of photoresist with C-MEMS process has good electrochemical performance. After electrodepositing nano-RuOx particles on its surface, the electrochemical detection of insulin was realized. This is the first time that C-MEMS was applied to insulin measurement after glassy carbon electrode, carbon paste electrode and carbon fiber microelectrode. The advantage is that the electrode structure and ruler can be flexibly designed according to the actual detection needs, which is conducive to the miniaturization and integration of insulin sensors.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R318.6

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