發(fā)布時(shí)間：2017-12-30 21:32

  本文關(guān)鍵詞：新型生物傳感器及其在相關(guān)生物物理研究中的應(yīng)用　出處：《中國(guó)科學(xué)技術(shù)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 微納米機(jī)電系統(tǒng) 生物材料導(dǎo)熱系數(shù) 細(xì)胞跨膜水傳輸 胞內(nèi)冰形成概率 血小板功能檢測(cè)

【摘要】：低溫生物學(xué)和細(xì)胞力學(xué)是現(xiàn)代生物醫(yī)學(xué)工程領(lǐng)域中的兩個(gè)重要分支。本博士論文以冷凍保存血小板為主線(xiàn),闡述了作者為解決血小板冷凍保存這一世界性難題,在上述兩個(gè)領(lǐng)域中所做的研究工作。本文第一部分主要針對(duì)血小板冷凍保存環(huán)節(jié)中所面臨的如何更快速精確的測(cè)量生物材料導(dǎo)熱系數(shù)、如何更深入的認(rèn)識(shí)低溫冷凍期間水的跨膜傳輸特性和如何更準(zhǔn)確的預(yù)測(cè)胞內(nèi)冰形成概率這三個(gè)問(wèn)題。筆者詳細(xì)介紹了一種基于微納米加工技術(shù),可以對(duì)生物材料的微觀(guān)熱物性進(jìn)行高精度自動(dòng)化測(cè)量的微電子機(jī)械系統(tǒng)的研發(fā)工作;同時(shí),還詳細(xì)闡述了使用該套系統(tǒng)在探索生物材料微尺度傳熱以及細(xì)胞冷凍損傷過(guò)程中所進(jìn)行的基礎(chǔ)性研究。筆者通過(guò)微納米加工技術(shù)將傳統(tǒng)的熱物性探針微型化,并將熱探針的制造工藝工業(yè)化,將熱探針的精度校準(zhǔn)和測(cè)量過(guò)程自動(dòng)化和標(biāo)準(zhǔn)化。筆者成功的在4英寸硅晶圓上將作為微探針能量發(fā)生單元和導(dǎo)熱系數(shù)測(cè)量單元的尺寸進(jìn)一步降低成線(xiàn)寬僅為3微米、線(xiàn)間距僅為5微米的蛇形高密度金屬熱絲。同時(shí),微探針的分割切片技術(shù)也升級(jí)成為自由度及加工精度都更高的感應(yīng)耦合等離子體深硅刻蝕技術(shù),能夠更快更容易的制造出尺寸和形狀都更為多樣的微型熱物性探針(單針局部測(cè)量或陣列式多針多點(diǎn)測(cè)量)。通過(guò)對(duì)不同低溫保護(hù)劑和不同生物材料在不同溫度下進(jìn)行大量且系統(tǒng)的導(dǎo)熱系數(shù)測(cè)量,筆者發(fā)現(xiàn)使用微納米加工工藝制造出的微型探針擁有更小的熱容,更緊湊的探針陣列,能更直接地與被測(cè)樣品接觸,通過(guò)使用不同結(jié)構(gòu)的熱探針可以更準(zhǔn)確、更靈敏、更可靠的對(duì)生物及其相關(guān)材料進(jìn)行單點(diǎn)微創(chuàng)式熱傳導(dǎo)系數(shù)的測(cè)量,或者獲得某個(gè)特定區(qū)域內(nèi)的熱物性分布數(shù)據(jù)。整套芯片的制備過(guò)程已經(jīng)完成了從微納米加工與傳統(tǒng)精密制造工藝的混合制造到純微納米加工的轉(zhuǎn)型,能夠像生產(chǎn)集成電路一樣,實(shí)現(xiàn)高良品率、高物理和電學(xué)相似性的大批量生產(chǎn),完美解決了傳統(tǒng)熱傳導(dǎo)系數(shù)測(cè)量設(shè)備與方法在臨床應(yīng)用和科學(xué)研究上的缺陷與不足(只能對(duì)材料進(jìn)行離體檢測(cè),設(shè)備內(nèi)部結(jié)構(gòu)復(fù)雜,繁瑣的測(cè)量前準(zhǔn)備工作,靈敏度低,制造難度大等)。與此同時(shí),我們的實(shí)驗(yàn)結(jié)果還揭示出一直被廣泛采用的熱探針精度標(biāo)定過(guò)程存在嚴(yán)重缺陷,當(dāng)測(cè)量溫度和標(biāo)定溫度不一致時(shí),通過(guò)常規(guī)標(biāo)定過(guò)程所得出的熱探針系統(tǒng)精度校準(zhǔn)常數(shù)會(huì)導(dǎo)致最終測(cè)量結(jié)果出現(xiàn)較大誤差。因此,我們提出了一套新的標(biāo)定與測(cè)量流程,有效確保了熱探針系統(tǒng)精度校準(zhǔn)常數(shù)的準(zhǔn)確性,使得最終測(cè)量結(jié)果的分析與處理過(guò)程標(biāo)準(zhǔn)化、統(tǒng)一化、精準(zhǔn)化。并且根據(jù)所獲得的實(shí)驗(yàn)數(shù)據(jù),通過(guò)使用聯(lián)合擬合、方程組合等數(shù)據(jù)分析方法,筆者還首次提出了一種基于Second-order Polynomial和Filippov equation的可用于預(yù)測(cè)不同濃度的二元低溫保護(hù)劑溶液在不同溫度下導(dǎo)熱系數(shù)的雙因素(溫度和濃度)理論公式。通過(guò)使用研發(fā)出來(lái)的微納米熱探針和低溫顯微鏡平臺(tái),筆者還對(duì)細(xì)胞的跨膜水傳輸特性和胞內(nèi)冰形成概率進(jìn)行了深入的學(xué)習(xí)。由于實(shí)驗(yàn)條件的限制,筆者使用了人子宮頸癌細(xì)胞作為概念證明實(shí)驗(yàn)里的實(shí)驗(yàn)對(duì)象。對(duì)人子宮頸癌細(xì)胞在不同降溫速率下體積的變化情況進(jìn)行了仔細(xì)深入的觀(guān)察與分析,對(duì)人子宮頸癌細(xì)胞的冷凍響應(yīng)特性(跨膜水傳輸性質(zhì)和胞內(nèi)冰形成概率)進(jìn)行了系統(tǒng)的研究。使用Mazur提出的跨膜水傳輸模型,聯(lián)合擬合得出人子宮頸癌細(xì)胞細(xì)胞膜的水傳輸參數(shù),從而進(jìn)一步預(yù)測(cè)在其他冷凍環(huán)境下細(xì)胞跨膜水傳輸?shù)捻憫?yīng)規(guī)律。筆者在實(shí)驗(yàn)中發(fā)現(xiàn)胞內(nèi)冰的形成主要分為Darkening和Twitching兩種類(lèi)型。這兩種不同類(lèi)型的胞內(nèi)冰形成機(jī)制很可能與最初所形成的胞內(nèi)冰大小密切相關(guān)。通過(guò)使用Toner提出的胞內(nèi)冰生成概率模型對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行擬合,筆者還發(fā)現(xiàn)該模型存在較為嚴(yán)重的弊端和局限性,因此筆者通過(guò)引入臨界體積這一新參數(shù),提出了一種新的能更準(zhǔn)確預(yù)測(cè)胞內(nèi)冰生成概率的改進(jìn)型理論模型。利用微納米加工技術(shù)大規(guī)模生產(chǎn)微尺度熱探針是現(xiàn)代科學(xué)技術(shù)與傳統(tǒng)生物熱物理測(cè)量技術(shù)的完美結(jié)合。在傳統(tǒng)宏觀(guān)生物材料熱傳導(dǎo)系數(shù)測(cè)量技術(shù)的基礎(chǔ)上,從微尺度、微創(chuàng)和集成的角度上,更加準(zhǔn)確、可靠和全面地研究生物活體材料及其相關(guān)材料的微尺度傳熱及損傷特性,這不僅可以幫助認(rèn)識(shí)和發(fā)現(xiàn)生物材料在微觀(guān)傳熱過(guò)程中的本質(zhì)規(guī)律,還可以在生物材料或器官的低溫冷凍保存、低溫或聚能外科手術(shù)及相關(guān)傳熱領(lǐng)域中,提供必要的研究測(cè)量技術(shù)和理論依據(jù)。本文的第二部分主要針對(duì)冷凍血小板復(fù)溫后所面臨的如何快速可靠的對(duì)血小板功能進(jìn)行定量測(cè)量的問(wèn)題。筆者詳細(xì)闡述了一種基于BioMEMS技術(shù)對(duì)對(duì)血小板收縮力進(jìn)行定量檢測(cè)的細(xì)胞微力測(cè)量系統(tǒng)的研發(fā)和使用該系統(tǒng)對(duì)血小板綜合性功能進(jìn)行評(píng)估的探索過(guò)程。激活后的血小板,其中后期的功能表達(dá)主要表現(xiàn)在用產(chǎn)生的血小板收縮力來(lái)強(qiáng)化和鞏固前期形成的松散的止血拴,讓止血栓能更牢固地粘附在受損血管的裂口處,繼續(xù)起到止血,維護(hù)血管壁完整性的作用。在本文中,我們提出用血小板收縮力來(lái)做為檢驗(yàn)其中后期功能的新指標(biāo),并詳細(xì)闡述了一種通過(guò)利用陣列微柱式微力感測(cè)系統(tǒng)對(duì)血小板收縮力直接進(jìn)行微力測(cè)量的技術(shù)。筆者通過(guò)微納米加工技術(shù)制造出具有不同直徑(600納米-3微米)、不同高度(5-15微米)和不同微柱間距(1-3微米)的微柱陣列,而且用該微力感測(cè)系統(tǒng)首次測(cè)量并研究了處于納牛頓量級(jí)上半靜態(tài)下的血小板收縮力與外界生物蛋白、刺激劑和刺激時(shí)間等參數(shù)之間的關(guān)系,更首次在高倍微顯條件下動(dòng)態(tài)觀(guān)測(cè)并分析了血小板收縮力的整個(gè)產(chǎn)生、發(fā)展和變化過(guò)程。通過(guò)陣列微柱式微力感測(cè)系統(tǒng),血小板收縮力首次被高精度的測(cè)量出來(lái),由此,為解決血小板功能性檢測(cè)這一世界性難題,提供了新的解決思路和辦法。為早期發(fā)現(xiàn)血小板功能異常,指導(dǎo)血小板抗凝藥物開(kāi)發(fā)和闡明相關(guān)疾病的形成機(jī)理,提供了必要的技術(shù)設(shè)備和奠定了一定的理論基礎(chǔ)�？偟恼f(shuō)來(lái),本論文針對(duì)血小板在低溫保存和復(fù)溫后功能檢測(cè)這兩個(gè)方面進(jìn)行了系統(tǒng)深入的研究。成功研發(fā)出的兩種新型微納米生物傳感器,可以很好的用于檢測(cè)生物材料的熱物理和力學(xué)特性。在其他相關(guān)生物醫(yī)學(xué)領(lǐng)域的研究中,我們提出的新的微納米生物醫(yī)學(xué)微全分析系統(tǒng)系統(tǒng)與檢測(cè)方法也有著廣泛的應(yīng)用前景。
[Abstract]:Low temperature biology and cell mechanics are two important branches of modern biomedical engineering. This thesis takes the cryopreserved platelets as the main line, elaborated the author to solve the cryopreservation of platelets which is a worldwide difficult problem, the research work done in the above two areas. The first part of this paper mainly for the cryopreservation of platelets facing link in how to quickly and accurately measuring thermal conductivity of biomaterials, how the transmembrane transport characteristics of water during freezing and a deeper understanding of how to more accurately predict intracellular ice formation probability of these three questions. The author introduces a kind of micro nano processing technology based on micro electro mechanical system, can high precision automatic measurement the micro thermal properties of biological materials research and development work; at the same time, also elaborated the system used in the exploration of micro scale biological material Based on the heat and cell freezing injury process. Through the micro nano processing technology will be thermal probe micro traditional manufacturing technology, industrialization and heat probe, the precision of the calibration and measurement of process automation and thermal probe. The standard of success in 4 inch silicon wafers, as micro the probe energy generating unit and the thermal conductivity measurement unit to further reduce the size of Cheng Xiankuan is only 3 microns, line spacing is only a serpentine high density metal wire 5 microns. At the same time, the segmentation technology of micro probe also upgraded inductively coupled plasma deep silicon etching technology has become a degree of freedom and accuracy are higher, faster and easier to create the size and shape of more micro thermal probe diversity (local measurement or single needle array of multi needle multipoint measurement). According to the different cryoprotectants and different students By measuring the thermal conductivity of a material and system at different temperatures, the author found that the heat capacity with smaller use of micro probe micro nano processing technology to produce the probe array is more compact, more direct contact with the sample, through the thermal probe using different structure can be more accurate, more sensitive measurement single point of minimally invasive thermal conductivity of biological and related materials are more reliable, or obtain the thermal distribution data of a specific area of the whole chip. The preparation process has been completed from the mixture of micro nano processing and traditional precision manufacturing technology to manufacture pure micro nano processing transformation, as production the integrated circuit, to achieve high yields, high physical and electrical properties similar to mass production, the perfect solution to the traditional heat conduction coefficient measuring device and method in the clinical application and scientific research. Defects and shortcomings (only the materials were measured in vitro, the internal structure of complex equipment, preparation work, tedious before the measurement sensitivity is low, difficult to manufacture etc.). At the same time, our results reveal that the thermal probe has been widely used in precision calibration process in serious defects, when the measured temperature and temperature calibration consistent, through the conventional calibration precision calibration of thermal probe system constant the process will lead to the final measurement result error. Therefore, we put forward a new set of calibration and measurement process, effectively ensure the accuracy of thermal probe system precision calibration constant, standard analysis and process of making the final measurement results, unified and accurate. And according to the experimental data, by using the combined fitting equation, combined data analysis methods, the author also proposed a method based on Second-or Der Polynomial and Filippov equation can be used in two factor two yuan of cryoprotectant solutions at different temperatures and thermal conductivity prediction of different concentration (temperature and concentration). By using the theoretical formula developed by the micro nano scaled heat and low temperature probe microscope platform, the author on the cell membrane water transport properties and intracellular ice formation the probability of in-depth study. Due to the limitation of experimental conditions, the author used the human cervical cancer cells as a proof of concept in the experimental subjects. Observation and in-depth analysis carefully carried on a human cervical cancer cell in the volume changes at different cooling rates, on human cervical cancer cell freezing response (characteristics of transmembrane water transport and intracellular ice formation probability) was studied. Mazur proposed transmembrane water transport model, combined with the fitted cells of human cervical carcinoma cells Water transmission parameters of membrane, thus predicting response of cells in other frozen environment of transmembrane water transport. The further experiments found that the intracellular ice formation is mainly divided into two types of Darkening and Twitching. The formation mechanism of these two different types of intracellular ice is likely to be closely related to the initial form intracellular ice size. By using the Toner of intracellular ice formation probability model to fit the experimental results, the author also found that the model has disadvantages and limitations of the more serious, so the author introduces a new parameter of the critical volume, proposes a new improved theoretical model can more accurately predict intracellular ice generation probability. Using the micro nano processing technology for large-scale production of micro scale thermal probe is a perfect combination of modern science and technology and traditional bio physical measurement technology. In the traditional macro biological material thermal conductivity Based on the measurement technique, from micro scale, minimally invasive and integrated perspective, more accurate, reliable and comprehensive study of biological materials and related materials in micro scale heat transfer and damage characteristics, which can not only help understand and find the essence of the law of biological materials in micro heat transfer process, can also be frozen in biology material or organ preservation, field surgery and related energy at low temperature or heat transfer, provide the necessary measurement technology research and theoretical basis. The second part mainly for frozen platelets after rewarming faced by how fast and reliable quantitative measurement of platelet function. The author elaborates a kind of BioMEMS technology research and development quantitative detection of platelet contractile force on cells based on micro force measuring system and use the system to evaluate the comprehensive functions of the explored platelet Cheng. After activation of platelet function, the late expression mainly in loose platelet contractile force generated to strengthen and consolidate the early formation of the hemostatic plug to let the more firmly in the gap of damaged blood vessels, continue to maintain hemostasis, integrity of vessel wall in this role., we propose to use platelet contractile force to test a new index which later function, and elaborated a micro column array by using the micro force sensing system of platelet contractile force directly to micro force measurement technology. The micro nano processing technology to produce with different diameter (600 nm -3 micron). Different height (5-15 microns) and different micro column spacing (1-3 microns) micro column array, and the micro force sensing system for the first time measurement and study in nano platelet contractile force and semi static under Newton on Bound protein, the relationship between stimuli and stimulus parameters such as time, for the first time in high times slightly under the condition of dynamic observation and analysis of platelet contractile force of the whole generation, development and change process. The micro column array micro force sensing system for the first time, platelet contractile force was measured with high precision, thus, in order to solve the detection of platelet function which is a worldwide difficult problem, provides new ideas and solutions. For the early detection of abnormal platelet function, the formation mechanism of guidance of anticoagulant drug development and clarify the related diseases, provides the necessary technical equipment and laid a theoretical foundation. In general, this paper aresystematically platelet function during cold storage and rewarming after these two aspects. Successfully developed two new micro nano bio sensor, can be very good for the detection of students The thermophysical and mechanical properties of materials and materials have been widely applied in other related biomedical fields.

【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：TP212.3;R33

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