【摘要】：基于納米孔的第三代基因測(cè)序儀以及相關(guān)的關(guān)鍵技術(shù)對(duì)于保護(hù)我國(guó)自己的基因資源具有戰(zhàn)略意義。要實(shí)現(xiàn)該類(lèi)基因測(cè)序技術(shù),目前存在的主要問(wèn)題:一是DNA分子過(guò)孔速度過(guò)快,由于過(guò)孔速度過(guò)快,通過(guò)測(cè)試得到的過(guò)孔電流的特征來(lái)識(shí)別過(guò)孔的堿基還具有較大困難;二是四種堿基與生物納米孔壁相互作用機(jī)理尚不清晰,進(jìn)一步的研究表明,DNA堿基與納米孔孔壁之間的相互作用力越大,那么納流體中離子的熱運(yùn)動(dòng)就越小,而噪聲信號(hào)主要就來(lái)源于離子的熱運(yùn)動(dòng),增加DNA堿基與納米孔壁之間的相互作用力有助于提高信噪比。因此研究DNA堿基與壁面的特異性作用對(duì)于第三代基因測(cè)序具有重大的意義。本文對(duì)DNA兩種堿基的結(jié)構(gòu)進(jìn)行了分析,從機(jī)械特性出發(fā),研究了單晶硅的納米力學(xué)性能,并以單晶硅為基體材料,實(shí)現(xiàn)DNA堿基生物分子的組裝,系統(tǒng)的研究修飾機(jī)理、固定率的影響因素以及受限條件下修飾表面的力學(xué)行為。表征結(jié)果表明,腺嘌呤分子A和胸腺嘧啶分子T均有區(qū)別于其他堿基的特征性譜峰。接觸角測(cè)量結(jié)果顯示,硅基體完全硅烷化的時(shí)間為30min左右;醛基化反應(yīng)在20min時(shí)初次形成完整的分子膜,并隨著時(shí)間延長(zhǎng)逐層生長(zhǎng)。DNA堿基鏈的活性氨基基團(tuán)與硅基體表面的醛基基團(tuán)發(fā)生反應(yīng)從而固定在硅基體上。熒光試驗(yàn)結(jié)果表明,影響熒光背景強(qiáng)度的四個(gè)因素中,硅烷化試劑的濃度影響最大,硅烷化時(shí)間影響最小。當(dāng)硅烷化和醛基化試劑濃度較低時(shí),堿基A和堿基T的固定率隨著時(shí)間的增加均呈現(xiàn)線性增長(zhǎng);當(dāng)試劑濃度較大時(shí),堿基A和堿基T的固定率均出現(xiàn)極大值。當(dāng)探針的緩沖溶液pH為弱堿性時(shí),堿基固定率較大;探針點(diǎn)樣濃度為50μmol/L時(shí)固定率較高;在同一條件下堿基A固定率高于堿基T。單晶硅的納米力學(xué)研究結(jié)果表明:對(duì)于既定的最大加載力,隨著壓頭尖端半錐角的增大,單晶硅彈性回復(fù)率逐漸增大,而彈性回復(fù)量基本恒定;在同一實(shí)驗(yàn)條件下,相較于(100)晶面,單晶硅(111)晶面具有較小的硬度和彈性模量值;隨著最大加載力的增加,單晶硅壓痕周?chē)牧蠒?huì)出現(xiàn)堆積和表面隆起現(xiàn)象,壓痕的彈性回復(fù)量增大,塑性區(qū)的范圍隨著加載力的增大而增大,出現(xiàn)明顯的尺寸效應(yīng)。修飾面的力學(xué)行為研究結(jié)果表明:在大氣環(huán)境下,對(duì)于10nm以上的間隙,探針與硅片表面法向作用力受表面化學(xué)形態(tài)影響較小;在7nm以下間隙,經(jīng)堿基修飾后的表面與探針作用力明顯減小,且探針與堿基A和堿基T的作用力受加載速率的影響較大。隨著法向力的增加,探針與硅基體的橫向作用力增加,摩擦系數(shù)逐漸減小;探針與不同修飾條件下硅片表面產(chǎn)生了不同的相互作用力。此外,與堿基T相比,由堿基A修飾后表面所產(chǎn)生的橫向力較大。
[Abstract]:The third generation gene sequencer based on nano-pore and related key technologies are of strategic significance for protecting our own gene resources. In order to realize this kind of gene sequencing technology, the main problems are as follows: first, the DNA molecule passes through the hole too fast, because of the passing through speed too fast, it is difficult to identify the base through the pore by testing the characteristic of the passing hole current; Second, the interaction mechanism between the four bases and the biological nano-pore wall is not clear. Further studies show that the greater the interaction force between DNA bases and the nano-pore wall, the smaller the thermal movement of ions in the nano fluid. The noise signal is mainly derived from the thermal movement of ions, and increasing the interaction force between DNA base and nano-pore wall is helpful to improve the signal-to-noise ratio (SNR). Therefore, it is of great significance for the third generation gene sequencing to study the wall specificity of DNA base. In this paper, the structure of two bases of DNA is analyzed, and the mechanical properties of monocrystalline silicon are studied based on the mechanical properties. The monocrystalline silicon is used as the substrate material to realize the assembly of DNA base biomolecules, and the modification mechanism is studied systematically. The influence factors of the fixed rate and the mechanical behavior of the modified surface under limited conditions. The characteristic peaks of adenine A and thymine T are different from those of other bases. The results of contact angle measurement showed that the time of complete silanization of silicon substrate was about 30min, and the complete molecular membrane was formed in the first time during the 20min reaction. The active amino group of DNA base chain reacts with the aldehyde group on the surface of silicon substrate and is immobilized on the silicon substrate. The results of fluorescence test showed that the concentration of silanization reagent had the greatest influence on the intensity of fluorescence background and the time of silanization was the least. The fixation rates of base A and T increased linearly with the increase of time when the concentration of silanized and aldehydized reagents was low, and the immobilization rates of base A and T showed maximum values when the concentration of reagents was higher. When the pH of the buffer solution of the probe is weak basic, the base fixation rate is larger, the probe sample concentration is 50 渭 mol/L, and the fixed rate of base A is higher than that of base T under the same condition. The results of nanomechanical study of monocrystalline silicon show that the elastic recovery rate of monocrystalline silicon increases with the increase of the half cone angle at the tip of the head for a given maximum loading force, and the elastic recovery is basically constant under the same experimental conditions. Compared with the (100) crystal face, the single crystal silicon (111) crystal mask has smaller hardness and elastic modulus, and with the increase of the maximum loading force, the material around the indentation of single crystal silicon will appear the phenomenon of stacking and surface bulging, and the elastic recovery of the indentation will increase. The range of plastic zone increases with the increase of loading force, and there is obvious size effect. The results of the mechanical behavior of the modified surface show that the normal force between the probe and the surface of the silicon wafer is less affected by the surface chemical morphology for the gap above 10nm in the atmospheric environment, and the gap below the 7nm is less affected by the normal force on the surface of the wafer. The interaction force between the surface and the probe was obviously decreased after the modification of the base, and the force between the probe and the base A and T was greatly affected by the loading rate. With the increase of the normal force, the transverse force between the probe and the silicon substrate increases, and the friction coefficient decreases gradually, and there are different interaction forces between the probe and the silicon wafer surface under different modification conditions. In addition, compared with base T, the surface modified by base A produces larger transverse force.
【學(xué)位授予單位】：中國(guó)礦業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：TN304.12

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