【作者】 刘祥；

【导师】 肖守军；

【作者基本信息】 南京大学 ， 无机化学， 2011， 博士

【摘要】 近年来,人们对平面硅表面进行了各种方法的化学改性,以期满足其在生物芯片、分子器件、微电子等领域对平面硅界面特殊性能的要求：通过化学方法对单晶硅表面的化学修饰,可以赋予它新的物理化学特性,可以用它来制备生物传感器、蛋白质和DNA的检测芯片。化学修饰方法可用硅烷偶联剂与表面Si-OH作用以及Grignard试剂与表面Si-Cl或Si-Br的反应实现,前者虽然容易进行,但是生成的Si-O-Si键在碱性溶液中,容易进一步裂解：后者则需要比较苛刻的反应条件,且由于Grignard试剂种类有限,并不能满足多种实际的要求。实际应用过程中,要求在硅表面能够生成稳定的共价键,能够经受不同的化学环境,可以方便地引入不同功能的官能团。Si-C键化学性能稳定,而平面硅材料本身能够耐各种有机溶剂、酸和弱碱；Si-C键形成可以通过40%NH4F水溶液的腐蚀,在Si(111)的表面首先形成垂直于硅平面的Si-H键,再与一端带有功能性官能团同时另一端带有C=C双键的有机物反应,形成末端功能化的单分子膜,还可以对其进行进一步的化学修饰直至满足一定的要求为止,反应过程中Si(111)仍然保持原子级的平整。这些预示着Si(111)晶片在生物、分子器件等及其它领域会有广泛的应用前景。本文涉及如下方面：首先,在微波加热的条件下,通过Si-H与10-烯-1-酸的直接反应形成末端带有羧基的单分子膜,羧基与N-羟基丁二酰亚胺(NHS)酯化以后,与含有伯胺基的氨基丁基三乙酸胺(ANTA)在pH=8.0的水溶液中发生亲核反应,产生末端形成三乙酸胺(NTA),利用其很强的配位能力,使Ni(Ⅱ)与之配位。再以配位键将His-tagged蛋白(thioredoxin-urodilatin)固定在平面硅的表面。同样,同时带有FITC和Histidine标记的蛋白也可以固定在平面硅的表面。为了进一步提高活性官能团的密度,我们将上一步得到的末端NTA在0.1M的HCl中酸化,然后再依次与NHS发生酯化和ANTA发生酰胺化反应,在硅表面生成第二代的NTA结构。这样NTA基团的密度会明显提高,因此通过这样的重复手段可以固定更多的Ni(Ⅱ),也就可以固定更多的蛋白质分子。化学修饰过程中的大多数反应都利用我们课题组开发的多次透射-反射红外(MTR-IR)附件来表征。硅表面固定蛋白数量的增加也通过MTR-IR、激光辅助解离/离子化-时间飞行质谱(MALDI-TOF-MS)和荧光扫描来表征,表面绑定的Ni(Ⅱ)量由X-射线光电子能谱(XPS)来表征。MTR-IR和荧光扫描证明：第二代NTA/Ni2+所固定的蛋白量是第一代NTA/Ni2+的1.63倍。为了进一步提高平面硅表面官能团NTA的数量,同时考虑在生物领域的应用,我们在平面硅表面接枝具有抗生物污染的聚(聚乙二醇单甲基丙烯酸甲酯)(PEGMA)聚合物刷(Si-g-P(PEGMA)),利用上述方法提高了被绑定的蛋白质的量。首先通过Si-H与10-烯-1-醇在微波加热条件下的反应,在平面硅表面组装末端羟基的基底；然后将原子转移自由基聚合反应(ATRP)所必需的引发剂2-溴-2-甲基丙酰溴(BMPB)通过酯化反应引入平面硅表面；单体PEGMA在表而进行接枝共聚,所产生的Si-g-P(PEGMA)用丁二酸酐活化产生末端羧基的结构(Si-g-P(PEGMA-COOH)).末端羧基与NHS反应产生NHS酯,再与ANTA反应产生末端NTA。继续与NHS反应,继而与ANTA反应,生成第二代的NTA结构；重复前述的方法,直至生成第三代的NTA结构。通过与Ni2+配位以后,将产生的三代NTA结构用于绑定Histidine修饰的Thioredoxin-urodilatin。采用MTR-IR跟踪上述的逐步反应。通过对红外谱图中v(NH)积分面积的计算,得到第一代三乙酸胺至第三代三乙酸胺固定的蛋白量的比值为1：1.7：2.3。此外,我们还报道了一种在硅表面聚合物刷上(Si-g-P(PEGMA-OH))合成树枝结构聚酰胺胺(PAMAM)的方法。首先通过ATRP方法在平面硅表面制备Si-g-P(PEGMA-OH)。其与SOCl2反应,末端的-OH被转化为-Cl,末端-Cl被乙二胺取代,得到末端伯胺基结构。每一个末端伯氨基理论上可以与两个丙烯酸甲酯发生Michael加成,理论上生成两个末端酯(-(C=O)-OCH3)。这两个末端酯基与乙二胺发生酰胺化反应,得到两个末端氨基。如此重复上面的Michael加成和酰胺化反应,在具有抵抗生物污染的Si-g-P(PEGMA-OH)末端得到树枝结构PAMAM。硅表面密集的氨基可以通过其与N-琥珀酰胺基-6-马来酰胺基己烷酯(N-succinimidyl-6-maleimidylhexanoate, SMH)的交联反应得到活化。生物分子如缩宫素就可以被绑定在如此功能化的平面硅表面。另外,这种硅表面树枝结构PAMAM可以作为一个平台合成三个氨基酸的小肽H-Arg-Gly-OH(RGD)。上述每一步反应均有MTR-IR监测跟踪,辅助以XPS、UV-Vis和MALDI-TOF-MS作进一步的证明。最后,我们在硅表面高分子刷Si-g-P(PEGMA)上制备了两种不同的基团,以期实现Click Chemistry反应。一方面,Si-g-P(PEGMA-OH)与SOC12反应,-OH被-Cl取代,接着-Cl被NaN3中的-N3基取代,得到Si-g-P(PEGMA-N3)聚合物刷；另一方面,通过Si-g-P(PEGMA-OH)与溴丙炔反应,得到Si-g-P(PEGMA-CH2C=CH)。末端含炔基的模型化合物炔丙基胺、丙炔酸和10-炔-1-酸可以与末端叠氮基的聚合物刷反应,而末端含叠氮基的模型化合物苄基叠氮则可以与末端炔基的聚合物刷反应。这些反应都在微波条件下,以Cu(Ⅱ)/L-抗坏血酸钠体系为催化剂,30℃下反应均在1h内完成。每一步的化学反应都采用界面敏感的技术：MTR-IR和XPS进行详细地分析。归属于三唑环上v(H-C=)的3139 cm-1吸收峰和XPS高分辨扫描N ls作为确凿的证据证明了Click chemistry反应。通过Click反应前后的定量红外分析,我们发现微波作用带来了很高的收率和效率。因此,微波辐射促使硅表面Click反应的技术可以用于功能化合物的组装和有机物/硅器件的制备。更多还原

【Abstract】 Various modifications have been carried out on silicon surface to satisfy the specific demands of different areas such as biochips, molecular devices and microelectronics in recent years. Chemical modifications on the silicon crystalline surface bring new chemical properties to prepare biosensors and biochips for protein and DNA assays.Chemical modifications can be realized including reactions between silane coupling reagents and the surface Si-OH as well as reactions between Grignard reagents and surface Si-Cl or Si-Br. However, the resulted Si-O-Si via the former reactions is apt to degrade under alkaline conditions. Meanwhile, the later reactions take place under stringent conditions and fewer sorts of Grignard reagents do not meet various practical demands.It demands robust covalent bonds between the interface of soft materials and silicon, which can suffer from diverse chemical environments and can be converted into different functional groups in practical use. The surface Si-C bond satisfies the requirement and silicon wafer is capable of enduring different organic solvents, acids and weak alkalis. Bond Si-C can be obtained by means of Si(111)-H, generated from etching with 40% NH4F solutions, coupling with terminal vinyl C=C of organic compounds with a functional group at the other end to fabricate monolayers on silicon surface. Further chemical modifications can be implemented until proper functionalized surface is yielded while the Si(111) surface remains atomic flat. All these properties of Si(111) forecast its extensive applications in biochemistry, molecular device and other areas. The research work described in this dissertation is as follows. First, a Si(111)-H surface was modified via a direct reaction between Si-H and 1-undecylenic acid (UA) under microwave irradiation to form molecular monolayers with terminal carboxyl groups. After esterifying carboxylic acid with N-hydroxysuccinimide (NHS), aminobutyl nitrilotriacetic acid (ANTA) was bound to the silicon surface through amidation (pH=8.0) between its primary amino group and NHS-ester, producing nitrilotriacetic acid (NTA) anions. Then hexa-histidine tagged thioredoxin-urodilatin (his-tagged protein) and FITC labeled hexa-histidine tagged thioredoxin-urodilatin (FITC-his-tagged protein) can be anchored after NTA was coordinated with Ni2+. Furthermore, the NTA-terminated chip was acidified with 0.1M HCl and subsequently esterified with NHS and then amidated with ANTA again to produce a second generation NTA. Thus the surface density of nitrilotriacetic acid anions was improved and resultantly that of anchored proteins was also enhanced through the iterative reactions. Both multiple transmission-reflection infrared spectroscopy (MTR-IR) and fluorescence scanning measurements demonstrated a proximate 1.63 times of anchored proteins on the second generation NTA/Ni2+ as that on the first generation NTA/Ni2+ monolayer.Apart from this, we developed a facile way of preparing a bioactive silicon surface through constructing multiple generation nitriloacetates from poly(poly(ethylene glycol)monomethacrylate) brushes on a planar silicon surface (Si-g-P(PEGMA)) to improve the capacity of binding proteins and biofouling repellence. Firstly the freshly etched Si(111)-H reacted with 10-undecen-l-ol (UO) under an optimized microwave irradiation condition to produce hydroxyl-terminated substrates. Secondly surface initiators for atom transfer radical polymerization (ATRP) were introduced by means of esterification between 2-bromo-2-methylpropionyl bromide (BMPB) and surface hydroxyl groups. Thirdly polymer brushes of Si-g-P(PEGMA) grown from surface initiators were carboxylated with succinic anhydride onto their side chains (Si-g-P(PEGMA-COOH)). Finally multiple generation nitriloacetates were obtained by iterative esterification of carboxylic acids with N-hydroxysuccinimide (NHS)/N,N’-dicyclohexylcarbodiimide (DCC) yielding amino-reactive NHS esters and successive amidation with aminobutyl nitrilotriacetic acid (ANTA) yielding nitrilotriacetic acid ligands for capturing histidine-tagged proteins. After coordinated with Ni(II), the first to third generation nitriloacetate surfaces were applied to bind histidine-tagged thioredoxin-urodilatin. All stepwise conversions were monitored with MTR-IR. By means of comparison of integrated peak areas of v(NH) from amide, we estimated the enhancement of protein loading by nitriloacetates as first generation/second generation/third generation= 1: 1.7:2.3.In addition, we reported a facile approach to building dendrons of polyamidoamine (PAMAM) derived from grafted polymer brushes of poly(poly(ethylene glycol) monomethacrylate) on planar silicon surface (Si-g-P(PEGMA-OH)). In the first place, Si-g-P(PEGMA-OH) brushes were built via surface-initiated atom transfer radical polymerization (ATRP) technique on silicon surface. The peripheral hydroxyl groups of Si-g-P(PEGMA-OH) were chlorinated with thionyl chloride to convert-OH into-Cl. Then the end chlorines were substituted with amino groups of ethylenediamine giving terminal primary amine. Each amino group reacted with methyl acrylate via Michael addition to produce two end esters of-(C=O)-OCH3 theoretically. The two resulted esters reacted with ethylenediamine (EDA) subsequently via amidation generating two terminal amino groups. We iterated such Michael addition to obtain terminal esters and subsequent amidation to achieve dendrons of PAMAM on such a biofouling-resistant surface of Si-g-P(PEGMA-OH). The dense amino groups could be activated via a cross-link reaction between peripheral amino groups of dendrons and N-succinimidyl-6-maleimidylhexanoate. Thus biomolecules such as oxytocins could be anchored on such functionalized surface, In addition, the dendrons of PAMAM on silicon surface could be used as a platform to synthesize a three amino acid peptide of H-Arg-Gly-Asp-OH (RGD). The modifications had been monitored and demonstrated by MTR-IR spectra, X-ray photoelectron spectroscopes (XPS), UV-Vis spectra and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).At last, two surface approaches were realized to complete click chemistry reactions at covalently grafted polymer brushes of poly(poly(ethylene glycol) monomethacrylate) on a planar silicon surface (Si-g-P(PEGMA-OH)). On one hand, the hydroxyls from Si-g-P(PEGMA-OH) brushes can be replaced by chlorines of thionyl chloride and then chlorines can be substituted with azides of sodium azide to achieve azide-terminated (Si-g-P(PEGMA-N3)) brushes. On the other hand, the terminal acetylene (Si-g-P(PEGMA-CH2C=CH)) brushes can be prepared easily by reaction between Si-g-P(PEGMA-OH) and propargyl bromide. Model compounds of propargylamine, propiolic acid and 10-undecynoic acid with terminal acetylene and of benzyl azide with terminal azide were chosen to investigate the surface click reactions catalyzed with Cu(Ⅱ)/sodium L-ascorbate by microwave irradiation under very mild conditions at 30℃for 1 h. The stepwise modifications were characterized by two surface-sensitive techniques, Multiple Transmission-Reflection Infrared Spectroscopy (MTR-IR) and XPS, and their spectra were analyzed in detail. The infrared band at 3139 cm-1 attributed to the triazole ring v(H-C=) and the XPS high-resolution scan of N 1s give the direct evidences to confirm the click reactions. By quantifying their infrared spectra before and after click reactions, we conclude that the click reactions on silicon surfaces by microwave irradiation possess high yield and efficiency. Hence, the microwave irradiated click reaction approaches on silicon might open convenient avenues to fabricate functional and hybrid organic/silicon devices.更多还原