【作者】 马慧；

【导师】 于丽；

【作者基本信息】 山东大学 ， 物理化学， 2019， 硕士

【摘要】 近年发展起来的液晶传感平台,具有构造简单、成本低、特异性强和灵敏度高等优势,为生物分子的检测开辟了一条重要途径。液晶传感平台利用加入目标物后微小的理化性质的变化来诱导液晶分子的排列取向发生变化,同时能够将这种变化以光学信号的方式输出,从而实现对目标物的检测。鉴于此,本论文以4-氰基-4’-戊基联苯(5CB)热致液晶为传感基底,基于主客体作用和静电作用等分子间弱相互作用构筑了液晶传感平台,并用于检测石胆酸、α-淀粉酶以及凝血酶等生物分子,主要采用偏光显微镜(POM)观察传感平台的光学响应信号,辅助使用等温滴定量热仪(ITC)和表面张力仪对生物分子的响应机理进行探究。论文的主要内容分为以下四个部分:第一章介绍了液晶、液晶传感平台和分子间弱相互作用等方面的相关背景知识,以及近年来液晶传感平台在检测领域的国内外研究现状,并提出了本论文的研究思路。第二章基于主客体作用构筑液晶传感平台,利用竞争的主客体作用实现了对石胆酸的特异性检测。当把十二烷基硫酸钠(SDS)/β-环糊精(β-CD)复合物加入到液体-液晶界面上的时候,由于SDS几乎被完全包覆在β-CD的内腔里,溶液本体中没有游离的SDS吸附在液晶界面上诱导液晶分子垂直排列,所以在偏光显微镜下观察到亮的图像。当加入石胆酸后,液晶的光学形貌发生了由亮到暗的变化。基于此可以检测石胆酸,其检测限约为2 μM。构筑的液晶传感平台对无机盐、葡萄糖、抗坏血酸、尿素和尿酸等物质没有响应。根据等温滴定量热的实验结果推测该方法检测石胆酸的可能机理是:相比于SDS,石胆酸与β-CD的结合作用更强,因此加入的石胆酸会把SDS从β-CD的内腔里替换出来,游离的SDS分子会吸附在液体-液晶界面上使液晶垂直排列。采用此方法还检测了实际样品人体尿液里面的石胆酸。本章研究工作的开展可为早期肝脏疾病的临床诊断提供依据。第三章是在第二章的研究基础之上,通过破坏SDS和β-CD之间的主客体作用检测了α-淀粉酶。发现只加入SDS/β-CD复合物的时候,液晶呈现亮的光学形貌;当同时加入α-淀粉酶和SDS/β-CD复合物时,在偏光显微镜下观察到暗的图像。通过表面张力数据分析,液晶呈现暗的光学形貌可能是由于α-淀粉酶水解β-CD释放SDS,随后SDS吸附在液体-液晶界面上诱导其垂直排列。在较宽的pH(3.0～11.0)和盐浓度(0～1.0mM)范围内,液晶图像依然能保持明亮,表明该传感平台具有较好的稳定性。构筑的液晶传感平台对α-淀粉酶的检测限约为15 U/L,对胰蛋白酶、胃蛋白酶及溶菌酶等其它酶没有响应。另外,还对稀释的唾液和尿液里的α-淀粉酶进行了检测。该方法在高灵敏、无标记检测α-淀粉酶领域具有潜在的应用价值。第四章利用静电作用构筑了液晶传感平台,用于凝血酶的检测。发现掺杂了十八烷基三甲基溴化铵(OTAB)的液晶分子在OTAB的诱导下呈垂直排列;加入凝血酶的适配子后,OTAB通过与适配子之间的静电作用,将其吸附在液体-液晶界面上,随后适配子的疏水碱基直接与液晶分子作用,同时降低了OTAB的界面密度,液晶分子的排列由垂直变为倾斜,对应的光学形貌发生由暗到亮的变化;当存在凝血酶的时候,适配子会优先和凝血酶结合,此时OTAB又会诱导液晶分子垂直排列,相应地在偏光显微镜下观察到暗的形貌,因此可以通过液晶的亮暗光学形貌的变化检测凝血酶,检测限约为136 nM。这种基于静电作用构筑液晶传感平台,并利用目标分子和适配子之间的特异性结合来检测生物分子的策略,为未来进行相关的临床诊断提供了一条新的途径。更多还原

【Abstract】 Recently,liquid crystals(LCs)sensing platform could open up a significant approach for detection of biomolecules due to its advantages such as simple construction,low cost,high specificity and sensitivity.LCs sensing platform can achieve monitoring analytes via the variation in orientational alignment of LC molecules triggered by slight changes in physical and chemical properties when adding analytes.It can transform such changes into amplified and visible optical signals.In view of this,we chose a thermotropic LC,namely 4-cyano-4’-pentylbiphenyl(5CB),as sensing substrate in this dissertation.Some biomolecules including lithocholic acid,a-amylase,and thrombin were detected by the LCs sensing platform designed based on the intermolecular weak interactions(e.g.host-guest interaction and electrostatic interaction).Polarized optical microscope(POM)was employed to observe the optical response signal of sensing platform.We also explained the responsive mechanism,aided by isothermal titration calorimetry(ITC)and surface tensiometer.There are four main parts in this dissertation as follows:Chapter 1 is an introduction of the relevant background knowledge of LCs,LCs sensing platforms and weak intermolecular interactions,as well as the recent research status of LCs sensing platform in detection at home and abroad,and the research ideas for this dissertation.In chapter 2,we constructed a LCs sensing platform based on host-guest interaction and detected lithocholic acid(LCA)by competitive host-guest inclusion.When sodium dodecyl sulfate(SDS)/β-cyclodextrin(β-CD)complex was added to the aqueous/LCs interface,a bright image was observed under POM,because almost all of SDS molecules were into the cavities of p-CD.In this case,no free SDS existed in the bulk of solution and adsorbed at the fluid interface to induce the homeotropic ordering of LCs.When injecting LCA into the aqueous/LCs interface,POM captured a bright-to-dark transformation of optical response.The as-prepared LCs sensing platform could detect LCA as low as～2 μM,without interference from other components such as inorganic salt,glucose,ascorbic acid,urea and uric acid.According to the experimental results of ITC,the possible LCA detection mechanism was speculated.The host-guest interaction between β-CD and LCA is much stronger than that of β-CD with SDS.Therefore,upon addition of LCA,SDS molecules which were excluded from the cavity of β-CD on account of competitive host-guest inclusion adsorbed at the aqueous/LCs interface and resulted in the orientational transition of LCs from tilted to homeotropic state.Moreover,the practicability of such approach was validated by monitoring the amount of LCA in human urine.The research work in this chapter can provide a basis for the early clinical diagnosis of hepatic disease.In chapter 3,we detected a-amylase by disruption of host-guest interaction between SDS and β-CD on the basis of chapter 2.Only in the presence of SDS/β-CD solution,a bright optical image was observed.While a black optical appearance was captured when the pre-incubated mixture containing SDS/β-CD complex and a-amylase was transferred onto the fluid interface.According to the data of surface tension,we inferred that a-amylase could hydrolyze β-CD and subsequently destroy the host-guest interaction between SDS and P-CD.SDS molecules escaping from the cavity of β-CDs adsorbed at the aqueous/LCs interface and evoked the homeotropic state of LCs.The POM images of LCs always kept bright within a wide range of pH(3.0～11.0)and ionic strength(0～1.0 mM),indicating high stability of the LCs sensing platform.Based on these,detection of α-amylase could be achieved and its detection limit was about 15 U/L.This sensing platform shew no response to other enzymes such as trypsin,pepsin and lysozyme.Moreover,it was successfully utilized to monitor α-amylase in dilute urine and saliva.This approach has a great potentiality in sensitive and label-free detection of a-amylase.In chapter 4,we designed a LCs sensing platform based on electrostatic interaction to analyze thrombin.LCs doped with octadecyl-trimethylammonium bromide(OTAB)adopted homeotropic alignment under the induction of OTAB.When thrombin aptamer was added,it was attached to aqueous/LCs interface because of the electrostatic interaction between aptamer and OTAB,and then the hydrophobic nucleobases of thrombin aptamer associate directly with the LCs,effectively competing with OTAB molecules for interfacial sites,which caused the orientational transition of LCs from homeotropic to tilted state corresponding to variation in the optical morphology of LCs from dark to bright.When thrombin was present,the aptamer was preferred to bind to it.After that,OTAB induced the vertical arrangement of LCs corresponding to a dark image under POM.Therefore,thrombin could be detected by observing the bright and dark change in the optical appearance of LCs.The detection limit of thrombin was about 136 nM.The LCs sensing platform constructed based on electrostatic interaction can detect biomolecules by the specific combination of target molecule and corresponding aptamer,which reveals a new way to relevant clinical application in the future.更多还原