DNA和石英晶体微天平为基的传感平台的构建及其在靶定生物分子和环境检测中的应用
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摘要
本论文的工作分为三部分,第一部分是关于针对河豚体中河蚌毒素快捷方便检测方法的研制;第二部分是关于石英晶体微天平的传感平台的建立以扩大仪器应用范围,针对生物小分子腺苷的检测方法的确立和蓝藻爆发时对藻毒素吸附材料表面设计提出建议;第三部分是基于纳米金的SPR效应所产生的颜色变化与具有pH响应的多聚腺嘌呤DNA分子进行结合,构建具有pH响应的可视化传感器。
     传感器是将对靶定分子具有特异性识别作用的识别元件与一些信号转换或放大元件相结合的分析工具。生物传感器则是对生物分子具有响应的传感器或者识别元件自身就是具有生物活性的材料如蛋白质(酶、抗原、抗体、)、DNA和生物膜等。近年来随着社会发展,人们对于食品安全和环境健康的问题越来越关注,对此类问题的检测手段的研制也是科研一个比较集中的课题。对于环境和食品检测,一些大型仪器可以提供很高的精密度但由于其价格昂贵、操作复杂、需要专人培训操作而使得这些大型仪器在日常生活和生产中的应用有很大的限制。因此开发一些操作简单容易制备且成本低廉的传感器可以有效提高公众在日常生活中对有毒有害物质的提前防护意识,同时由于操作方便不需培训而使大众更容易接受。在我硕博期间,我只要集中在对操作简单、制备方便的生物传感器的方法研究以使得任何没有专业知识背景的群众都可以操作;同时还对石英晶体微天平为基的传感平台的建立进行研究。
     河蚌毒素是一种具有生物富集作用的天然神经毒素。其可以通过阻碍钠泵运行而使人体内钠钾失衡导致人体瘫痪甚至死亡。这种毒素在水生物体中无法发挥毒副作用主要是由于水生物体中的钙泵作用更重要。由于这种毒素稳定性好因此无法通过简单杀菌消毒或者高温烹饪去除,且一旦食入中毒则无解药可救,因此对此毒素在未食用前的检测有很重大的意义。现在研发出的针对河蚌毒素的检测方法有七种,但是这些方法由于受到仪器复杂、动物实验的道德问题、生物提取物难度大、抗体稳定性差等的问题而不能大规模生产日常生活应用。我们通过研究此毒素结构,发现河蚌毒素结构中含有两个背靠背的胍基团,这种基团与DNA结构中的鸟嘌呤很相似,我们怀疑这种结构的相似性会影响有鸟嘌呤参与的一些结构形成比如G四联体的形成。通过圆二色谱我们发现河蚌毒素的存在会显著延缓-四联体的形成且G-四联体的量会减少,同时加入越多的河蚌毒素会诱导G-四联体的结构形成更少。而这种阻碍G-四联体形成的现象在其他类似物如具有线型胍基团的精氨酸、盐酸胍中则没有观察到。除了结构的研究,我们还进行的热力学方面的研究。利用等温滴定微量热仪,我们观察到DNA分子与河蚌毒素和其他类似物之间的结合比和结合常数,发现河蚌毒素与DNA分子的结合相比于其他类似物更强但弱于DNA与钾离子之间的作用,很好的解释了河蚌毒素可以阻碍和干扰G-四联体的形成但却不能使所有DNA分子处于单链状态。通过比较河蚌毒素及类似物与DNA之间相互作用发现,河蚌毒素与DNA的结合常数是其他两种类似物的5到10倍,解释了河蚌毒素可以干扰G-四联体的形成而类似物则不具有这种性质。利用圆二色谱和等温滴定微量热仪得出的结论,我们认为通过检测有毒素存在的DNA体系中G-四联体的量可以间接的测定体系中毒素的含量。我们采用了一个广泛应用且价格低廉的染料结晶紫作为荧光信号分子,有研究组报道此染料可以识别G-四联体和单链DNA因此可以作为一个很好的信号传导分子。我们发现此分子在与单链DNA结合时会荧光增强,因此当有毒素存在而使部分DNA不能形成G-四联体时,结晶紫可以与DNA结合荧光增强而达到对毒素检测的荧光增强效果。
     石英晶体微天平是利用石英震动时有固有的震动频率,震动频率会由于片子表面有吸附或修饰而产生变化,通过对片子震动时频率和耗散的变化可以达到对片子表面吸附材料的质量和性质测定的目的。因此仪器在使用初期经常用于吸附和机理研究,但用此仪器进行检测分析研究的则很有限。在本人硕博期间,由于检测的分子均为小分子,因此一直在考虑如何将此仪器应用于对小分子的检测中。通过DNA适配体对于生物小分子腺苷特异性识别作用和纳米金大的质量,利用DNA互补配对,当有小分子目标物存在时,由于DNA结合目标物而产生构象变化不能再与负载纳米金的配对链相结合,而没有目标物时负载纳米金的配对链可以配对到芯片表面而产生很大的频率信号。通过这种方法可以有效改善传统不能检测小分子的一个关键问题:即当小分子存在时由于小分子分子量小而产生很小的信号变化。通过纳米金的扩增,存在的小分子越少可以负载上去的纳米金就越多,那么信号就越大,从而使得对小分子的低限检测成为可能,且由于信号得到很大增强而使信号识别和数据处理更加方便。同时利用DNA分子可逆互补配对我们也达到了对芯片表面反复利用的目的。
     夏天时蓝藻爆发时产生的恶臭和水体中的藻毒素的去除是我们关系的问题之一。常用的去除吸附材料是活性炭。我们考虑到现在塑料产生的污染很大,如果可以通过对塑料表面进行改性,可以同时达到对水体中藻毒素吸附的去除和变废为宝清洁环境双赢的目的。我们以比较常用的树脂材料聚苯乙烯作为本体进行吸附材料设计的研究。由于藻毒素是一种具有等电点的非蛋白质毒素,其受环境pH的影响很大,在不同的pH会显现不同的电性。通过对聚苯乙烯表面基团设计,我们合成了聚苯乙烯树脂、季铵化聚苯乙烯和磺化聚苯乙烯三种具有不同电性的聚苯乙烯基体。通过旋涂将三种不同基底涂敷于石英晶体微天平的芯片上,利用仪器进行在线检测吸附过程。通过实验发现在中性水域,季铵化聚苯乙烯有最好的对于藻毒素的吸附效果,此结论可以作为以后对废弃塑料改性的一个理论依据。
     除了研制简单制备的荧光传感器,可视化传感器的研制也是我的一个研究课题。利用纳米金距离不同产生不同颜色的特点,将其与具有pH响应性的DNA分子相结合,达到了对特定pH范围检测的目的。由于产生颜色变化的范围很窄,因此可以作为潜在的pH报警器使用。
During the past several decades, we have witnessed the fast development in industry and technology. However, the pollution induced during the process is also severe. Many technical methods have been constructed to detect the pollutants. However, a large portion of them is sophisticated instruments which are difficult for daily usage and need long detection period due to instrument modulation. To make daily detection simple and convenient, the sensors for daily detection usage are in great need. During my phd studies, I mainly concentrate on the development of very simple, easy handling methods for detection and any one without training can conduct those experiments according to the developed protocols.
     The first part is on the development of saxitoxin (STX) detection method. Saxitoxin is a natural neurotoxin which can block the sodium channel inducing paralysis even death. It is often caused by the ingestion of shellfish with accumulated toxins from phytoplankton species. The toxin is nontoxic to shellfish due to the less importance of sodium channel in shellfish body. However, due to its high stability towards heat, acid and oxidation, this guanidinium based toxin is often difficult to get rid of by simple baking. Therefore, it is in great necessity that the toxin is detected before it is digested into human body. Seven methods have been applied for STX detection. The sodium channel based method is constraint due to the difficulty in extracting the sodium channel from biological bodies and this extraction procedure needs long time training in expertise. The antibody based detection method offers high specificity but the protein is less stable under non-biological conditions and is easily affected by environmental changes. The animal based method like the usage of mouse is not only under ethic issues but also individual differences and the HPLC post-column fluorescence method is accurate but the instrument needs training and long-time usage. Herein, we developed a method for saxitoxin detection based on DNA conformational changes using a commonly used dye crystal violet (CV) as reporter. Guanine-rich DNA is DNA strands with guanine bases. Four guanine bases can interact through hydrogen bonding to form a planar G-quartet. The hydrophobic stacking of those G quartets stabilized by a monovalent cation induces the formation of G-quadruplex. Since the saxitoxin has two guanidinium backbones which are similar with guanine in structure, we speculate that the addition of saxitioxn may interfere with the formation of cation induced G-quadruplex formation. In our study, we used one widely studied G-quadruplex sequence-thrombin binding aptamer (TBA) which has two layers of G-quartets and15bases. Using potassium ions as G-quadruplex formation inducer, we found that the addition of saxitoxin can reduce the formation of G-quadruplex and a linear relationship can be extrapolated using circular dichroism (CD) signal versus saxitoxin concentration. Since TBA is a very short sequence, the fluorescence change before and after TBA G-quadruplex formation is small, therefore, the inserted dye detection method was used to reflect the conformational changes. Crystal violet (CV) is an organic molecule and the hydrophobic interaction makes the molecules tend to combine with random coiled DNA so that the fluorescence is on. However, when metal ions exist, CV cannot expel ions from the cavity, therefore, the fluorescence is weak. With the addition of STX, the G-quadruplex becomes opened and due to the electrostatic interaction between the CV and DNA strand, the CV turns on. Therefore, a turn-on sensor has been constructed using TBA as a sensing element and CV as a reporter part. However, the CV based insertion dye detection method has a shortcoming in that the detection limit is usually too high for daily detection.
     Part two is the construction of biosensors based on quartz crystal microbalance-dissipation (QCM-D). QCM-D is a powerful tool for in-situ studies of interface interaction and label-free, real-time characterization and quantification of reaction mechanism because it can not only reflect the reaction in the interfaces the mass changes but also viscoelasticity character of the adsorbed layer. However, few efforts are concentrated on the development of QCM-D for sensing platform. Therefore, during my phd studies, I mainly concentrate on the development of QCM-D based sensing platform constructions. The first one is for small molecule detection. Since the frequency signal is proportional to mass change, the higher molecular weight the substance, the higher frequency making the detection of large molecular weight substances detection easier. However, to detect small molecular weight molecules is needed to explore in order to expand the application of QCM-D. In my work, I use gold nanoparticles (AuNPs) which have large mass and easy modification characteristics to enhance the detection resolution and lower the detection limit. The QCM-D crystal was first modified with a layer of immobilization DNA using mercapto-gold interaction and then through base pair interaction, the linker part containing sequence for adenosine detection was hybridized onto the chip. If adenosine contains in the sample, the adenosine sequence can capture the target hampering the further hybridization of reporter DNA loaded with AuNPs and induces large signal changes due to the AuNPs. With the enhancement of AuNPs, the detection limit is enhanced at least3orders of magnitude and the resolution is higher since the signal range lies in100Hz instead of1Hz which is difficult to differentiate between signal and noise.
     Another work uses QCM-D is to explore the adsorption of microcystein onto polymer modified QCM-D chips. The commonly studied adsorption substrate is activated carbon. However, the functional groups'influences on the adsorption are unknown for the difficulty in modification. Therefore, we found one substrate substituting the existing widely studied activated carbon with easy changing functional groups and convenient obtainable ways-the polystyrene based polymers. Many studies on adsorption are detected by detecting the mobile phase analyte concentration change to plot the adsorption kinetics. With the aid of QCM-D, we are able to monitor the kinetics in real time. Moreover, with the analysis of dissipation, the adsorption mechanism can be inferred and amine modified polystyrene in neutral solution is recommended for adsorption of microcystein.
     The third part is about the construction of sensors based on gold nanoparticles (AuNPs) colorimetric effects. AuNPs have surface plasmon resonance (SPR) effects which make them change color when the distances between particles changes. When they aggregate, the color changes from red to purple or even blue, gray, depending on the aggregation extent. Based on this phenomenon, a simple pH alarm was constructed using DNA as sensing element and AuNPs as reporter part. It is found that adenine base can base paired with each other under acidic conditions. When the pH is low, sequences full of adenines can base pair to form duplex while at high pH condition, the sequence remain in single stranded form. In the AuNPs system, DNA single strands have bare bases that can interact with AuNPs through nitrogen-AuNPs coordination. However, when formed in duplex, the base pair interaction is substituted by hydrogen bonding among DNA strands. Without the protection of DNA, AuNPs is susceptible to aggregation under high salt concentration. From the color change, the pH can be easily differentiated. Moreover, by changing the inherent influences, the detection range can be modulated.
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