电化学多肽生物传感器信号放大策略的研究

发布时间：2018-06-04 14:57

本文选题：电化学多肽传感器 + 蛋白酶　；参考：《西南大学》2017年硕士论文

【摘要】：蛋白酶是一种水解蛋白质或多肽的水解酶,它与人体代谢过程如细胞生长与死亡、组织重塑和免疫防御等有关,已作为生物标志物应用于临床检测。建立简单、灵敏、特异和快速的生物标志物检测方法,对疾病的预防、诊断和治疗具有十分重要的意义。电化学生物传感器是将特异性分子识别物质例如抗体、酶、适体、多肽等固定在换能器上,以电化学信号为检测信号的分析器件,在分析化学的研究领域中起着越来越重要的地位。将多肽作为分子识别物质,与抗体和酶相比,它稳定、可靠、成本低,并且具有高的亲和性,较强的生物活性等优点,在蛋白酶生物标志物的检测中得到人们的广泛关注。本论文以多肽作为分子识别物质,致力于设计不同的信号放大策略,构建了一系列操作简便、成本低廉的电化学多肽传感器,实现对前列腺抗原(一种丝氨酸蛋白酶)、基质金属蛋白酶-2、基质金属蛋白酶-7的高灵敏检测,具体工作如下:1.基于带正电荷的金纳米粒子为信号增强剂检测前列腺抗原的电化学多肽传感器研究大部分的电化学多肽传感器是基于目标物对标记信号标签的多肽进行剪切而构建的“信号衰减型”(signal-off)传感器,其灵敏度受到限制,且易产生假阳性信号。当前,金属纳米粒子由于可以克服一些酶生物材料固有的不稳定性问题,已被广泛用于信号放大。本工作中,基于正电荷的金纳米粒子作为信号增强剂构建了一种“信号增强型”(signal-on)的高灵敏的电化学多肽传感器。正电荷的金纳米粒子修饰在分子识别元件多肽上,它吸附测试底液中带负电荷的氧化还原探针铁氰化钾([Fe(CN)6]3-/4-),加速了电子间的传递,获得一个较低电化学阻抗值。在目标物前列腺抗原(PSA)存在的条件下,PSA特异性地剪切多肽,从而使得正电荷的金纳米粒子离开电极表面,在[Fe(CN)6]3-/4-底液中进行电化学阻抗分析时,得到了一个较大的电化学阻抗值。通过监测电化学阻抗值的变化,此传感器实现了对目标物PSA的高灵敏检测。此外,传感体系是基于目标物对多肽的特异性剪切所设计,具有较高的选择性和灵敏度,为其他蛋白质的分析提供了一个通用性的检测方法。2.基于核酸外切酶辅助的循环放大策略检测基质金属蛋白酶-2的电化学多肽传感器研究将目标物对肽链的剪切直接转化为输出DNA(output DNA),它可以进一步地结合各种DNA放大技术实现高效信号放大,提高传感器的灵敏度。本工作中将多肽剪切事件转换为DNA检测,并结合核酸外切酶III(EXO III)辅助目标物循环信号放大策略构建了一种超灵敏检测基质金属蛋白酶-2(MMP-2)的电化学传感器。所构建的传感体系可总结以下几个优点:首先,“信号增强型”(signal-on)传感器的设计,在一定程度上减小了由电极表面剥落或者污染导致的假阳性信号的产生;其次,把蛋白质分析与DNA放大技术结合,有效地提高了传感器的灵敏度;另外,基于金纳米粒子(depAu)良好的导电性和葫芦脲[7]与电活性物质亚甲基蓝(MB)之间强的主客体识别作用,采用CB[7]/depAu修饰的电极作为传感界面有效地促进了电活性物质的采集;最后,此方法还可以拓展到其他具有酶剪切活性的蛋白质的检测。3.基于多肽剪切诱导的级联信号放大策略检测基质金属蛋白酶-7的电化学多肽传感器研究多肽剪切事件可以直接转化为输出DNA(output DNA),然而,output DNA通常带有氨基酸残基,其位阻效应会影响DNA的放大效率。为了解决这个问题,在本工作中,以指数放大反应(EXPAR)作为模型,通过设计一个两段式的DNA模板,构建了一个高效的DNA放大体系用于高灵敏检测基质金属蛋白酶-7(MMP-7)。基于这个两段式的DNA模板,output DNA作为触发剂1(trigger 1)最先引发了一个低效率的EXPAR,反应过程中不仅获得部分产物DNA(S1),还产生了与output DNA具有相同核苷酸序列的触发剂2(trigger 2)。由于trigger 2不包含任何氨基酸残基,它继续诱导了一个额外的,高效的EXPAR放大,迅速地获得大量的产物S1。产物S1作为催化剂进一步诱导了催化发夹自组装反应(CHA),获得了级联放大的电化学信号,从而实现了对目标物MMP-7的超灵敏检测,检测限是0.02 pg·mL-1。本工作中,目标物对于多肽的剪切过程,即输入蛋白转换为output DNA的步骤是在溶液中进行的,避免了复杂的电极制备过程,同时也提高了目标物的转换效率。另外,多肽剪切诱导的级联放大反应包括EXPAR和CHA,它们不仅有较高的放大效率,而且均是等温反应过程,具有简单,低耗的优点,有较好的应用前景。
[Abstract]:Protease is a hydrolytic protein or polypeptide hydrolase, which is related to human metabolic processes such as cell growth and death, tissue remodeling and immune defense. It has been used as a biomarker for clinical detection. A simple, sensitive, specific and rapid method for biomarker detection has been established to prevent, diagnose and treat diseases. The electrochemical biosensor is an analytical device which is fixed on the transducer, such as antibodies, enzymes, aptamers, peptides, etc., with electrochemical signals as the detection signal, and plays a more and more important role in the research field of analytical chemistry. It has the advantages of stable, reliable, low cost, high affinity, strong biological activity and so on. People pay much attention to the detection of protease biomarkers. In this paper, polypeptides are used as molecular recognition materials and are devotes to design different signal amplification strategies. A series of simple and inexpensive electrochemical methods have been constructed. Peptide sensors, high sensitive detection of prostatic antigen (a serine protease), matrix metalloproteinase -2, matrix metalloproteinase -7, specific work is as follows: 1. electrochemical polypeptide sensors based on positive charged gold nanoparticles as signal enhancers for detection of prostate antigen most of the electrochemical polypeptide sensors The "signal attenuation" (signal-off) sensor based on the targeting of labeled signal labels is limited and is susceptible to false positive signals. At present, metal nanoparticles have been widely used in signal amplification because they can overcome the inherent instability of some enzyme biomaterials. The positive charged gold nanoparticles are used as signal enhancers to construct a highly sensitive electrochemical polypeptide sensor of "signal enhancement" (signal-on). The positive charged gold nanoparticles are modified on the molecular recognition element polypeptide. It adsorbs the negatively charged oxidation-reduction probe potassium ferricyanide ([Fe (CN) 6]3-/4-) in the test bottom solution. In the presence of the target prostate antigen (PSA), the PSA specific shear peptide, which makes the positive charged gold nanoparticles leave the surface of the electrode and the electrochemical impedance analysis in the [Fe (CN) 6]3-/4- bottom solution, obtains a larger electrochemical impedance value. By monitoring the changes in electrochemical impedance values, the sensor has achieved a high sensitivity detection of the target PSA. Furthermore, the sensing system is designed based on the specific shear of the peptide on the target. It has high selectivity and sensitivity, and provides a universal detection method for the analysis of other proteins based on the nucleic acid exonuclease supplemented. The electrochemical peptide sensor of matrix metalloproteinase -2 was detected by the assisted cyclic amplification strategy. The target substance was transformed directly into the output DNA (output DNA) by the target peptide chain shear. It could be further amplified by various DNA amplification techniques to improve the sensitivity of the sensor. The polypeptide shear event was converted to DNA in this work. An electrochemical sensor for ultra sensitive detection of matrix metalloproteinase -2 (MMP-2) was constructed by combining the nucleic acid exonuclease III (EXO III) assisted target circulation signal amplification strategy. The proposed sensor system can summarize the following advantages: first, the design of "signal enhancement" (signal-on) sensor is reduced to a certain extent. The false positive signals caused by the surface stripping or pollution of the electrode are produced. Secondly, the sensitivity of the sensor is effectively improved by combining the protein analysis with the DNA amplification technology. In addition, the good conductivity of the gold nanoparticles (depAu) and the recognition of the host and guest between the cucurbit [7] and the electroactive methylene blue (MB) are strong. The use of CB[7]/depAu modified electrode as a sensing interface effectively promotes the collection of electroactive substances; finally, this method can also be extended to the detection of other proteins with enzyme shear activity,.3. based on the cascade signal amplification strategy of peptide shear induced detection of the matrix metalloproteinase -7 by the electrochemical polypeptide sensor and the study of the polypeptide scissors The tangent events can be directly converted to the output DNA (output DNA), however, output DNA usually has amino acid residues, and its steric effect affects the amplification efficiency of DNA. In order to solve this problem, in this work, the exponential amplification reaction (EXPAR) is used as a model to construct an efficient DNA amplifier by designing a two segment DNA template. The system is used for highly sensitive detection of matrix metalloproteinase -7 (MMP-7). Based on this two stage DNA template, output DNA as a trigger 1 (trigger 1) first initiate a low efficiency EXPAR. The reaction process not only obtains a partial product DNA (S1), but also produces the trigger agent 2 (trigger 2) with the same nucleotide sequence as output DNA. Ger 2, which does not contain any amino acid residues, continues to induce an extra, efficient EXPAR amplification and rapidly obtain a large number of product S1. products S1 as a catalyst to further induce the catalytic hairpin self assembly reaction (CHA), and obtain a cascade of amplified electrochemical signals. The ultra sensitive detection of the target MMP-7 is realized and the detection limit is achieved. In the 0.02 pg. ML-1. work, the step of the target to the shear process of the polypeptide, that is, the step of the input protein to output DNA, is carried out in the solution, avoiding the complex electrode preparation process and improving the conversion efficiency of the target. In addition, the cascade amplification reaction induced by the polypeptide shear, including EXPAR and CHA, is not only more effective. High magnification efficiency, both of which are isothermal reaction processes, have the advantages of simplicity and low consumption, and have good application prospects.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3;O657.1

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