肽酶与核酸修饰酶检测新传感方法研究
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摘要
随着生命科学的发展，生物传感器技术作为一种简便、灵敏的生物分析检测技术，得到了长足的发展。它已成为生命科学分析化学的重要研究方法，在生物技术、临床诊断、环境监测、食品工业、医药和军事等领域具有广泛的应用前景。
本研究论文以重要的生物酶为研究核心，结合电化学与光学生物传感技术，发展了一些具有高灵敏度与选择性的生物传感技术用于快速检测地衣芽孢杆菌蛋白酶（第二、三章），组蛋白去乙酰化酶（第四、五章），DNA甲基化转移酶（第六章），DNA糖基化酶（第七章）。具体内容包括：
（1）基于高选择性的多肽构建用于检测细菌蛋白酶标志物检测的电化学生物传感器【第二章】
蛋白酶是一种普遍存在于生物体内的酶，可通过其对底物的消化来进行检测。由于蛋白酶在细菌中也广泛的存在，因此，细菌分泌的蛋白酶是一种理想的生物标志物，为微生物临床检测提供了快速、灵敏的目标物。基于文献报道，利用择性识别地衣芽孢杆菌分泌的蛋白酶的含D-型氨基酸多肽，结合电化学生物传感器的一些成熟手段，发展一种用于检测细菌蛋白酶的简单的电化学生物传感器。将选择性的多肽利用C端半胱氨酸残基固定到金电极上，链酶亲和素标记的碱性磷酸酶与标记在多肽N端的生物素结合，碱性磷酸酶催化无电活性的1-萘酚磷酸酯转化为有电活性的1-萘酚产生一个放大的电化学信号。当有标志物蛋白酶存在时，多肽将被选择性切割，标记有生物素的部分被移除，信号减小，这种信号的变化与蛋白酶的含量有关，从而实现对目标细菌蛋白酶定量检测的目的。当目标蛋白酶的浓度在0.7–10μg/mL范围内，氧化峰电流的大小与目标蛋白酶的浓度对数成反比，检测下限达0.16μg/mL。
（2）基于纳米金凝集变色对地衣芽孢杆菌蛋白酶的灵敏比色检测【第三章】
纳米金具有独特的光学性质及生物相容性，将其用于生物传感器的构建中，可较大提高传感器的性能。因此，我们基于纳米金凝集变色原理构建了一种用于便捷检测地衣芽孢杆菌蛋白酶的比色法。本文使用两端含有半胱氨酸残基的多肽为底物，此多肽中间包含能被目标蛋白酶选择性识别的D-型氨基酸区域。由于巯基与金的强作用力，多肽将纳米金拉近，使得纳米金靠近，发生凝集变色。当有目标蛋白酶存在时，目标蛋白酶将底物切断，切断的底物只有一端有半胱氨酸残基，不能将纳米金拉近，从而不能发生团聚变色，利用颜色的差异，即可裸眼进行观察比色，也可以采用紫外吸收测定对蛋白酶进行检测。该方法在0.1–5μg/mL浓度范围内与A520/A600的吸光度比值呈线性关系，检测下限可达0.09μg/mL。
（3）基于乙酰化的组蛋白H4多肽构建用于灵敏检测组蛋白去乙酰化酶的电化学生物传感器【第四章】
组蛋白的翻译后修饰在转录等过程有着非常重要的意义。组蛋白的翻译后修饰主要有甲基化、磷酸化、乙酰化、泛素化等。其中，组蛋白的乙酰化是一种十分重要的组蛋白翻译后修饰。生物体内控制乙酰化平衡是乙酰化酶和去乙酰化酶。这两种酶的活性对生物的很多过程有着重要的影响，例如：细胞的分化、凋亡等。研究表明，异常的去乙酰化酶活性与人类疾病有重大的关系。因此，我们以组蛋白去乙酰化酶SIRT2作为检测目标，将乙酰化的多肽通过N端的半胱氨酸残基固定到金电极表面，利用乙酰化位点的特异抗体去识别乙酰化多肽，标记有碱性磷酸酶的二抗去识别一抗并提供电化学信号。当样品中存在去乙酰化酶SIRT2时，底物上的乙酰基被移除，特异性抗体结合量减小，电化学信号随之减小，从而实现对去乙酰化酶的检测。在对实验条件优化后，该方法在的1–500nM浓度范围与电流信号值线性相关，检测下限可达0.1nM。
（4）基于纳米金淬灭荧光构建的荧光传感器用于组蛋白去乙酰化酶的灵敏检测【第五章】
结合纳米金对荧光染料的强淬灭性，利用标记有荧光素的乙酰化多肽对去乙酰化酶进行检测。将特异识别乙酰化的抗体标记到纳米金上，由于抗体特异识别乙酰化多肽，将标记有荧光分子的乙酰化多肽与纳米金拉近，使得荧光淬灭。当样品中存在去乙酰化酶时，多肽被去乙酰化后，不能被抗体识别，荧光信号增强。该方法在50–500nM浓度范围内SIRT2与荧光信号线性相关，检测下限为11.9nM。
（5）基于DNA甲基化敏感性内切酶和末端转移酶碱基延伸构建的电化学传感器用于灵敏检测DNA甲基化转移酶活性【第六章】
DNA甲基化是一种很重要的DNA修饰，与很多生命过程相关，例如：转录、基因印记、细胞分化、染色体结构和胚胎行程等。研究发现，很多疾病都与非正常的基因甲基化水平有关。DNA甲基化的过程是通过DNA甲基化转移酶催化甲硫亮氨酸上的甲基共价连接到DNA序列中的胞嘧啶或者腺嘌呤上而完成的。非正常的DNA甲基化转移酶活性会导致非正常的基因甲基化水平。因此，我们基于甲基化敏感的限制性内切酶的切割，利用末端转移酶进行末端碱基延伸，构建了一种用于灵敏检测甲基化转移酶的电化学传感器。将含有甲基化转移酶特异识别序列的DNA探针组装到金电极表面，甲基化的探针可以被甲基化敏感的限制性内切酶识别、切割，从而产生一个带有3`-OH端的新引物，末端转移酶可以将生物素化的dUTP延伸到新产生引物的3`-OH末端。之后，偶联有碱性磷酸酶的链霉亲和素（SA-ALP）与新延伸的生物素结合。碱性磷酸酶可以催化非电活性物质1-萘酚磷酸酯水解，生成电活性物质1-萘酚在电极表面氧化产生电信号。从而实现对甲基化转移酶的灵敏检测。该方法在0.1-20U/mL浓度范围与电流值呈线性关系，检测下限为0.04U/mL。（6）基于Lambda外切酶构建用于灵敏检测DNA糖基化酶的DNAzyme催化显色法【第七章】
基因的损伤是不可避免的，如果这种损伤得不到不修复，会导致基因突变、细胞凋亡，甚至会诱导癌症发生。生物体有自身的修复系统，可以对DNA的损伤进行识别和修复。碱基切补修复是对碱基损伤的一种修复手段，这过程是由很多酶共同作用实现的。在这碱基切补修复过程中，DNA糖基化酶对识别、除去受损碱基，起着至关重要的作用。非正常的DNA糖基化酶活性可能会导致疾病的发生，这就为疾病的检测提供了一种生物标志物。因此，我们利用具有辣根过氧化物酶活性的DNAzyme催化底物显色，并结合DNA外切酶的高活性，发展一种比色法对DNA糖基化酶进行方便、灵敏的检测。将含有受损碱基（8-oxo-dG）的DNA与含有血红素核酸适体的DNA互补杂交。糖基化酶hOGG1能识别受损碱基8-oxo-dG并将其从DNA序列中移除，产生一个5`磷酸端的缺口，然后利用Lambda核酸外切酶选择性消化5`磷酸端DNA的特性，将血红素核酸适体的抑制链消化分解，使得血红素核酸适体释放，与血红素结合，形成血红素-核酸适体的DNAzyme复合物结构，催化无色底物ABTS2-转化为有色的ABTS-，通过简单的比色就能达到对DNA糖基化酶的检测。该方法在418nm紫外吸收值与hOGG10.05-16U/mL浓度对数值在呈线性关系，检测下限为0.01U/mL。
With the development of life sciences, as a simple and sensitive biological analysistechnology, biosensing technology has made remarkable progress. It has become animportant research method for analytical chemistry of life sciences. It has extensiveapplication prospects in many fields, such as biology technique, clinical diagnosis,environmental protection, food industry, medicine and military.
Enzymes are biological molecules that catalyze chemical reactions. Enzymes serve awide variety of functions inside living organisms. Almost all chemical reactions in abiological cell need enzymes in order to occur at rates sufficient for life. They areindispensable for metabolism, signal transduction and cell regulation. In addition, reactionsclosely connected with life process are almost all enzyme-catalyzed reactions. Aberrantactivity of important enzyme would lead to critical disease. Many human diseases are relatedto aberrant enzyme activity. Herein, enzymes are perfect biomarkers for quick and sensitivedisease detection in clinical diagnosis.
This dissertation focuses on developing a series of biosensing techniques for detection ofprotease biomarker from Bacillus licheniformis (in chapter2,3), histone deacetylase (inchapter4,5), DNA methyltransferase (in chapter6), DNA glycosylase (in chapter7) usingthe electrochemical and optical biosensing techniques. The details are listed as follows:
(1) A novel electrochemical biosensor for highly selective detection of proteasebiomarker from Bacillus licheniformis with D-amino acid containing peptide (in chapter2)
Protease is a kind of the common enzymes in the living organisms and accessible fordetection based on substrates cleavage. There are abundant proteases in bacteria and itssecretion. Herein, bacterial protease is an ideally biomarker for quick and sensitiveidentification of microorganisms in clinical samples. Therefore, an electrochemicalbiosensor was constructed for the detection of the protease biomarker from the Bacilluslicheniformis, a model of Bacillus anthracis. The D-amino acid containing peptide labelledwith biotin was used as the specific substrate. Firstly, the specific peptide was assembled onthe gold electrode through the C-terminal Cys interacting with gold. Upon the addition of theprotease, the substrate was selectively recognized and cleaved. Subsequently, thestreptavidin-alkaline phosphatase (SA-ALP) could specifically bind the remaining biotinmoieties on the electrode surface. ALP could catalyze the conversion of an electrochemicallyinactive1-naphthyl phosphate into an electrochemically active naphenol, generating anamplified signal for electrochemical readouts. Differential pulse voltammetry (DPV) was employed to detect the signal which could be used for quantifying the proteases of Bacilluslicheniformis. Electrochemical response arising from the oxidation of enzymatic product of1-naphthyl phosphate was observed to be inversely proportional to the logarithmic value ofprotease concentrtion in the range from0.7to10μg/mL with a detection limit as low as0.16μg/mL.
(2) Gold nanoparticle aggregation-based colorimetric assay of protease biomarker fromBacillus licheniformis
Gold nanoparticle is attractive due to its novel optical and biocompatibility properties.With the introduction of gold nanoparticle into biosensor fabrication, biosensors will begreatly improved. Therefore, we developed a simple colorimetric method based on goldnanoparticle aggregation for the detection of protease biomarker from Bacillus licheniformis.A C-and N-terminal Cys peptide substrate was exploited in the assay, which contained twoD-amino acids for selective recognition and cleavage by the target protease. Cys couldstrongly interact with gold nanoparticle, leading to the aggregation of gold nanoparticles andthe color change of its solution. Whereas, cleaved peptides are unable to induce goldnanoparticle aggregation; and as a result the solution color would not change. We couldobserve the change of color by naked eye, or monitor the changes of the absorption spectrumby a UV-Vis spectrophotometer. There is a linear correlation between the ratio of theabsorbance of the system at520to600nm (A520nm/A600nm) and the logarithm of adenosineconcentration range from0.1μg/mL to5μg/mL, with a detection limit of0.09μg/mL.
(3) A sensitive electrochemical biosensor for the detection of histone deacetylase with ahistone H4Lys16acetylated peptide
Histone post-translationally modifications play important roles in transcription. Oneprevalent modification is acetylation, which is related to the gene activity. The steady-statebalance of the acetylation and deacetylation of histone is achieved through two species ofenzyme: histone acetyltransferase and histone deacetylase. The activity of histoneacetyltransferase and histone deacetylase affects the angiogenesis, cell-cycle arrest,apoptosis and terminal differentiation of different cell types. Herein, we developed a simple,sensitive electrochemical biosensor for detecting the activity of sirtuin2one of the histonedeacetylase and screening its inhibitor. An acetylated peptide with a cystein residual atN-terminal as the substrate is self-assembled on the electrode surface by the interactionbetween cystein residual and gold. The rabbit anti-acetylated peptide antibody is employedto specifically bind with the acetylated peptide. The alkaline phosphatase labelled goatanti-rabbit antibody is captured through selective interaction with the rabbit anti-acetylatedpeptide antibody. The alkaline phosphatase catalyzes the conversion of the electrochemically inactive1-naphthyl phosphate into an electrochemically active naphenol for generating theamplified electrochemical signal. In the presence of sirtuin2, the peptide substrate isdeacetylated, resulting in the decrease of electrochemical signal. Therefore, the change of thecurrent signal could reflect the concentration of sirtuin2. Under the optimum conditions, thedetection limit is achieved down to0.1nM with a linear range from1nM to500nM.
(4) A simple gold nanoparticle-based fluorescence biosensor for sensitive assay ofhistone deacetylase activity
Gold nanoparticle, as a class of nanomaterials with many specific properties, such asconductivity, colorimetric and nonlinear optical properties, has been extensivy applied inbiomolecular research. Herein, we developed a fluorescence assay using gold nanopartilce asthe quencher. An acetylated peptide labeled with a fluorophore at C-terminal was used as thesubstrate of histone deacetylase sirtuin2. Gold nanoparticle labeled with anti-acetylatedpeptide antibody acted as the quenching probe. The fluorescence would be efficientlyquenched by the gold nanoparticle, when the acetylated peptide captured by its antibody. Inthe presence of sirtuin2, the acetyl was removed from the substrate, resulting in the increaseof fluorescence signal. The fluorescence intensity was proportional to logarithmic value ofthe sirtuin2concentration ranging from50nM to500nM, with a detection limit of11.9nM.
(5) A sensitive electrochemical biosensor for the detection of DNA methyltransferaseactivity by combining DNA methylation-sensitive cleavage and terminaltransferase-mediated extension
DNA methylation plays an important role in many biological processes includingtranscription, genomic imprinting, cellular differentiation, chromatin structure andembryogenesis. A number of human diseases have been found to be associated with aberrantgene methylation. The DNA methylation process was regulated by DNA methyltransferasecatalyzing covalent addition of a methyl group to cytosine or adenine in DNA sequences.The aberrant DNA methyltransferase activity was reported to be related to pathogenesis ofcancer, such relationship provides a potential target in disease diagnosis and therapy. Herein,we developed a sensitive electrochemical biosensor based on methylation sensitive cleavageusing terminal transferase-mediated extension for the detection of the methyltransferaseactivity. A DNA probe contained the sequence which could be recognized by DNAmethyltransferase, was self-assembled on the surface of gold electrode by3’-SH. After themethylation reaction by DNA methyltransferase, the methylation sequence was specificallyrecognized and cleaved by methylation sensitive restrictive endonuclease, that produced anew primer with a3`-OH on the electrode surface. Subsequently, the dUTP-biotin wasincoporated into the3`-OH terminal of the new primer by TdTase-mediated extension. Then, the SA-ALP was added and bound with the labeled biotin on the primer. The amplifiedelectrochemical signal was obtained by ALP catalyzing1-NP convertion. Differential pulsevoltammetry (DPV) was employed to detect the signal which can be used for quantifying theDam MTase activity and the concentration of its inhibitor. The DPV current is proportionalto the logarithmic value of DNA methyltransferase concentration ranging from0.1to20U/mL with a detection limitation of0.04U/mL.
(6) DNAzyme-based label-free colorimetric method for sensitive detection of DNAglycosylase
DNA damage occurs by various mechanisms, either as a by-product of normal cellularmetabolism or as a result of exposure to environmental and biological mutagens. DNAdamage that is not repaired leads to apoptosis or mutation, which in turn may lead toinduction of carcinogenesis. Multiple systems are in place to respond to this damage, whichrecognize and repair DNA damage. DNA base lesion is a kind of DNA damage. Baseexcision repair (BER) is the primary defense against oxidative and alkylating DNA damage.The process of BER needs many kinds of DNA enzyme. The glycosylase is one of thoseenzymes which recognize and cleave damage base. Aberrant activity of DNA glycosylasemay be related to many diseases and a potential biomarker for disease cline. Herein, wedeveloped a label-free colorimetric method for sensitive detection of hOGG1, an importantDNA glycosylase. A dsDNA was used as substrate, one of its signal strand contains thelesion base (8-oxo-dG) which could be recognized by hOGG1. The other signal strandcontains hemin aptamer. When the lesion base was removed by hOGG1, a new recesseddsDNA with5`-PO4end was produced. The5`-PO4DNA strand would be digested byLambda exonuclease, which resulted in the release of the hemin aptamer then combined withhemin. The hemin/aptamer complex could catalyze ABTS2--H2O2and give color readout.The absorbance at418is proportional to the logarithmic value of hOGG1concentrationranging from0.05to16U/mL with a detection limitation of0.01U/mL.
本研究论文以重要的生物酶为研究核心，结合电化学与光学生物传感技术，发展了一些具有高灵敏度与选择性的生物传感技术用于快速检测地衣芽孢杆菌蛋白酶（第二、三章），组蛋白去乙酰化酶（第四、五章），DNA甲基化转移酶（第六章），DNA糖基化酶（第七章）。具体内容包括：
（1）基于高选择性的多肽构建用于检测细菌蛋白酶标志物检测的电化学生物传感器【第二章】
蛋白酶是一种普遍存在于生物体内的酶，可通过其对底物的消化来进行检测。由于蛋白酶在细菌中也广泛的存在，因此，细菌分泌的蛋白酶是一种理想的生物标志物，为微生物临床检测提供了快速、灵敏的目标物。基于文献报道，利用择性识别地衣芽孢杆菌分泌的蛋白酶的含D-型氨基酸多肽，结合电化学生物传感器的一些成熟手段，发展一种用于检测细菌蛋白酶的简单的电化学生物传感器。将选择性的多肽利用C端半胱氨酸残基固定到金电极上，链酶亲和素标记的碱性磷酸酶与标记在多肽N端的生物素结合，碱性磷酸酶催化无电活性的1-萘酚磷酸酯转化为有电活性的1-萘酚产生一个放大的电化学信号。当有标志物蛋白酶存在时，多肽将被选择性切割，标记有生物素的部分被移除，信号减小，这种信号的变化与蛋白酶的含量有关，从而实现对目标细菌蛋白酶定量检测的目的。当目标蛋白酶的浓度在0.7–10μg/mL范围内，氧化峰电流的大小与目标蛋白酶的浓度对数成反比，检测下限达0.16μg/mL。
（2）基于纳米金凝集变色对地衣芽孢杆菌蛋白酶的灵敏比色检测【第三章】
纳米金具有独特的光学性质及生物相容性，将其用于生物传感器的构建中，可较大提高传感器的性能。因此，我们基于纳米金凝集变色原理构建了一种用于便捷检测地衣芽孢杆菌蛋白酶的比色法。本文使用两端含有半胱氨酸残基的多肽为底物，此多肽中间包含能被目标蛋白酶选择性识别的D-型氨基酸区域。由于巯基与金的强作用力，多肽将纳米金拉近，使得纳米金靠近，发生凝集变色。当有目标蛋白酶存在时，目标蛋白酶将底物切断，切断的底物只有一端有半胱氨酸残基，不能将纳米金拉近，从而不能发生团聚变色，利用颜色的差异，即可裸眼进行观察比色，也可以采用紫外吸收测定对蛋白酶进行检测。该方法在0.1–5μg/mL浓度范围内与A520/A600的吸光度比值呈线性关系，检测下限可达0.09μg/mL。
（3）基于乙酰化的组蛋白H4多肽构建用于灵敏检测组蛋白去乙酰化酶的电化学生物传感器【第四章】
组蛋白的翻译后修饰在转录等过程有着非常重要的意义。组蛋白的翻译后修饰主要有甲基化、磷酸化、乙酰化、泛素化等。其中，组蛋白的乙酰化是一种十分重要的组蛋白翻译后修饰。生物体内控制乙酰化平衡是乙酰化酶和去乙酰化酶。这两种酶的活性对生物的很多过程有着重要的影响，例如：细胞的分化、凋亡等。研究表明，异常的去乙酰化酶活性与人类疾病有重大的关系。因此，我们以组蛋白去乙酰化酶SIRT2作为检测目标，将乙酰化的多肽通过N端的半胱氨酸残基固定到金电极表面，利用乙酰化位点的特异抗体去识别乙酰化多肽，标记有碱性磷酸酶的二抗去识别一抗并提供电化学信号。当样品中存在去乙酰化酶SIRT2时，底物上的乙酰基被移除，特异性抗体结合量减小，电化学信号随之减小，从而实现对去乙酰化酶的检测。在对实验条件优化后，该方法在的1–500nM浓度范围与电流信号值线性相关，检测下限可达0.1nM。
（4）基于纳米金淬灭荧光构建的荧光传感器用于组蛋白去乙酰化酶的灵敏检测【第五章】
结合纳米金对荧光染料的强淬灭性，利用标记有荧光素的乙酰化多肽对去乙酰化酶进行检测。将特异识别乙酰化的抗体标记到纳米金上，由于抗体特异识别乙酰化多肽，将标记有荧光分子的乙酰化多肽与纳米金拉近，使得荧光淬灭。当样品中存在去乙酰化酶时，多肽被去乙酰化后，不能被抗体识别，荧光信号增强。该方法在50–500nM浓度范围内SIRT2与荧光信号线性相关，检测下限为11.9nM。
（5）基于DNA甲基化敏感性内切酶和末端转移酶碱基延伸构建的电化学传感器用于灵敏检测DNA甲基化转移酶活性【第六章】
DNA甲基化是一种很重要的DNA修饰，与很多生命过程相关，例如：转录、基因印记、细胞分化、染色体结构和胚胎行程等。研究发现，很多疾病都与非正常的基因甲基化水平有关。DNA甲基化的过程是通过DNA甲基化转移酶催化甲硫亮氨酸上的甲基共价连接到DNA序列中的胞嘧啶或者腺嘌呤上而完成的。非正常的DNA甲基化转移酶活性会导致非正常的基因甲基化水平。因此，我们基于甲基化敏感的限制性内切酶的切割，利用末端转移酶进行末端碱基延伸，构建了一种用于灵敏检测甲基化转移酶的电化学传感器。将含有甲基化转移酶特异识别序列的DNA探针组装到金电极表面，甲基化的探针可以被甲基化敏感的限制性内切酶识别、切割，从而产生一个带有3`-OH端的新引物，末端转移酶可以将生物素化的dUTP延伸到新产生引物的3`-OH末端。之后，偶联有碱性磷酸酶的链霉亲和素（SA-ALP）与新延伸的生物素结合。碱性磷酸酶可以催化非电活性物质1-萘酚磷酸酯水解，生成电活性物质1-萘酚在电极表面氧化产生电信号。从而实现对甲基化转移酶的灵敏检测。该方法在0.1-20U/mL浓度范围与电流值呈线性关系，检测下限为0.04U/mL。（6）基于Lambda外切酶构建用于灵敏检测DNA糖基化酶的DNAzyme催化显色法【第七章】
基因的损伤是不可避免的，如果这种损伤得不到不修复，会导致基因突变、细胞凋亡，甚至会诱导癌症发生。生物体有自身的修复系统，可以对DNA的损伤进行识别和修复。碱基切补修复是对碱基损伤的一种修复手段，这过程是由很多酶共同作用实现的。在这碱基切补修复过程中，DNA糖基化酶对识别、除去受损碱基，起着至关重要的作用。非正常的DNA糖基化酶活性可能会导致疾病的发生，这就为疾病的检测提供了一种生物标志物。因此，我们利用具有辣根过氧化物酶活性的DNAzyme催化底物显色，并结合DNA外切酶的高活性，发展一种比色法对DNA糖基化酶进行方便、灵敏的检测。将含有受损碱基（8-oxo-dG）的DNA与含有血红素核酸适体的DNA互补杂交。糖基化酶hOGG1能识别受损碱基8-oxo-dG并将其从DNA序列中移除，产生一个5`磷酸端的缺口，然后利用Lambda核酸外切酶选择性消化5`磷酸端DNA的特性，将血红素核酸适体的抑制链消化分解，使得血红素核酸适体释放，与血红素结合，形成血红素-核酸适体的DNAzyme复合物结构，催化无色底物ABTS2-转化为有色的ABTS-，通过简单的比色就能达到对DNA糖基化酶的检测。该方法在418nm紫外吸收值与hOGG10.05-16U/mL浓度对数值在呈线性关系，检测下限为0.01U/mL。
With the development of life sciences, as a simple and sensitive biological analysistechnology, biosensing technology has made remarkable progress. It has become animportant research method for analytical chemistry of life sciences. It has extensiveapplication prospects in many fields, such as biology technique, clinical diagnosis,environmental protection, food industry, medicine and military.
Enzymes are biological molecules that catalyze chemical reactions. Enzymes serve awide variety of functions inside living organisms. Almost all chemical reactions in abiological cell need enzymes in order to occur at rates sufficient for life. They areindispensable for metabolism, signal transduction and cell regulation. In addition, reactionsclosely connected with life process are almost all enzyme-catalyzed reactions. Aberrantactivity of important enzyme would lead to critical disease. Many human diseases are relatedto aberrant enzyme activity. Herein, enzymes are perfect biomarkers for quick and sensitivedisease detection in clinical diagnosis.
This dissertation focuses on developing a series of biosensing techniques for detection ofprotease biomarker from Bacillus licheniformis (in chapter2,3), histone deacetylase (inchapter4,5), DNA methyltransferase (in chapter6), DNA glycosylase (in chapter7) usingthe electrochemical and optical biosensing techniques. The details are listed as follows:
(1) A novel electrochemical biosensor for highly selective detection of proteasebiomarker from Bacillus licheniformis with D-amino acid containing peptide (in chapter2)
Protease is a kind of the common enzymes in the living organisms and accessible fordetection based on substrates cleavage. There are abundant proteases in bacteria and itssecretion. Herein, bacterial protease is an ideally biomarker for quick and sensitiveidentification of microorganisms in clinical samples. Therefore, an electrochemicalbiosensor was constructed for the detection of the protease biomarker from the Bacilluslicheniformis, a model of Bacillus anthracis. The D-amino acid containing peptide labelledwith biotin was used as the specific substrate. Firstly, the specific peptide was assembled onthe gold electrode through the C-terminal Cys interacting with gold. Upon the addition of theprotease, the substrate was selectively recognized and cleaved. Subsequently, thestreptavidin-alkaline phosphatase (SA-ALP) could specifically bind the remaining biotinmoieties on the electrode surface. ALP could catalyze the conversion of an electrochemicallyinactive1-naphthyl phosphate into an electrochemically active naphenol, generating anamplified signal for electrochemical readouts. Differential pulse voltammetry (DPV) was employed to detect the signal which could be used for quantifying the proteases of Bacilluslicheniformis. Electrochemical response arising from the oxidation of enzymatic product of1-naphthyl phosphate was observed to be inversely proportional to the logarithmic value ofprotease concentrtion in the range from0.7to10μg/mL with a detection limit as low as0.16μg/mL.
(2) Gold nanoparticle aggregation-based colorimetric assay of protease biomarker fromBacillus licheniformis
Gold nanoparticle is attractive due to its novel optical and biocompatibility properties.With the introduction of gold nanoparticle into biosensor fabrication, biosensors will begreatly improved. Therefore, we developed a simple colorimetric method based on goldnanoparticle aggregation for the detection of protease biomarker from Bacillus licheniformis.A C-and N-terminal Cys peptide substrate was exploited in the assay, which contained twoD-amino acids for selective recognition and cleavage by the target protease. Cys couldstrongly interact with gold nanoparticle, leading to the aggregation of gold nanoparticles andthe color change of its solution. Whereas, cleaved peptides are unable to induce goldnanoparticle aggregation; and as a result the solution color would not change. We couldobserve the change of color by naked eye, or monitor the changes of the absorption spectrumby a UV-Vis spectrophotometer. There is a linear correlation between the ratio of theabsorbance of the system at520to600nm (A520nm/A600nm) and the logarithm of adenosineconcentration range from0.1μg/mL to5μg/mL, with a detection limit of0.09μg/mL.
(3) A sensitive electrochemical biosensor for the detection of histone deacetylase with ahistone H4Lys16acetylated peptide
Histone post-translationally modifications play important roles in transcription. Oneprevalent modification is acetylation, which is related to the gene activity. The steady-statebalance of the acetylation and deacetylation of histone is achieved through two species ofenzyme: histone acetyltransferase and histone deacetylase. The activity of histoneacetyltransferase and histone deacetylase affects the angiogenesis, cell-cycle arrest,apoptosis and terminal differentiation of different cell types. Herein, we developed a simple,sensitive electrochemical biosensor for detecting the activity of sirtuin2one of the histonedeacetylase and screening its inhibitor. An acetylated peptide with a cystein residual atN-terminal as the substrate is self-assembled on the electrode surface by the interactionbetween cystein residual and gold. The rabbit anti-acetylated peptide antibody is employedto specifically bind with the acetylated peptide. The alkaline phosphatase labelled goatanti-rabbit antibody is captured through selective interaction with the rabbit anti-acetylatedpeptide antibody. The alkaline phosphatase catalyzes the conversion of the electrochemically inactive1-naphthyl phosphate into an electrochemically active naphenol for generating theamplified electrochemical signal. In the presence of sirtuin2, the peptide substrate isdeacetylated, resulting in the decrease of electrochemical signal. Therefore, the change of thecurrent signal could reflect the concentration of sirtuin2. Under the optimum conditions, thedetection limit is achieved down to0.1nM with a linear range from1nM to500nM.
(4) A simple gold nanoparticle-based fluorescence biosensor for sensitive assay ofhistone deacetylase activity
Gold nanoparticle, as a class of nanomaterials with many specific properties, such asconductivity, colorimetric and nonlinear optical properties, has been extensivy applied inbiomolecular research. Herein, we developed a fluorescence assay using gold nanopartilce asthe quencher. An acetylated peptide labeled with a fluorophore at C-terminal was used as thesubstrate of histone deacetylase sirtuin2. Gold nanoparticle labeled with anti-acetylatedpeptide antibody acted as the quenching probe. The fluorescence would be efficientlyquenched by the gold nanoparticle, when the acetylated peptide captured by its antibody. Inthe presence of sirtuin2, the acetyl was removed from the substrate, resulting in the increaseof fluorescence signal. The fluorescence intensity was proportional to logarithmic value ofthe sirtuin2concentration ranging from50nM to500nM, with a detection limit of11.9nM.
(5) A sensitive electrochemical biosensor for the detection of DNA methyltransferaseactivity by combining DNA methylation-sensitive cleavage and terminaltransferase-mediated extension
DNA methylation plays an important role in many biological processes includingtranscription, genomic imprinting, cellular differentiation, chromatin structure andembryogenesis. A number of human diseases have been found to be associated with aberrantgene methylation. The DNA methylation process was regulated by DNA methyltransferasecatalyzing covalent addition of a methyl group to cytosine or adenine in DNA sequences.The aberrant DNA methyltransferase activity was reported to be related to pathogenesis ofcancer, such relationship provides a potential target in disease diagnosis and therapy. Herein,we developed a sensitive electrochemical biosensor based on methylation sensitive cleavageusing terminal transferase-mediated extension for the detection of the methyltransferaseactivity. A DNA probe contained the sequence which could be recognized by DNAmethyltransferase, was self-assembled on the surface of gold electrode by3’-SH. After themethylation reaction by DNA methyltransferase, the methylation sequence was specificallyrecognized and cleaved by methylation sensitive restrictive endonuclease, that produced anew primer with a3`-OH on the electrode surface. Subsequently, the dUTP-biotin wasincoporated into the3`-OH terminal of the new primer by TdTase-mediated extension. Then, the SA-ALP was added and bound with the labeled biotin on the primer. The amplifiedelectrochemical signal was obtained by ALP catalyzing1-NP convertion. Differential pulsevoltammetry (DPV) was employed to detect the signal which can be used for quantifying theDam MTase activity and the concentration of its inhibitor. The DPV current is proportionalto the logarithmic value of DNA methyltransferase concentration ranging from0.1to20U/mL with a detection limitation of0.04U/mL.
(6) DNAzyme-based label-free colorimetric method for sensitive detection of DNAglycosylase
DNA damage occurs by various mechanisms, either as a by-product of normal cellularmetabolism or as a result of exposure to environmental and biological mutagens. DNAdamage that is not repaired leads to apoptosis or mutation, which in turn may lead toinduction of carcinogenesis. Multiple systems are in place to respond to this damage, whichrecognize and repair DNA damage. DNA base lesion is a kind of DNA damage. Baseexcision repair (BER) is the primary defense against oxidative and alkylating DNA damage.The process of BER needs many kinds of DNA enzyme. The glycosylase is one of thoseenzymes which recognize and cleave damage base. Aberrant activity of DNA glycosylasemay be related to many diseases and a potential biomarker for disease cline. Herein, wedeveloped a label-free colorimetric method for sensitive detection of hOGG1, an importantDNA glycosylase. A dsDNA was used as substrate, one of its signal strand contains thelesion base (8-oxo-dG) which could be recognized by hOGG1. The other signal strandcontains hemin aptamer. When the lesion base was removed by hOGG1, a new recesseddsDNA with5`-PO4end was produced. The5`-PO4DNA strand would be digested byLambda exonuclease, which resulted in the release of the hemin aptamer then combined withhemin. The hemin/aptamer complex could catalyze ABTS2--H2O2and give color readout.The absorbance at418is proportional to the logarithmic value of hOGG1concentrationranging from0.05to16U/mL with a detection limitation of0.01U/mL.
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