Novel Biosensor For Activity Assay Of Peptide Enzymes And DNA Modified Enzymes

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 licheniformisGold 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 peptideHistone 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 activityGold 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 extensionDNA 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 DNAglycosylaseDNA 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.