Novel And High Sensitive Electrochemical Biosensors For Researching Enzyme Activity

Enzyme and biological small molecules are indispensable important substances which can maintain the metabolic in plants and animals. Almost all cells activity processes require the participation of enzyme. It is a catalyst for metabolism of organisms. The normal activities of the organism can maintain only in the presence of enzyme. Organisms will get diseases and even dead if biological enzymes in the body lack or its synthesis suffer obstacles. Therefore, using biological sensors to detect the enzyme activity is very significant. Electrochemical biosensor is one of the sensors which have the advantage of high sensitivity electrochemical method and the advantage of biological specificity. At the same time, designing electrochemical biosensor is simple and easy to implement. The cost of test is relatively low. So in this paper, electrochemical biosensors to detect small molecules and biological activity of enzymes were proposed, which includes three chapters:1. A novel electrochemical assay for non-enzyme detection of glucose was proposed based on higher biospecific binding affinity of concanavalin A (Con A) for D-glucose over thiolated P-Cyclodextrins (β-SH-CDs), coupled with gold nanoparticles (AuNPs) signal amplification. In this method, β-SH-CDs could bind to both Con A which was covalently immobilized on the pretreated glassy carbon electrode and AuNPs onto which thionine (TH) could be attached. However, upon the addition of Con A, the electrode response of the TH moiety decreased which was attributed to the glucose competing with β-SH-CDs at the binding sites of Con A. Consequently, a highly stable sensing platform was developed. A linear response to the D-glucose concentration ranging from 5.0 × 10-7 to 1.55 × 10-5 M and a low detection limit of 2.2 × 10-7 M (3 σ) are obtained.2. A novel electrochemical strategy for simple and accurate monitoring of the activity and the inhibition of T4 polynucleotide kinase (PNK) is developed by coupling the λ exonuclease (λ exo) cleavage reaction with a dual-signaling amplification method. In the presence of PNK and adenosine triphosphate, the 5’-hydroxyl group of the substrate DNA is phosphorylated, initiating the hydrolysis of the 5’-phosphoryl termini catalyzed by λ exo, and by combining the resulting G-quadruplex-hemin signal-on probe and the ferrocene signal-off probe, a dual-signaling amplification strategy is realized. This approach exhibits a low detection limit of 0.02 U/mL (3σ) for PNK activity analysis. Additionally, the inhibition effects of two known PNK inhibitors, namely ammonium sulfate and sodium hydrogen phosphate, have also been successfully detected. This new dual-signaling electrochemical biosensor provides a facile platform for the detection of the activity and inhibition of nucleotide kinases.3. A novel and highly sensitive homogeneous electrochemical assay was developed for the detection of Dam MTase based on methylation-responsive exonuclease Ⅲ-assisted signal amplification. Upon the action of MTase/endonuclease on hairpin probe 1 (HP 1) containing the methylation-responsive sequence, single-stranded DNA segments are generated to hybridize with methylene blue (MB)-labeled hairpin probe 2 (HP 2). Then the digestion of HP 2 from the blunt 3’ terminus by exonuclease Ⅲ is activated, resulting in the release of MB-labeled mononucleotides and the complementary DNA segment which could hybridize with another HP 2 to initiate the signal amplification process. The MB-labeled mononucleotide, due to its less negative charge and smaller size, diffuses easily to the negatively charged indium tin oxide (ITO) electrode, generating an amplified electrochemical signal. The detection limit of the proposed assay was estimated to be 0.04 U/mL (3σ). To the best of our knowledge, it is the first time to adopt exonuclease Ⅲ-assisted signal amplification for homogeneous electrochemical assay of MTase activity, and this strategy exhibits the advantages of high sensitivity as well as simplicity. Since this assay is carried out in a homogeneous solution phase instead of on an electrode/solution interface, sophisticated probe immobilization processes could be avoided.