| T4 polynucleotide kinase?T4 PNK? is a member of 5'-kinase family, which can catalyze the transfer of the ?-phosphate residue of ATP to 5'-hydroxyl termini of single or double chain of DNA, RNA, oligonucleotides or mononucleotide with 3'-phosphate groups. Abnormal PNK behaviors is directly related to some vital human diseases including Werner syndrome, loom's syndrome and Rothmund-Thomson syndrome. Adenosine-5'-triphosphate?ATP? is a major fuel molecule for all living species and is generally acknowledged as“energy currency”. ATP Participates in the synthesis of protein, sugar, nucleic acid and fat. It has been reported that aberrant ATP levels are associated with particular diseases such as angiocardiopathy, Alzheimer's disease and Parkinson's disease. So it is of great value to develop high sensitive and high selective determination methods for T4 PNK and ATP in biochemical study and clinical diagnosis.Fluorescent biosensing technology is a determination method which relys on fluorescence signal changes,including fluoscence quenching, enhancement or migration of emission wavelength. With high sensitivity and rapid response time,fluorescent biosensing technology has been widely applied in the detection of small molecules, protein, enzyme activity and nucleic acids.Nowadays, graphene oxide?GO?, a robust carbon nanomaterial with unique electronic and structure properties has gained significant attention. As a super quencher for a wide range of fluorophores, GO could be used to absorb single stranded DNA effectively while the adsorption between GO and DNA fragments is very weak. Cytochrome c?Cyt c? is a kind of hemoproteins. The isoelectric point?p I? of Cyt c is about 10.0–10.5. Cyt c could interact with single stranded or double stranded DNA by electrostatic and hydrophobic interactions in slightly basic solutions. Furthermore,the heme moiety of Cyt c can be also applied as a efficient fluorescence quencher through a electron transfer process.According to the considerations above and previous reports, we developed a series of novel biosensors for the detection of T4 polynucleotide kinase and Adenosine-5'-triphosphate in this research paper.?1?In this chapter, we present a Exo ? aided fluorescence method for ATP based on the following three factors. First, the high recognition ability between ATP and its aptamer. Second, Exo ? performs differently toward different substrate. Third, GO could absorb single stranded DNA effectively while the adsorption between GO and DNA fragments is ignorable. This developed assay showed a dynamic range of 0-200 ?M with a detection limit of 0.1 ?M. Furthermore, our presented platform works well in relatively complex biological solutions such as human serum.?2?Based on the fluorescence quenching capacity of cytochrome c and the huge differences in the adsorption ability toward doubled stranded DNA and DNA fragments, in this chapter, we proposed a new and simple strategy for the detection of T4 PNK. In the absent of T4 PNK, the fluorescence of ds DNA-FAM will be quenched upon the addition of Cyt which ascribed to the fact that Cyt c would strongly adsorb double stranded DNA and effective electron transfer between dye and Cyt c would take place. While, in the present of T4 PNK, ds DNA-FAM was phosphorylated by PNK at the 5'-hydroxyl end and followed by ? Exo cleave, yielding FAM labeled DNA fragments. The fluorecence can not be quenched after the addition of Cyt c. Fluorescence signals were used to quantify the activity of T4 PNK. This novel Fluorescence biosensor showed fairly good selectivity, as well as extraordinary stability.?3? This chapter we developed a amplified fluorescence detection method for T4 polynucleotide kinase activity and inhibition which based on ?exonuclease reaction and exonuclease ? digestion. First, the substrate is Phosphorylated by T4 PNK and followed by ? Exo cleave, resulting in the release of trigger DNA. Then, trigger DNA will free P1-FAM from P2-Dabcyl through a toehold-mediated strand displacement reaction?SRD?. Finally an amplified fluorescence signal can be achieved through Exo ? aided trigger DNA recycling. The fluorescence biosensor platform showed a dynamic range of 0-1 U m L-1 with a detection limit of 0.01 U m L-1. |
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