Study On DNA Biosensor Based On The Enzyme Activity And Small Molecule

Biosensors which are one of the most important subjects in analytical chemistryhave been an unprecedented development and wide application. Especially, nucleicacid biosensors have been the hot topic of large number of scholars in the recent pastyears. DNA biosensors, analyzing the DNA sequences biosensors, would convert thereaction products into recognizable signals such as electricity/light/sound, which arerelied on the specificity of complementary coupling about the DNA molecules andthe DNA as a bio-recognition element. DNA biosensors have been used in a widerange of applications from medical diagnostics, food quality assurance,environmental monitoring because of their high-speed, high sensitivity, prefectstability, and low cost.Enzyme, dominating the process of the livings’ metabolism, nutrition andenergy transition, is an biocatalyst. The livings’ life activities such as growth,propagate, inheritance, movement, nerve conduction, relating to enzymaticprocedure, most of which are existing in tissue cells, erythrocytes, plasma and arerelevant for cell damage, oxygen-poor, poisoning, aging and multiple diseases.Uracil-DNA glycosylase (UDG), one of the major enzymes to maintain the geneticintensity and prevent genetic mutation, which exists ubiquitously among prokaryotesand eukaryotes, is a highly conserved damage repair protein. The uracil residues inDNA that are caused by various factors in cells can be readily removed by the uracilexcision repair which is initiated by UDG. UDG plays a key role in the process ofrepairing abnormal base. In the recent decades, the researches of uracil removinghave had increasingly attentions. The researches are not only exploring themechanism of DNA-UDG interaction but also directly measuring the activity ofUDG, which is momentous significance in biology. Additionally, small moleculescould conjoin with DNA greatly through non-covalent bond, hydrogen bond, ionicbond, van der Waals force, hydrophobic bond. According on this theory, installing anovel method could make a solution on food safety being emphasized, which will bea breakthrough on solving practical problems by theory. In this paper, there arekinds of convenient, high-speed, sensitivity DNA biosensors to detect the activity ofenzymes and small molecules. The contents are carried out as follows: (1) A low-cost, high-speed, sensitivity, simple DNA biosensor to detect theactivity of UDG has been proposed. The biosensor, designing the hairpin DNA, isbased on the fluorescence of SYBR Green I will strengthen hugely after combiningwith double-stranded DNA and the enzymatic mechanism of UDG. When there areUDGs, the uracil will be removed, hairpin DNA being damaged, then the fluorescentof SYBR Green I will be decreased. With the reaction mixture consisted of200nMDNA,5×SG,15U/mL Endo IV, containing1×NEBuffer3(5mM Tris-HCl,10mMNaCl,1mM MgCl2,0.1mM Dithiothreitol, pH7.9), the fluorescence of SYBR GreenI decreases as the concentration of UDG increasing. Then, the working curve ofUDG is established, with0.05U/mL of the detection limit. Above all, while theconcentration of UDG is between0.05U/mL and3U/mL, the fluorescence of SYBRGreen I changes apparently. The linear regression equation were Y=－1562.9X+8190.2(r~2=0.992). This DNA biosensor opens up a new method about detection theactivity of UDG.(2) There is a DNA biosensor based on colorimetric analysis to control theactivity of UDG. The gold nanoparticles have lots of merits such as simplepreparation, stability, homogeneous size, high-conductivity, well bio-compatibilityand unique optical effect. The biosensor which is based on the merits of goldnanoparticles and the aggregative gold nanoparticles having varies wavelength andabsorbance could measure the activity of UDG and evaluate the inhibitor of UDG.To optimize the biosensor’s working conditions, with the50nM DNA,25U/mLEndo IV,5μL DNA1-AuNPs, DNA2-AuNPs in the condition of25mM Tris-HCl,50mM NaCl,5mM MgCl2,0.5mM EDTA, pH7.9, the absorbance will be decreasingand the wavelength will have a red shift as the concentration of UDG increase. Thelinear relationship of the absorbance and the concentration of UDG is good, thelinear regression equation were Y=－0.024X+0.642(r~2=0.997), with1U/mL ofthe detection limit. The novel, high-sensitivity biosensor expands another newdetection of the activity of UDG.(3) A DNA biosensor for detection melamine is presented. The fluorescence ofthe SYBR Green I with single or double strand DNA is different, and the mechanismof the graphene oxide with single or double strand DNA is diverse, and the melaminecan induce the poly Tn to the folding structure T-Melamine-T. The biosensor isbased on them to detect the melamine rapidly through detecting the fluorescence ofSYBR Green I. The SYBR Green I is hardly any fluorescent while there are onlypoly Tn, graphene oxide and SYBR Green I. However, after adding in melamine, the fluorescence of SYBR Green I strengthens greatly due to folding structureT-melamine-T. The fluorescent intensity strengthens quickly along with theconcentration of melamine raising continually, with nM of the detection limit. Thisbiosensor gets to the purpose of detecting melamine rapidly, sensitively, low-costly.