Novel Methods Of Optical Analysis For Enzyme And Protein Biological Markers

Biosensing technology has become an important research method for analyticalchemistry and life sciences and possesses extensive application prospects in manyfields. Optical biosensor is endowed with the strongpoints of low detection limit, fastanalysis speed, real-time and non-destructive analysis. As the material basis of lifeactivity, the content of enzyme and protein has become an important index of theclinical diagnosis of some diseases. Therefore, establishing analysis method s ofsimple and rapid detection of enzyme, protein biological markers have importantbiological significance. This thesis takes two important types of the enzyme, thymineDNA glycosylase and histone methylation enzyme, and C-reactive protein as studyobjects, developing a novel biological sensing technology based on optical method.The concrete content includes:(1) Thymine DNA glycosylase (TDG) plays a key role in sustaining the genomeintegrity and epigenetic regulation. This thesis reports a sensitive homogeneousfluorescence assay of TDG activity with exonuclease-mediated amplification.Thymine base excision from G/T mispair by TDG initiates two consecutive enzymedigestion reactions which are catalyzed by Endonuclease IV and T7exonucleaserespectively. The results reveal that the enzyme activity assay shows dynamicresponse to the concentration of TDG ranging from0to0.083U/μL with a detectionlimit of0.00018U/μL. The developed strategy offers desirable sensitivity, highselectivity, excellent reproducibility, low cost and simplified experiment sets,indicating that this strategy might hold considerable potential for TDG activityanalysis and associated biochemical function studies.(2) The development of a novel assay platform based on surface enhanced Ramanscattering (SERS) using formaldehyde-selective reactive probe (Purpald) formonitoring the activity of histone demethylases (HDMs) by direct observation ofby-product formaldehyde is reported. Our biosensing strategy relies on the additionreaction between Purpald and formaldehyde followed by covalently binding to thesurfaces of AuNPs for SERS transduction. This nanosensor has the advantages in itshigh signal-to-noise ratio, superb specificity, and rapid, convenient, and reproducibledetection with homogeneous single-phase reaction. This work is the first example ofdetecting of HDMs by SERS technique using formaldehyde-selective reactive probe. Thus, the developed strategy can provide a simple and high-performance platform forthe characterization of HDMs and related researches into their biological functions.(3) Using CRP as an analysis model, We have developed a new colorimetricanalysis based on gold nanoparticle, achieving simple, rapid and sensitive detectionof protein. After DPPC crosslinking CTAB which has been stabilized on goldnanoparticles, Au surface adsorbs a large number of phosphatidyl choline. while inthe presence of Ca2+, coordination reaction between phosphatidyl choline and CRPoccurs. Then, the color of gold nanoparticles changes owing to shortened spacedistance, UV-Vis absorption spectrum changed simultaneously. This can be used forquantitative detection of CRP with the linear range from0to5pM and the detectionlimit of8.2fM. Holding the merits of homogeneous analysis and high sensitivity, thisstrategy is easier to achieve high flux. Also, it provides a desirable platform for theanalysis of other biological molecules, for example, protein, nucleic acid andpolypeptide.