Quantitative DNA And Myoglobin Detection Using Portable POCT Analyzer

Since POCT analyzers have many significant advantages, such as convenient,inexpensive, time-saving and easy to operate, this technology has a wide field ofapplication with good prospects. So far, personal glucose meter (PGM) and personaluric acid meter (PUAM) are the most common POCT analyzers. However, they canonly detect one target (glucose or uric acid), so it can not meet people’s demands. Inthis thesis, enzyme-DNA conjugates which can catalyze hydrolysis of substrate(xanthine or sucrose) to “signal molecule”(uric acid or glucose), were used as abridge between the POCT analyzer (PUAM or PGM) and other targets. A new strategyof DNA and Myoglobin detection method based on enzyme-DNA conjugates usingPUAM and PGM was developed. The main researches are as followed:1. Using personal uric acid meter and xanthine oxidase-DNA conjugate forportable and sensitive DNA detectionIn this system, xanthine oxidase-DNA conjugate was synthesized firstly. Takingp53DNA and HBV DNA for examples, a new DNA detection method based on PUAMwas developed. Capture DNA was self-assembled on the Au film with Au–S linkages.When target DNA was added, capture DNA and xanthine oxidase-DNA conjugateformed a “sandwich” structure through hybridization with target DNA. As a result, thexanthine oxidase was dragged to the Au film. Since xanthine oxidase could catalyzehydrolysis of xanthine to uric acid, the concentration of target DNA in the samplecould be quantified by monitoring uric acid using PUAM. A good specificity andsensitivity was obtained in this detection method, the detection limit was ca.10pmol/L. Moreover, this method can be easily used for DNA detection in human serum.Due to the use of micro-chamber array, many samples could be detected at the sametime.2. Using personal glucose meter and invertase-aptamer conjugate for portableand sensitive myoglobin detectionIn this system, we firstly optimized the aptamer of myoglobin according to thespecificity and affinity. A new myoglobin detection method based on invertase-DNAconjugate and personal glucose meter was then developed. Anti-myoglobin wasself-assembled on the microplate. When myoglobin was added, anti-myoglobin andinvertase-aptamer would formed a “sandwich” structure. Invertse-aptamer conjugatecould catalyze hydrolysis of sucrose to glucose, and the concentration of Myoglobin could be quantified by monitoring glucose using PGM. This enzyme-based assay washighly sensitive and specific, and the detection limit was50pmol/L. Furthermore, thisdetection method can be used for myoglobin detection in human serum.