Research On Microfluidic Bioassay Based On G-quadruplex DNAzyme-enhanced Chemiluminescence

Functional nucleic acids, which bind ligands with high specificity and exhibit multiple catalytic activities, have attracted extensively attention in recent years. Aptamer, ribozyme/deoxyribozyme and aptazyme are functional nucleic acids. Some metal ions can induce special G-rich oligonucleotide to fold into G-quadruplex and bind hemin to form deoxyribozyme (DNAzyme), which shows HRP-like catalytic activity.Chemiluminescence (CL) combined with microfluidic sequential injection analysis (μSIA) has advantages of high sensitivity, wide calibration range, fast dynamic response, low background interference and high throughput. Moreover, since miniaturization has been a long-term trend in clinical diagnostics instrumentation, miniaturizable CL-μSIA system with low sample and reagent consumption may have a wide range of application prospect in biochemical assay.The thesis is mainly composed of two parts:Recent studies on some functional nucleic acids are reviewed in the first chapter. Especially, the composition and activity of the HRP-like DNAzyme, potassium analysis based on DNAzyme-enhanced assay, and signal-amplification detection of DNAzyme-based aptasensor are stressed. Furthermore, both μSIA and CL as well as their coupling technology have also been discussed.The research described in the second chapter is focused on the microfluidic bioassay method based on G-quadruplex DNAzyme-enhanced chemiluminescence.First, DNAzyme synthesis through the formation of K+-guanine quartet complex and binding with hemin is studied, and the peroxidase-like activity of the DNAzyme is identified. Then, a microfluidic method based on G-Quadruplex DNAzyme-enhanced chemiluminescence for determination of K+in human serum is developed. Finally, DNAzyme amplification by target triggered hybridization chain reaction (HCR) is investigated for further sensitivity improvement and for presumptive genetic diagnosis of specific Alzheimer’s disease.In the experiment, the interaction between G-rich oligonucleotide and hemin in the absence or presence of K+was characterized by UV-Vis absorption spectra. AGRO100was selected as the ideal oligonucleotide due to the rather high HRP-like activity induced by K+and with hemin. The DNAzyme incubation and chemiluminescence reaction conditions were optimized. And the conformation stability and HRP-like activity of the DNAzyme were found long-time preserved below water-freezing point. The accuracy of the developed K+-bioassay approach was demonstrated by compared the analysis results with common potentiometry. Moreover, by taking the coding sequence rs242557as the target, which associates with Alzheimer’s disease, the triggered HRC was applied to amplify the DNAzyme amount for further sensitivity improvement of the developed method. The method, which achieved the detection limit at nmol L-1level, can be used for SNP typing of the corresponding genetic diagnosis.