Study Of New Electrogenerated Chemiluminescence Bioenzyme Sensor Based On Carbon Nanomaterial-luminol

In this thesis, the basic principles, condition, main reaction types of electrogenerated chemiluminescence(ECL) were reviewed, moreover, the application was reported about the ECL emitter connected to nanomaterial for biosensor analysis. Graphene quantum dots(GQDs), the recently emerging carbon-based materials, have been conducted as cathodic ECL reagent for biosensing. Electrochemiluminescence resonance energy transfer(ECL-RET) has been used for DNA detection, cytosensing, and immunoassay. In this work, this thesis mainly focuses on constructing novel electrochemiluminescence biosensors for the DNA adenine methylation methyltransferase assay and protein kinase activity detection and the related inhibitors research by utilizing nanomaterials-based signal enhancement. The main works was composed of the following two aspects:1. Herein, we firstly synthetized GO/AgNPs/luminol composites as ECL reagent and developed a novel ECL biosensor for sensitive DNA adenine methylation(Dam) methyltransferase(MTase) assay. The alkynyl functionalized GO/AgNPs/luminol composites were assembled on the azide-terminated dsDNA modified electrode surface via click chemistry for ECL signal production. Once the Dam and Dpn I(a site-specific endonuclease cleaving the duplex symmetrical sequence of 5′-GAmTC-3′) were added to the above electrode, GO/AgNPs/luminol composites released from the electrode surface to the solution, leading to significant decrease of the ECL signal. Therefore, we can utilize this strategy for detecting with Dam activity in the range of 0.1 to 20 U/mL, and the detection limit was calculated to be 0.03 U/mL. This method can be successfully used in the assay of Dam MTase inhibitors and the inhibition efficiency of 5-fluorouracil was the most serious one, moreover, the IC50 of 5-fluorouracil was calculated to be 18.2 μM. Based on these, this method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.2. Herein, for the first time, we present a novel dual-potential electrochemiluminescence(ECL) ratiometric sensing approach for monitoring the activity of protein kinases by using graphene quantum dots(GQDs) and luminol. The Au NPs were captured thiophosphorylated peptide/GQDs modified electrode surface for regulating the two ECL reagents. ECL from GQDs coated on glassy carbon electrode at-1.25 V could be quenched by closely contacted Au NPs, while ECL from luminol in solution at +0.55 V could be enhanced by the same Au NPs, in the presence of the common coreactant of H2O2. At a higher concentration of kinase, the more Au NPs could give higher electrocatalytic efficiency to the luminol ECL reaction and better ECL resonance energy transfer with the GQDs. The ratio of luminol ECL enhancement degree to GQDs ECL quenching degree was positively correlated with the PKA activity. Therefore, the activity of protein kinases was detected with wide linear range(0.01-10 U/mL) and low detection limit(0.005 U/mL). This method can be successfully used in the assay of PKA inhibitors and the inhibition efficiency of ellagic acid was the most serious one, moreover, the IC50 of ellagic acid was calculated to be 0.15 μM. This biosensor can also be used for quantitative analysis of PKA activity in serum samples and MCF-7 cell lysates, indicating that the developed method has the potential application in biochemical fundamental research and clinical diagnosis.