| As a kind of redox enzymes containing a heme cofactor in their actives sites, aperoxidase can effectively promote the oxidation of a wide range of substrates in animals,plants and micro-organisms. A peroxidase acts as catalysts to allow variety of biologicalprocesses to take place, and it is widely used in multiple biochemical detection project andvarious immune kit. Howewers, as a kind of natural enzyme, a peroxidase has the seriousdisadvantage of instability to high temperature, high pressure, extreme pH, heavy metals andorganic solvents, etc. At the same time, the content of peroxidase is low in living body, andtheir preparation, purification and storage are usually time-consuming and expensive. Thesefactors limit their practical application. Therefore, it has become a hot research topic in themuti-disciplinary and cross-cutting area that exploit artificial mimic enzymes with highstability and low cost to take the place of natural enzymes. Over the past few decades, theresearch of artificial mimic enzymes has made significant achievements, and many peroxidemimic enzymes have been found, such as porohyrins, hemin/G-quadruplex DNAzymes andmolecularly imprinted polymers etc. In addition, some nanomaterials which have aperoxidase-like activity have aroused great interest among scientists, such as some carbonnanomaterials, metal nanomaterials, metal oxide nanomaterials, and nanomaterials displayedintrinsic peroxidase-like activity have been used in biology, medicine and environment.Today, the further application of artificial mimic enzymes in analytical chemistry have alsosuffer from low catalytic activity and selectivity. It has become a very realistic and full ofchallenge task to exploit the application of artificial mimic enzyme in the fields of biologicalassay and environmental monitor。This paper is focused on the development of luminescence sensor by using artificialmimic enzyme for signal amplifer, mainly in the following areas.1）we developed aconvenient signal amplification strategy for sensitive and selective chemiluminescencedetection employed hemin/G-quadruplex DNAzymes as signal amplifier. Our resultsindicated that DNAzymes had far higher catalytic activity to luminol-H2O2chemiluminescence system than natural enzyme HRP in strong alkaline medium. Therefore,these unique properties of DNAzymes would allow them to become a promising alternativeto HRP for the chemiluminescence immnoassay of various proteins in strong alkalinemedium.2）As some nanomaterials which possess highly-efficient peroxidase-like catalytic activity and excellent photoluminescence, we tried to integrate catalyst and fluorescent probeinto the same site in sensing system. In this way, not only the constitute elements of thesensor can be simplified, but also the distend between catalysis and fluorescent probes canbe shorten and the sensitivity of seasoning system can be enhanced, which successfullyimplement high sensitivity, high selectivity of fluorescence analysis of target molecules.The thesis consists of six chapters. In chapter1, the development of artifialperoxidase-like activity is summarized, such as porphyrins, DNAzymes, and nanozymes.In chapter2, we investigated the catalytic activity of hemin/G-quadruplex DNAzymesto catalyze the chemiluminescent reaction of luminol with H2O2. Our results indicated thatDNAzymes had far higher catalytic activity to luminol-H2O2chemiluminescent system thannatural enzyme HRP in strong alkaline medium. A sensitive chemiluminescentimmunosensor to detect leptin was fabricated by using hemin/G-quadruplex DNAzymes toamplify detection signal. Under the optimum conditions, the proposed immunosensorshowed high sensitivity and selectivity with a low detection limit of1.9pg mL1and a widelinear response range of human leptin from10to1000pg mL1.In chapter3, a highly sensitive and selective sandwich chemiluminescenceimmunosensor was fabricated based on the enlargement of catalytic hemin/G-quadruplexDNAzymes and the enrichment of functional superparamagnetic nanocomposites. Toconstruct such a sensing interface, core-shell structural Fe3O4/polydopamine(PD)/Ausuperparamagnetic nanocomposites were synthesized by an in-situ method with as asubstrate adhesion. In this immunosensor, the monoclonal anti-human leptin antibody(capture antibody) was bound to the Fe3O4@Au nanocomposites. Human leptin, biotinylateddetection antibodies and streptavidin-hemin/G-quadruplex DNAzymes were successivelycombined to form sandwich-type immune complexes through specific interactions. Themagnetic particles loading with immunocomplex were separated by an external magnet.Chemiluminescence was generated by the hemin/G-quadruplex DNAzymes-catalyzedreaction between luminol and hydrogen peroxide. The immunosensor exhibited highsensitivity and a wide linear range for human leptin detection from1.0to8.0×102pg mL1with a low detection limit of0.3pg mL1.In chapter4, the hemin-functionalized graphene quantum dots were synthesise throughto the noncovalent assembly between hemin and GQDs. The nano-composites had bothhighly-efficient peroxidase-like catalytic activity and excellent photoluminescence, and weresensitive to H2O2. Based on this effect, a novel fluorescent platform is proposed for thesensing of H2O2and glucose. Under the optimized conditions, the linear range of H2O2is from1to300μM, and the limit of detection is0.1μM. The linear range of glucose is from9to300μM, and the limit of detection is0.1μM. The proposed method provides a newpathway to further design the biosensors based on the assembly of GQDs with hemin fordetection of biomolecules.In chapter5, a novel fluorescence sensor for hydroquinone was developed by usinggraphene quantum dots which simultaneously functions as a peroxidase-mimicking catalyst,a photoluminescence indicator, and and better surface grafting. In the presence of dissolvedoxygen, graphene quantum dots with intrinsic peroxidase-mimicking catalytic activity cancatalyze the oxidation of hydroquinone to produce p-benzoquinone, an intermediate, whichcan efficiently quench graphene quantum dots’ photoluminescence. Based on this effect, afluorescent sensing platform is proposed for the sensing of hydroquinone. A wide linearresponse range of hydroquinone was from0.01to30μM, and the detection limit is as low as0.5nM.In chapter6, a simple, sensitive and enzyme-free detection method for catechins wasdeveloped based on versatile GQDs which simultaneously functions asperoxidase-mimicking catalyst, a fluorescence probe, and better surface grafting. As GQDshave peroxidase-mimicking catalytic activity, catechins could be oxidized to quinone in thepresence of dissolved oxygen. Consequently, the fluorescence of GQDs was efficientlyquenched by the quinone generated by the above-mentioned reaction. The method has highselectivity, and the reducing agents in green tea, such and vitamins, amino acids and sugars,have no interference. We challenged the proposed method by five tea samples. This detectedamount was comparable to that obtained from the Folin-Ciocalteu method. The strongagreement between our method and Folin-Ciocalteu method indicates that the proposedbiosensor in the study can be used in real green tea samples for accurate catechins detection. |
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