Development Of Novel Biosensors Based On Flow Injection Chemiluminescence

The main idea of the research was to develop the sensitive biosensors based onthe combination of flow injection chemiluminescence (FI-CL) and some novelnanoparticles. It provided the alternate ways for the clinical diagnoses of manydiseasesatearlystage.Theexperimentalprotocolscouldbesummarized asfollows:1.Anovelflowinjectionchemiluminescence bienzyme-channelingbiosensorwasdeveloped for the determination of cholesterol. The biosensor was constructed byentrappingcholesterol oxidase (COD) and horseradish peroxidase (HRP) in thehybridmaterialswhich was synthesized bygold nanoparticles andmesopores silica structuresSBA-15 (GNPs/SBA-15). In the presence of cholesterol, the enzymatic reaction ofCOD-cholesterol-dissolved oxygen system generated hydrogen peroxide in thebienzyme-entrapped mesopores, which was immediately catalyzed by the sameentrapped HRP to lead to a sensitive and fast CL response of Luminol-H2O2-HRP.With well-ordered hexagonal mesoporous silica structures, SBA-15 mesoporousmaterials could not only adsorb COD and HRP firmly, but also enlarge CL reactioninterface because of the large specific surface area. The doped gold nanoparticles inSBA-15increasedtheabsorptionefficiencyofenzymemoleculeslargelyandhelpedtokeep bioactivity of enzyme molecules. Under the optimized experimental conditions,the detection limit of cholesterol was down to 5×10?7 mol/L (3σ) with a very widelinear range from 1.0×10?6 to 1×10?3 mol/L. The method has been satisfactorilyappliedtothedeterminationofthe cholesterollevel inserum samples.Theconstructedbienzyme channeling biosensor provided a strategy for CL detection of oxidasesubstratesbyusingthemesoporous materials.2. Bienzymatic biosensor for the determination of glucose by flow injectionchemiluminescence detection was proposed. Hybrids of Gold nanoparticles (GNPs) and chitosan were chosen as the immobilization matrix of glucose oxidase (GOD) andhorseradish peroxidase to fabricate the biosensors with silane-pretreated glassmicrobeads. After the enzyme catalyzing oxidation of glucose in GOD biosensor, theproduced H2O2 flowed into HRP biosensor to react with luminol. The doped GNPs inchitosan were found to enhance the classical CL reaction of Luminol-H2O2-HRP. TheCLenhancement was investigated indetail byCLandUV-visiblespectrum.Undertheoptimizedexperimental conditions, glucosecouldbe determinedinalinear rangefrom0.01 to 6.0 mmol/L with a detection limit of 5.0μmol/L at 3σ. The accuracy of theproposed method was examined by detecting the glucose level in four clinical serumsamples from the hospital. The proposed method provides a new alternative todetermineglucose.3. A novel flow-through chemiluminescent immunosensor was designed for thesimultaneous detection ofα-fetoprotein (AFP) and carcinoembryonic antigen (CEA).Two transparent microtubes immobilized with monoclonal antibodies were set in amobile flow cell to fabricate the multi-analyte immunoassay channels. Based on asandwich immunoassay format, two mixtures of the sample antigens andcorresponding horseradish peroxidase labeled antibodies were introduced into thechannels for on-line incubation. Upon injection of luminol and H2O2, the different CLsignals from the two channels were sequentially detected by moving the handle of themobile flow cell. Under the optimal experimental conditions, AFP and CEA could bedetected inthelinear ranges of1.25 ~50 and 1.25~40ng/mLwithdetectionlimits of1.06 and 1.0 ng/mL, respectively. The accuracy of the proposed system was assessedby human serum samples. The novel multi-analyte strategy provided an automated,simple,andlow-costapproachwithoutusingofexpensivearraydetector.4. Asimple flow injection chemiluminescence procedure for the determination ofandrographolide (AD) was proposed. The method was based on the CL emittingreaction between andrographolide and potassium permanganate in acidic medium withformaldehyde as the CL enhancer. The surfactant of cetyl-trimethyl ammoniumbromide (CTAB) could further enhance the signal of the above CLsystem. Under theoptimized experimental conditions, andrographolide could be determined in a linearrange from 5.0×10-6 to 2.5×10-4 g/mLwith a correlation coefficient of 0.999 (n = 7)and a detection limit of 1.0×10-6 g/mL. The relative standard deviation of theproposed method was calculated as 0.9% from 12 repetitive injections of 1.0×10-5g/mL andrographolide. The sample throughput was 150/h. The method has been satisfactorily applied to the determination of andrographolide in pharmaceuticalformulations. The result was shown that there was no obvious interference from thecommon medicaments. In addition, the CL reaction mechanism of KMnO4-HCHO-ADsystem was also explored by the fluorescence and CL spectrum. The proposed methodprovided an alternative strategy for the rapid determination of andrographolide.