Electrochemiluminescence Immunosensor Based On Ferro Magnetic Nano-probe Modified Screen Printed Carbon Electrode

In this thesis, some ultra-sensitive and renewable electrochemiluminescence immunosensorsfor the determination of alpha-fetoprotein （AFP） were constructed, which were based on somemagnetic nano-probes and a series of luminous efficiency and high stability of the composite nano-signal labels（RuL@SiO₂^-Au^RuL-Ab2, RuL-MWNTs@Au^RuL-Ab2and PDDA-G@Lu-Au^HRP-Ab2）. To fabricate the immunosensor, the magnetic nanoprobes were mixed with alpha-fetoprotein （AFP） and signal lable complexes, then the resulting magnetic sandwich-typeimmunocomplexes were absorbed on screen printed carbon electrode （SPCE） for detection. Theproposed method is also simple, avoiding the complicated process modification on the electrodes’surface, and which simultaneously realize separation, enrichment and determination, re-utilizing,the preparation process is simple, save time with high sensitivity, which would be valuable forclinical immunoassay for tumor markers in human serum.The main works are as follows:1. The synthesis and characterization of Fe₃O₄（core）@Au（shell） magnetic nanoprobes.Fe₃O₄NPs were initially prepared by co-precipitation method and subsequently coated withgold nanoparticles （Au NPs） by reducing HAuCl₄on the surface of Fe₃O₄NPs to formFe₃O₄（core）@Au（shell） NPs （GMPs）. To obtaine GMPs-anti-AFP （GMPs-Ab1）, the primaryantibody （Ab1） was immobilized on the GMPs and incubated with1.0%BSA for1h to block theunreacted and nonspecific sites, then which were stored in pH7.4PBS at4°C. Thecharacterization results showed that the size and morphology of Fe₃O₄NPs and GMPs arerelatively homogeneous, and the preparation process is simple, save time with a mild reactioncondition.2. An electrochemiluminescence immunosensor based on Ru（bpy）₃²⁺（RuL） doped silica-goldnanoparticle signal label and magnetic beads nanoprobe modified SPCEs.An ultra-sensitive and renewable electrochemiluminescence immunosensor for thedetermination of AFP was constructed by using films of magnetic nano-prode （SMPs/Biotin-Ab1）and double nano-signal label. The double nano-signal label （RuL@SiO₂^-Au^RuL-Ab2） wasprepared by loading Ru（bpy）₃²⁺（RuL）-conjuged signal antibodies （RuL-Ab2） on Ru（bpy）₃²⁺（RuL）doped silica-gold nanoparticles （RuL@SiO₂^-Au）. To fabricate the immunosensor, SMPs were mixed with biotinylated AFP primary antibody （Biotin-Ab1）, AFP and RuL@SiO₂^-Au^RuL-Ab2complexes, then the resulting SMPs/Biotin-Ab1/AFP/RuL@SiO₂^-Au^RuL-Ab2（SBAR） magneticsandwich-type immunocomplexes were absorbed on screen printed carbon electrode （SPCEs） forECL detection. The present immunosensor showed a wide linear range of0.05–100ng mL^-1fordetecting AFP, with a low detection limit of0.02ng mL^-1（defined as S/N=3）. Thus, the presentimmunosensor can simultaneously realize separation, enrichment and determination, re-utilizing,the preparation process is simple, save time with high sensitivity, which would be valuable forclinical immunoassay for AFP in human serum.3. An electrochemiluminescence immunosensor based on RuL-MWNTs@Au^Ru-Ab2signallabel and GMPs magnetic nanoprobe modified SPCEs.An ultra-sensitive and renewable electrochemiluminescence （ECL） immunosensor for alpha-fetoprotein （AFP） was fabricated by using RuL-MWNTs （Tris（2,2’-bipyridine） ruthenium（II）complex immobilized on multiwall carbon nanotubes through Ï€-Ï€ stacking function between thecarbon aromatic systems） doped Au （ultra-sensitive and renewable） composite as labels andGMPs^Ab1. To fabricate the immunosensor, GMPs^Ab1were mixed with, AFP, and RuL-MWNTs@Au^Ru-Ab2complexes, then the resulting RuL-MWNTs@Au^RuLAb2/AFP/anti-AFP-GMP magnetic sandwich-type immunocomplexes were absorbed on screen printed carbonelectrode （SPCE） for detection. The present immunosensor showed a wide linear range of0.01–50ng mL^-1for detecting AFP, with a low detection limit of3pg mL^-1（defined as S/N=3）.Compared with the above RuL@SiO₂^-Au complexes, the introduction of carbon nanotubes in theimmunosensor can greatly improve the electrical conductivity and load capacity of the signal label,which can significantly reduce the detection limit; the GMPs fixed capture antibody formation ofmagnetic probe, which is easily prepared, and GMPs surface of gold nanoparticles can maintain theantibody activity and improve the probe conductivity.4. An electrochemiluminescence immunosensor based on PDDA-G@Lu-Au signal label andGMP^Ab1modified SPCEs.An ultrasensitive multiplexed electrochemiluminescence immunoassay method wasdeveloped for the detection of tumor markers by combining a functionalized graphene nanosheetsand gold-coated magnetic Fe₃O₄nanoparticles （GMPs） labeled alpha-fetoprotein （AFP） antibody（GMP^Ab1）. The functionalized graphene nanosheets （PDDA-G） with poly（diallyldimethylammonium chloride）（PDDA） were synthesized and used to combine withluminol-capped gold nanoparticles （Lu-Au NPs）. The resulting PDDA-G@Lu-Au compositedisplayed an enhanced capability for the immobilization of horseradish peroxidase （HRP） andsignal antibody （Ab2） to realize its electrochemiluminescence immunoassay. To fabricate theimmunosensor, GMPs^Ab1were mixed with, AFP, and PDDA-G@Lu-Au^HRP-Ab2complexes, then the resulting PDDA-G@Lu-Au^HRP-Ab2/AFP/anti-AFP-GMP magnetic sandwich-typeimmunocomplexes were absorbed on SPCE for detection. The present immunosensor showed awide linear range of0.002–20ng mL^-1ng mL^-1for detecting AFP, with a low detection limit of0.2pg mL^-1（defined as S/N=3）. Compared with the above complexes, the introduction ofgraphene nanosheets in the immunosensor can greatly improve the electrical conductivity, loadcapacity of the reporter molecules （Lu-Au）. Besides gold nanoparticles were not only used ascarriers of Ab2and HRP but also catalyzed the ECL reaction of luminol, which further amplifiedthe ECL signal of luminol in the presence of H2O2and significantly reduce the detection limit.