Recently, magnetic nanomaterials, quantum dots, catalytic nanomaterials have gained increasing attention because of their unique structure and properties. Besides the universal nature of nanomaterial (like quantum size effect、surface effect and macroscopic quantum tunneling effect), magnetic nanoparticles possess other properties such as magnetic guidance and good biocompatibility, and thus are often employed in bioassay; semiconductor quantum dots are viewed as candidates of Ab2 label owing to their high ECL efficiency; The nanomaterials with catalytic activity have overwhelming superiority, mainly for their relationship with electroactive molecules released from organism. The target of this thesis is to construct sensitive and selective ECL immunosensors with these functional nanomaterials. However, there are still some challenges: (1) Although the magnetic property could facilitate the wash process during immune recognition and avoid nonspecific adsorption, the conductivity of these materials is not good enough, which will affect the final sensitivity of the immunosensor. (2) The ECL behavior of semiconductor quantum dots is excellent, but the existence of Cd will make the protein denatured easily and thereby influence the results; (3) Now, most of the ECL immunosensors are fabricated on the glassy carbon electrode, the incubation process needs specialists, and the instrument is expensive. Thus how to utilize the advantages of these materials, make up for the deficiency, confirm the interaction between these nanoparticles and protein, and improve the sensitivity are our research content. In brief, this thesis includes four parts as followed.
(1) General strategy to fabricated Electrochemiluminescence sandwich-type nanoimmunosensors using CdTe@ZnS as luminescent labels and Fe3O4@SiO2 nanoparticles as magnetic separable scaffolds. A series of low-toxic CdTe@ZnS QDs with different core diameters are prepared via hydrothermal method. And their ECL behaviors are compared. The difference of the ECL efficiency mainly comes from their bandgaps and surface trap. The QDs with best ECL performance are used as label to conjugate with detection antibody (Ab2), and the successful conjugation is verified by running agarose gel electrophoresis. Meanwhile, Fe3O4@SiO2 nanoparticles with large surface area and good magnetic performance are employed to immobilize the capture antibody (Ab1). Under the optimum condition, this ECL immunosensor achieves a wide linear range of 0.01-125 ng mL-1 with a detection limit of 3.0 pg mL-1. Also, this immunosensor displays good selectivity, stability and reproducibility.
(2) Sandwich-structured electrogenerated chemiluminescence immunosensor based on dual-stabilizers-capped CdTe quantum dots as signal probes and Fe3O4-Au nanocomposites as magnetic separable carriers. Dual-stabilizers-capped CdTe quantum dots are synthesized with 3-mercaptopropionic acid (MPA) and sodium hexametaphosphate (HMP) as cappers, and used to conjugate with Ab2. The ECL quantum efficiency of these QDs is high due to the less surface traps and high-passivated surface. Fe3O4-Au nanocomposites are used to immobilize the Ab1 for the better electro-conductivity than previous Fe3O4@SiO2. The successful fabrication of the immunosensor is confirmed by CV, EIS, TEM and EDS. For CEA detection, the linear range is 0.005-80 ng mL-1, and the detection limit is 1 pg mL-1. Meanwhile, this sensor is also applied in real sample.
(3) Graphite paper-based bipolar electrode (BPE) electrochemiluminescence sensing platform. Graphite paper is firstly used as BPE for its good conductivity, uniform composition, and ease of operation. This platform is used for H2O2 and CEA detection. For H2O2 detection, Pt NPs are electrodeposited at BPE cathode to catalyze the reduction of H2O2. The linear range is 0.001-15 mM and the detection limit is 0.5 μM. For CEA detection, CS-MWCNTs are utilized to supply a hydrophilic platform for the immobilization of Ab1. Au@Pt nanostructures are employed to conjugate with Ab2 as the catalyst of H2O2 reduction in the cathodic cell. Under the optimum condition, this immunosensor achieves wide linear range of 0.01-60 ng mL-1 with a low detection limit of 5.0 pg mL-1.
(4) Patchy gold coated Fe3O4 nanospheres with enhanced catalytic activity applied for paper-based bipolar electrode-Electrochemiluminescence aptasensors. In this part, patchy gold coated Fe3O4 nanospheres (PG-Fe3O4) are synthesized for the first time in aqueous solution via a simple adsorption-reduction process. The formation mechanism is discussed detail. This nanomaterial possesses good magnetic property and catalytic activity toward H2O2 electro-reduction. Chronoamperometric and amperometric experiments indicate a relatively high catalytic rate constant of 3.13×105 M-1s-1, a high sensitivity of 578.87 μA·mM-1·cm-2 and a low Michaelis-Menten constant of 462 μM. The introduction of patchy gold facilitates the biofunctionalization. Thiol-terminated aptamers are immobilized onto the patchy gold part as signal probe to detect CEA. A related paper-based BPE aptasensor is fabricated. To enhance the sensitivity of the sensor, Au nanodendrites are electrodeposited at BPE cathode in the porous paper. The aptasensor achieves wide linear range of 0.1 pg mL-1-15 ng mL-1 with detection limit of 0.03 pg mL-1, suggesting the good catalytic activity of PG-Fe3O4 and their potential application in bioassay.
In summary, this thesis introduces four ECL immunosensor based on different functional nanomaterials. One novel nanomaterial is applied in biosensors.