Studies On New Electrochemiluminescence Immunoassay Methods Based On Nanomaterials

A detailed review on the electrochemiluminescence （ECL）, electrochemiluminescenceimmunoassay （ECLIA） and their applications was carried out. Quantum dots （QDs） have beenwidely used in constructing immunosensors due to their excellent luminescent properties and goodbiocompatibilities. Graphene and its corresponding nano-composites have excellent electrical andoptical properties, so they have become a hot topic in developing high-performance analyticaldevices. In this study, a series of ECL immunosensors for ultrasensitive determination of tumormarkers were fabricated by combining biotechnology, nanotechnology with ECL technology. Themajor contents were described as follows:1. Electrochemiluminescence immunosensor based on graphene–CdS quantumdots–agarose composite for the ultrasensitive detection of alpha fetoproteinA novel strategy for the enhancement of electrochemiluminescence （ECL） was developed bycombining CdS quantum dots （QDs）, graphene （G） and agarose. This enhanced ECL was exploitedto develop a label-free ECL immunosensor for the ultrasensitive detection of Alpha fetoprotein（AFP）. The novel G-CdS QDs-agarose composite was first coated on the glassy carbon electrodesurface to form a robust film, which exhibited high ECL intensity, good biocompatibility and highstability. After that3-aminopropyl-triethoxysilane （APS）, as a binding linker, was conjugated tothe G-CdS QDs-agarose composite film on the electrode, the ECL signal was significantlyenhanced. The fabrication of ECL immunosensor was successfully completed by immobilizing theAFP-antibody （Ab） onto the electrode through glutaric dialdehyde （GLD）. The specificimmunoreaction between AFP and antibody resulted in the decrease in ECL intensity and theintensity decreased linearly with the logarithm of AFP concentration in the range of0.0005–50pg/mL with a detection limit of0.2fg/mL. The immunosensor exhibits high sensitivity, specificity,stability, reproducibility and good regeneration, thus has the potential to be used in clinical application. Besides, the highly enhanced ECL from the G-CdS QDs-agarose composite filmopened new avenues to apply graphene and QDs ECL in analytical systems and ECL biosensors.2. Ultrasensitive detection of carcinoembryonic antigen based onelectrochemiluminescence quenching of Ru（bpy）3²⁺by quantum dotsAn ultrasensitive electrochemiluminescence （ECL） immunosensor for the detection ofcarcinoembryonic antigen （CEA） in serum and saliva was developed. Ru（bpy）3²⁺-graphene-Nafioncomposite was first coated on the glassy carbon electrode surface in this ECL immunosensor. Thesandwich-type immunoreactions between the first antibody and the second antibody bridged thedonors Ru（bpy）3²⁺and acceptors QDs, which led to the occurrence of ECL quenching ofRu（bpy）3²⁺by QDs. Based on the efficient ECL quenching, ultrasensitive CEA detection wasrealized. Under optimal conditions, the ECL signal depended linearly on the logarithm of the CEAconcentration within a range from0.005to0.5pg/mL, and the detection limit of CEA was0.002pg/mL. Moreover, the proposed ECL immunosensor provides a stable, specific, and highlysensitive immunoassay protocol for CEA detection at very low levels, which might hold a promisefor clinical application.3. Electrochemiluminescence immunosensor for the determination of ag alphafetoprotein based on energy scavenging of quantum dotsAn electrochemiluminescence （ECL） immunosensor for ultrasensitive detection of alphafetoprotein （AFP） was fabricated using graphene-CdS quantum dots-alginate （G-CdS QDs-AL） asthe immobilizing support and CdSe/ZnS QDs as the label. CdSe/ZnS QDs could effectivelyscavenge the ECL of G-CdS QDs-AL composite, and the quenched ECL intensity dependedlinearly on the logarithm for AFP concentration in the range from0.05to500fg/mL. The detectionlimit was20ag/mL. The proposed ECL immunoassay protocol for AFP detection is stable, specific,highly sensitive and promising for clinical application.4. Multiplex electrochemiluminescence immunoassay of two tumor markers usingmulticolor quantum dots as labels and graphene as conducting bridgeA multiplex electrochemiluminescence （ECL） immunoassay for simultaneous determinationof two different tumor markers, alpha-fetoprotein （AFP） and carcinoembryonic antigen （CEA）,using multicolor quantum dots as labels and graphene as conducting bridge was developed. Herein,a typical sandwich immune complex was constructed on the glassy carbon electrode, with QDs525 and QDs625labeled on secondary anti-AFP and anti-CEA antibodies, respectively, thus to obtaindistinguishable ECL signals. Because most of those QDs labeled on secondary antibodies werebeyond the space domain of the ECL reaction, graphene was used as a conducting bridge topromote the electron transfer between QDs and the electrode, leading to about30-foldenhancement of the ECL intensity. Experimental results revealed that the multiplexelectrochemiluminescence immunoassay enabled the simultaneous monitoring of AFP and CEA ina single run with a working range of0.001–0.1pg/mL. The detection limit （LOD） for bothanalytes at0.4fg/mL was very low. No obvious cross-reactivity was found. Precision, recoveryand stability were satisfactory. This novel multiplex ECL immunoassay provided a simple,sensitive, specific and reliable alternative for the detection of tumor markers in clinical laboratory.