Electrochemiluminescent Immunoassay With CdSe And CdZnSe Nanocrystals As Labels

Electrochemiluminescence (ECL) analysis has great potential in biosensing and medical diagnose applications due to its simple setups and high noise to signal ratios, compared with fluorescent analysis. Although the limit of detection (LOD) for ECL has been improved to femto molar level by now, the overwhelming majority of ECL fundamental researches and applications were carried out by directly detecting the total ECL intensity, as there is no dispersion device in the conventional ECL setups. Herein, with mercaptopropionic acid (MPA) and sodium hexametaphosphate (HMP) as dual capping agents, systematic spectral exploration on the ECL of dual-stabilizers-capped CdSe and CdZnSe nanocrystals (NCs) and corresponding sensing application were carried out with carcinoembryonic antigen (CEA) and alpha-fetoproteinantigen (AFP) as model proteins.1. ECL immunoassay with dual-stabilizers-capped CdSe NCs as Labels. The capping agents, i.e. MPA and HMP, can facilitate the electrochemical involved hole (or electron) injecting process and improve stability of the dual-stabilizers-capped CdSe NCs, so that the CdSe NCs could be electrochemically and repeatly inspired to excited states by giving off ECL in a cyclic pattern. With the CdSe NCs as ECL label and CEA as target molecule, a convenient single molecular immunoassay was proposed by simply detecting ECL intensity of the dual-stabilizers-capped CdSe NCs in a sandwich-typed immune-complex. The limit of detection is 0.1 fg/mL at S/N=3, which is corresponded to about 6-8 CEA molecules in 20 μ L serum sample. Importantly, ECL spectra of both CdSe NCs and its conjugate with probe antigen in the immune-complex were almost identical to the photoluminescence spectrum of bare CdSe NCs, indicating that all emissions were originated from the same excited species. The molecular-counting-free and ECL based immunoassay might be a promising alternative to the fluorescent single molecule detection strategy.2. Spectrum-based ECL immunoassay with lowtoxic ternary CdZnSe NCs as labels. The ternary CdZnSe NCs could be repeatedly injected with electrons via some electrochemical ways and then result in strong cathodic ECL with the coupling of ammonium persulfate. ECL spectrum of the CdZnSe NCs was almost identical to corresponding photoluminescence spectrum, indicating that the excited states of CdZnSe NCs in ECL were essentially the same as those in photoluminescence. Importantly, after being labeled to the probe antibody (Ab2) of alpha fetal protein (AFP) antigen, the ternary NCs in the Ab2｜NCs conjugates could preserve their ECL spectrum very well. A spectrum-based ECL immunoassay was consequently proposed with the limit of detection of 0.010 pg/mL, indicating a sensitive ECL sensing strategy different from the conventional ones. This work might open a way to the spectral resolved ECL analysis with even higher signal-to-noise ratio than the fluorescent analysis.