A novel dual-template molecularly imprinted electrochemiluminescence immunosensor array using Ru(bpy)32+-Silica@Poly-L-lysine-Au composite nanoparticles as labels for near-simultaneous detection of tumor markers

Abstract

A novel electrochemiluminescence (ECL) immunosensor array was fabricated on a screen-printed electrode (SPE) to perform multiplexed immunoassay of tumor markers (TMs), such as carcinoembryonic antigen (CEA) and carbohydrate antigen-199 (CA199). The SPE substrate consisted of a common Ag/AgCl reference electrode, a common carbon counter electrode and two gold nanoparticles modified carbon working electrodes. Firstly, dopamine (DA) as the functional monomer along with the corresponding TMs (CEA or CA199) as template molecules was electro-polymerized on the surface of different working electrodes. Then, the dual-template molecularly imprinted polydopamine (MIP-PDA) film was formed as the capture probe to recognize CEA and CA199. Secondly, a novel label was fabricated by conjugating the antibodies of TMs with Ru(bpy)32+-Silica@Poly-L-lysine-Au (Ru-Si@PLL-Au) nanocomposites, in which gold colloids (Au NPs) were doped on the surface of Ru-Silica (Ru(bpy)32+-doped Silica) using poly-L-lysine (PLL) as a bridging agent. PLL was also employed as a co-reactant of the luminophore. Based on a sandwich-type immunoassay, the MIP-PDA film, the corresponding antigen and labels were conjugated to produce the immunocomplex. The signals from the ECL immunosensor array were near-simultaneously detected by a photomultiplier tube (PMT) using a homemade single-pore-two-throw switch, which could avoid crosstalk between adjacent working electrodes. The obtained concentrations were 0.05-100pgmL−1 for CEA and 0.03-80 U L−1 for CA199, with detection limits of 0.02pgmL−1 and 0.01 U L−1, respectively. This novel ECL strategy provides a simple, economical, fast and sensitive approach for multiplexed immunoassay of CEA and CA199, and has significant potential for protein detection in a clinical laboratory setting.