Proximity Hybridization-Regulated Immunoassay for Cell Surface Protein and Protein-Overexpressing Cancer Cells via Electrochemiluminescence

Abstract

A simple electrochemiluminescence (ECL) immunoassay based on a proximity hybridization-regulated strategy was developed for highly sensitive and specific detection of cell surface protein and protein-overexpressing cancer cells. A biosensor was fabricated by self-assembling a thiolated capture ss-DNA3 (partially hybridize with ss-DNA1 and ss-DNA2) and blocking with 6-mercapto-1-hexanol on a gold electrode surface. Target protein was simultaneously bound by two ss-DNA-tagged antibody probes (DNA1-Ab1 and DNA2-Ab2), while DNA1 and DNA2 were brought in sufficient proximity and hybridized with capture DNA3 on the surface of the biosensor. After ECL signal reagent Ru(phen) was intercalated into the hybridized ds-DNAs, ECL measurement was performed in the coreactant solution. A "signal on" proximity hybridization-regulated ECL immunoassay for alpha-fetoprotein (AFP) was developed. The ECL intensity increased with the increase of AFP concentration in the range of 0.05-20.0 ng/mL with a detection limit of 6.2 pg/mL. Moreover, the developed ECL method was successfully used to detect AFP-overexpressing cancer cells (MCF-7 cancer cells as model) with a detection limit of 620 cells/mL (∼60 MCF-7 cells in 100 μL of cell suspension) and discriminate AFP expression on different types of the living cell surface. This work for the first time reports a proximity hybridization-regulated ECL immunoassay for the detection of the cell surface protein on a living cell surface with good specificity and sensitivity. This simple, specific, and sensitive strategy is greatly promising for the detection of proteins and specific cells.

Surface-Confined Electrochemiluminescence Microscopy of Cell Membranes

“…[1][2][3][4][5] For example, the selective measurement of overexpressed membrane proteins on tumor cells is critical for diagnosis and dedicated disease treatment. 3,[6][7][8] These proteins are of great interest, particularly because they constitute targets for antibodies in immunoassays and immunohistochemistry. The study of protein transport and trafficking is required to understand the function and organization of the cell.…”

Recent advances in electrogenerated chemiluminescence biosensing methods for pharmaceuticals

“…The relative ECL intensity from the first wave is significant, especially in diluted Ru(bpy) 3 2+ solutions (less than approximately micromolar) containing ∼0.1 M TPA. Thus, for the low concentrations of analytes such as in immunoassays and detection of drugs and biomakers with Ru(bpy) 3 2+ as an ECL label, the bulk of the ECL signal obtained in this system probably originates from the first ECL wave at Au electrode or from the second ECL wave at carbon electrode [27] , [28] , [29] , [30] , [31] , [32] . In recent years, cyclometalated iridium (III)-complexes have received much attention, since these complexes have high ECL efficiencies and low emission potentials compared with Ru(II) complexes [33] , [34] .…”

Electrochemiluminescence Single‐Cell Analysis: Intensity‐ and Imaging‐Based Methods

“…[31] Therefore, for most ECL cytosensors, the ECL signal must be amplified. There are a lot of amplification strategies for ECL biosensing, including the usage of new ECL luminophores and co-reactant, [32] using nanoparticles (NPs) to catalyze ECL reactions or load ECL tags, [33] DNA-amplification technique, [34] etc. Some excellent reviews have summarized these strategies [35] and we only list the works with high sensitivity that is quite close to single cell analysis in Table 1.…”