Dual Electrochemiluminescence Signal System for In Situ and Simultaneous Evaluation of Multiple Cell-Surface Receptors

Abstract

A mutiplex cytosensor based on a dual electrochemiluminescence (ECL) signal system was fabricated for in situ and simultaneous detection of the expression levels of multiple cell-surface receptors, mannose and epidermal growth factor receptor (EGFR), using luminol-capped gold nanoparticles (Au@luminol) and CdS quantum dots (CdS QDs) as potential-resolved ECL nanoprobes. Two spatially resolved areas on indium tin oxide (ITO) electrodes were modified with polyaniline (PANI) by electropolymerization, on which gold nanoparticles (AuNPs) were attached to strengthen conductivity and stability of the sensing interface. Human mucin1 protein (MUC1) aptamer was immobilized onto AuNPs for capturing MUC1-positive MCF-7 cells. Au@luminol and CdS QDs as ECL nanoprobes were covalently linked with concanavalin A (ConA) and epidermal growth factor (EGF) to label MCF-7 cells on the two areas of the cytosensor separately. Compared to conventional multiplex biosensor, we demonstrated a novel analysis platform for the simultaneous detection of multiple cell-surface receptors; it could provide two sensitive and potential-resolved ECL signals during one potential scanning and avoid cross-reactivity between the two nanoprobes. The quantification of MCF-7 cells on the two spatially resolved areas could be achieved over the linear range from 10 to 1.0 × 10 cells mL with a detection limit of 20 cells mL. This multiplex cytosensor was further applied for simultaneous quantitative evaluation of the expression levels of mannose and EGFR on MCF-7 cells, revealed that the average numbers of mannose and EGFR per captured MCF-7 cell were 1.2 × 10 and 0.86 × 10 with the relative standard deviation of 5.3% and 4.2%, respectively. The multiplex cytosensor was capable of evaluating multiple cell-surface receptors, which would be beneficial to developing a better diagnostic tool for diseases.

Recent Advances in Electrochemiluminescence-Based Systems for Mammalian Cell Analysis

“…The two ECL probes were introduced to label the cells on two spatially resolved areas of a single sensing interface. During the potential sweep, well-separated and sensitive ECL signals at +0.5 V and −1.2 V were observed, demonstrating the sensitive and sequential detection of multiple receptors on the cell surface [76]. Recently, Ding et al proposed a bimodal ECL system based on an internal standard method [77].…”

“…Schematic illustration of the dual ECL signal system. ECL signals at cathodic and anodic potentials can be obtained using CdS quantum dots (QDs) and luminol as ECL emitters, respectively.Reproduced with permission from[76].…”

Recent Development of Nanomaterials-Based Cytosensors for the Detection of Circulating Tumor Cells

“…During the past decades, several types of nanomaterials have been exploited with good ECL property. For example, QDs have been used as the ECL emitters since they can be excited by electrical injection of an electron-hole pair to generate strong ECL signal [240]. For this view, Jiang et al reported a label-free method for the assays of folate receptor (+) HeLa cells by molecular recognition (Figure 15A) [241].…”

Quantitative Real‐Time Monitoring of Antibody‐Induced Internalization of Epidermal Growth Factor Receptor on Single Living Mammalian Cells Using Scanning Electrochemical Microscopy

“…Various measurement and labeling methods have been applied in analyzing the internalization process of EGFR. Radioactive materials,, nanoparticles,, chemiluminescent materials,, and fluorescent materials with immunostaining methods have been widely applied in the detection of cell‐surface EGFR, while many types of toxins, antibodies, inhibitors, genetic mutation samples, and gene knockout samples have been used to identify internalization pathways ,. To clarify the internalization mechanisms via comparison of conditions such as difference in cell type, it is important to develop quantitative measurement methods with low levels of invasiveness (e. g., time‐dependent methods) applicable at the single‐cell level.…”

“…However, there are few methods that can be measured in a minimally invasive, quantitative and single cell level. Electrochemical methods, for instance cyclic voltammetry, impedance analysis and electrochemiluminescence, have been applied due to rapid, continues and quantitative analysis under physiological conditions. However, single cell level measurements were not easy, because sizes of electrodes usually using in these measurements are much larger than that of mammalian cells.…”