A new technology for cell imaging: Professor Huangxian Ju proposed an electrochemiluminescence method for cell membrane protein imaging


Electrochemiluminescence (ECL) integrates the advantages of high sensitivity of chemiluminescence and potential controllability of electrochemistry, and has been widely used in analytical chemistry and clinical medicine. ECL immunoassay based on ruthenium derivatives is one of the main methods to detect disease markers. To meet the demand of biological detection and life science research, the development of novel ECL systems has become a long-term research topic in this field.

In 2002, quantum dots (QDs) were found to be ideal ECL emitters with size-controlled luminescence, high fluorescence quantum yields and narrow emission spectra (Science 2002, 296, 1293−1297). Huangxian Ju’s group overcame the condensation of QDs with simple methods to realize the ECL of QD in aqueous systems and develop the QDs-based ECL chemical sensing method for the first time (Anal. Chem. 2004, 76, 6871), and prepared the first QDs-based ECL biosensor (Chem. Commun. 2007, 404). Theyconstructed a series of new QDs-based ECL systems with energy and electron transfer mechanisms and found novel coreactant to propose ECL detection methods for small molecules, DNA, protein and glycosyl groups (Anal. Chem. 2007, 79, 6690; 2007, 79, 8055; 2008, 80, 5377; Chem. Commun. 2010, 46, 5446). Moreover, Ju’s group also developed new QDs with low ECL potential (Anal. Chem. 2010, 82, 3359), and new immunoassay methods for biomarkers (Anal. Chem. 2010, 82, 7351; 2011, 83, 5214; 2013, 83, 5390).

However, the toxicity of QDs limits their applications in cytosensing and in vivo sensing. In recent years, Ju’s group has focused their attention on polymer dots (Pdots) with low toxicity and high biocompatibility to design new Pdots with high ECL efficiency and expand their biomedical applications. They firstly synthesized silole-carbazole coupled Pdots (Anal. Chem. 2016, 88, 845) and ruthenium doped Pdots to propose a dual ECL enhancement strategy (Anal. Chem. 2017, 89, 7659), and then designeddonor−acceptor and aggregation-induced emission-active Pdots (J. Phys. Chem. Lett. 2018, 9, 5296) and dual resonance energy transfer ECL system (Chem. Sci. 2019, 10, 6815) to construct a series of high-throughput and visual imaging methods for metal ions (Anal. Chem. 2018, 90, 1202) and multiple disease markers (Anal. Chem. 2018, 90, 7708).

Due to the demand for high concentration of coreactant, the short life time of intermediates, and their damage to cells, ECL technology is limited in cytosensing and in vivo detection. Therefore, it is urgent to develop novel ECL systems with high ECL efficiency, low cytotoxicity but no additional coreactant. Recently, Ju’s group designed a coreactant-embedded Pdots (TEA-Pdots) along with dual intramolecular electron transfer to develop an excellent ECL emitter, which showed 132 times stronger ECL in PBS than that in intermolecular ECL system at equivalent amounts. The ECL efficiency was even higher than that of classical ruthenium/coreactant system, leading to a reagentless ECL imaging method for membrane protein on single living cell.

Firstly, 2,2-(9,9-bis(6-bromohexyl)-9H-fluorene-2,7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborane) and 4,7-dibromobenzo[c][1,2,5]thiadiazol were polymerized at 100 oC to form poly(4-(9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)benzo[c][1,2,5], which further reacted with diethylamine to obtain diethylamine-conjugated PFBT (TEA-PFBT). After mixing TEA-PFBT withpoly(styrene-co-maleic anhydride) (PSMA) for nanoprecipitation, coreactant-embedded Pdots could be synthesized (Figure 1A). The TEA-Pdots with rich modification sites, low cytotoxicity and high ECL efficiency were conjugated with streptavidin (SA) and further labeled to the cell surface through double recognitions of biotin labeled antibody with the protein and SA, which realized in situ detection of membrane proteins without permeability treatment and extra coreactant (Figure. 1B). Through the reagentless ECL imaging of human epidermal growth factor receptor-2 (HER2) on the surface of living cells, the technique has been successfully used to evaluate the regulation of drugs on membrane proteins. This work opens a new avenue for ECL applications in single cell analysis and dynamic study of biological events.

The above results entitled “Dual Intramolecular Electron Transfer for In Situ Coreactant-Embedded Electrochemiluminescence Microimaging of Membrane Protein” were published online in Angew. Chem. Int. Ed. (DOI: 10.1002/anie.202011176) on September 21. Ningning Wang is the first author of this work, and Professor Huangxian Ju is the corresponding author.


Figure 1. (A) Synthetic route of TEA-PFBT and preparation of TEA-Pdots. (B) Schematic illustration of dual intramolecular electron transfer of TEA-Pdots for ECL microimaging of HER2 on single cells.