An AIE‐based enzyme‐activatable fluorescence indicator for Western blot assay: Quantitative expression of proteins with reproducible stable signal and wide linear range

Abstract

Western blot is a commonly used experimental method to analyze the protein expression. However, the most commonly used chromogenic indicator based on chemiluminescence is limited by narrow linear range and unstable quantitative reproducibility, whereas the recently developed fluorescent indicator suffers from poor detection limit. Herein, we report an enzyme‐activatable fluorescence indicator to quantify proteins with reproducible stable signal and wide linear range, through introducing the hydrophilic alkaline phosphatase (ALP)‐triggered phosphoric acid moiety into our established aggregation‐induced emission (AIE) building block of quinoline‐malononitrile (QM). In this strategy, the indicator DQM‐ALP disperses well in both aqueous and lipid environments to exhibit initial “off” fluorescence, but when exposing to the ALP‐coupled secondary antibody on the PVDF membrane, the specific enzymatic turnover would liberate hydrophobic AIE luminogen (AIEgen) QM‐OH to emit strong luminescence, thereby achieving an ideal “off‐on” state for sensitively imaging proteins with high signal‐to‐noise (S/N) ratio. Moreover, benefiting from the excellent signal stability of AIE fluorophore, DQM‐ALP indicator exhibits superior quantitative analysis of protein expression with high reproducibility. Upon taking advantage of the AIEgens to reduce high concentration‐induced luminance quenching, the linear quantification range is extremely expanded. In contrast with the traditional chemiluminescent indicator, the AIE‐based enzyme‐activatable indicator DQM‐ALP not only greatly improves the signal stability for quantitative reproducibility, but also expands the linear quantification range, and further provides a practical alternative reagent for fluorescence Western blot assay.

AIE-active luminogens as highly efficient free-radical ROS photogenerator for image-guided photodynamic therapy

“…24 In addition, AIEgens with "off-on" fluorescence could realize high-fidelity imaging compared to ACQ fluorophore, especially for decreasing false fluorescence signal. [25][26][27][28] Our group have reported a novel AIE building block TCM, 29,30 with high fluorescence in aggregation state, multiple ARTICLE Journal Name 2 | J. Name., 2012, 00, [1][2][3] This journal is © The Royal Society of Chemistry 20xx…”

“…24 In addition, AIEgens with “off–on” fluorescence could realize higher-fidelity imaging compared to ACQ fluorophores, especially in terms of decreasing false fluorescence signals. 25–28…”

“…24 In addition, AIEgens with "off-on" uorescence could realize higher-delity imaging compared to ACQ uorophores, especially in terms of decreasing false uorescence signals. [25][26][27][28] Our group has reported the novel AIE building block TCM, 29,30 which has high uorescence in the aggregated state, multiple modication sites, and good photostability; TCM is expected to be useful in the construction of photosensitizers through structural regulation (Fig. 1).…”

Fluorescence and stimuli-responsive performance of polymer composites filled with tetraphenylethene derivatives

“…Stimuli-responsive materials, which undergo structural or property changes in response to external triggers, have shown enhanced use in complex applications and have become a hot topic of increasing interest. 1,2 Among them, fluorescent molecules with unique optical properties have been widely applied as functional materials in biological probes, 3 optoelectronics, 4 chemical sensing, 5,6 and monitoring the damage process in composites. 7 Most fluorescent molecules are used in their aggregated states and suffer from the pernicious aggregationcaused quenching effect, which limits their applications.…”

Metal‐Based Aggregation‐Induced Emission Theranostic Systems

“…[17][18][19][20] Besides the high quantum yield in the aggregate state, AIEgens also boast large Stokes shifts, high photostability and satisfactory biocompatibility, which render them ideal for biomedical applications. [21][22][23][24][25][26] Moreover, some AIEgens can serve as efficient therapeutic agents. Typically, the nonradiative decay of AIEgens will be suppressed in the aggregate state, which facilitates the generation of reactive oxygen species (ROS) because of the efficient intersystem crossing (ISC) and the formation of stable triplet exciton.…”