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.
Development of “ultrahigh contrast” fluorogenic probes for trapping alkaline phosphatase (ALP) activities in human serum is highly desirable for clinical auxiliary diagnosis for hepatobiliary diseases. However, the intrinsic dilemma of incomplete ionization of intramolecular charge transfer (ICT)‐based ALP fluorophores and autofluorescence interference of serum result in low sensitivity and accuracy. Given that unique halogen effects could lead to a drastic decrease in the pKa value and a significant enhancement in the fluorescence quantum yield, herein we report an enzyme‐activatable near‐infrared probe based on a difluoro‐substituted dicyanomethylene‐4H‐chromenep for achieving fluorescent quantification of human serum ALP. Rational design strategy is demonstrated by altering the substituted halogen groups to well regulate the pKa for meeting the physiological precondition. Owing to the complete ionization at pH 7.4 with tremendous fluorescence enhancement, the difluoro‐substituted DCM‐2F‐HP manifests a linear relationship between the emission intensity and ALP concentration in both solution and serum samples. Along with measuring 77 human serum samples, the DCM‐2F‐HP based fluorescence method not only exhibits significant correlations with clinical colorimetry, but also distinguishes ALP patients from healthy volunteers, as well as assessing the progress of liver disease, thus providing a potential toolbox for quantitatively detecting ALP and warning the stage of hepatopathy.
Photosensitizers equipped with high reactive oxygen species (ROS) generation capability and bright emission are essential for accurate tumor imaging and precise photodynamic therapy (PDT). However, traditional aggregation-caused quenching (ACQ) photosensitizers cannot simultaneously produce desirable ROS and bright fluorescence, resulting in poor image-guided therapy effect. Herein, we report an aggregation-induced emission (AIE) photosensitizer TCM-Ph with a strong donor–π–acceptor (D–π–A) structure, which greatly separates the HOMO–LUMO distribution and reduces the ΔEST, thereby increasing the number of triplet excitons and producing more ROS. The AIE photosensitizer TCM-Ph has bright near-infrared emission, as well as a higher ROS generation capacity than the commercial photosensitizers Bengal Rose (RB) and Chlorine e6 (Ce6), and can effectively eliminate cancer cells under image guidance. Therefore, the AIE photosensitizer TCM-Ph has great potential to replace the commercial photosensitizers.
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