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.
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