“Zero” Intrinsic Fluorescence Sensing‐Platforms Enable Ultrasensitive Whole Blood Diagnosis and In Vivo Imaging

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re‐engineered far‐red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of “zero” PIF sensing‐platforms were created by systematically regulating the open‐loop/spirocyclic forms. Leveraging these advancements, we devised various ultra‐sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300‐fold). Among these indicators, 8‐LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8‐LAP with an endoplasmic reticulum‐targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases. The present work reports a versatile strategy involving engineering long‐wavelength rhodol derivatives by systematically regulating the open‐loop/spirocyclic forms, thereby aiding in eliminating probe intrinsic fluorescence (PIF) of far‐red to NIR II probes and creating ultra‐sensitive fluorescence probes with a robust turn‐on response (190 to 1300‐fold). These novel probes enable accurate detection and quantification of biomarkers in abnormal physiological processes (p53 abnormalities) and diseases (tumor metastases).