Powerful Chemiluminescence Probe for Rapid Detection of Prostate Specific Antigen Proteolytic Activity: Forensic Identification of Human Semen

[Image: see text] The prostate specific antigen (PSA), a serine protease with chymotrypsin-like activity, is predominantly expressed in the prostate and is considered as the most common marker in use to identify and follow the progress of prostate cancer. In addition, it is also now accepted as a marker for detecting semen in criminal cases. Here, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for detection of PSA enzymatic activity. The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane luminophore, that then undergoes efficient chemiexcitation to emit a green photon. The probe exhibits a significant turn-on response upon reaction with PSA and produces strong light emission signal with an extremely high signal-to-noise ratio. Comparison of the chemiluminescence probe with an analogous fluorescence probe showed superior detection capability in terms of response time and sensitivity. In addition, the probe was able to efficiently detect and image human semen traces on fabric, even after 3 days from sample preparation. The advantageous sensitivity and simplicity of a chemiluminescence assay to detect seminal fluid was effectively demonstrated by on-site measurements using a small portable luminometer. It is expected that the new chemiluminescence probe would be broadly useful for numerous applications in which PSA detection or imaging is required.