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

Abstract

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.

Direct and Indirect Chemiluminescence: Reactions, Mechanisms and Challenges

“…The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane intermediate affording a chemiexcited benzoate that emits a green photon. The benefit and simplicity of a CL assay to detect seminal fluid was effectively demonstrated by on-site measurements using a small portable luminometer [64].…”

Universal Access to Protease Chemiluminescent Probes through Solid-Phase Synthesis

“…Recently, our group developed chemiluminescent luminophores that are highly emissive under physiological conditions. − Molecular probes composed of these luminophores were found to be suitable for monitoring enzymatic activity with great sensitivity, both in vitro and in vivo . − The general design and activation pathway for such chemiluminescent probes is illustrated in Figure . Remarkably, protease probes based on our chemiluminescent dioxetane luminophore exhibited up to 16,000-fold higher sensitivity in comparison to analogous fluorescent probes based on AMC dye. − …”

Peptide probes for proteases – innovations and applications for monitoring proteolytic activity

“…As such it shows real potential as a point-of-care sensing platform, as do the PSA probes ( 61 ) developed by Shabat that allows on-site measurements using a small portable luminometer. 184,185…”