Towards sarcosine determination in urine for prostatic carcinoma detection

Abstract

Sarcosine, a potential biomarker for prostate cancer, can be detected in a solid state enzyme based biosensor using sarcosine oxidase, with particle immobilised reagents. A novel fusion protein of the fluorescent protein, mCherry, sarcosine oxidase (SOx), and the polypeptide R5 (R52-mCherry-SOx-R5-6H), was explored, which allowed self-immobilization on silica microparticles and long-term (90 days +) retention of activity, even at room temperature. In contrast, commercial wildtype SOx lost activity in a few days. A silica-R52-mCherry-SOx-R5-6H microparticle sensor for determination of sarcosine in urine, linked the SOx coproduct, H2O2, to a measurement catalysed by horseradish peroxidase (HRP) immobilised on silica, in the presence of Amplex Ultrared (AR) to generate fluorescence at 582 nm. Silica microparticles carrying all the reagents (R52-mCherry-SOx-R5-6H, HRP and AR) were used to produce a silica-microparticle biosensor which responded to sarcosine at micromolar levels. Interference by amino acids and uric acid was examined and it was found that the silica-reagent carrying system could be calibrated in urine and responded across the clinically relevant concentration range. This contrasted with similar assays using commercial SOx, where interference inhibited the sarcosine signal measurement in urine. The microparticle biosensor was tested in urine from healthy volunteers and prostate cancer patients, showing higher concentrations of sarcosine in cancer patients consistent with previous reports of elevated sarcosine levels.