Colorimetric paper-based sarcosine assay with improved sensitivity

• Published in: Analytical and bioanalytical chemistry Vol. 414; no. 1; pp. 691 - 701
• Main Authors: Masumoto, Mayu, Ohta, Sera, Nakagawa, Misaki, Hiruta, Yuki, Citterio, Daniel
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 2022; Springer; Springer Nature B.V
• Subjects: ABCs 20th Anniversary; Amino acids; Analysis; Analytical Chemistry; Assaying; Bicarbonates; Biochemistry; Biological markers; Biomarkers; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Colorimetry; Colorimetry - methods; Diagnosis; Filter paper; Food Science; Glycine; Horseradish Peroxidase; Humans; Hydrogen Peroxide; Hydrophobicity; Identification and classification; Laboratory Medicine; Limit of Detection; Male; Methods; Monitoring/Environmental Analysis; Oxidation; Peroxidase; pH effects; Polymers; Prostate cancer; Reagents; Research Paper; Sarcosine; Sarcosine oxidase; Sarcosine Oxidase - chemistry; Selectivity; Shelf life; Storage stability; Substrates; Japan; Artificial urine; Prostate cancer biomarker; Cationically charged polyamine; Hydrogen peroxide; Sarcosine oxidase; Enzymatic sarcosine assay
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-021-03682-0

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)–catalyzed conversion of the colorless indicator 3,3’,5,5’-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at − 20 °C for a period of 50 days were confirmed. Graphical abstract