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Colorimetric paper-based sarcosine assay with improved sensitivity

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Abstract

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.

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Acknowledgements

We thank Mr. Kogi Kaizu of Keio University for his support with SEM image recording.

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Author notes

  1. Sera Ohta and Misaki Nakagawa contributed equally to this work.

Authors and Affiliations

  1. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan

    Mayu Masumoto, Sera Ohta, Misaki Nakagawa, Yuki Hiruta & Daniel Citterio

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  1. Mayu Masumoto
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  2. Sera Ohta
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Correspondence to Daniel Citterio.

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Masumoto, M., Ohta, S., Nakagawa, M. et al. Colorimetric paper-based sarcosine assay with improved sensitivity. Anal Bioanal Chem 414, 691–701 (2022). https://doi.org/10.1007/s00216-021-03682-0

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  • DOI: https://doi.org/10.1007/s00216-021-03682-0

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