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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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References

  1. Couzin J. Metabolite in urine may point to high-risk prostate cancer. Science. 2009;323:865.

    Article  CAS  PubMed  Google Scholar 

  2. Katz A. Ce: early localized prostate cancer. Am J Nurs. 2015;115:34–44.

    Article  PubMed  Google Scholar 

  3. Merriel SWD, Funston G, Hamilton W. Prostate cancer in primary care. Adv Ther. 2018;35:1285–94.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Schiffer E. Biomarkers for prostate cancer. World J Urol. 2007;25:557–62.

    Article  CAS  PubMed  Google Scholar 

  5. Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu J, et al. Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature. 2009;457:910–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jiang Y, Cheng X, Wang C, Ma Y. Quantitative determination of sarcosine and related compounds in urinary samples by liquid chromatography with tandem mass spectrometry. Anal Chem. 2010;82:9022–7.

    Article  CAS  PubMed  Google Scholar 

  7. Narwal V, Kumar P, Joon P, Pundir CS. Fabrication of an amperometric sarcosine biosensor based on sarcosine oxidase/chitosan/CuNPs/c-MWCNT/au electrode for detection of prostate cancer. Enzym Microb Technol. 2018;113:44–51.

    Article  CAS  Google Scholar 

  8. Liu T, Fu B, Chen J, Li K. An electrochemical sarcosine sensor based on biomimetic recognition. Microchim Acta. 2019;186:136.

    Article  Google Scholar 

  9. Lan J, Xu W, Wan Q, Zhang X, Lin J, Chen J, et al. Colorimetric determination of sarcosine in urine samples of prostatic carcinoma by mimic enzyme palladium nanoparticles. Anal Chim Acta. 2014;825:63–8.

    Article  CAS  PubMed  Google Scholar 

  10. Uhlirova D, Stankova M, Docekalova M, Hosnedlova B, Kepinska M, Ruttkay-Nedecky B, et al. A rapid method for the detection of sarcosine using Spions/Au/CS/SOX/NPs for prostate cancer sensing. Int J Mol Sci. 2018;19:3722.

    Article  PubMed Central  Google Scholar 

  11. Burton C, Gamagedara S, Ma Y. A novel enzymatic technique for determination of sarcosine in urine samples. Anal Methods. 2012;4:141–6.

    Article  CAS  Google Scholar 

  12. Heger Z, Cernei N, Krizkova S, Masarik M, Kopel P, Hodek P, et al. Paramagnetic nanoparticles as a platform for fret-based sarcosine picomolar detection. Sci Rep. 2015;5:8868.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Henderson CJ, Pumford E, Seevaratnam DJ, Daly R, Hall EAH. Gene to diagnostic: self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase. Biomaterials. 2019;193:58–70.

    Article  CAS  PubMed  Google Scholar 

  14. Jornet-Martínez N, Henderson CJ, Campíns-Falcó P, Daly R, Hall EAH. Towards sarcosine determination in urine for prostatic carcinoma detection. Sensors Actuators B Chem. 2019;287:380–9.

    Article  Google Scholar 

  15. Martinez AW, Phillips ST, Butte MJ, Whitesides GM. Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angew Chem Int Ed. 2007;46:1318–20.

    Article  CAS  Google Scholar 

  16. Yamada K, Shibata H, Suzuki K, Citterio D. Toward practical application of paper-based microfluidics for medical diagnostics: state-of-the-art and challenges. Lab Chip. 2017;17:1206–49.

    Article  CAS  PubMed  Google Scholar 

  17. Cate DM, Adkins JA, Mettakoonpitak J, Henry CS. Recent developments in paper-based microfluidic devices. Anal Chem. 2015;87:19–41.

    Article  CAS  PubMed  Google Scholar 

  18. Chen X, Chen J, Wang F, Xiang X, Luo M, Ji X, et al. Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices. Biosens Bioelectron. 2012;35:363–8.

    Article  CAS  PubMed  Google Scholar 

  19. Dungchai W, Chailapakul O, Henry CS. Electrochemical detection for paper-based microfluidics. Anal Chem. 2009;81:5821–6.

    Article  CAS  PubMed  Google Scholar 

  20. Feng Q-M, Pan J-B, Zhang H-R, Xu J-J, Chen H-Y. Disposable paper-based bipolar electrode for sensitive electrochemiluminescence detection of a cancer biomarker. Chem Commun. 2014;50:10949–51.

    Article  CAS  Google Scholar 

  21. Chen X, Luo Y, Shi B, Liu X, Gao Z, Du Y, et al. Chemiluminescence diminishment on a paper-based analytical device: high throughput determination of β-agonists in swine hair. Anal Methods. 2014;6:9684–90.

    Article  CAS  Google Scholar 

  22. Yamada K, Henares TG, Suzuki K, Citterio D. Distance-based tear lactoferrin assay on microfluidic paper device using interfacial interactions on surface-modified cellulose. ACS Appl Mater Interfaces. 2015;7:24864–75.

    Article  CAS  PubMed  Google Scholar 

  23. Martinez AW, Phillips ST, Carrilho E, Thomas SW, Sindi H, Whitesides GM. Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis. Anal Chem. 2008;80:3699–707.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Morbioli GG, Mazzu-Nascimento T, Stockton AM, Carrilho E. Technical aspects and challenges of colorimetric detection with microfluidic paper-based analytical devices (μpads)- a review. Anal Chim Acta. 2017;970:1–22.

    Article  CAS  PubMed  Google Scholar 

  25. Mazzu-Nascimento T, Gomes Carneiro Leão PA, Catai JR, Morbioli GG, Carrilho E. Towards low-cost bioanalytical tools for sarcosine assays for cancer diagnostics. Anal Methods. 2016;8:7312–8.

    Article  CAS  Google Scholar 

  26. de Tarso GP, Garcia Cardoso TM, Garcia CD, Carrilho E, Tomazelli Coltro WK. A handheld stamping process to fabricate microfluidic paper-based analytical devices with chemically modified surface for clinical assays. RSC Adv. 2014;4:37637–44.

    Article  Google Scholar 

  27. Parween S, Asthana A. An affordable, rapid determination of total lipid profile using paper-based microfluidic device. Sensors Actuators B Chem. 2019;285:405–12.

    Article  CAS  Google Scholar 

  28. Evans E, Moreira Gabriel EF, Benavidez TE, Tomazelli Coltro WK, Garcia CD. Modification of microfluidic paper-based devices with silica nanoparticles. Analyst. 2014;139:5560–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wang S, Ge L, Song X, Yu J, Ge S, Huang J, et al. Paper-based chemiluminescence elisa: lab-on-paper based on chitosan modified paper device and wax-screen-printing. Biosens Bioelectron. 2012;31:212–8.

    Article  PubMed  Google Scholar 

  30. Gabriel EFM, Garcia PT, Cardoso TMG, Lopes FM, Martins FT, Coltro WKT. Highly sensitive colorimetric detection of glucose and uric acid in biological fluids using chitosan-modified paper microfluidic devices. Analyst. 2016;141:4749–56.

    Article  CAS  PubMed  Google Scholar 

  31. Kudo H, Yamada K, Watanabe D, Suzuki K, Citterio D. Paper-based analytical device for zinc ion quantification in water samples with power-free analyte concentration. Micromachines. 2017;8:127.

    Article  PubMed Central  Google Scholar 

  32. Ota R, Yamada K, Suzuki K, Citterio D. Quantitative evaluation of analyte transport on microfluidic paper-based analytical devices (μPADs). Analyst. 2018;143:643–53.

    Article  CAS  PubMed  Google Scholar 

  33. Liu S, Cao R, Wu J, Guan L, Li M, Liu J, et al. Directly writing barrier-free patterned biosensors and bioassays on paper for low-cost diagnostics. Sensors Actuators B Chem. 2019;285:529–35.

    Article  CAS  Google Scholar 

  34. Alila S, Boufi S, Belgacem MN, Beneventi D. Adsorption of a cationic surfactant onto cellulosic fibers I. Surface charge effects. Langmuir. 2005;21:8106–13.

    Article  CAS  PubMed  Google Scholar 

  35. Carrilho E, Martinez AW, Whitesides GM. Understanding wax printing: a simple micropatterning process for paper-based microfluidics. Anal Chem. 2009;81:7091–5.

    Article  CAS  PubMed  Google Scholar 

  36. Lu Y, Shi W, Jiang L, Qin J, Lin B. Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay. Electrophoresis. 2009;30:1497–500.

    Article  CAS  PubMed  Google Scholar 

  37. Brooks T, Keevil CW. A simple artificial urine for the growth of urinary pathogens. Lett Appl Microbiol. 1997;24:203–6.

    Article  CAS  PubMed  Google Scholar 

  38. Porstmann T, Kiessig ST. Enzyme immunoassay techniques an overview. J Immunol Methods. 1992;150:5–21.

    Article  CAS  PubMed  Google Scholar 

  39. Harpaz D, Eltzov E, Ng TSE, Marks RS, Tok AIY. Enhanced colorimetric signal for accurate signal detection in paper-based biosensors. Diagnostics. 2020;10:28.

    Article  CAS  PubMed Central  Google Scholar 

  40. Zhang X, Yang Q, Lang Y, Jiang X, Wu P. Rationale of 3,3’,5,5’-tetramethylbenzidine as the chromogenic substrate in colorimetric analysis. Anal Chem. 2020;92:12400–6.

    Article  CAS  PubMed  Google Scholar 

  41. Currie LA. Limits for qualitative detection and quantitative determination. Application to radiochemistry. Anal Chem. 1968;40:586–93.

    Article  CAS  Google Scholar 

  42. Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29(Suppl 1):S49–52.

    PubMed  PubMed Central  Google Scholar 

  43. Suzuki M. Purification and some properties of sarcosine oxidase from Corynebacterium sp. U-96. J Biochem. 1981;89:599–607.

    Article  CAS  PubMed  Google Scholar 

  44. Katoh A, Maejima K, Hiruta Y, Citterio D. All-printed semiquantitative paper-based analytical devices relying on QR code array readout. Analyst. 2020;145:6071–8.

    Article  CAS  PubMed  Google Scholar 

  45. Zhou W, Sun J, Li X. Low-cost quantitative photothermal genetic detection of pathogens on a paper hybrid device using a thermometer. Anal Chem. 2020;92:14830–7.

    Article  CAS  PubMed  Google Scholar 

  46. Chattopadhyay K, Mazumdar S. Structural and conformational stability of horseradish peroxidase: effect of temperature and pH. Biochemistry. 2000;39:263–70.

    Article  CAS  PubMed  Google Scholar 

  47. Saito M, Itoh A, Suzuki H. Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme–substrate complex is a reactive species. J Biochem. 2012;151:633–42.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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

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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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