Salivary diagnostics on paper microfluidic devices and their use as wearable sensors for glucose monitoring

  • Lucas F. de Castro
  • Soraia V. de Freitas
  • Lucas C. Duarte
  • João Antônio C. de Souza
  • Thiago R. L. C. Paixão
  • Wendell K. T. ColtroEmail author
Research Paper
Part of the following topical collections:
  1. Young Investigators in (Bio-)Analytical Chemistry


Microfluidic paper-based devices (μPADs) and wearable devices have been highly studied to be used as diagnostic tools due to their advantages such as simplicity and ability to provide instrument-free fast results. Diseases such as periodontitis and diabetes mellitus can potentially be detected through these devices by the detection of important biomarkers. This study describes the development of μPADs through craft cutter printing for glucose and nitrite salivary diagnostics. In addition, the use of μPADs integrated into a mouthguard as a wearable sensor for glucose monitoring is also presented. μPADs were designed to contain two detection zones for glucose and nitrite assays and a sampling zone interconnected by microfluidic channels. Initially, the analytical performance of the proposed μPADs was investigated and it provided linear behavior (r2 ≥ 0.994) in the concentration ranges between 0 to 2.0 mmol L−1 and 0 to 400 μmol L−1 for glucose and nitrite, respectively. Under the optimized conditions, the limits of detection achieved for glucose and nitrite were 27 μmol L−1 and 7 μmol L−1, respectively. Human saliva samples were collected from healthy individuals and patients previously diagnosed with periodontitis or diabetes and then analyzed on the proposed μPADs. The results found using μPADs revealed higher glucose concentration values in saliva collected from patients diagnosed with diabetes mellitus and greater nitrite concentrations in saliva collected from patients diagnosed with periodontitis, as expected. The results obtained on μPADs did not differ statistically from those measured by spectrophotometry. With the aim of developing paper-based wearable sensors, μPADs were integrated, for the first time, into a silicone mouthguard using a 3D-printed holder. The proof of concept was successfully demonstrated through the monitoring of the glucose concentration in saliva after the ingestion of chocolate. According to the results reported herein, paper-based microfluidic devices offer great potential for salivary diagnostics, making their integration into a silicone mouthguard possible, generating simple, low-cost, instrument-free, and powerful wearable sensors.


Clinical diagnostics Diabetes mellitus Instrument-free portable sensors Periodontitis Microfluidic paper-based analytical devices 



This study was supported by CNPq (grants 426496/2018-3 and 308140/2016-8), CAPES (grant 3363/2014), INCTBio (grant 465389/2014-7), and FAPESP (2017/10522-5). CNPq and CAPES are also thanked for the scholarships granted to L.F.C., S.F.V., and L.C.D.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflict of interest.

Supplementary material

216_2019_1788_MOESM1_ESM.pdf (1.9 mb)
ESM 1 (PDF 1923 kb)


  1. 1.
    Klasner SA, Price AK, Hoeman KW, Wilson RS, Bell KJ, Culbertson CT. Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva. Anal Bioanal Chem. 2010;397:1821–9. Scholar
  2. 2.
    Choi S, Kim SK, Lee GJ, Park HK. Paper-based 3D microfluidic device for multiple bioassays. Sensors Actuators B Chem. 2015;219:245–50. Scholar
  3. 3.
    Martinez AW, Phillips ST, Butte MJ, Whitesides GM. NIH public access. Angew Chem Int Ed Eng. 2007;46:1318–20. Scholar
  4. 4.
    de Oliveira RAG, Camargo F, Pesquero NC, Faria RC. A simple method to produce 2D and 3D microfluidic paper-based analytical devices for clinical analysis. Anal Chim Acta. 2017;957:40–6. Scholar
  5. 5.
    Li X, Ballerini DR, Shen W. A perspective on paper-based microfluidics: current status and future trends. Biomicrofluidics. 2012;6.Google Scholar
  6. 6.
    Park TS, Li W, McCracken KE, Yoon J-Y. Smartphone quantifies Salmonella from paper microfluidics. Lab Chip. 2013;13:4832–40. Scholar
  7. 7.
    Lopez-Ruiz N, Curto VF, Erenas MM, Benito-Lopez F, Diamond D, Palma AJ, et al. Smartphone-based simultaneous pH and nitrite colorimetric determination for paper microfluidic devices. Anal Chem. 2014;86:9554–62. Scholar
  8. 8.
    Martinez AW, Phillips ST, Carrilho E, Iii SWT, 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. Scholar
  9. 9.
    Hu J, Wang SQ, Wang L, Li F, Pingguan-Murphy B, Lu TJ, et al. Advances in paper-based point-of-care diagnostics. Biosens Bioelectron. 2014;54:585–97. Scholar
  10. 10.
    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. Scholar
  11. 11.
    Tomazelli Coltro WK, Cheng CM, Carrilho E, de Jesus DP. Recent advances in low-cost microfluidic platforms for diagnostic applications. Electrophoresis. 2014;35:2309–24. Scholar
  12. 12.
    Yetisen AK, Martinez-Hurtado JL, Ünal B, Khademhosseini A, Butt H. Wearables in medicine. Adv Mater. 2018;30.
  13. 13.
    O’Donoghue J, O’Connor KA, O’Donovan T, O’Reilly P, Sammon D, Sreenan C (2012) A context aware wireless body area network (BAN). doi:
  14. 14.
    O’Donoghue J, Herbert J. Data management within mHealth environments. J Data Inf Qual. 2012;4:1–20. Scholar
  15. 15.
    Mukhopadhyay SC. Wearable sensors for human activity monitoring: a review. IEEE Sensors J. 2015;15:1321–30. Scholar
  16. 16.
    Kwak YH, Kim W, Park KB, Kim K, Seo S. Flexible heartbeat sensor for wearable device. Biosens Bioelectron. 2017;94:250–5. Scholar
  17. 17.
    Costa J, Adams AT, Jung MF, Guimbretiere F, Choudhury T. EmotionCheck: a wearable device to regulate anxiety through false heart rate feedback. Mob Comput Commun Rev. 2017;21:22–5. Scholar
  18. 18.
    Yilmaz T, Foster R, Hao Y. Detecting vital signs with wearable wireless sensors. Sensors. 2010;10:10837–62. Scholar
  19. 19.
    Gong MM, Sinton D. Turning the page: advancing paper-based microfluidics for broad diagnostic application. Chem Rev. 2017;117:8447–80. Scholar
  20. 20.
    Vella SJ, Beattie P, Cademartiri R, Laromaine A, Martinez AW, Phillips ST, et al. Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick. Anal Chem. 2012;84:2883–91. Scholar
  21. 21.
    Yang X, Forouzan O, Brown TP, Shevkoplyas SS. Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices. Lab Chip. 2012;12:274–80. Scholar
  22. 22.
    Tseng CC, Yang RJ, Ju WJ, Fu LM. Microfluidic paper-based platform for whole blood creatinine detection. Chem Eng J. 2018;348:117–24. Scholar
  23. 23.
    Rossini EL, Milani MI, Carrilho E, Pezza L, Pezza HR. Simultaneous determination of renal function biomarkers in urine using a validated paper-based microfluidic analytical device. Anal Chim Acta. 2018;997:16–23. Scholar
  24. 24.
    Farandos NM, Yetisen AK, Monteiro MJ, Lowe CR, Yun SH. Contact lens sensors in ocular diagnostics. Adv Healthc Mater. 2015;4:792–810. Scholar
  25. 25.
    Munje RD, Muthukumar S, Prasad S. Lancet-free and label-free diagnostics of glucose in sweat using zinc oxide based flexible bioelectronics. Sensors Actuators B Chem. 2017;238:482–90. Scholar
  26. 26.
    Lee H, Song C, Hong YS, Kim MS, Cho HR, Kang T, et al. Wearable/disposable sweat-based glucose monitoring device with multistage transdermal drug delivery module. Sci Adv. 2017;3:1–9. Scholar
  27. 27.
    Bhakta SA, Borba R, Taba M Jr, Garcia CD, Carrilho E. Determination of nitrite in saliva using microfluidic paper-based analytical devices. Anal Chim Acta. 2014;809:117–22. Scholar
  28. 28.
    Villiger M, Stoop R, Vetsch T, Hohenauer E, Pini M, Clarys P, et al. Evaluation and review of body fluids saliva, sweat and tear compared to biochemical hydration assessment markers within blood and urine. Eur J Clin Nutr. 2018;72:69–76. Scholar
  29. 29.
    Liu J, Duan Y. Saliva: a potential media for disease diagnostics and monitoring. Oral Oncol. 2012;48:569–77. Scholar
  30. 30.
    Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human periodontal disease. Oral Dis. 2002;8:254–60. Scholar
  31. 31.
    Bejeh-mir AP, Parsian H, Khoram MA, Ghasemi N, Bijani A, Khosravi-samani M, et al. Diagnostic role of salivary and GCF nitrite, nitrate and nitric oxide to distinguish healthy periodontium from gingivitis and periodontitis. Int J Mol Cell Med. 2014;3:138–45.Google Scholar
  32. 32.
    Gupta S, Sandhu SV, Bansal H, Sharma D. Comparison of salivary and serum glucose levels in diabetic patients. J Diabetes Sci Technol. 2015;9:91–6. Scholar
  33. 33.
    Yang K, Peretz-Soroka H, Liu Y, Lin F. Novel developments in mobile sensing based on the integration of microfluidic devices and smartphones. Lab Chip. 2016;16:943–58. Scholar
  34. 34.
    Griess P. Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt ???Ueber einige Azoverbindungen??? Ber Dtsch Chem Ges. 1879;12:426–8. Scholar
  35. 35.
    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. Scholar
  36. 36.
    De Freitas SV, De Souza FR, Rodrigues Neto JC, Vasconcelos GA, Abdelnur PV, Vaz BG, et al. Uncovering the formation of color gradients for glucose colorimetric assays on microfluidic paper-based analytical devices by mass spectrometry imaging. Anal Chem. 2018;90:11949–54. Scholar
  37. 37.
    Demirel G, Babur E. Vapor-phase deposition of polymers as a simple and versatile technique to generate paper-based microfluidic platforms for bioassay applications. Analyst. 2014;139:2326–31. Scholar
  38. 38.
    Yetisen AK, Martinez-Hurtado JL, Garcia-Melendrez A, Da Cruz Vasconcellos F, Lowe CR. A smartphone algorithm with inter-phone repeatability for the analysis of colorimetric tests. Sensors Actuators B Chem. 2014;196:156–60. Scholar
  39. 39.
    Chun HJ, Park YM, Han YD, Jang YH, Yoon HC. Paper-based glucose biosensing system utilizing a smartphone as a signal reader. Biochip J. 2014;8:218–26. Scholar
  40. 40.
    Zhu WJ, Feng DQ, Chen M, Chen ZD, Zhu R, Fang HL, et al. Bienzyme colorimetric detection of glucose with self-calibration based on tree-shaped paper strip. Sensors Actuators B Chem. 2014;190:414–8. Scholar
  41. 41.
    Xiao L, Liu X, Zhong R, Zhang K, Zhang X, Zhou X, et al. A rapid, straightforward, and print house compatible mass fabrication method for integrating 3D paper-based microfluidics. Electrophoresis. 2013;34:3003–7. Scholar
  42. 42.
    Jayawardane BM, Wei S, McKelvie ID, Kolev SD. Microfluidic paper-based analytical device for the determination of nitrite and nitrate. Anal Chem. 2014;86:7274–9. Scholar
  43. 43.
    Li B, Fu L, Zhang W, Feng W, Chen L. Portable paper-based device for quantitative colorimetric assays relying on light reflectance principle. Electrophoresis. 2014;35:1152–9. Scholar
  44. 44.
    Miller JC 21202653-Miller-M-James-Statistics-and-Chemometrics-for-Analytical-Chemistry-5th-Ed.pdf.Google Scholar
  45. 45.
    Satish BNVS, Srikala P, Maharudrappa B, Awanti SM, Prashant Kumar DH. Saliva : a tool in assessing glucose levels in diabetes mellitus. J Int Oral Heal. 2014;6:114–7.Google Scholar
  46. 46.
    RP A. Noninvasive method for glucose level estimation by saliva. J Diabetes Metab. 2013;04.
  47. 47.
    Zhang W, Du Y, Wang ML. Noninvasive glucose monitoring using saliva nano-biosensor. Sens Bio-Sensing Res. 2015;4:23–9. Scholar
  48. 48.
    Noiphung J, Nguyen MP, Punyadeera C, Wan Y, Laiwattanapaisal W, Henry CS. Development of paper-based analytical devices for minimizing the viscosity effect in human saliva. Theranostics. 2018;8:3797–807. Scholar
  49. 49.
    Sánchez GA, Miozza VA, Delgado A, Busch L. Total salivary nitrates and nitrites in oral health and periodontal disease. Nitric Oxide Biol Chem. 2014;36:31–5. Scholar
  50. 50.
    Rajkumar D, Gokulanathan S, Shanmugasundaram N, Lakshmigandhan M, Kavin T. Diabetes and periodontal disease. Dent Sci. 2012;4:280–2. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Lucas F. de Castro
    • 1
  • Soraia V. de Freitas
    • 1
  • Lucas C. Duarte
    • 1
  • João Antônio C. de Souza
    • 2
  • Thiago R. L. C. Paixão
    • 3
    • 4
  • Wendell K. T. Coltro
    • 1
    • 3
    Email author
  1. 1.Instituto de QuímicaUniversidade Federal de GoiásGoiâniaBrazil
  2. 2.Faculdade de OdontologiaUniversidade Federal de GoiásGoiâniaBrazil
  3. 3.Instituto Nacional de Ciência e Tecnologia de BioanalíticaCampinasBrazil
  4. 4.Instituto de QuímicaUniversidade de São PauloSão PauloBrazil

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