Analytical and Bioanalytical Chemistry

, Volume 403, Issue 6, pp 1567–1576 | Cite as

Multiplexed paper test strip for quantitative bacterial detection

  • S. M. Zakir Hossain
  • Cory Ozimok
  • Clémence Sicard
  • Sergio D. Aguirre
  • M. Monsur Ali
  • Yingfu Li
  • John D. BrennanEmail author
Original Paper


Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (β-galactosidase (B-GAL) or β-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-β-d-glucuronide sodium salt (XG), chlorophenol red β-galactopyranoside (CPRG) or both and FeCl3 were entrapped using sol–gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl3 zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.


Pathogen Sensing Paper: Paper strips with ink-jet printed sensing zones can detect low levels of pathogenic or non-pathogenic bacteria. Incorporation of an immunomagnetic separation step results in selective detection of ~25 cfu of H7:O157 bacteria in under 1 h.


Bacteria detection Bioactive paper sensor Colorimetric 





Color intensity


Colony-forming units


Chlorophenol red β-galactopyranoside

E. coli

Escherichia coli




Hydrophobic barrier


Immunomagnetic separation


Limit of detection


Antibody-derivatized magnetic beads




5-Bromo-4-chloro-3-indolyl-β-d-glucuronide sodium salt



The authors thank the Natural Sciences and Engineering Research Council of Canada and the Sentinel-Canadian Network for the Development and Use of Bioactive Paper for funding. The authors also thank the Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation for support of this work. The authors thank Puneet Chavan for his contribution to experimental data. YL holds the Canada Research Chair in Nucleic Acids Chemistry. JDB holds the Canada Research Chair in Bioanalytical Chemistry and Biointerfaces.

Supplementary material

216_2012_5975_MOESM1_ESM.pdf (210 kb)
ESM 1 (PDF 209 kb)


  1. 1.
    World Health Organization (2007) Food safety and foodborne illness. Accessed on March 20, 2012
  2. 2.
    Edberg SC, Rice EW, Karlin RJ, Allen MJ (2000) J Appl Microbiol 88:106S–116SGoogle Scholar
  3. 3.
    Ho J-AA, Hsu H-W (2003) Anal Chem 75:4330–4334CrossRefGoogle Scholar
  4. 4.
    Lee W, Park K-S, Kim Y-W, Lee WH, Choi J-W (2005) Biosen Bioelectron 20:2292–2299CrossRefGoogle Scholar
  5. 5.
    Pal S, Alocilja EC, Downes FP (2007) Biosen Bioelectron 22:2329–2336CrossRefGoogle Scholar
  6. 6.
    Waswa J, Irudayaraj J, Debroy C (2007) LWT - Food Sci Technol 40:187–192CrossRefGoogle Scholar
  7. 7.
    Huang C-J, Dostalek J, Sessitsch A, Knoll W (2011) Anal Chem 83:674–677CrossRefGoogle Scholar
  8. 8.
    Wang L-J, Wu C-S, Hu Z-Y, Zhang Y-F, Li R, Wang P (2008) J Zhejiang Univ Sci B 9:121–131CrossRefGoogle Scholar
  9. 9.
    Seo S, Kim HC, Cheng M, Ruan X, Ruan W (2006) J Vac Sci Technol B 24:3133CrossRefGoogle Scholar
  10. 10.
    Hahn Ma, Tabb JS, Krauss TD (2005) Anal Chem 77:4861–4869CrossRefGoogle Scholar
  11. 11.
    Kalele SA, Kundu AA, Gosavi SW, Deobagkar DN, Deobagkar DD, Kulkarni SK (2006) Small 2:335–338CrossRefGoogle Scholar
  12. 12.
    Zhao X, Hilliard LR, Mechery SJ, Wang Y, Bagwe RP, Jin S, Tan W (2004) Proc Nat Acad Sci U S A 101:15027–15032CrossRefGoogle Scholar
  13. 13.
    Heo J, Hua SZ (2009) Sensors 9:4483–4502CrossRefGoogle Scholar
  14. 14.
    Yeung S-W, Lee TM-H, Cai H, Hsing I-M (2006) Nucl Acids Res 34:e118CrossRefGoogle Scholar
  15. 15.
    Lee H-J, Kim BC, Kim K-W, Kim YK, Kim J, Oh M-K (2009) Biosen Bioelectron 24:3550–3555CrossRefGoogle Scholar
  16. 16.
    Tu S-I, Reed S, Gehring A, He Y, Paoli G (2009) Sensors 9:717–730CrossRefGoogle Scholar
  17. 17.
    Chapman PA (2000) World J Microbiol Biotechnol 16:733–740CrossRefGoogle Scholar
  18. 18.
    Guan J, Levin RE (2002) Food Biotechnol 16:135–144CrossRefGoogle Scholar
  19. 19.
    Miranda OR, Li X, Garcia-Gonzalez L, Zhu Z-J, Yan B, Bunz UHF, Rotello VM (2011) J Am Chem Soc 133:9650–9653CrossRefGoogle Scholar
  20. 20.
    Ngom B, Guo Y, Wang X, Bi (2010) Anal Bioanal Chem 397:1113–1135CrossRefGoogle Scholar
  21. 21.
    Abe K, Suzuki K, Citterio D (2008) Anal Chem 80:6928–6934CrossRefGoogle Scholar
  22. 22.
    Zhao W, Ali MM, Aguirre SD, Brook MA, Li Y (2008) Anal Chem 80:8431–8437CrossRefGoogle Scholar
  23. 23.
    Martinez AW, Phillips ST, Whitesides GM, Carrilho E (2010) Anal Chem 82:3–10CrossRefGoogle Scholar
  24. 24.
    Apilux A, Dungchai W, Siangproh W, Praphairaksit N, Henry CS, Chailapakul O (2010) Anal Chem 82:1727–1732CrossRefGoogle Scholar
  25. 25.
    Luckham RE, Brennan JD (2010) Analyst 135:2028–2035CrossRefGoogle Scholar
  26. 26.
    Khan MS, Thouas G, Shen W, Whyte G, Garnier G (2010) Anal Chem 82:4158–4164CrossRefGoogle Scholar
  27. 27.
    Wutor VC, Togo CA, Pletschke BI (2009) WaterSA 35:85–88Google Scholar
  28. 28.
    Kilian M, Bülow P (1976) Acta Patholo Microbiol Scandi B 84:245–251Google Scholar
  29. 29.
    Ratnam S, March SB, Ahmed R, Bezanson GS, Kasatiya S (1988) J Clin Microbiol 26:2006–2012Google Scholar
  30. 30.
    Ali MM, Aguirre SD, Lazim H, Li Y (2011) Angew Chem Int Ed 50:3751–3754CrossRefGoogle Scholar
  31. 31.
    Hossain SMZ, Luckham RE, Smith AM, Lebert JM, Davies LM, Pelton RH, Filipe CDM, Brennan JD (2009) Anal Chem 81:5474–5483CrossRefGoogle Scholar
  32. 32.
    Hossain SMZ, Luckham RE, McFadden MJ, Brennan JD (2009) Anal Chem 81:9055–9064CrossRefGoogle Scholar
  33. 33.
    Delisle GJ, Ley A (1989) J Clin Microbiol 27:778–779Google Scholar
  34. 34.
    Wutor VC, Togo CA, Limson JL, Pletschke BI (2007) Enzyme Microb Technol 40:1512–1517CrossRefGoogle Scholar
  35. 35.
    Wutor VC, Togo CA, Pletschke BI (2007) Chemosphere 68:622–627CrossRefGoogle Scholar
  36. 36.
    Cotson S, Holt SJ (1958) Proc R Soc Lond B 148:506–519CrossRefGoogle Scholar
  37. 37.
    Kim D-H, Jin Y-H, Jung E-A, Han MJ, Kobashi K (1995) Biol Pharm Bull 18:1184–1188CrossRefGoogle Scholar
  38. 38.
    Buehler HJ, Katzman PA, Doisy EA (1951) Proc Soc Exp Biol Med 76:672–676Google Scholar
  39. 39.
    Tenu J-P, Viratelle OM, Garnier J, Yon J (1971) Eur J Biochem 20:363–370CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • S. M. Zakir Hossain
    • 1
  • Cory Ozimok
    • 1
  • Clémence Sicard
    • 1
  • Sergio D. Aguirre
    • 2
  • M. Monsur Ali
    • 2
  • Yingfu Li
    • 2
  • John D. Brennan
    • 1
    Email author
  1. 1.Department of Chemistry and Chemical BiologyMcMaster UniversityHamiltonCanada
  2. 2.Department of Biochemistry and Biomedical SciencesMcMaster UniversityHamiltonCanada

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