Analytical and Bioanalytical Chemistry

, Volume 408, Issue 8, pp 2083–2093 | Cite as

Fluorescent molecularly imprinted polymer based on Navicula sp. frustules for optical detection of lysozyme

  • Guat Wei Lim
  • Jit Kang Lim
  • Abdul Latif Ahmad
  • Derek Juinn Chieh ChanEmail author
Research Paper


The direct correlation between disease and lysozyme (LYZ) levels in human body fluids makes the sensitive and convenient detection of LYZ the focus of scientific research. Fluorescent molecularly imprinted polymer has emerged as a new alternative for LYZ detection in order to resolve the limitation of immunoassays, which are expensive, unstable, require complex preparation, and are time consuming. In this study, a novel fluorescence molecularly imprinted polymer based on Navicula sp. frustules (FITC-MIP) has been synthesized via post-imprinting treatment for LYZ detection. Navicula sp. frustules were used as supported material because of their unique properties of moderate surface area, reproducibility, and biocompatibility, to address the drawbacks of nanoparticle core material with low adsorption capacity. The FITC acts as recognition signal and optical readout, whereas MIP provides LYZ selectivity. The synthesized FITC-MIP showed a response time as short as 5 min depending on the concentration of LYZ. It is found that the LYZ template can significantly quench the fluorescence intensity of FITC-MIP linearly within a concentration range of 0 to 0.025 mg mL–1, which is well described by Stern-Volmer equation. The FITC-MIP can selectively and sensitively detect down to 0.0015 mg mL–1 of LYZ concentration. The excellent sensing performance of FITC-MIP suggests that FITC-MIP is a potential biosensor in clinical diagnosis applications.


Fluorescent Molecularly imprinted polymer Navicula sp. frustules FITC Lysozyme recognition 



This work is supported by Postgraduate Research Grant Scheme (grant no. 8046003), FRGS grant (6071271), and Research University Grant (814209). G.W.L. is financially assisted by MyPhD scholarship from the Ministry of Higher Education of Malaysia. All authors are affiliated with Membrane Science and Technology cluster USM.

Compliance with ethical standards

Conflict of interest

The authors declare that there have no conflicts of interest.

Supplementary material

216_2015_9298_MOESM1_ESM.pdf (247 kb)
ESM 1 (PDF 246 kb)


  1. 1.
    Akinalp AS, Asan M, Ozcan N. Expression of T4 lysozyme gene (gene e) in Streptococcus salivarius subsp. Thermophilus. Afr J Biotechnol. 2007;6:963–6.Google Scholar
  2. 2.
    Li S, Mulloor JJ, Wang L, Ji Y, Mulloor CJ, Micic M, et al. Strong and selective adsorption of lysozyme on graphene oxide. ACS Appl Mater Interfaces. 2014;6:5704–12.CrossRefGoogle Scholar
  3. 3.
    Ou SH, Wu MC, Chou TC, Liu CC. Polyacrylamide gels with electrostatic functional groups for the molecular imprinting of lysozyme. Anal Chim Acta. 2004;504:163–6.CrossRefGoogle Scholar
  4. 4.
    Pascual R, Gee B, Finch S. Usefulness of serum lysozyme analysis in diagnosis and evaluation of sarcoidosis. N Engl J Med. 1973;289:1074–6.CrossRefGoogle Scholar
  5. 5.
    Perillie PE, Kaplan SS, Lefkowitz E, Rogaway W, Finch SC. Studies of muramidase (lysozyme) in leukemia. J Am Med Assoc. 1968;203:317–22.CrossRefGoogle Scholar
  6. 6.
    Osserman EF, Lawlor DP. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med. 1966;124:921–52.CrossRefGoogle Scholar
  7. 7.
    Prockop DJ, Davidson WD. A study of urinary and serum lysozyme in patients with renal disease. N Engl J Med. 1964;270:269–74.CrossRefGoogle Scholar
  8. 8.
    Fleming A. On a remarkable bacteriolytic element found in tissues and secretions. Proc R Soc Lond Ser B. 1922;93:306–17.CrossRefGoogle Scholar
  9. 9.
    Newman J, Cacatian A, Josephson A, Tsang A. Spinal-fluid lysozyme in the diagnosis of central-nervous-system tumours. Lancet. 1974;304:756–7.CrossRefGoogle Scholar
  10. 10.
    Ding Z, Annie Bligh SW, Tao L, Quan J, Nie H, Zhu L, et al. Molecularly imprinted polymer based on MWCNT-QDs as fluorescent biomimetic sensor for specific recognition of target protein. Mater Sci Eng C. 2015;48:469–79.CrossRefGoogle Scholar
  11. 11.
    Rachkov A, McNiven S, El’skaya A, Yano K, Karube I. Fluorescence detection of β-estradiol using a molecularly imprinted polymer. Anal Chim Acta. 2000;405:23–9.CrossRefGoogle Scholar
  12. 12.
    Zourob M. Recognition receptors in biosensors. New York: Springer Science Business Media; 2010.CrossRefGoogle Scholar
  13. 13.
    Garcia R, Cabrita MJ, Costa Freitas AM. Application of molecularly imprinted polymers for the analysis of pesticide residues in food-A highly selective and innovative approach. Am J Anal Chem. 2011;2:16–25.CrossRefGoogle Scholar
  14. 14.
    Fu G, He H, Chai Z, Chen H, Kong J, Wang Y, et al. Enhanced lysozyme imprinting over nanoparticles functionalized with carboxyl groups for noncovalent template sorption. Anal Chem. 2011;83:1431–6.CrossRefGoogle Scholar
  15. 15.
    He H, Fu G, Wang Y, Chai Z, Jiang Y, Chen Z. Imprinting of protein over silica nanoparticles via surface graft copolymerization using low monomer concentration. Biosens Bioelectron. 2010;26:760–5.CrossRefGoogle Scholar
  16. 16.
    Qian L, Hu X, Guan P, Wang D, Li J, Du C, et al. The effectively specific recognition of bovine serum albumin imprinted silica nanoparticles by utilizing a macromolecularly functional monomer to stabilize and imprint template. Anal Chim Acta. 2015;884:97–105.CrossRefGoogle Scholar
  17. 17.
    Chen H, Yuan D, Li Y, Dong M, Chai Z, Kong J, et al. Silica nanoparticle supported molecularly imprinted polymer layers with varied degrees of crosslinking for lysozyme recognition. Anal Chim Acta. 2013;779:82–9.CrossRefGoogle Scholar
  18. 18.
    Liu D, Yang Q, Jin S, Song Y, Gao J, Wang Y, et al. Core–shell molecularly imprinted polymer nanoparticles with assistant recognition polymer chains for effective recognition and enrichment of natural low-abundance protein. Acta Biomater. 2014;10:769–75.CrossRefGoogle Scholar
  19. 19.
    Gai Q-Q, Qu F, Liu Z-J, Dai R-J, Zhang Y-K. Superparamagnetic lysozyme surface-imprinted polymer prepared by atom transfer radical polymerization and its application for protein separation. J Chromatogr A. 2010;1217:5035–42.CrossRefGoogle Scholar
  20. 20.
    Jia X, Xu M, Wang Y, Ran D, Yang S, Zhang M. Polydopamine-based molecular imprinting on silica-modified magnetic nanoparticles for recognition and separation of bovine hemoglobin. Analyst. 2013;138:651–8.CrossRefGoogle Scholar
  21. 21.
    Lee M-H, Thomas JL, Ho M-H, Yuan C, Lin H-Y. Synthesis of magnetic molecularly imprinted poly(ethylene-co-vinyl alcohol) nanoparticles and their uses in the extraction and sensing of target molecules in urine. ACS Appl Mater Interfaces. 2010;2:1729–36.CrossRefGoogle Scholar
  22. 22.
    Li L, He X, Chen L, Zhang Y. Preparation of core-shell magnetic molecularly imprinted polymer nanoparticles for recognition of bovine hemoglobin. Chem Asian J. 2009;4:286–93.CrossRefGoogle Scholar
  23. 23.
    Li Y, Yang H-H, You Q-H, Zhuang Z-X, Wang X-R. Protein recognition via surface molecularly imprinted polymer nanowires. Anal Chem. 2006;78:317–20.CrossRefGoogle Scholar
  24. 24.
    Wang J, Gao L, Han D, Pan J, Qiu H, Li H, et al. Optical detection of λ-cyhalothrin by core-shell fluorescent molecularly imprinted polymers in chinese spirits. J Agric Food Chem. 2015;63:2392–9.CrossRefGoogle Scholar
  25. 25.
    Sunayama H, Ooya T, Takeuchi T. Fluorescent protein recognition polymer thin films capable of selective signal transduction of target binding events prepared by molecular imprinting with a post-imprinting treatment. Biosens Bioelectron. 2010;26:458–62.CrossRefGoogle Scholar
  26. 26.
    Zhao Y, Ma Y, Li H, Wang L. Composite QDs@MIP nanospheres for specific recognition and direct fluorescent quantification of pesticides in aqueous media. Anal Chem. 2012;84:386–95.CrossRefGoogle Scholar
  27. 27.
    Wu X, Zhang Z, Li J, You H, Li Y, Chen L. Molecularly imprinted polymers-coated gold nanoclusters for fluorescent detection of bisphenol A. Sensors Actuators B: Chem. 2015;211:507–14.CrossRefGoogle Scholar
  28. 28.
    Campbell PA, Canono BP, Drevets DA (2001) Measurement of bacterial ingestion and killing by macrophages. Current protocols in immunology. Hoboken NJ: John Wiley and Sons, Inc.; 2001.Google Scholar
  29. 29.
    Grunberg E, Cleeland R. Fluorescence and viability of proteus mirabilis stained directly with fluorescein isothiocyanate. J Bacteriol. 1966;92:23–7.Google Scholar
  30. 30.
    Miller JS, Quarles JM. Flow cytometric identification of microorganisms by dual staining with FITC and PI. Cytometry. 1990;11:667–75.CrossRefGoogle Scholar
  31. 31.
    Kelly KA, Reynolds F, Weissleder R, Josephson L. Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions. Anal Biochem. 2004;330:181–5.CrossRefGoogle Scholar
  32. 32.
    McClatchey KD. Clinical laboratory medicine. Philadelphia, PA: Lippincott Wiliams and Wilkins; 2002.Google Scholar
  33. 33.
    Huang J, Liu H, Men H, Zhai Y, Xi Q, Zhang Z, et al. Molecularly imprinted polymer coating with fluorescence on magnetic particle. Macromol Res. 2013;21:1021–8.CrossRefGoogle Scholar
  34. 34.
    Chen L, Li J, Wang S, Lu W, Wu A, Choo J, et al. FITC functionalized magnetic core-shell Fe3O4/Ag hybrid nanoparticle for selective determination of molecular biothiols. Sensors Actuators B Chem. 2014;193:857–63.CrossRefGoogle Scholar
  35. 35.
    Feng H, Wang N, Tran TT, Yuan L, Li J, Cai Q. Surface molecular imprinting on dye-(NH2)-SiO2 NPs for specific recognition and direct fluorescent quantification of perfluorooctane sulfonate. Sensors Actuators B Chem. 2014;195:266–73.CrossRefGoogle Scholar
  36. 36.
    Stobiecka M, Chalupa A. Modulation of plasmon-enhanced resonance energy transfer to gold nanoparticles by protein survivin channeled-shell gating. J Phys Chem B. 2015;119:13227–35.CrossRefGoogle Scholar
  37. 37.
    Stobiecka M. Novel plasmonic field-enhanced nanoassay for trace detection of proteins. Biosens Bioelectron. 2014;55:379–85.CrossRefGoogle Scholar
  38. 38.
    Lim GW, Lim JK, Ahmad AL, Chan DJC. Molecularly imprinted polymer layers using Navicula sp. frustule as core material for selectively recognition of lysozyme. Chem Eng Res Des. 2015;101:2–14.CrossRefGoogle Scholar
  39. 39.
    Fowler CE, Buchber C, Lebeau B, Patarin J, Delacôte C, Walcarius A. An aqueous route to organically functionalized silica diatom skeletons. Appl Surf Sci. 2007;253:5485–93.CrossRefGoogle Scholar
  40. 40.
    Hildebrand M. Biological processing of nanostructured silica in diatoms. Prog Org Coat. 2003;47:256–66.CrossRefGoogle Scholar
  41. 41.
    Yu Y, Addai-Mensah J, Losic D. Functionalized diatom silica microparticles for removal of mercury ions. Sci Technol Adv Mater. 2012;13:1–11.CrossRefGoogle Scholar
  42. 42.
    Lemonas JF. Diatomite. Am Ceram Soc Bull. 1997;76:92–5.Google Scholar
  43. 43.
    Lim GW, Lim JK, Ahmad AL, Chan DJC. Influences of diatom frustule morphologies on protein adsorption behavior. J Appl Phycol. 2015;27:763–75.CrossRefGoogle Scholar
  44. 44.
    Baumgärtel T, von Borczyskowski C, Graaf H. Selective surface modification of lithographic silicon oxide nanostructures by organofunctional silanes. Beilstein J Nanotechnol. 2013;4:218–26.CrossRefGoogle Scholar
  45. 45.
    Bergmann NM (2005) Molecularly imprinted polyacrylamide polymers and copolymers with specific recognition for serum proteins. Ph.D. Dissertation, The University of Texas.Google Scholar
  46. 46.
    Pang S, Liu S, Su X. A novel fluorescence assay for the detection of hemoglobin based on the G-quadruplex/hemin complex. Talanta. 2014;118:118–22.CrossRefGoogle Scholar
  47. 47.
    Tan L, Kang C, Xu S, Tang Y. Selective room temperature phosphorescence sensing of target protein using Mn-doped ZnS QDs-embedded molecularly imprinted polymer. Biosens Bioelectron. 2013;48:216–23.CrossRefGoogle Scholar
  48. 48.
    Zhang C, Cui H, Cai J, Duan Y, Liu Y. Development of fluorescence sensing material based on CdSe/ZnS quantum dots and molecularly imprinted polymer for the detection of carbaryl in rice and chinese cabbage. J Agric Food Chem. 2015;63:4966–72.CrossRefGoogle Scholar
  49. 49.
    Loucaides S, Behrends T, Van Cappellen P. Reactivity of biogenic silica: surface versus bulk charge density. Geochim Cosmochim Acta. 2010;74:517–30.CrossRefGoogle Scholar
  50. 50.
    Tozak KÖ, Erzengin M, Sargın İ, Ünlü N. Sorption of DNA by diatomite-Zn (II) embedded supermacroporous monolithic p(HEMA) cryogels. EXCLI J. 2013;12:670–80.Google Scholar
  51. 51.
    Bergmann NM, Peppas NA. Configurational biomimetic imprinting for protein recognition: structural characteristics of recognitive hydrogels. Ind Eng Chem Res. 2008;47:9099–107.CrossRefGoogle Scholar
  52. 52.
    Siyam T, Abd-Elatif ZH. Gamma radiation-induced preparation of poly(acrylamide-acrylic acid-dimethylaminoethylmethacrylate) as exchanger. J Macromol Sci. 1999;36:417–28.CrossRefGoogle Scholar
  53. 53.
    Ghosh SK, Ali M, Chatterjee H. Studies on the interaction of fluorescein isothiocyanate and its sugar analogues with cetyltrimethylammonium bromide. Chem Phys Lett. 2013;561(562):147–52.CrossRefGoogle Scholar
  54. 54.
    Jiang L, Li X, Liu L, Zhang Q. Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles. Int Jf Nanomed. 2013;8:1825–34.Google Scholar
  55. 55.
    Ma LY, Wang HY, Xie H, Xu LX. A long lifetime chemical sensor: study on fluorescence property of fluorescein isothiocyanate and preparation of pH chemical sensor. Spectrochim Acta A. 2004;60:1865–72.CrossRefGoogle Scholar
  56. 56.
    Sjöback R, Nygren J, Kubista M. Absorption and fluorescence properties of fluorescein. Spectrochim Acta A. 1995;51:L7–21.CrossRefGoogle Scholar
  57. 57.
    Harz S, Schimmelpfennig M, Tse Sum Bui B, Marchyk N, Haupt K, Feller K-H. Fluorescence optical spectrally resolved sensor based on molecularly imprinted polymers and microfluidics. Eng Life Sci. 2011;11:559–65.CrossRefGoogle Scholar
  58. 58.
    Deng Q, Wu J, Zhai X, Fang G, Wang S. Highly selective fluorescent sensing of proteins based on a fluorescent molecularly imprinted nanosensor. Sensors. 2013;13:12994.CrossRefGoogle Scholar
  59. 59.
    Verheyen E, Schillemans JP, van Wijk M, Demeniex M-A, Hennink WE, van Nostrum CF. Challenges for the effective molecular imprinting of proteins. Biomaterials. 2011;32:3008–20.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Guat Wei Lim
    • 1
  • Jit Kang Lim
    • 1
    • 2
  • Abdul Latif Ahmad
    • 1
  • Derek Juinn Chieh Chan
    • 1
    Email author
  1. 1.School of Chemical Engineering, Engineering CampusUniversiti Sains Malaysia, Seri AmpanganNibong Tebal, Seberang Perai SelatanMalaysia
  2. 2.Department of PhysicsCarnegie Mellon UniversityPittsburghUSA

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