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Cell analysis using a multiple internal reflection photonic lab-on-a-chip


Here we present a protocol for analyzing cell cultures using a photonic lab-on-a-chip (PhLoC). By using a broadband light source and a spectrometer, the spectrum of a given cell culture with an arbitrary population is acquired. The PhLoC can work in three different regimes: light scattering (using label-free cells), light scattering plus absorption (using stained cells) and, by subtraction of the two former regimes, absorption (without the scattering band). The acquisition time of the PhLoC is 30 ms. Hence, it can be used for rapid cell counting, dead/live ratio estimation or multiparametric measurements through the use of different dyes. The PhLoC, including microlenses, micromirrors and microfluidics, is simply fabricated in a single-mask process (by soft lithographic methods) using low-cost materials. Because of its low cost it can easily be implemented for point-of-care applications. From raw substrates to final results, this protocol can be completed in 29 h.

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Figure 1: Schematic representation of the merging of micro-optics and microfluidics.
Figure 2: Layout of the mask used for implementing the PhLoC system.
Figure 3: Fabrication steps.
Figure 4: Picture of the completed PhLoC, having self-alignment microchannels for accurate fiber optics positioning, microlenses and air mirrors.
Figure 5: Characterization in the LS regime.
Figure 6: Characterization in the LS + ABS regime.
Figure 7: Characterization in the ABS regime obtained by subtraction of the spectra obtained in LS and LS + ABS regimes.
Figure 8: Measurement of the monocytes dead/live ratio.


  1. Yeo, L.Y., Chang, H.C., Chan, P.P.Y. & Friend, J.R. Microfluidic devices for bioapplications. Small 7, 12–48 (2011).

    CAS  Article  Google Scholar 

  2. Dittrich, P.S. & Manz, A. Lab-on-a-chip: microfluidics in drug discovery. Nat. Rev. Drug. Discov. 5, 210–218 (2006).

    CAS  Article  Google Scholar 

  3. Nagrath, S. et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450, 1235–1239 (2007).

    CAS  Article  Google Scholar 

  4. Gilleland, C.L., Rohde, C.B., Zeng, F. & Yanik, M.F. Microfluidic immobilization of physiologically active Caenorhabditis elegans. Nat. Protoc. 5, 1888–1902 (2010).

    CAS  Article  Google Scholar 

  5. Yager, P. et al. Microfluidic diagnostic technologies for global public health. Nature 442, 412–418 (2006).

    CAS  Article  Google Scholar 

  6. Martinez, A.W., Phillips, S.T. & Whitesides, G.M. Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal. Chem. 82, 3–10 (2010).

    CAS  Article  Google Scholar 

  7. Zhao, W. & van den Berg, A. Lab on paper. Lab. Chip 8, 1988–1991 (2008).

    CAS  Article  Google Scholar 

  8. Centers for Disease Control and Prevention. Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and Adults. Morbidity and Mortality Weekly Report 1–19 (18 December 1992).

  9. Myers, F.B. & Lee, L.P. Innovations in optical microfluidic technologies for point-of-care diagnostics. Lab. Chip 8, 2015–2031 (2008).

    CAS  Article  Google Scholar 

  10. Mayer, G. et al. Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Nat. Protoc. 5, 1993–2004 (2010).

    CAS  Article  Google Scholar 

  11. Llobera, A., Demming, S., Wilke, R. & Büttgenbach, S. Multiple internal reflection poly(dimethylsiloxane) systems for optical sensing. Lab. Chip 7, 1560–1566 (2007).

    CAS  Article  Google Scholar 

  12. Godin, J. et al. Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip. J. Biophoton. 1, 355–376 (2008).

    CAS  Article  Google Scholar 

  13. Chung, T.D. & Kim, H.C. Recent advances in miniaturized microfluidic flow cytometry for clinical use. Electrophoresis 28, 4511–4520 (2007).

    CAS  Article  Google Scholar 

  14. Qin, D., Xia, Y. & Whitesides, G.M. Soft lithography for micro- and nanoscale patterning. Nat. Protoc. 5, 491–502 (2010).

    CAS  Article  Google Scholar 

  15. Llobera, A. et al. Monolithic PDMS passband filters for fluorescence detection. Lab. Chip 10, 1987–1992 (2010).

    CAS  Article  Google Scholar 

  16. Llobera, A., Wilke, R. & Büttgenbach, S. Enhancement of the response of poly(dimethylsiloxane) hollow prisms through air mirrors for absorbance-based sensing. Talanta 75, 473–479 (2008).

    CAS  Article  Google Scholar 

  17. Ibarlucea, B. et al. Cell screening using disposable photonic lab on a chip systems. Anal. Chem. 82, 4246–4251 (2010).

    CAS  Article  Google Scholar 

  18. Merbs, S.L. & Nathans, J. Absorption-spectra of human cone pigments. Nature 356, 433–435 (1992).

    CAS  Article  Google Scholar 

  19. Kajihara, D. et al. FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nat. Methods 3, 923–929 (2006).

    CAS  Article  Google Scholar 

  20. Gao, X.H., Cui, Y.Y., Levenson, R.M., Chung, L.W.K. & Nie, S.M. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 22, 969–976 (2004).

    CAS  Article  Google Scholar 

  21. Llobera, A., Wilke, R. & Büttgenbach, S. Poly(dimethylsiloxane) hollow Abbe prism with microlenses for detection based on absorption and refractive index shift. Lab. Chip 4, 24–27 (2004).

    CAS  Article  Google Scholar 

  22. Mayers, B.T., Vezenov, D.V., Vullev, V.I. & Whitesides, G.M. Arrays and cascades of fluorescent liquid-liquid waveguides: broadband light sources for spectroscopy in microchannels. Anal. Chem. 77, 1310–1316 (2005).

    CAS  Article  Google Scholar 

  23. Llobera, A., Wilke, R. & Büttgenbach, S. Optimization of poly(dimethylsiloxane) hollow prisms for optical sensing. Lab. Chip 5, 506–511 (2005).

    CAS  Article  Google Scholar 

  24. Bhattacharya, S., Datta, A., Berg, J.M. & Gangopadhyay, S. Studies on surface wettability of poly(dimethyl)siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength. J. Microelectromech. Syst. 14, 590–597 (2005).

    CAS  Article  Google Scholar 

  25. Blanchard, D., Ligrani, P., Gale, B. & Harvey, I. Micro-structure mechanical failure characterization using rotating Couette flow in a small gap. J. Micromech. Microeng. 15, 792–801 (2005).

    Article  Google Scholar 

  26. Martin, C. et al. Stress and aging minimization in photoplastic AFM probes. Microel. Eng. 86, 1226–1229 (2009).

    CAS  Article  Google Scholar 

  27. Feng, R. & Farris, R.J. Influence of processing conditions on the thermal and mechanical properties of SU-8 negative photoresist coatings. J. Micromech. Microeng. 13, 80–88 (2003).

    CAS  Article  Google Scholar 

  28. Cheng, L.L., Luk, Y.Y., Murphy, C.J., Israel, B.A. & Abbott, N.L. Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses. Biomaterials 26, 7173–7182 (2005).

    CAS  Article  Google Scholar 

  29. Llobera, A. Device for particle analysis and counting. WIPO patent application no. PCT/ES2011/070011 (11 January 2010).

  30. Demming, S. et al. Poly(dimethylsiloxane) photonic microbioreactors based on segmented waveguides for local absorbance measurement. Electrophoresis 32, 431–439 (2011).

    CAS  Article  Google Scholar 

  31. Long, G.L. & Wineforder, J.D. Limit of detection: a closer look at the IUPAC definition. Anal. Chem. 55, A712–A724 (1983).

    Article  Google Scholar 

  32. Cui, X. et al. Lensless high-resolution on-chip optofluidic microscopes for Caenorhabditis elegans and cell imaging. PNAS 105, 10670–10675 (2008).

    CAS  Article  Google Scholar 

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The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 209243 and also from the IST Programme (P. CEZANNE, IST-2-IP-031867). We thank the research group FOR 856, mikroPART 'Mikrosysteme für partikuläre Life-Science-Produkte', for support of this work. Discussions with A. Voigt and G. Gruetzner of Microresist (Germany) regarding the fabrication steps are highly appreciated.


J.V.-P., E.F.-R. and B.I. conducted the experiments. E.F.-R. and C.N. prepared the cell culture. S.D., S.B. and A.L. designed and fabricated the PhLoC. J.A.P., C.D., S.B. and A.L. defined the broadband screening protocol. A.L. supervised the project at all stages.

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Correspondence to Andreu Llobera.

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Vila-Planas, J., Fernández-Rosas, E., Ibarlucea, B. et al. Cell analysis using a multiple internal reflection photonic lab-on-a-chip. Nat Protoc 6, 1642–1655 (2011).

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