Analytical and Bioanalytical Chemistry

, Volume 411, Issue 1, pp 157–170 | Cite as

Luminescent nanomaterials for droplet tracking in a microfluidic trapping array

  • Manibarathi Vaithiyanathan
  • Khashayar R. Bajgiran
  • Pragathi Darapaneni
  • Nora Safa
  • James A. DormanEmail author
  • Adam T. MelvinEmail author
Paper in Forefront


The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped β-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform.

Graphical abstract


Single-cell analysis Microfluidics Rare earth elements Nanoparticles, high-throughput screening 



This work was supported by grants from the National Institute of Biomedical Imaging and Bioengineering, R03EB02935 (ATM); the National Science Foundation, CBET1509713 (ATM); and the Louisiana Board of Regents, LEQSF (2016-19)-RD-A-03 (JAD/PD). The authors would like to thank Dr. Nancy Albritton (University of North Carolina) for providing the GFP-expressing HeLa cells and Dr. Elizabeth Martin (LSU) for providing the MDA-MB-231 cells and RFP-expressing MDA-MB-231 cells. The authors would like to thank Riad Elkhanoufi (LSU) for the assistance with device fabrication.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

216_2018_1448_MOESM1_ESM.pdf (638 kb)
ESM 1 (PDF 638 kb)


  1. 1.
    Saadatpour A, Lai S, Guo G, Yuan G-C. Single-cell analysis in cancer genomics. Trends Genet. 2015;31(10):576–86. Scholar
  2. 2.
    Tellez-Gabriel M, Ory B, Lamoureux F, Heymann M-F, Heymann D. Tumour heterogeneity: the key advantages of single-cell analysis. Int J Mol Sci. 2016;17(12):2142. Scholar
  3. 3.
    Tsoucas D, Yuan G-C. Recent progress in single-cell cancer genomics. Curr Opin Genet Dev. 2017;42:22–32. Scholar
  4. 4.
    Schneider T, Kreutz J, Chiu DT. The potential impact of droplet microfluidics in biology. Anal Chem. 2013;85(7):3476–82. Scholar
  5. 5.
    Shang L, Cheng Y, Zhao Y. Emerging droplet microfluidics. Chem Rev. 2017;117(12):7964–8040. Scholar
  6. 6.
    Newell EW, Cheng Y. Mass cytometry: blessed with the curse of dimensionality. Nat Immunol. 2016;17:890. Scholar
  7. 7.
    O'Donnell EA, Ernst DN, Hingorani R. Multiparameter flow cytometry: advances in high resolution analysis. Immune Netw. 2013;13(2):43–54. Scholar
  8. 8.
    Wyatt Shields Iv C, Reyes CD, Lopez GP. Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation. Lab Chip. 2015;15(5):1230–49. Scholar
  9. 9.
    Hu P, Zhang W, Xin H, Deng G. Single cell isolation and analysis. Front Cell Dev Biol. 2016;4:116. Scholar
  10. 10.
    Spitzer MH, Nolan GP. Mass cytometry: single cells, many features. Cell. 2016;165(4):780–91. Scholar
  11. 11.
    Chattopadhyay PK, Gierahn TM, Roederer M, Love JC. Single-cell technologies for monitoring immune systems. Nat Immunol. 2014;15(2):128–35. Scholar
  12. 12.
    Zhou Y, Basu S, Wohlfahrt KJ, Lee SF, Klenerman D, Laue ED, et al. A microfluidic platform for trapping, releasing and super-resolution imaging of single cells. Sensors Actuator B Chem. 2016;232:680–91. Scholar
  13. 13.
    Holton AB, Sinatra FL, Kreahling J, Conway AJ, Landis DA, Altiok S. Microfluidic biopsy trapping device for the real-time monitoring of tumor microenvironment. PLoS One. 2017;12(1):e0169797. Scholar
  14. 14.
    Zilionis R, Nainys J, Veres A, Savova V, Zemmour D, Klein AM, et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc. 2017;12(1):44–73. Scholar
  15. 15.
    Ma J, Zhan L, Li RS, Gao PF, Huang CZ. Color-encoded assays for the simultaneous quantification of dual cancer biomarkers. Anal Chem. 2017;89(16):8484–9. Scholar
  16. 16.
    Lan F, Haliburton JR, Yuan A, Abate AR. Droplet barcoding for massively parallel single-molecule deep sequencing. Nat Commun. 2016;7:11784.
  17. 17.
    Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015;161(5):1187–201. Scholar
  18. 18.
    Chen C-H, Miller MA, Sarkar A, Beste MT, Isaacson KB, Lauffenburger DA, et al. Multiplexed protease activity assay for low volume clinical samples using droplet based microfluidics and its application to endometriosis. J Am Chem Soc. 2013;135(5):1645–8. Scholar
  19. 19.
    Rane TD, Zec HC, Wang TH. A barcode-free combinatorial screening platform for matrix metalloproteinase screening. Anal Chem. 2015;87(3):1950–6. Scholar
  20. 20.
    Fan J, Hu M, Zhan P, Peng X. Energy transfer cassettes based on organic fluorophores: construction and applications in ratiometric sensing. Chem Soc Rev. 2013;42(1):29–43. Scholar
  21. 21.
    Abdel-Mottaleb MMA, Beduneau A, Pellequer Y, Lamprecht A. Stability of fluorescent labels in PLGA polymeric nanoparticles: quantum dots versus organic dyes. Int J Pharm. 2015;494(1):471–8. Scholar
  22. 22.
    Montón H, Nogués C, Rossinyol E, Castell O, Roldán M. QDs versus Alexa: reality of promising tools for immunocytochemistry. J Nanobiotechnol. 2009;7(1):4. Scholar
  23. 23.
    Wen C-Y, Xie H-Y, Zhang Z-L, Wu L-L, Hu J, Tang M, et al. Fluorescent/magnetic micro/nano-spheres based on quantum dots and/or magnetic nanoparticles: preparation, properties, and their applications in cancer studies. Nanoscale. 2016;8(25):12406–29. Scholar
  24. 24.
    Leng Y, Wu W, Li L, Lin K, Sun K, Chen X, et al. Magnetic/fluorescent barcodes based on cadmium-free near-infrared-emitting quantum dots for multiplexed detection. Adv Funct Mater. 2016;26(42):7581–9. Scholar
  25. 25.
    Gao X, Cui Y, Levenson RM, Chung LWK, Nie S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol. 2004;22:969.
  26. 26.
    Duan N, Wu S, Yu Y, Ma X, Xia Y, Chen X, et al. A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels. Anal Chim Acta. 2013;804:151–8. Scholar
  27. 27.
    Buranda T, Wu Y, Sklar LA. Quantum dots for quantitative flow cytometry. Methods Mol Biol. 2011;699:67–84. Scholar
  28. 28.
    Ghrera AS, Pandey CM, Ali MA, Malhotra BD. Quantum dot-based microfluidic biosensor for cancer detection. Appl Phys Lett. 2015;106(19):193703. Scholar
  29. 29.
    Wegner KD, Hildebrandt N. Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors. Chem Soc Rev. 2015;44(14):4792–834. Scholar
  30. 30.
    Gai SL, Li CX, Yang PP, Lin J. Recent progress in rare earth micro/nanocrystals: soft chemical synthesis, luminescent properties, and biomedical applications. Chem Rev. 2014;114(4):2343–89. Scholar
  31. 31.
    Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R, Nann T. Quantum dots versus organic dyes as fluorescent labels. Nat Methods. 2008;5:763.
  32. 32.
    Dong H, Du SR, Zheng XY, Lyu GM, Sun LD, Li LD, et al. Lanthanide nanoparticles: from design toward bioimaging and therapy. Chem Rev. 2015;115(19):10725–815. CrossRefPubMedGoogle Scholar
  33. 33.
    Haase M, Schafer H. Upconverting nanoparticles. Angew Chem Int Ed. 2011;50(26):5808–29. Scholar
  34. 34.
    Eliseeva SV, Bunzli JCG. Lanthanide luminescence for functional materials and bio-sciences. Chem Soc Rev. 2010;39(1):189–227. Scholar
  35. 35.
    Oliveira E, Bértolo E, Núñez C, Pilla V, Santos HM, Fernández-Lodeiro J, et al. Green and red fluorescent dyes for translational applications in imaging and sensing analytes: a dual-color flag. ChemistryOpen. 2018;7(1):9–52. Scholar
  36. 36.
    Lee H, Park HJ, Park C-S, Oh E-T, Choi B-H, Williams B, et al. Response of breast cancer cells and cancer stem cells to metformin and hyperthermia alone or combined. PLoS One. 2014;9(2):e87979. Scholar
  37. 37.
    Zhou J, Liu Q, Feng W, Sun Y, Li F. Upconversion luminescent materials: advances and applications. Chem Rev. 2015;115(1):395–465. Scholar
  38. 38.
    Chen G, Qiu H, Prasad PN, Chen X. Upconversion nanoparticles: design, nanochemistry, and applications in theranostics. Chem Rev. 2014;114(10):5161–214. Scholar
  39. 39.
    Li CX, Yang J, Quan ZW, Yang PP, Kong DY, Lin J. Different microstructures of ss-NaYF4 fabricated by hydrothermal process: effects of pH values and fluoride sources. Chem Mater. 2007;19(20):4933–42. Scholar
  40. 40.
    Wang F, Liu XG. Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals. Chem Soc Rev. 2009;38(4):976–89. Scholar
  41. 41.
    Dorman JA, Choi JH, Kuzmanich G, Bargar JR, Chang JP. Optimizing the crystal environment through extended x-ray absorption fine structure to increase the luminescent lifetimes of Er3+ doped Y2O3 nanoparticles. J Appl Phys. 2012;111(8).
  42. 42.
    Sabnis A, Rahimi M, Chapman C, Nguyen KT. Cytocompatibility studies of an in situ photopolymerized thermoresponsive hydrogel nanoparticle system using human aortic smooth muscle cells. J Biomed Mater Res Part B Appl Biomater. 2009;91(1):52–9. CrossRefGoogle Scholar
  43. 43.
    Wong DY, Ranganath T, Kasko AM. Low-dose, long-wave UV light does not affect gene expression of human mesenchymal stem cells. PLoS One. 2015;10(9):e0139307. Scholar
  44. 44.
    Naczynski DJ, Tan MC, Zevon M, Wall B, Kohl J, Kulesa A, et al. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat Commun. 2013;4:2199. Scholar
  45. 45.
    Yang NJ, Hinner MJ. Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods Mol Bio. 2015;1266:29–53. Scholar
  46. 46.
    Wu P, Yan X-P. Doped quantum dots for chemo/biosensing and bioimaging. Chem Soc Rev. 2013;42(12):5489–521. Scholar
  47. 47.
    Alivisatos AP, Gu W, Larabell C. Quantum dots as cellular probes. Annu Rev Biomed Eng. 2005;7:55–76. Scholar
  48. 48.
    Chan WCW, Maxwell DJ, Gao X, Bailey RE, Han M, Nie S. Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol. 2002;13(1):40–6. Scholar
  49. 49.
    Collins DJ, Neild A, deMello A, Liu A-Q, Ai Y. The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. Lab Chip. 2015;15(17):3439–59. Scholar
  50. 50.
    Kern KM, Schroeder JR. Comparison of cantharidin toxicity in breast cancer cells to two common chemotherapeutics. Int J Breast Cancer. 2014;2014:423059. Scholar
  51. 51.
    Muluneh M, Kim B, Buchsbaum G, Issadore D. Miniaturized, multiplexed readout of droplet-based microfluidic assays using time-domain modulation. Lab Chip. 2014;14(24):4638–46. Scholar
  52. 52.
    Gu S-Q, Zhang Y-X, Zhu Y, Du W-B, Yao B, Fang Q. Multifunctional picoliter droplet manipulation platform and its application in single cell analysis. Anal Chem. 2011;83(19):7570–6. Scholar
  53. 53.
    Bui M-PN, Li CA, Han KN, Choo J, Lee EK, Seong GH. Enzyme kinetic measurements using a droplet-based microfluidic system with a concentration gradient. Anal Chem. 2011;83(5):1603–8. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Cain Department of Chemical EngineeringLouisiana State UniversityBaton RougeUSA

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