Skip to main content
SpringerLink
Log in

Direct separation and enumeration of CTCs in viscous blood based on co-flow microchannel with tunable shear rate: a proof-of-principle study

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Circulating tumor cells (CTCs), which have extremely low density in whole blood, are an important indicator of primary tumor metastasis. Isolation and enumeration of these cells are critical for clinical applications. Separation of CTCs from massive blood cells without labeling and addition of synthetic polymers is challenging. Herein, a novel well-defined co-flow microfluidic device is presented and used to separate CTCs in viscous blood by applying both inertial and viscoelastic forces. Diluted blood without any synthetic polymer and buffer solution were used as viscoelastic fluid and Newtonian fluid, respectively, and they were co-flowed in the designed chip to form a sheath flow. The co-flow system provides the function of particle pre-focusing and creates a tunable shear rate region at the interface to adjust the migration of particles or cells from the sample solution to the buffer solution. Successful separation of CTCs from viscous blood was demonstrated and enumeration was also conducted by image recognition after separation. The statistical results indicated that a recovery rate of cancer cells greater than 87% was obtained using the developed method, which proved that the direct separation of CTCs from diluted blood can be achieved without the addition of any synthetic polymer to prepare viscoelastic fluid. This method holds great promise for the separation of cells in viscous biological fluid without either complicated channel structures or the addition of synthetic polymers.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Wan L, Liu Q, Liang D, Guo Y, Liu G, Ren J, He Y, Shan B. Circulating Tumor Cell and Metabolites as Novel Biomarkers for Early-Stage Lung Cancer Diagnosis. Front Oncol. 2021;11:630672. https://doi.org/10.3389/fonc.2021.630672.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Bidard FC, Proudhon C, Pierga JY. Circulating tumor cells in breast cancer. Mol Oncol. 2016;10(3):418–30. https://doi.org/10.1016/j.molonc.2016.01.001.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Gallo M, De Luca A, Maiello MR, D’Alessio A, Esposito C, Chicchinelli N, Forgione L, Piccirillo MC, Rocco G, Morabito A, Botti G, Normanno N. Clinical utility of circulating tumor cells in patients with non-small-cell lung cancer. Transl Lung Cancer Res. 2017;6(4):486–98. https://doi.org/10.21037/tlcr.2017.05.07.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Karabacak NM, Spuhler PS, Fachin F, Lim EJ, Pai V, Ozkumur E, Martel JM, Kojic N, Smith K, Chen PI, Yang J, Hwang H, Morgan B, Trautwein J, Barber TA, Stott SL, Maheswaran S, Kapur R, Haber DA, Toner M. Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat Protoc. 2014;9(3):694–710. https://doi.org/10.1038/nprot.2014.044.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Shen S, Yi Z, Li X, Xie S, Shui L. Flow-Field-Assisted Dielectrophoretic Microchips for High-Efficiency Sheathless Particle/Cell Separation with Dual Mode. Anal Chem. 2021;93(21):7606–15. https://doi.org/10.1021/acs.analchem.1c00018.

    Article  CAS  PubMed  Google Scholar 

  6. Thomas RSW, Mitchell PD, Oreffo ROC, Morgan H, Green NG. Image-based sorting and negative dielectrophoresis for high purity cell and particle separation. Electrophoresis. 2019;40(20):2718–27. https://doi.org/10.1002/elps.201800489.

    Article  CAS  PubMed  Google Scholar 

  7. Li S, Li M, Bougot-Robin K, Cao W, Yeung Yeung Chau I, Li W, Wen W. High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip. Biomicrofluidics. 2013;7(2):24106. https://doi.org/10.1063/1.4795856.

  8. Li S, Li M, Hui YS, Cao W, Li W, Wen W. A novel method to construct 3D electrodes at the sidewall of microfluidic channel. Microfluid. Nanofluidics. 2012;14(3-4):499–508. https://doi.org/10.1007/s10404-012-1068-6.

    Article  CAS  Google Scholar 

  9. Cao Q, Li Z, Wang Z, Qi F, Han X. Disaggregation and separation dynamics of magnetic particles in a microfluidic flow under an alternating gradient magnetic field. J. Phys. D. 2018;51(19):195002. https://doi.org/10.1088/1361-6463/aab9dd.

    Article  CAS  Google Scholar 

  10. Nistler A, Niessner R, Seidel M. Magnetic nanocomposites: versatile tool for the combination of immunomagnetic separation with flow-based chemiluminescence immunochip for rapid biosensing of Staphylococcal enterotoxin B in milk. Anal Bioanal Chem. 2019;411(19):4951–61. https://doi.org/10.1007/s00216-019-01808-z.

    Article  CAS  PubMed  Google Scholar 

  11. Errarte A, Martin-Mayor A, Aginagalde M, Iloro I, Gonzalez E, Falcon-Perez JM, Elortza F, Bou-Ali MM. Thermophoresis as a technique for separation of nanoparticle species in microfluidic devices. Int J Therm Sci. 2020;156:106435. https://doi.org/10.1016/j.ijthermalsci.2020.106435.

    Article  CAS  Google Scholar 

  12. Zhou Y, Yang C, Lam YC, Huang X. Thermophoresis of charged colloidal particles in aqueous media – Effect of particle size. Int. J. Heat Mass Transf. 2016;101:1283–91. https://doi.org/10.1016/j.ijheatmasstransfer.2016.05.109.

    Article  CAS  Google Scholar 

  13. Ma Z, Collins DJ, Guo J, Ai Y. Mechanical Properties Based Particle Separation via Traveling Surface Acoustic Wave. Anal Chem. 2016;88(23):11844–51. https://doi.org/10.1021/acs.analchem.6b03580.

    Article  CAS  PubMed  Google Scholar 

  14. Zhang Y, Chen X. Particle separation in microfluidics using different modal ultrasonic standing waves. Ultrason Sonochem. 2021;75:105603. https://doi.org/10.1016/j.ultsonch.2021.105603.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ahmed H, Destgeer G, Park J, Jung JH, Sung HJ. Vertical Hydrodynamic Focusing and Continuous Acoustofluidic Separation of Particles via Upward Migration. Adv Sci (Weinh). 2018;5(2):1700285. https://doi.org/10.1002/advs.201700285.

    Article  CAS  PubMed  Google Scholar 

  16. Sehgal P, Kirby BJ. Separation of 300 and 100 nm Particles in Fabry-Perot Acoustofluidic Resonators. Anal Chem. 2017;89(22):12192–200. https://doi.org/10.1021/acs.analchem.7b02858.

    Article  CAS  PubMed  Google Scholar 

  17. Jeon H, Jundi B, Choi K, Ryu H, Levy BD, Lim G, Han J, Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics. Lab Chip. 2020;20:3612-3624. https://doi.org/10.1039/d0lc00675k.

  18. Condina MR, Dilmetz BA, Bazaz SR, Meneses J, Warkiani ME, Hoffmann P. Rapid separation and identification of beer spoilage bacteria by inertial microfluidics and MALDI-TOF mass spectrometry. Lab Chip. 2019; 19: 1961-1970. https://doi.org/10.1039/C9LC00152B.

  19. Syed MS, Rafeie M, Vandamme D, Asadnia M, Henderson R, Taylor RA, Warkiani ME. Selective separation of microalgae cells using inertial microfluidics. Bioresour Technol. 2018;252:91–9. https://doi.org/10.1016/j.biortech.2017.12.065.

    Article  CAS  PubMed  Google Scholar 

  20. Zhang J, Yuan D, Zhao Q, Teo AJT, Yan S, Ooi CH, Li W, Nguyen NT. Fundamentals of Differential Particle Inertial Focusing in Symmetric Sinusoidal Microchannels. Anal Chem. 2019;91(6):4077–84. https://doi.org/10.1021/acs.analchem.8b05712.

    Article  CAS  PubMed  Google Scholar 

  21. Ren H, Zhu Z, Xiang N, Wang H, Zheng T, An H, Nguyen N-T, Zhang J. Multiplexed serpentine microchannels for high-throughput sorting of disseminated tumor cells from malignant pleural effusion. Sens. Actuators B Chem. 2021;337. https://doi.org/10.1016/j.snb.2021.129758.

  22. Bilican I. Cascaded contraction-expansion channels for bacteria separation from RBCs using viscoelastic microfluidics. J Chromatogr A. 2021;1652:462366. https://doi.org/10.1016/j.chroma.2021.462366.

    Article  CAS  PubMed  Google Scholar 

  23. Mahboubidoust A, Ramiar A, Sedighi K. Design of an optimized ECCA microchannel for particle manipulation utilizing dean flow coupled elasto-inertial method. Adv Powder Technol. 2021;32(5):1688–709. https://doi.org/10.1016/j.apt.2021.03.029.

    Article  Google Scholar 

  24. Fan L-L, Yan Q, Guo J, Zhao H, Zhao L, Zhe J. Inertial particle focusing in microchannels with gradually changing geometrical structures. J Micromech Microeng. 2017;27(1):015027. https://doi.org/10.1088/1361-6439/27/1/015027.

    Article  Google Scholar 

  25. Sim TS, Kwon K, Park JC, Lee JG, Jung HI. Multistage-multiorifice flow fractionation (MS-MOFF): continuous size-based separation of microspheres using multiple series of contraction/expansion microchannels. Lab Chip. 2011;11(1):93–9. https://doi.org/10.1039/c0lc00109k.

    Article  CAS  PubMed  Google Scholar 

  26. Zhou J, Kulasinghe A, Bogseth A, O'Byrne K, Punyadeera C, Papautsky I. Isolation of circulating tumor cells in non-small-cell-lung-cancer patients using a multi-flow microfluidic channel. Microsyst Nanoeng. 2019;5:8. https://doi.org/10.1038/s41378-019-0045-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lu X, Liu C, Hu G, Xuan X. Particle manipulations in non-Newtonian microfluidics: A review. J Colloid Interface Sci. 2017;500:182–201. https://doi.org/10.1016/j.jcis.2017.04.019.

    Article  CAS  PubMed  Google Scholar 

  28. Tian F, Zhang W, Cai L, Li S, Hu G, Cong Y, Liu C, Li T, Sun J. Microfluidic co-flow of Newtonian and viscoelastic fluids for high-resolution separation of microparticles. Lab Chip. 2017;17(18):3078–85. https://doi.org/10.1039/c7lc00671c.

    Article  CAS  PubMed  Google Scholar 

  29. Liu P, Liu H, Yuan D, Jang D, Yan S, Li M. Separation and Enrichment of Yeast Saccharomyces cerevisiae by Shape Using Viscoelastic Microfluidics. Anal Chem. 2021;93(3):1586–95. https://doi.org/10.1021/acs.analchem.0c03990.

    Article  CAS  PubMed  Google Scholar 

  30. Karimi A, Yazdi S, Ardekani AM. Hydrodynamic mechanisms of cell and particle trapping in microfluidics. Biomicrofluidics. 2013;7(2):21501. https://doi.org/10.1063/1.4799787.

    Article  CAS  PubMed  Google Scholar 

  31. Nam J, Namgung B, Lim CT, Bae JE, Leo HL, Cho KS, Kim S. Microfluidic device for sheathless particle focusing and separation using a viscoelastic fluid. J Chromatogr A. 2015;1406:244–50. https://doi.org/10.1016/j.chroma.2015.06.029.

    Article  CAS  PubMed  Google Scholar 

  32. Nam J, Yoon J, Jee H, Jang WS, Lim CS. High-Throughput Separation of Microvesicles from Whole Blood Components Using Viscoelastic Fluid. Adv. Mater. 2020;5(12):2000612. https://doi.org/10.1002/admt.202000612.

    Article  CAS  Google Scholar 

  33. Tan J, Park SY, Leo HL, Kim S. Continuous Separation of White Blood Cells From Whole Blood Using Viscoelastic Effects. IEEE Trans Biomed Circuits Syst. 2017;99:1–7. https://doi.org/10.1109/TBCAS.2017.2748232.

    Article  Google Scholar 

  34. Nam J, Jang WS, Lim CS. Non-electrical powered continuous cell concentration for enumeration of residual white blood cells in WBC-depleted blood using a viscoelastic fluid. Talanta. 2019;197:12–9. https://doi.org/10.1016/j.talanta.2018.12.102.

    Article  CAS  PubMed  Google Scholar 

  35. Shi X, Liu L, Cao W, Zhu G, Tan W. A Dean-flow-coupled interfacial viscoelastic fluid for microparticle separation applied in a cell smear method. Analyst. 2019;144(20):5934–46. https://doi.org/10.1039/c9an01070j.

    Article  CAS  PubMed  Google Scholar 

  36. Lim H, Back SM, Hwang MH, Lee DH, Choi H, Nam J. Sheathless High-Throughput Circulating Tumor Cell Separation Using Viscoelastic non-Newtonian Fluid. Micromachines (Basel). 2019;10(7):462. https://doi.org/10.3390/mi10070462.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Narayana IS, Kumar T, Martensson G, Russom A. High resolution and rapid separation of bacteria from blood using elasto-inertial microfluidics. Electrophoresis. 2021;42(23):2538–51. https://doi.org/10.1002/elps.202100140.

    Article  CAS  Google Scholar 

  38. Liu C, Xue C, Chen X, Shan L, Tian Y, Hu G. Size-Based Separation of Particles and Cells Utilizing Viscoelastic Effects in Straight Microchannels. Anal Chem. 2015;87(12):6041–8. https://doi.org/10.1021/acs.analchem.5b00516.

    Article  CAS  PubMed  Google Scholar 

  39. Zhou J, Papautsky I. Viscoelastic microfluidics: progress and challenges. Microsyst Nanoeng. 2020;6:113. https://doi.org/10.1038/s41378-020-00218-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Thompson MS, Vadala TP, Vadala ML, Lin Y, Riffle JS. Synthesis and applications of heterobifunctional poly(ethylene oxide) oligomers. Polymer. 2008;49(2):345–73. https://doi.org/10.1016/j.polymer.2007.10.029.

    Article  CAS  Google Scholar 

  41. Bodratti AM, Sarkar B, Alexandridis P. Adsorption of poly(ethylene oxide)-containing amphiphilic polymers on solid-liquid interfaces: Fundamentals and applications. Adv Colloid Interface Sci. 2017;244:132–63. https://doi.org/10.1016/j.cis.2016.09.003.

    Article  CAS  PubMed  Google Scholar 

  42. Liu X, Xu Y, Wu Z, Chen H. Poly(N-vinylpyrrolidone)-modified surfaces for biomedical applications. Macromol Biosci. 2013;13(2):147–54. https://doi.org/10.1002/mabi.201200269.

    Article  CAS  PubMed  Google Scholar 

  43. Berret JF. Local viscoelasticity of living cells measured by rotational magnetic spectroscopy. Nat Commun. 2016;7:10134. https://doi.org/10.1038/ncomms10134.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Brust M, Schaefer C, Doerr R, Pan L, Garcia M, Arratia PE, Wagner C. Rheology of Human Blood Plasma: Viscoelastic Versus Newtonian Behavior. Phys. Rev. Lett. 2013;110:078305. https://doi.org/10.1103/PhysRevLett.110.078305.

    Article  CAS  PubMed  Google Scholar 

  45. Indei T, Schieber JD, Córdoba A. Competing effects of particle and medium inertia on particle diffusion in viscoelastic materials, and their ramifications for passive microrheology. Phys. Rev. E. 2012;85(4). https://doi.org/10.1103/PhysRevE.85.041504.

  46. Li D, Lu X, Xuan X. Viscoelastic Separation of Particles by Size in Straight Rectangular Microchannels: A Parametric Study for a Refined Understanding. Anal Chem. 2016;88(24):12303–9. https://doi.org/10.1021/acs.analchem.6b03501.

    Article  CAS  PubMed  Google Scholar 

  47. Manshadi MKD, Mohammadi M, Monfared LK, Sanati-Nezhad A. Manipulation of micro- and nanoparticles in viscoelastic fluid flows within microfluid systems. Biotechnol Bioeng. 2020;117(2):580–92. https://doi.org/10.1002/bit.27211.

    Article  CAS  PubMed  Google Scholar 

  48. Tian F, Cai L, Chang J, Li S, Liu C, Li T, Sun J. Label-free isolation of rare tumor cells from untreated whole blood by interfacial viscoelastic microfluidics. Lab Chip. 2018;18(22):3436–45. https://doi.org/10.1039/c8lc00700d.

    Article  CAS  PubMed  Google Scholar 

  49. Liu C, Guo J, Tian F, Yang N, Yan F, Ding Y, Wei J, Hu G, Nie G, Sun J. Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows. ACS Nano. 2017;11(7):6968–76. https://doi.org/10.1021/acsnano.7b02277.

    Article  CAS  PubMed  Google Scholar 

  50. Jaensson NO, Mitrias C, Hulsen MA, Anderson PD. Shear-Induced Migration of Rigid Particles near an Interface between a Newtonian and a Viscoelastic Fluid. Langmuir. 2018;34(4):1795–806. https://doi.org/10.1021/acs.langmuir.7b03482.

    Article  CAS  PubMed  Google Scholar 

  51. Lu X, Xuan X. Inertia-Enhanced Pinched Flow Fractionation. Anal. Chem. 2015;87:4560–5. https://doi.org/10.1021/acs.analchem.5b00752.

    Article  CAS  PubMed  Google Scholar 

  52. Zhou J, Tu C, Liang Y, Huang B, Fang Y, Liang X, Papautsky I, Ye X. Isolation of cells from whole blood using shear-induced diffusion. Sci Rep. 2018;8(1):9411. https://doi.org/10.1038/s41598-018-27779-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Nam J, Lim H, Kim D, Jung H, Shin S. Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid. Lab Chip. 2012;12(7):1347–54. https://doi.org/10.1039/c2lc21304d.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

S. Li and Y. Xu would like to acknowledge the financial support from the National Key Research and Development Program of China (2020YFB2009001) and the National Natural Science Foundation of China (61904021, 62071072).

Author information

Authors and Affiliations

  1. Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education & Key Disciplines Laboratory of Novel Micro-Nano Devices and System Technology, College of Optoelectronic Engineering, Chongqing University, Chongqing, 400044, China

    Mengnan Li, Yuping Yang, Minshan Gan, Yi Xu, Li Chen & Shunbo Li

  2. International R & D center of Micro-nano Systems and New Materials Technology, Chongqing University, Chongqing, 400044, China

    Mengnan Li, Minshan Gan, Yi Xu, Li Chen & Shunbo Li

  3. Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, 400030, China

    Chuang Ge

Authors
  1. Mengnan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chuang Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuping Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Minshan Gan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Li Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shunbo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization and methodology, Shunbo Li and Yi Xu; Experiments, Mengnan Li and Chuang Ge; Data analysis, Mengnan Li and Yuping Yang; Validation, Minshan Gan and Li Chen; Writing, review & editing, all authors.

Corresponding authors

Correspondence to Yi Xu or Shunbo Li.

Ethics declarations

The studies were approved by the Chongqing University Cancer Hospital ethics committee (Ethical code: CZLS2021267-A) and were performed in accordance with ethical standards.

The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(DOCX 1642 kb)

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, M., Ge, C., Yang, Y. et al. Direct separation and enumeration of CTCs in viscous blood based on co-flow microchannel with tunable shear rate: a proof-of-principle study. Anal Bioanal Chem 414, 7683–7694 (2022). https://doi.org/10.1007/s00216-022-04299-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-022-04299-7

Keywords

Search

Navigation