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Microfluidic integration of regeneratable electrochemical affinity-based biosensors for continual monitoring of organ-on-a-chip devices

Nature Protocols volume 16, pages 2564–2593 (2021)Cite this article

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Abstract

Organs-on-chips have emerged as viable platforms for drug screening and personalized medicine. While a wide variety of human organ-on-a-chip models have been developed, rarely have there been reports on the inclusion of sensors, which are critical in continually measuring the microenvironmental parameters and the dynamic responses of the microtissues to pharmaceutical compounds over extended periods of time. In addition, automation capacity is strongly desired for chronological monitoring. To overcome this major hurdle, in this protocol we detail the fabrication of electrochemical affinity-based biosensors and their integration with microfluidic chips to achieve in-line microelectrode functionalization, biomarker detection and sensor regeneration, allowing continual, in situ and noninvasive quantification of soluble biomarkers on organ-on-a-chip platforms. This platform is almost universal and can be applied to in-line detection of a majority of biomarkers, can be connected with existing organ-on-a-chip devices and can be multiplexed for simultaneous measurement of multiple biomarkers. Specifically, this protocol begins with fabrication of the electrochemically competent microelectrodes and the associated microfluidic devices (~3 d). The integration of electrochemical biosensors with the chips and their further combination with the rest of the platform takes ~3 h. The functionalization and regeneration of the microelectrodes are subsequently described, which require ~7 h in total. One cycle of sampling and detection of up to three biomarkers accounts for ~1 h.

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Fig. 1: Schematic representation of the electrode fabrication and functionalization.
Fig. 2: Effect of Au electrode thickness on reproducibility, sensitivity and selectivity upon multiple regeneration cycles.
Fig. 3: Assembly and valve configuration of microfluidic EC affinity-based biosensor chips (see Supplementary Data 1 for designs).
Fig. 4: Diagrams of the assembly of the breadboard and its connections with side modules (see Supplementary Data 1 for designs).
Fig. 5: Examples of EC affinity-based biosensor detection capabilities in multi-organ-on-a-chip configurations.

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Data availability

Most data associated with this protocol can be found in previous publications13,21,22. Additional datasets that support this protocol are available from the corresponding authors upon reasonable request. All requests for raw and analyzed data and materials will be promptly reviewed by the Brigham and Women’s Hospital to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a Material Transfer Agreement.

Code availability

The MATLAB codes can be accessed through previous publications13,21,22 and the Zhang Lab website (Electrochemical Biosensing; https://shrikezhang.com/publications/opensource). The running of these codes is standardized, and additional instructions can be found in the readme file in the package.

References

  1. Ahadian, S. et al. Organ-on-a-chip platforms: a convergence of advanced materials, cells, and microscale technologies. Adv. Healthc. Mater. https://doi.org/10.1002/adhm.201800734 (2018).

  2. Wang, Y. I., Carmona, C., Hickman, J. J. & Shuler, M. L. Multiorgan microphysiological systems for drug development: strategies, advances, and challenges. Adv. Healthc. Mater. 7, 1701000 (2018).

    Article  Google Scholar 

  3. Ronaldson-Bouchard, K. & Vunjak-Novakovic, G. Organs-on-a-chip: a fast track for engineered human tissues in drug development. Cell Stem Cell 22, 310–324 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang, Y. S. et al. From cardiac tissue engineering to heart-on-a-chip: beating challenges. Biomed. Mater. 10, 034006 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Arrigoni, C., Lopa, S., Candrian, C. & Moretti, M. Organs-on-a-chip as model systems for multifactorial musculoskeletal diseases. Curr. Opin. Biotechnol. 63, 79–88 (2020).

    Article  CAS  PubMed  Google Scholar 

  6. Mobini, S., Song, Y. H., McCrary, M. W. & Schmidt, C. E. Advances in ex vivo models and lab-on-a-chip devices for neural tissue engineering. Biomaterials 198, 146–166 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Bein, A. et al. Microfluidic organ-on-a-chip models of human intestine. Cell. Mol. Gastroenterol. Hepatol. 5, 659–668 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Ashammakhi, N., Wesseling-Perry, K., Hasan, A., Elkhammas, E. & Zhang, Y. S. Kidney-on-a-chip: untapped opportunities. Kidney Int. 94, 1073–1086 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Conant, G. et al. High-content assessment of cardiac function using heart-on-a-chip devices as drug screening model. Stem Cell Rev. Rep. 13, 335–346 (2017).

    Article  PubMed  Google Scholar 

  10. Ribas, J. et al. Biomechanical strain exacerbates inflammation on a progeria-on-a-chip model. Small https://doi.org/10.1002/smll.201603737 (2017).

  11. Zhang, Y. S., Zhang, Y. N. & Zhang, W. Cancer-on-a-chip systems at the frontier of nanomedicine. Drug Discov. Today 22, 1392–1399 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Skardal, A., Shupe, T. & Atala, A. Organoid-on-a-chip and body-on-a-chip systems for drug screening and disease modeling. Drug Discov. Today 21, 1399–1411 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, Y. S. et al. Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors. Proc. Natl Acad. Sci. USA 114, E2293–E2302 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Riahi, R. et al. Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers. Sci. Rep. 6, 24598 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Skardal, A. et al. Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Sci. Rep. 7, 8837 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Skardal, A. et al. Drug compound screening in single and integrated multi-organoid body-on-a-chip systems. Biofabrication 12, 025017 (2020).

    Article  CAS  PubMed  Google Scholar 

  17. Edington, C. D. et al. Interconnected microphysiological systems for quantitative biology and pharmacology studies. Sci. Rep. 8, 4530 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Oleaga, C. et al. Multi-organ toxicity demonstration in a functional human in vitro system composed of four organs. Sci. Rep. 6, 20030 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Novak, R. et al. Robotic fluidic coupling and interrogation of multiple vascularized organ chips. Nat. Biomed. Eng. 4, 407–420 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Kratz, S. R. A., Holl, G., Schuller, P., Ertl, P. & Rothbauer, M. Latest trends in biosensing for microphysiological organs-on-a-chip and body-on-a-chip systems. Biosensors (Basel) https://doi.org/10.3390/bios9030110 (2019).

  21. Shin, S. R. et al. Label-free and regenerative electrochemical microfluidic biosensors for continual monitoring of cell secretomes. Adv. Sci. (Weinh.) 4, 1600522 (2017).

    Google Scholar 

  22. Shin, S. R. et al. Aptamer-based microfluidic electrochemical biosensor for monitoring cell-secreted trace cardiac biomarkers. Anal. Chem. 88, 10019–10027 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang, Y. S. et al. Google Glass-directed monitoring and control of microfluidic biosensors and actuators. Sci. Rep. 6, 22237 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Rusling, J. F. Multiplexed electrochemical protein detection and translation to personalized cancer diagnostics. Anal. Chem. 85, 5304–5310 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Goode, J. A., Rushworth, J. V. H. & Millner, P. A. Biosensor regeneration: a review of common techniques and outcomes. Langmuir 31, 6267–6276 (2015).

    Article  CAS  PubMed  Google Scholar 

  26. Rajan, S. A. P. et al. Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform. Acta Biomater. 106, 124–135 (2020).

    Article  CAS  PubMed  Google Scholar 

  27. Aleman, J. & Skardal, A. A multi-site metastasis-on-a-chip microphysiological system for assessing metastatic preference of cancer cells. Biotechnol. Bioeng. 116, 936–944 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Aleman, J. et al. Deconstructed microfluidic bone marrow on-a-chip to study normal and malignant hemopoietic cell-niche interactions. Small 15, e1902971 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Soucy, J. R., Bindas, A. J., Koppes, A. N. & Koppes, R. A. Instrumented microphysiological systems for real-time measurement and manipulation of cellular electrochemical processes. IScience 21, 521–548 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Pourmand, A. et al. Fabrication of whole-thermoplastic normally closed microvalve, micro check valve, and micropump. Sens. Actuat. B-Chem. 262, 625–636 (2018).

    Article  CAS  Google Scholar 

  31. Shaegh, S. A. M. et al. Rapid prototyping of whole-thermoplastic microfluidics with built-in microvalves using laser ablation and thermal fusion bonding. Sens. Actuat. B-Chem. 255, 100–109 (2018).

    Article  CAS  Google Scholar 

  32. Lee, Y.-S., Bhattacharjee, N. & Folch, A. 3D-printed Quake-style microvalves and micropumps. Lab Chip 18, 1207–1214 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  PubMed  Google Scholar 

  34. Dauphin-Ducharme, P., Arroyo-Currás, N. Y. & Plaxco, K. W. High-precision electrochemical measurements of the guanine-, mismatch-, and length-dependence of electron transfer from electrode-bound DNA are consistent with a contact-mediated mechanism. J. Am. Chem. Soc. 141, 1304–1311 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kurnik, M., Pang, E. Z. & Plaxco, K. W. An electrochemical biosensor architecture based on protein folding supports direct real‐time measurements in whole blood. Angew. Chem. (2020).

  36. Kang, E., Shin, S.-J., Lee, K. H. & Lee, S.-H. Novel PDMS cylindrical channels that generate coaxial flow, and application to fabrication of microfibers and particles. Lab Chip 10, 1856–1861 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. Kang, E. et al. Digitally tunable physicochemical coding of material composition and topography in continuous microfibres. Nat. Mater. 10, 877–883 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. MacDonald, M. A. & Andreas, H. A. Method for equivalent circuit determination for electrochemical impedance spectroscopy data of protein adsorption on solid surfaces. Electrochim. Acta 129, 290–299 (2014).

    Article  CAS  Google Scholar 

  39. McCauley, T. G., Hamaguchi, N. & Stanton, M. Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. Anal. Biochem. 319, 244–250 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Kara, P. et al. Aptamers based electrochemical biosensor for protein detection using carbon nanotubes platforms. Biosens. Bioelectron. 26, 1715–1718 (2010).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors acknowledge support by the National Institutes of Health (R00CA201603 and UG3TR003274) and the Brigham Research Institute. This work was also partially sponsored by the Office of the Secretary of Defense through the Advanced Regenerative Manufacturing Institute (ARMI|BioFabUSA) and was accomplished under Agreement Number W911NF-17-3-003. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Office of the Secretary of Defense or the US Government. The US Government is authorized to reproduce and distribute reprints for government purposes notwithstanding any copyright notation here. T.K. is grateful to the Swiss National Science Foundation for the Postdoc Mobility fellowship (no. P400PM_180788/1).

Author information

Author notes

  1. These authors contributed equally: Julio Aleman, Tugba Kilic.

Authors and Affiliations

  1. Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA

    Julio Aleman

  2. Center for Systems Biology and Department of Radiology, Massachusetts General Hospital Research Institute, Harvard Medical School, Boston, MA, USA

    Tugba Kilic

  3. Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, USA

    Luis S. Mille, Su Ryon Shin & Yu Shrike Zhang

Authors
  1. Julio Aleman
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  2. Tugba Kilic
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Contributions

Y.S.Z. and S.R.S. conceived the project; J.A., T.K. and Y.S.Z. drafted the initial manuscript; L.S.M. created the figures; J.A., T.K., S.R.S. and Y.S.Z. revised the manuscript. All authors approved the manuscript.

Corresponding authors

Correspondence to Su Ryon Shin or Yu Shrike Zhang.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Xingyu Jiang, Arben Merkoçi and José Pingarrón for their contribution to the peer review of this work.

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Related links

Key reference using this protocol:

Zhang, Y. S. et al. Proc. Natl Acad. Sci. USA 114, E2293–E2302 (2017): https://doi.org/10.1073/pnas.1612906114

Shin, S. R. et al. Adv. Sci. (Weinh) 4, 1600522 (2017): https://doi.org/10.1002/advs.201600522

Shin, S. R. et al. Anal. Chem. 88, 10019–10027 (2016): https://doi.org/10.1021/acs.analchem.6b02028

Supplementary information

Reporting Summary

Supplementary Data 1

AutoCAD designs of breadboard and EC sensing chip with regular, simplified and multiplexed channel configurations

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Aleman, J., Kilic, T., Mille, L.S. et al. Microfluidic integration of regeneratable electrochemical affinity-based biosensors for continual monitoring of organ-on-a-chip devices. Nat Protoc 16, 2564–2593 (2021). https://doi.org/10.1038/s41596-021-00511-7

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