Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Bar-coded hydrogel microparticles for protein detection: synthesis, assay and scanning

Nature Protocols volume 6, pages 1761–1774 (2011)Cite this article

Subjects

Abstract

This protocol describes the core methodology for the fabrication of bar-coded hydrogel microparticles, the capture and labeling of protein targets and the rapid microfluidic scanning of particles for multiplexed detection. Multifunctional hydrogel particles made from poly(ethylene glycol) serve as a sensitive, nonfouling and bio-inert suspension array for the multiplexed measurement of proteins. Each particle type bears a distinctive graphical code consisting of unpolymerized holes in the wafer structure of the microparticle; this code serves to identify the antibody probe covalently incorporated throughout a separate probe region of the particle. The protocol for protein detection can be separated into three steps: (i) synthesis of particles via microfluidic flow lithography at a rate of 16,000 particles per hour; (ii) a 3–4-h assay in which protein targets are captured and labeled within particles using an antibody sandwich technique; and (iii) a flow scanning procedure to detect bar codes and quantify corresponding targets at rates of 25 particles per s. By using the techniques described, single- or multiple-probe particles can be reproducibly synthesized and used in customizable multiplexed panels to measure protein targets over a three-log range and at concentrations as low as 1 pg ml−1.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Synthesis device and particle design.
Figure 2: Schematic of the sandwich assay.
Figure 3: Scanning device and data analysis.
Figure 4: Microscope setup for synthesis and scanning.
Figure 5: Optimization of polymerization conditions.
Figure 6: Quality of scan data.
Figure 7: Single-probe particles used in a calibration of IL-2 detection.

References

  1. De Angelis, D., Rittenhouse, H.G., Mikolajczyk, S.D., Blair Shamel, L. & Semjonow, A. Twenty years of PSA: from prostate antigen to tumor marker. Rev. Urol. 9, 113–123 (2007).

    PubMed  PubMed Central  Google Scholar 

  2. Gorelik, E. et al. Multiplexed immunobead-based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol. Biomarkers Prev. 14, 981–987 (2005).

    Article  CAS  Google Scholar 

  3. Wulfkuhle, J.D., Liotta, L.A. & Petricoin, E.F. Proteomic applications for the early detection of cancer. Nat. Rev. Cancer 3, 267–275 (2003).

    Article  CAS  Google Scholar 

  4. Trieschmann, L. et al. Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy. BMC Biotechnol. 5, 26 (2005).

    Article  Google Scholar 

  5. Mirkin, C.A., Thaxton, C.S. & Rosi, N.L. Nanostructures in biodefense and molecular diagnostics. Expert Rev. Mol. Diagn. 4, 749–751 (2004).

    Article  Google Scholar 

  6. Verrills, N.M. Clinical proteomics: present and future prospects. Clin. Biochem. Rev. 27, 99–116 (2006).

    PubMed  PubMed Central  Google Scholar 

  7. Petricoin, E.F. & Liotta, L.A. Clinical applications of proteomics. J. Nutr. 133, 2476S–2484S (2003).

    Article  CAS  Google Scholar 

  8. Zichi, D.B., Eaton, B., Singer, B. & Gold, L. Proteomics and diagnostics: let's get specific, again. Curr. Opin. Chem. Biol. 12, 78–85 (2008).

    Article  CAS  Google Scholar 

  9. Engvall, E. & Perlman, P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874 (1971).

    Article  CAS  Google Scholar 

  10. Giljohann, D.A. & Mirkin, C.A. Drivers of biodiagnostic development. Nature 462, 461–464 (2009).

    Article  CAS  Google Scholar 

  11. Wilson, R., Cossins, A.R. & Spiller, D.G. Encoded microcarriers for high-throughput multiplexed detection. Angew. Chem. Int. Ed. 45, 6104–6117 (2006).

    Article  CAS  Google Scholar 

  12. Ellington, A.A., Kullo, I.J., Bailey, K.R. & Klee, G.G. Antibody-based protein multiplex platforms: technical and operational challenges. Clin. Chem. 56, 186–193 (2010).

    Article  CAS  Google Scholar 

  13. Fu, Q., Zhu, J. & Van Eyk, J.E. Comparison of multiplex immunoassay platforms. Clin. Chem. 56, 314–318 (2010).

    Article  CAS  Google Scholar 

  14. Meiring, J.E. et al. Hydrogel biosensor array platform indexed by shape. Chem. Mater. 16, 5574–5580 (2004).

    Article  CAS  Google Scholar 

  15. Lee, W., Choi, D., Kim, J. & Koh, W. Suspension arrays of hydrogel microparticles prepared by photopatterning for multiplexed protein-based bioassays. Biomed. Microdevices 10, 813–822 (2008).

    Article  CAS  Google Scholar 

  16. Moorthy, J., Burgess, R., Yethiraj, A. & Beebe, D. Microfluidic based platform for characterization of protein interactions in hydrogel nanoenvironments. Anal. Chem. 79, 5322–5327 (2007).

    Article  CAS  Google Scholar 

  17. Rubina, A.Y., Kolchinsky, A., Makarov, A.A. & Zasedatelev, A.S. Why 3-D? Gel-based microarrays in proteomics. Proteomics 8, 817–831 (2008).

    Article  CAS  Google Scholar 

  18. Zubtsov, D.A. et al. Comparison of surface and hydrogel-based protein microchips. Anal. Biochem. 368, 205–213 (2007).

    Article  CAS  Google Scholar 

  19. Pregibon, D.C., Toner, M. & Doyle, P.S. Multifunctional encoded particles for high-throughput biomolecule analysis. Science 315, 1393–1396 (2007).

    Article  CAS  Google Scholar 

  20. Bong, K.W., Bong, K.T., Pregibon, D.C. & Doyle, P.S. Hydrodynamic focusing lithography. Angew. Chem. Int. Ed. 49, 87–90 (2010).

    Article  CAS  Google Scholar 

  21. Chung, S.E. et al. Optofluidic maskless lithography system for real-time synthesis of photopolymerized microstructures in microfluidic channels. Appl. Phys. Lett. 91, 041106 (2007).

    Article  Google Scholar 

  22. Dendukuri, D., Pregibon, D.C., Collins, J., Hatton, T.A. & Doyle, P.S. Continuous flow lithography for high-throughput microparticle synthesis. Nat. Mater. 5, 365–369 (2006).

    Article  CAS  Google Scholar 

  23. Birtwell, S. & Morgan, H. Microparticle encoding technologies for high-throughput multiplexed suspension assays. Integr. Biol. 1, 345–362 (2009).

    Article  CAS  Google Scholar 

  24. Broder, G.R. et al. Diffractive micro bar codes for encoding of biomolecules in multiplexed assays. Anal. Chem. 80, 1902–1909 (2008).

    Article  CAS  Google Scholar 

  25. Fan, R. et al. Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nat. Biotech. 26, 1373–1378 (2008).

    Article  CAS  Google Scholar 

  26. Brunker, S.E., Cederquist, K.B. & Keating, C.D. Metallic barcodes for multiplexed bioassays. Nanomedicine 2, 695–710 (2007).

    Article  CAS  Google Scholar 

  27. Hwang, D.K. et al. Stop-flow lithography for the production of shape-evolving degradable microgel particles. J. Am. Chem. Soc. 131, 4499–4504 (2009).

    Article  CAS  Google Scholar 

  28. Kloxin, A.M., Kasko, A.M., Salinas, C.N. & Anseth, K.S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59–63 (2009).

    Article  CAS  Google Scholar 

  29. Fotin, A.V., Drobyshev, A.L., Proudnikov, D.Y., Perov, A.N. & Mirzabekov, A.D. Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips. Nucleic Acids Res. 26, 1515–1521 (1998).

    Article  CAS  Google Scholar 

  30. Pregibon, D.C. & Doyle, P.S. Optimization of encoded hydrogel particles for nucleic acid quantification. Anal. Chem. 81, 4873–4881 (2009).

    Article  CAS  Google Scholar 

  31. Appleyard, D.C., Chapin, S.C. & Doyle, P.S. Multiplex protein quantification with barcoded hydrogel microparticles. Anal. Chem. 83, 193–199 (2011).

    Article  CAS  Google Scholar 

  32. Helgeson, M.E., Chapin, S.C. & Doyle, P.S. Hydrogel microparticles from lithographic processes: novel materials for fundamental and applied colloid science. Curr. Opin. Colloid Interface Sci. 16, 106–117 (2011).

    Article  CAS  Google Scholar 

  33. Dedukuri, D. & Doyle, P.S. The synthesis and assembly of polymeric microparticles using microfluidics. Adv. Mater. 21, 4071–4086 (2009).

    Article  Google Scholar 

  34. Panda, P. et al. Stop-flow lithography to generate cell-laden microgel particles. Lab Chip 8, 1056–1061 (2008).

    Article  CAS  Google Scholar 

  35. Bong, K.W., Chapin, S.C. & Doyle, P.S. Magnetic barcoded hydrogel microparticles for multiplexed detection. Langmuir 26, 8008–8014 (2010).

    Article  CAS  Google Scholar 

  36. Lee, H., Kim, J., Kim, H., Kim, J. & Kwon, S. Colour-barcoded magnetic microparticles for multiplexed bioassays. Nat. Mater. 9, 745–749 (2010).

    Article  CAS  Google Scholar 

  37. Bong, K.W., Pregibon, D.C. & Doyle, P.S. Lock release lithography for 3D and composite microparticles. Lab Chip 9, 863–866 (2009).

    Article  CAS  Google Scholar 

  38. Lewis, C.L. et al. Microfluidic fabrication of hydrogel microparticles containing functionalized viral nanotemplates. Langmuir 26, 13436–13441 (2010).

    Article  CAS  Google Scholar 

  39. Lee, S.A., Chung, S.E., Park, W., Lee, S.H. & Kwon, S. Three-dimensional fabrication of heterogeneous microstructures using soft membrane deformation and optofluidic maskless lithography. Lab Chip 9, 1670–1675 (2009).

    Article  CAS  Google Scholar 

  40. Finkel, N.H., Lou, X.H., Wang, C.Y. & He, L. Barcoding the microworld. Anal. Chem. 76, 352A–359A.

  41. Cederquist, K.B., Dean, S.L. & Keating, C.K. Encoded anisotropic particles for multiplexed bioanalysis. Rev. Nanomed. Nanobiotechnol. 2, 578–600 (2010).

    Article  CAS  Google Scholar 

  42. Chowdhury, F., Williams, A. & Johnson, P. Validation and comparison of two multiplex technologies, Luminex and mesoscale discovery, for human cytokine profiling. J. Immunol. Methods 340, 55–64 (2009).

    Article  CAS  Google Scholar 

  43. Nechansky, A., Grunt, S., Roitt, I.M. & Kircheis, R. Comparison of the calibration standards of three commercially available multiplex kits for human cytokine measurement to WHO standards reveals striking differences. Biomarker Insights 3, 227–235 (2008).

    Article  CAS  Google Scholar 

  44. Chapin, S.C., Appleyard, D.C., Pregibon, D.C. & Doyle, P.S. Rapid microRNA profiling on encoded gel microparticles. Angew. Chem. Int. Ed. 50, 2289–2293 (2011).

    Article  CAS  Google Scholar 

  45. Dendukuri, D., Gu, S.S., Pregibon, D.C., Hatton, T.A. & Doyle, P.S. Stop-flow lithography in a microfluidic device. Lab Chip 7, 818–828 (2007).

    Article  CAS  Google Scholar 

  46. Bong, K.W. et al. Compressed-air flow control system. Lab Chip 11, 743–747 (2011).

    Article  CAS  Google Scholar 

  47. Chapin, S.C., Pregibon, D.C. & Doyle, P.S. High-throughput flow alignment of barcoded hydrogel microparticles. Lab Chip 9, 3100–3109 (2009).

    Article  CAS  Google Scholar 

  48. He, M. & Herr, A.E. Microfluidic polyacrylamide gel electrophoresis with in situ immunoblotting for native protein analysis. Anal. Chem. 81, 8177–8184 (2009).

    Article  CAS  Google Scholar 

  49. Kandow, C.E., Georges, P.C., Janmey, P.A. & Beningo, K.A. Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses. Meth. Cell Bio. 83, 29–46 (2007).

    Article  CAS  Google Scholar 

  50. Bailey, R.C., Kwong, G.A., Radu, C.G., Witte, O.N. & Heath, J.R. DNA-encoded antibody libraries: a unified platform for multiplexed cell sorting and detection of genes and proteins. J. Am. Chem. Soc. 129, 1959–1967 (2007).

    Article  CAS  Google Scholar 

  51. de Jager, W., te Velthuis, H., Prakken, B.J., Kuis, W. & Rijkers, G.T. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin. Diagn. Lab Immunol. 10, 133–139 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge financial support from the Ragon Institute of Massachusetts General Hospital, the Massachusetts Institute of Technology and Harvard University; grant R21EB008814 from the National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health; and National Science Foundation grant DMR-1006147. R.L.S. is supported by a US National Institutes of Health/National Institute of General Medical Sciences Biotechnology Training Grant.

Author information

Author notes

  1. David C Appleyard and Stephen C Chapin: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    David C Appleyard, Stephen C Chapin, Rathi L Srinivas & Patrick S Doyle

Authors
  1. David C Appleyard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen C Chapin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rathi L Srinivas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patrick S Doyle
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.C.A. and S.C.C. contributed equally to this work. D.C.A., S.C.C. and P.S.D. designed instrumentation and experiments. D.C.A. and R.L.S. conducted the experiments. S.C.C. designed microfluidics. D.C.A., S.C.C., R.L.S. and P.S.D. wrote the paper.

Corresponding author

Correspondence to Patrick S Doyle.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

The zip file contains designs for the scanning channel, synthesis channel, and particle mask. For the scanning channel design, see provided AutoCAD file (Flow Focusing Device.dwg in AutoCAD Files.zip) for design. For the synthesis channel design. see the provided AutoCAD file (Synthesis Wafer.dwg in AutoCAD Files.zip) for a wafer containing various types of synthesis channels. For the particle masks, see provided AutoCAD file (Particle Masks.dwg in AutoCAD Files.zip) or Adobe Acrobate file (Particle Mask Sheet.pdf) for a variety of particle masks. (ZIP 153 kb)

Supplementary Movie 1

See provided AVI file (Supplementary Movie 1 (Doyle).avi). Synthesis of a 4-chemistry particle with monoclonal capture antibodies against Interferon-Îł (R&D systems, MAB2852). From top to bottom, the stream compositions are: fluorescent code monomer, blank monomer, IFN-Îł antibody probe monomer, blank monomer. Blank monomer is colored with blue food coloring for visible contrast of streams. The antibody concentration in the probe monomer is 2 mg/mL. The code on the mask is "02223". Throughput is approximately 5 particles every second. Recording and playback is set so that the synthesis occurs in real time. (AVI 3288 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Appleyard, D., Chapin, S., Srinivas, R. et al. Bar-coded hydrogel microparticles for protein detection: synthesis, assay and scanning. Nat Protoc 6, 1761–1774 (2011). https://doi.org/10.1038/nprot.2011.400

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.400

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing