Clinical Applications of Capillary Electrophoresis pp 233-248

Part of the Methods in Molecular Biology book series (MIMB, volume 919)

Chip-Based Immunoassays



Microfluidic immunoassay techniques offer advantages in speed, automation, and portability over ­bench-top gold standard counterparts. In particular, on-chip immunosubtraction is a rapid homogeneous immunoassay used for reporting both protein native mobility and binding specificity. Immunosubtraction is performed by removing antibody-bound target proteins from electrophoretic detection via a size-based exclusion filter, while unbound nontarget proteins are able to pass through the filter for downstream detection. Immunosubtraction is achieved on-chip by fabrication of discrete patterned polyacrylamide (PA) gel regions. Additionally, PA gel regions are used to define on-chip sample preparation regions for protein enrichment, fluorescent labeling, and antibody-target binding prior to immunosubtraction. Here we describe the immunosubtraction device fabrication technique as well as the electrophoretic assay protocol for determining target protein mobility and binding specificity within complex biological samples including cerebrospinal fluid.

Key words

Microfluidic immunoassay Immunosubtraction Sample preparation PAGE Microfabrication 


  1. 1.
    Ng AH, Uddayasankar U, Wheeler AR (2010) Immunoassays in microfluidic systems. Anal Bioanal Chem 397:991–1007PubMedCrossRefGoogle Scholar
  2. 2.
    Weigl B et al (2008) Towards non- and minimally instrumented, microfluidics-based diagnostic devices. Lab Chip 8:1999–2014PubMedCrossRefGoogle Scholar
  3. 3.
    Herr AE et al (2007) Microfluidic immunoassays as rapid saliva-based clinical diagnostics. Proc Natl Acad Sci U S A 104:5268–5273PubMedCrossRefGoogle Scholar
  4. 4.
    Hatch AV et al (2006) Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels. Anal Chem 78:4976–4984PubMedCrossRefGoogle Scholar
  5. 5.
    Sun XH et al (2008) Affinity monolith integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis. Anal Chem 80:5126–5130PubMedCrossRefGoogle Scholar
  6. 6.
    Beyor N et al (2009) Integrated capture, concentration, polymerase chain reaction, and capillary electrophoretic analysis of pathogens on a chip. Anal Chem 81:3523–3528PubMedCrossRefGoogle Scholar
  7. 7.
    Hutchison CA, Basnayake K, Cockwell P (2009) Serum free light chain assessment in monoclonal gammopathy and kidney disease. Nat Rev Nephrol 5:621–627PubMedCrossRefGoogle Scholar
  8. 8.
    Keren DF (1999) Procedures for the evaluation of monoclonal immunoglobulins. Arch Pathol Lab Med 123:126–132PubMedGoogle Scholar
  9. 9.
    Keren DF (2003) Protein electrophoresis in clinical diagnostics. Arnold Publishers, LondonGoogle Scholar
  10. 10.
    Clark R et al (1998) Differential diagnosis of gammopathies by capillary electrophoresis and immunosubtraction: analysis of serum samples problematic by agarose gel electrophoresis. Electrophoresis 19:2479–2484PubMedCrossRefGoogle Scholar
  11. 11.
    Palfrey S (1999) Immunosubtraction as a means of typing monoclonal and other proteins in serum and urine. In: Palfrey S (ed) Clinical applications of capillary electrophoresis. Humana Press, Totowa, NJ, pp 39–46CrossRefGoogle Scholar
  12. 12.
    Apori AA, Herr AE (2011) Homogeneous immunosubtraction integrated with sample preparation enabled by a microfluidic format. Anal Chem 83:2691–2698PubMedCrossRefGoogle Scholar
  13. 13.
    Hughes AJ, Herr AE (2010) Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography. Anal Chem 82:3803–3811PubMedCrossRefGoogle Scholar
  14. 14.
    He M, Herr AE (2010) Polyacrylamide gel photopatterning enables automated protein immunoblotting in a two-dimensional microdevice. J Am Chem Soc 132:2512–2513PubMedCrossRefGoogle Scholar
  15. 15.
    Hou CL, Herr AE (2010) Ultrashort separation length homogeneous electrophoretic immunoassays using on-chip discontinuous polyacrylamide gels. Anal Chem 82:3343–3351PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of BioengineeringUniversity of California BerkeleyBerkeleyUSA

Personalised recommendations