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Multiplexed protein detection using an affinity aptamer amplification assay

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Abstract

Affinity probe capillary electrophoresis (APCE) is potentially one of the most versatile technologies for protein diagnostics, offering an excellent balance between robustness, analysis speed and sensitivity. Combining the immunosensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification, aptamers can further push the analytical limits of APCE to offer ultrasensitive, multiplexed detection of protein biomarkers, even when differences in electrophoretic mobility between the different aptamer–target complexes are limited. It is demonstrated how, through careful selection of experimental parameters, simultaneous detection of picomolar levels of three target proteins can be achieved even with aptamers that were initially selected under very different conditions and further taking into account that the aptamers need to be modified to allow successful PCR amplification. Aptamer-enhanced APCE offers limits of detection that are orders of magnitude lower than those that can be achieved through traditional capillary electrophoresis-based immunosensing. With recent developments in aptamer selection that for the first time realise the promise of aptamers as easily accessible, high affinity recognition molecules, it can therefore be envisioned that aptamer-enhanced APCE on parallel microfluidic platforms can be the basis for a truly high-throughput multiplexed proteomics platform, rivalling genetic screening for the first time.

Multiplexed affinity probe capillary electrophoresis (APCE) is enhanced through the use of PCR affinity aptamer amplification, improving both the limits of detection as well as the resolution of a typical APCE assay.

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Acknowledgments

The authors would like to thank The Fund for Scientific Research Flanders (FWO), EFRO financing Interreg NanoSensEU and EU FP7-KBBE-2009-3-245137 Marex. JS acknowledges the support from the KU Leuven—IOF Knowledge Platform.

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Authors and Affiliations

  1. BIOSYST—MeBioS, KU Leuven, Willem De Croylaan 42, 3001, Leuven, Belgium

    Kris P. F. Janssen, Karel Knez, Dragana Spasic & Jeroen Lammertyn

  2. Department of Metallurgy and Materials Engineering, KU Leuven, Kasteelpark Arenberg 44, PB2450, 3001, Leuven, Belgium

    Jan Schrooten

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  1. Kris P. F. Janssen
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Correspondence to Jeroen Lammertyn.

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Janssen, K.P.F., Knez, K., Spasic, D. et al. Multiplexed protein detection using an affinity aptamer amplification assay. Anal Bioanal Chem 404, 2073–2081 (2012). https://doi.org/10.1007/s00216-012-6252-8

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  • DOI: https://doi.org/10.1007/s00216-012-6252-8

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