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DNA immobilization and detection using DNA binding proteins

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Abstract

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin’s, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

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Abbreviations

CNP:

Carbon nanoparticles

dHP:

Dimeric headpiece domain of the Escherichia coli LacI repressor

ELISA:

Enzyme-linked immunosorbent assay

ELONA:

Enzyme-linked oligonucleotide assay

EMSA:

Electrophoretic mobility shift assay

LFA:

Lateral flow assay

scCro:

Single-chain bacteriophage Cro repressor

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Acknowledgments

GBA acknowledges the Universitat Rovira i Virgili for the doctoral fellowship. The authors thank Dr. Aart van Amerongen (Wageningen University and Research, The Netherlands) for the carbon nanoparticles.

Funding

This work was supported financially by the FP7-PEOPLE-2011-CIG DeCoDeB project grant awarded to LM.

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Authors and Affiliations

  1. Departament d’Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain

    Gülsen Betül Aktas, Arnau Ribera, Vasso Skouridou & Lluis Masip

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  1. Gülsen Betül Aktas
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  2. Arnau Ribera
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  3. Vasso Skouridou
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  4. Lluis Masip
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Correspondence to Lluis Masip.

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Aktas, G.B., Ribera, A., Skouridou, V. et al. DNA immobilization and detection using DNA binding proteins. Anal Bioanal Chem 413, 1929–1939 (2021). https://doi.org/10.1007/s00216-021-03162-5

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  • DOI: https://doi.org/10.1007/s00216-021-03162-5

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