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Improving the sensitivity of immunoassays by reducing non-specific binding of poly(acrylic acid) coated upconverting nanoparticles by adding free poly(acrylic acid)

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Abstract

Upconverting nanoparticles (UCNPs) are attractive reporters in immunoassays because of their outstanding detectability. However, non-specific binding of antibody-UCNP conjugates on protein coated solid support results in background, which limits the immunoassay sensitivity. Thus, the full potential of UCNPs as reporters cannot be fully exploited. The authors report here a method to improve the sensitivity of UCNP-based immunoassays by reducing the non-specific binding of antibody-UNCP conjugates on the protein coated solid support. In the assays studied here, poly(acrylic acid) (PAA) coated NaYF4:Yb3+,Er3+ type UCNPs were conjugated to two different antibodies against cardiac troponin I (cTnI) and thyroid stimulating hormone (TSH). The two-step heterogeneous sandwich immunoassays were performed in microtitration wells, and the green luminescence of antibody-UCNP conjugates was measured at 540 nm upon 980 nm excitation. Non-specific binding of antibody-UCNP conjugates was reduced by mixing free PAA with PAA coated UCNPs before adding the UCNPs to the wells. The free PAA in the buffer reduced the background in both cTnI and TSH immunoassays (compared to the control assay without free PAA). The limits of detection decreased from 2.1 ng·L−1 to 0.48 ng·L−1 in case of cTnI and from 0.070 mIU·L−1 to 0.020 mIU·L−1 in case of TSH if PAA is added to the buffer. Presumably, the effect of free PAA is due to blocking of the surface areas where PAA coated UCNP would bind proteins non-specifically. The method introduced here is likely to be applicable to other kinds of PAA-coated nanoparticles, and similar approaches conceivably work also with other nanoparticle coatings.

The presence of free poly(acrylic acid) (PAA) in a buffer solution prevents aggregation and non-specific protein binding of PAA-coated upconverting nanoparticles (UCNPs) in heterogeneous sandwich immunoassays. The decrease in non-specific binding enables distinctly more sensitive assays to be performed.

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Acknowledgments

This study was supported by Tekes, the Finnish Funding Agency for Innovation and the Doctoral Programme of Molecular Life Sciences. The authors wish to thank Jessica Rosenholm from Åbo Akademi for assisting with the DLS measurements and Raili Kronström for the technical assistance.

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Authors and Affiliations

  1. Department of Biotechnology, University of Turku, Tykistökatu 6A, 20520, Turku, Finland

    Satu Lahtinen, Annika Lyytikäinen, Nina Sirkka, Henna Päkkilä & Tero Soukka

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  1. Satu Lahtinen
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  2. Annika Lyytikäinen
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  3. Nina Sirkka
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  4. Henna Päkkilä
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  5. Tero Soukka
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Correspondence to Satu Lahtinen.

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Lahtinen, S., Lyytikäinen, A., Sirkka, N. et al. Improving the sensitivity of immunoassays by reducing non-specific binding of poly(acrylic acid) coated upconverting nanoparticles by adding free poly(acrylic acid). Microchim Acta 185, 220 (2018). https://doi.org/10.1007/s00604-018-2756-z

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  • DOI: https://doi.org/10.1007/s00604-018-2756-z

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