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Determination of protein surface excess on a liquid/solid interface by single-molecule counting

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Abstract

Determination of protein surface excess is an important way of evaluating the properties of biomaterials and the characteristics of biosensors. A single-molecule counting method is presented that uses a standard fluorescence microscope to measure coverage of a liquid/solid interface by adsorbed proteins. The extremely low surface excess of lysozyme and bovine serum albumin (BSA), in a bulk concentration range from 0.3 nmol L−1 (0.02 μg mL−1) to 3 nmol L−1 (0.2 μg mL−1), were measured by recording the counts of spatially isolated single molecules on either hydrophilic (glass) or hydrophobic (polydimethylsiloxane, PDMS) surfaces at different pH. The differences observed in amounts of adsorbed proteins under different experimental conditions can be qualitatively explained by the combined interactions of electrostatic and hydrophobic forces. This, in turn, implies that single-molecule counting is an effective way of measuring surface coverage at a liquid/solid interface.

Adsorption fraction of proteins on different surfaces changed with pH.

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Acknowledgements

The authors are grateful to the Natural Science Foundation of China (NSFC, 20705007, 30570479, 30670532) and the Hunan University “985” Fund for financial support.

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Authors and Affiliations

  1. Biomedical Engineering Center, Hunan University, Changsha, Hunan, 410082, China

    Nan Li, Hui Tang, Hongwei Gai & Edward S. Yeung

  2. State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410082, China

    Hongwei Gai

  3. Dalian the Third Municipal Hospital, Dalian, 116033, China

    **uling Dong

  4. The Second Hospital Affiliated to Dalian Medical University, Dalian, 116023, China

    Qi Wang

  5. Department of Chemistry and Ames Laboratory, Iowa State University, Ames, IA, 50011, USA

    Edward S. Yeung

  6. # A202, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, 410082, China

    Hongwei Gai

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  1. Nan Li
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  3. Hongwei Gai
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  4. **uling Dong
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  5. Qi Wang
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  6. Edward S. Yeung
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Correspondence to Hongwei Gai.

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Li, N., Tang, H., Gai, H. et al. Determination of protein surface excess on a liquid/solid interface by single-molecule counting. Anal Bioanal Chem 394, 1879–1885 (2009). https://doi.org/10.1007/s00216-009-2888-4

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  • DOI: https://doi.org/10.1007/s00216-009-2888-4

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