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Intensity Histogram-Based Reliable Image Analysis Method for Bead-Based Fluorescence Immunoassay

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Abstract

Bead-based fluorescence immunoassay is drawing attention as a next-generation technology in disease diagnosis owing to its high sensitivity and multiplexing capability. Fluorescence imaging of beads is typically used to determine their mean fluorescence intensity. However, the mean intensity can be evaluated differently depending on the analysis methods [such as the shape and size of the region of interest (ROI)]. To address these problems, this study proposes a highly reliable and reproducible image analysis method utilizing a fluorescence intensity-based effective pixel extraction technique. Various potential sources of defective signals (e.g., fluorescence aggregation, non-specific antigen–antibody reactions, and bead defects) can be prevented from contributing to the average value by selectively extracting pixels representing the specific reactions of antigens and antibodies in the ROI. In this study, we fabricated a microfluidic chip composed of multiple bead-based detection lines, performed fluorescence immunoassay, and then compared the mean fluorescence intensity calculated from the fluorescence images with that of a conventional analysis method. Using the conventional method, the evaluated average mean intensity value of beads varied significantly based on the size of the ROI with the coefficients of variation ranging from approximately 29–95%. In contrast, the effective pixel extraction method resulted in a coefficient of variation of approximately 3–7% under varying ROI size. Furthermore, the coefficients of variation for four detection lines containing various types of defective signals significantly decreased from approximately 7.1% to 2.6%. The proposed technique will help in minimizing the analysis deviation caused by different ROI selections or defective signals in fluorescent image-based immunoassays.

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Data availability

The authors declare that the data supporting the findings of this study are available within the paper.

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Acknowledgements

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (NRF-2021M3H4A4079557 and RS-2023-00213140).

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Authors and Affiliations

  1. Division of Mechanical, Automotive and Robot Component Engineering, Dong-eui University, 176 Eomgwang-ro, Busanjin-gu, Busan, 47340, Republic of Korea

    Sanghyun Lee & Sangmin Lee

  2. Graduate School of Energy/Bio-Engineering, Dongseo University, 47 Jurye-ro, Sasang-gu, Busan, 47011, Republic of Korea

    Juyeong Kim & Hojin Kim

  3. BioNano Health Guard Research Center (H-GUARD), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, South Korea

    Pankee Bae

  4. Department of Mechanical Engineering, Dongseo University, 47 Jurye-ro, Sasang-gu, Busan, 47011, Republic of Korea

    Hojin Kim

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Lee, S., Kim, J., Bae, P. et al. Intensity Histogram-Based Reliable Image Analysis Method for Bead-Based Fluorescence Immunoassay. BioChip J 18, 137–145 (2024). https://doi.org/10.1007/s13206-023-00137-9

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