Immunodetection of a Cell-Surface Biomarker via the Freezing of Supercooled Water Droplets
- 1Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, Canada, V6T 1Z1
Abstract
Immunolabels traditionally detect biomarkers via a color change or fluorescence. The frequently weak signals from low biomarker concentrations prompted the development of numerous enzymatic amplification strategies. Although very effective for signal generation, these strategies increase the complexity, cost, and time required for immunoassays. What if water itself could generate and self-amplify a signal to detect a biomarker? Here, we demonstrate that it is possible to detect a cell-surface protein biomarker by inducing the phase change from water to ice in supercooled water droplets. Relative to enzymatic amplification, this phase change has the potential for orders-of-magnitude larger amplification factors on orders-of-magnitude shorter time scales. We engineered a recombinant ice-nucleating protein (rINpro), based on the bacterial protein InaZ, that binds to an antigen via a tetrameric antibody complex. Breast cancer cells expressing the Human Epidermal Growth Factor Receptor (HER2) antigen were adopted as a model system. Arrays with 96 × 10 μL droplets containing cells immunolabeled with rINpro froze at a higher average median freezing temperature (–11.2 ± 0.5 °C) than droplets with unlabeled cells (–23.4 ± 1.3 °C) and non-target cells (–25.1 ± 0.6 °C). High-speed videos of freezing droplets confirmed that the labeled cells triggered the freezing and nucleated macroscopic ice formation. As few as ca. 500 target cells were successfully detected among 10,000 non-target cells, and selective naked-eye detection of target cells was possible with a household freezer and fixed cooling period. The “cryo-assay” in this study represents a completely new approach to sensitive biomarker detection. With further development, it has vast potential for the detection of low-abundance biomarkers in research, clinical, and point-of-care immunoassay applications.
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BibTeX
@misc{bieber2026immunodetection,
title = {{Immunodetection of a Cell-Surface Biomarker via the Freezing of Supercooled Water Droplets}},
author = {Paul Bieber and Ghinwa H. Darwish and Paris Salamon and Katherine S. Ryan and W. Russ Algar and Nadine Borduas-Dedekind},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010489/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010489/v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Immunodetection of a Cell-Surface Biomarker via the Freezing of Supercooled Water Droplets
AU - Paul Bieber
AU - Ghinwa H. Darwish
AU - Paris Salamon
AU - Katherine S. Ryan
AU - W. Russ Algar
AU - Nadine Borduas-Dedekind
PY - 2026
DA - 2026/10/15
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010489/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010489/v1
AB - Immunolabels traditionally detect biomarkers via a color change or fluorescence. The frequently weak signals from low biomarker concentrations prompted the development of numerous enzymatic amplification strategies. Although very effective for signal generation, these strategies increase the complexity, cost, and time required for immunoassays. What if water itself could generate and self-amplify a signal to detect a biomarker? Here, we demonstrate that it is possible to detect a cell-surface protein biomarker by inducing the phase change from water to ice in supercooled water droplets. Relative to enzymatic amplification, this phase change has the potential for orders-of-magnitude larger amplification factors on orders-of-magnitude shorter time scales. We engineered a recombinant ice-nucleating protein (rINpro), based on the bacterial protein InaZ, that binds to an antigen via a tetrameric antibody complex. Breast cancer cells expressing the Human Epidermal Growth Factor Receptor (HER2) antigen were adopted as a model system. Arrays with 96 × 10 μL droplets containing cells immunolabeled with rINpro froze at a higher average median freezing temperature (–11.2 ± 0.5 °C) than droplets with unlabeled cells (–23.4 ± 1.3 °C) and non-target cells (–25.1 ± 0.6 °C). High-speed videos of freezing droplets confirmed that the labeled cells triggered the freezing and nucleated macroscopic ice formation. As few as ca. 500 target cells were successfully detected among 10,000 non-target cells, and selective naked-eye detection of target cells was possible with a household freezer and fixed cooling period. The “cryo-assay” in this study represents a completely new approach to sensitive biomarker detection. With further development, it has vast potential for the detection of low-abundance biomarkers in research, clinical, and point-of-care immunoassay applications.
ER -