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Next-generation oxygen sensing: fluorescent hemoglobin nanosensors for biological systems
Letch, Elizabeth Cash, Kevin J.
Letch, Elizabeth
Cash, Kevin J.
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2025-04
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Abstract
Hemoglobin is a protein found in red blood cells that binds and releases oxygen, allowing it to circulate throughout the bloodstream and body. Oxygen nanosensors can be created by leveraging hemoglobin’s natural oxygen-exchanging capabilities. A hemoglobin molecule can be tagged with a fluorescent dye through NHS ester bioconjugation techniques, allowing us to monitor the oxygenation status of hemoglobin through spectroscopy. However, hemoglobin can only exchange oxygen in the ferrous Fe2+ oxidation state. To keep protein in its desired oxidation state, the nanosensors are created in a solution of ascorbic acid (Vitamin C) and bovine hemoglobin dissolved in PBS, and the solution reacts in the fridge overnight. An amine-reactive dye (AZDye™ 350 NHS Ester) is added to the solution the next day, and through ester-activated crosslinking, the dye conjugates to the surface of the protein. The oxygen-exchange reversibility of the hemoglobin nanosensors is assessed by sequentially bubbling the solution with nitrogen (N₂) and air. The solution is deoxygenated by bubbling N₂ for 30 minutes, followed by reoxygenation with air for 30 minutes. This is repeated for at least three cycles of each gas mixture, with fluorescent intensity measurements and an absorbance spectra recorded after each cycle on our plate reader. The results demonstrate that the hemoglobin nanosensors exhibit reversible oxygenation changes upon alternating N₂ and air exposure.
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