{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T12:27:18Z","timestamp":1793708838445,"version":"4.6.1"},"posted":{"date-parts":[[2026,9,23]]},"group-title":"Bioengineering","reference-count":66,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,10,30]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Measuring the precise dynamics of specific neuromodulators in neurons is key to understanding how information is transmitted and processed in the brain. Despite the growing use of intensity-based neurochemical sensors such as dLight in recent years, it remains challenging to monitor the dynamics of multiple neurochemicals simultaneously in live neurons. In contrast to intensiometric sensors that are susceptible to artifacts caused by changes in sensor expression and rapid photobleaching, fluorescence lifetime-based sensors are less affected by these factors and can provide more stable measurements. Here we introduce dHaloLife\n                  <jats:sub>635<\/jats:sub>\n                  , a chemigenetic fluorescence-lifetime-based dopamine (DA) sensor that exhibits a fluorescence lifetime decrease of 0.21 ns upon dopamine binding. dHaloLife\n                  <jats:sub>635<\/jats:sub>\n                  consists of a G protein-coupled dopamine receptor (hDRD1), a circularly permuted HaloTag (cpHaloTag), and a HaloTag ligand-functionalized cell-permeable dye (JF\n                  <jats:sub>635<\/jats:sub>\n                  -HTL). Dopamine binding to dHaloLife\n                  <jats:sub>635<\/jats:sub>\n                  induces a conformational change in the cpHaloTag that alters the local environment of the conjugated dye, thereby changing its fluorescence lifetime. In live HEK293T cells, dHaloLife\n                  <jats:sub>635<\/jats:sub>\n                  responds to dopamine but not to serotonin, L-DOPA, or GABA, and only marginally to norepinephrine, with selectivity for dopamine over norepinephrine confirmed in primary neurons, demonstrating excellent specificity. Together, these results establish dHaloLife\n                  <jats:sub>635<\/jats:sub>\n                  as an initial step toward a broader family of chemigenetic lifetime sensors that exhibit distinct fluorescence lifetime signatures upon binding their respective analytes, thereby potentially enabling highly specific and sensitive monitoring of multiple neurochemical dynamics\n                  <jats:italic>in vivo<\/jats:italic>\n                  over extended timescales.\n                <\/jats:p>","DOI":"10.64898\/2026.09.19.752778","type":"posted-content","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:55:16Z","timestamp":1790211316000},"source":"Crossref","is-referenced-by-count":0,"title":["A Chemigenetic Fluorescence Lifetime Biosensor for Dopamine Sensing"],"prefix":"10.64898","author":[{"given":"Anh-Thu","family":"Nguyen","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, 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