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A structure-optimized BRET biosensor for rapid, dilution-free quantification of high-strength nitrate in wastewater
Kanglin Zhao1, Hao Bai2, Ziqi Sha1
1State Key Laboratory of Water Pollution Control and Green Resource Recycling, School of Environment, Nanjing University, Nanjing, Jiangsu, 210023, China.
None:
A bioluminescence resonance energy transfer (BRET) biosensor was developed for rapid, dilution-free quantification of high-strength nitrate in wastewater. The sensor integrates the Escherichia coli nitrate receptor NarX with a high-brightness NLuc/VenusΔC10 reported pair and was optimized by computationally guided mutagenesis (NarXV138W), structure-informed terminal truncation, and assay parameter tuning. The optimized construct, VenusΔC12-N5NarXV138WΔC3-ΔN4NLuc, enabled accurate quantification from 0.0781 to 2.5710 g/L (as N) with a four-parameter logistic fit (R2 = 0.999) and a ΔBRET of 0.28 after ∼500 s equilibration. Measurements proceeded under mild aqueous conditions and showed strong agreement with UV-visible spectrophotometry in both simulated and real water samples, eliminating the need for extensive dilution. Circular dichroism and mass spectrometry analyses revealed nitrate-dependent structural changes that are supportive of ligand-induced signaling mechanisms. To our knowledge, this represents the first BRET-based biosensing platform tailored to industrial-range nitrate detection, offering a simple, ratiometric, and reagent-efficient approach suitable for in situ monitoring of industrial and municipal effluents.
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