Fig. 4: A ratiometric luminescent assay can sense nitazenes specifically in relevant diagnostic scenarios.

A Cartoon of the luminescence assay. A mNeonGreen-NanoBiT “calibrator” protein enables ratiometric detection of samples, fluorescing at a wavelength of 520 nm independent of ligand concentration. PYR1 to ΔN-HAB1^T+ dimerization results in an increased ratio of the relative luminescence units per second (RLU/s) at 450 nm versus the RLU/s at 520 nm. B Ratiometric assay of PYR1^nitav2.1 in buffer or in urine using the indicated ligands. EC₅₀ measurements and limit of detection (LOD) for the sensor are colored for the appropriate condition. 200 nM SmBiT-ΔNHAB1, 10 nM LgBiT-PYR1, and 512 pM calibrator are used. Benzylfentanyl, codeine, and heroin were tested in buffer. The nitazene-specific EC₅₀ was 5.7 nM (95% c.i. 4.0– 8.3 nM) in the urine matrix and 2.8 nM (95% c.i. 2.3–3.4 nM) in buffer. C Luminescence assay results using PAN^nita.1 show sensitivity against a panel of nitazene variants and synthetic opioids across multiple concentrations. Analysis of variance was calculated using an ordinary 2-way ANOVA and Dunnett’s multiple comparison test, with a single pooled variance (****p < 0.0001, **p < 0.01, ns not statistically significant). 4 nM SmBit-ΔNHAB1 and 4 nM LgBiT-PYR1 are used. Data is shown as the average of n = 3 (B) or n = 4 (C) replicates. Error bars represent the standard error of the mean and in some cases are smaller than the symbols. EC_50s were calculated by fitting the data to a four-parameter logistical sigmoidal model with nonlinear least-squares regression. LODs were calculated using the 3σ method, which is equivalent to the drug concentration that yields a signal equal to 3 times the standard deviation of the blank after subtraction. Source Data are provided with this paper as Source Data.