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. 2026 Apr 25;11(18):26706–26712. doi: 10.1021/acsomega.5c13412

Impact of BHT Additive on the Optical and Reactive Behavior of Fluorescent Peroxynitrite Probes

An V Pham †, Emile J Morin †, Shivshankar R Mane †,‡, Andrea S Carlini †,‡,§,*
PMCID: PMC13177213  PMID: 42146236

Abstract

Peroxynitrite (ONOO–) is a reactive oxygen and nitrogen species that is central to oxidative stress and pathophysiological signaling. Fluorescent small-molecule chemicals have been widely employed for detecting peroxynitrite, also known as peroxynitrite-responsive probes (PRPs). However, the influence of common solvent stabilizers on sensor performance remains insufficiently examined. Here, we report the impact of a ubiquitous stabilizer, trace amounts of butylated hydroxytoluene (BHT), a frequently used antioxidant in organic solvents (i.e., tetrahydrofuran, diethyl ether, and isopropyl ether), which significantly alters the baseline optical properties and the apparent reactivity of model fluorescent probes with peroxynitrite. Utilizing UV–visible spectroscopy, fluorescence spectroscopy, and nuclear magnetic resonance (NMR), our results demonstrate that BHT has an impact at concentrations as low as 0.25 μM, which is ∼103–104-fold lower than the typical 900–1800 μM found in standard 4 L solvent bottles. The stabilizer (1) induces absorption baseline elevation near 310 nm, (2) partially distorts the probe’s emission band, and results in a (3) loss of phenolic proton signal consistent with oxidation products in CD3CN after exposure to ONOO–. These effects lead to an artificially suppressed apparent sensor response, corresponding to a 98% decrease in the expected signal. Our findings demonstrate that the presence of BHT in commercial solvents can compromise analytical peroxynitrite sensor assays, especially those reliant on short-wavelength (≤350 nm) excitations. This further emphasizes the need to consider stabilizer impurities when fabricating PRPs for both fundamental studies and translational applications.

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Introduction

Peroxynitrite (ONOO–) is a short-lived, biologically potent oxidant generated by the near-diffusion-controlled reaction between superoxide (O2·2–) and nitric oxide (NO·) in oxidatively stressed tissues. Its production in the body contributes to neurodegenerative disorders, cardiovascular dysfunction, inflammation, and cancer through oxidative and nitrative modification to proteins, lipids, and DNA. , Consequently, the selective and quantitative detection of ONOO– remains a central goal in chemical biology.

Peroxynitrite-responsive probes (PRPs) offer the potential for spatially resolved, real-time detection of ONOO– and have been developed using a diverse toolbox of chemistries, reliant on benzopyrylium dyes, boronic acids and esters, hydrazide functionalities, α-ketoamides, indolines, and phosphinates. − These chemistries, when coupled within or to fluorescent dyes, achieve ratiometric and selective detection of ONOO– over the other reactive oxygen and nitrogen species (RONS). Despite these advanced capabilities, the transient nature of ONOO– (half-life on the order of milliseconds under physiological conditions) and its cross-reactivity with other RONS complicate accurate quantification. The presence of antioxidants (i.e., TEMPOL) or natural scavengers (i.e., uric acid) further impacts its activity in biological environments. , Even under rigorously controlled conditions, PRP responses are often irreproducible across experiments, reflecting the strong dependence of ONOO– readouts on local microenvironments, RONS fluxes, and assay design. , While discrepancies are typically attributed to intrinsic PRP chemistry, extrinsic variables such as trace metal contaminants, temperature, pH, buffer composition, and solvent stabilizers can equally influence outcomes. −

Among solvent stabilizers, butylated hydroxytoluene (BHT) is ubiquitous in commercial etherated solvents, typically formulated at 0.02–0.04% w/v (900–1800 μM) in 4 L bottles of diethyl ether, tetrahydrofuran (THF), and related ethers due to its ability to suppress autoxidation and peroxide formation in storage. BHT inhibits peroxide formation by quenching peroxyl and alkoxyl radicals through phenolic hydrogen atom transfer. This pervasive presence of BHT in solvent stocks, spanning reagent supplies used for small-molecule synthesis and fluorescent probe preparations, can interfere with redox-triggered probe chemistries. −

Herein, we establish a systematic framework to evaluate how a widely used industrial solvent stabilizer influences the availability of a disease-relevant biomarker, ONOO–, for analytical detection and performance evaluations by PRPs. A previously reported benzopyrylium-based ratiometric probe (BZP), which provides dual-emission properties, was employed as a representative reliable model to optically interrogate changes to the ratiometric and kinetic responsiveness to ONOO–. Combined UV–vis absorption spectroscopy, fluorescence measurements, and nuclear magnetic resonance (NMR) spectroscopy were used to analytically probe the mechanisms by which coincubated BHT reacts with ONOO– and alters benzopyrylium-based probe (BZP) reactivity.

Materials and Methods

Chemicals and Reagents

N-Boc-ethylenediamine (≥98.0%), 4-(dimethylamino)­pyridine (DMAP) (≥99%), norbornene-exo-2,3-dicarboxylic anhydride (97%) and trifluoroacetic acid (TFA) (99%), pyridine, manganese­(IV) oxide (MnO2), hydrogen peroxide (H2O2), hydrochloric acid (HCl), sulfuric acid (H2SO4), perchloric acid (HClO4), sodium nitrite (NaNO2), sodium hydroxide (NaOH) (Thermo Fisher Scientific), nicotinamide adenine dinucleotide hydrogen (NADH) disodium salt, grade II (98%) (Roche), butylated hydroxytoluene (BHT), 1-ethyl-3-(3-(dimethylamino)­propyl)­carbodiimide hydrochloride (EDC·HCl), 3-morpholinosydnonimine hydrochloride (SIN-1·HCl) (Sigma-Aldrich), acetonitrile (MeCN) and deuterated NMR solvents (Sigma-Aldrich and Cambridge Isotope Laboratories), 4-(diethylamino)­salicylaldehyde, ethyl acetoacetate, and 2′-carboxy-4-diethylamino-2-hydroxybenzophenon (TCI America) were purchased and used without further purification. Anhydrous DCM (Thermo Fisher Scientific) was obtained from a Vacuum Atmospheres Patented Solvent Purifier System (SPS) and dried over activated molecular sieves. Water was obtained from a Satorius Arium Mini system.

Synthesis of 7-(Diethylamino)-2-oxo-2H-chromene-3-carboxylic Acid (Coumarin)

Coumarin was synthesized according to previously reported literature and stored in the dark until use.

Synthesis of Benzopyryllium-Based Probe

A 7,7′-bis­(diethylamino) benzopyrylium dye bearing a para-substituted piperazinyl–imide conjugate (BZP) was synthesized according to previously reported literature. BZP was stored in the dark until its use.

Peroxynitrite Stock Solution

The synthesis of peroxynitrite was carried out according to literature reported by Robinson and Beckman. Three freshly prepared solutions of 0.7 M HCl and 0.6 M H2O2, 0.6 M sodium nitrite, and 3 M sodium hydroxide were made at room temperature. Using 30 mL BD syringes, the pumps were run at a rate of 30 mL/min, and the resulting yellow solution was collected into a glass Erlenmeyer flask in an ice bath. 500 mg of MnO2 was added to the Erlenmeyer flask and was left to quench any excess H2O2 for 10 min. The final solution (stabilized at pH 11.0) was then aliquoted into Eppendorf tubes through a 0.22 μm syringe filter. The aliquots are subsequently flash frozen in liquid nitrogen before being stored at −80 °C until use. The concentration of the prepared peroxynitrite was determined by testing the absorption of the solution at 302 nm. The extinction coefficient of the ONOO– solution in 0.1 M NaOH is 1670 M–1 cm–1 at 302 nm, [ONOO–] (mM) = A302/(ε × l), with ε = 1670 M–1 cm–1 and l = 1 cm.

Upon usage, each aliquot of ONOO– was removed from the −80 °C freezer and thawed in an ice bucket. This stock solution was then diluted 10× and 100× with water, and the absorbance at 302 nm was measured to determine the actual concentration.

Nuclear Magnetic Resonance

NMR spectroscopic data were recorded on either a Bruker Avance NEO 500 MHz or a Varian Unity Inova AS600 600 MHz at 25 °C. A sample of 1.14 mM BHT was prepared by using 550 μL of CD3CN with 0.1% TMS. 2 μL of ONOO– in water was added to the NMR sample containing BHT to get a final concentration of 1.14 mM ONOO–. Spectra were analyzed using MestreNova version 15.1.0-37919.

Absorbance and Fluorescence Spectroscopy

Absorbance and fluorescence measurements were done on a SpectraMax iD5 multimode microplate reader with a high-power Xenon flash lamp at room temperature. Absorbance spectra were measured from 200 to 800 nm (10 nm step size) in polypropylene Costar 96-well plates. Fluorescence spectra were measured with excitation wavelengths of 340 nm, 400 nm, and an emission range between λex + 50 and 800 nm with samples in black polypropylene Costar 96-well plates. Data are reported as the mean of n = 4 samples ± standard deviation.

Kinetic measurements were performed using an integrated dual-injector module of the iD5. The injector system enables automated reagent addition during data acquisition and was used for rapid mixing and time-resolved assays. Each injector operates independently and dispenses defined reagent volumes in microliter increments with injection parameters programmed through the SoftMax Pro software. The injectors are compatible with absorbance, fluorescence, and luminescence read modes and are optimized for use across standard microplate formats. Prior to experiments, injector lines were primed with the assay solution to minimize dead volume and ensure reproducible delivery. All measurements were conducted at ambient temperature, unless otherwise noted.

Fluorescence emission spectra that required excitation below 300 nm utilized a PerkinElmer FL 6500 fluorescence spectrophotometer (PerkinElmer, Korea) with samples in a 4 mL quartz cuvette (Starna Cell).

Determining the Amount of BHT in Solvent

According to the manufacturer’s specification, a 4 L bottle of Sigma-Aldrich tetrahydrofuran (THF) contains up to 0.025% (w/w) butylated hydroxytoluene (BHT) as a stabilizer, corresponding to a maximum BHT concentration of approximately 1.14 mM. This concentration was within the range evaluated in the NADH assay. To assess the inhibitory effects of trace levels of BHT under conditions relevant to fluorescence measurements, we typically employed BHT concentrations of 0.25 and 25 μM, corresponding to BHT: BZP molar ratios of 1:400 and 1:4, respectively.

Peroxynitrite Response Assays

Ratiometric Assays

Briefly, BZP dissolved in 50:50 H2O:MeCN was treated with 0–5 equiv of ONOO– dissolved in water to achieve a final concentration of 100 μM BZP and incubated for 15 min prior to absorbance and fluorescence measurements. Data are reported as the mean of n = 4 samples ± standard deviation. Solutions were fluorescently excited at 400 nm and have an emission peak at 730 nm.

Kinetic Assays

Solutions of BZP were prepared in 50:50 H2O:MeCN and BHT dissolved in 50:50 H2O:MeCN was added to get a final concentration of 100 μM BZP and either 0.25 μM or 25 mM BHT. The addition of ONOO– was done by injecting 10 μL of 1 mM ONOO– solution diluted in Milli-Q water. The ONOO– solution was kept on ice inside the injector compartment throughout the assay to minimize degradation. Measurements were taken every 30 s. Data are reported as the mean of n = 4 samples ± standard deviation. In the case of coumarin release, the fluorescence signature was monitored at 480 nm.

NADH Oxidation with SIN-1 Control Assays

Solutions containing NADH and BHT when applicable (1.14 mM of each) were prepared in 50:50 H2O:MeCN. 10 μL of 11.4 mM SIN-1 was added immediately prior to the fluorescence measurements. These solutions were excited at 340 nm, and emission was monitored at 460 nm for NADH oxidation to NAD+. The t 1/2 values calculated from a one-phase decay model of samples with and without BHT were used to compare the effectiveness of BHT as a radical scavenger.

Results and Discussion

Quantitative Assessment of ONOO– Scavenging by BHT

The presence of ONOO– was evaluated by using an ONOO–-selective benzopyrylium-based probe (BZP) (Figures , S1 and S2). To optimize the solubility of both BHT and BZP, we introduced MeCN to our solvent system and found that a 50:50 mixture with water minimized ONOO– degradation (Figure S3). This solvent system was used for all measurements unless noted otherwise. Upon treatment with ONOO–, the near-IR emitting xanthane benzopyrylium functionality was oxidatively cleaved to yield a visible-range emitting fluorescent 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (7-DCCA) in a ratiometric manner (Figure a). Peroxynitrite treatment yielded a dual-emission response (λex = 400 nm) characterized by the attenuation of the benzopyrylium emission at 730 nm and the emergence of a strong coumarin band at 480 nm (Figure b). This spectroscopic change is accompanied by a visible color change of the solution from teal to yellow (Figure S4). BZP consumption was reproducibly achieved after addition of ∼1 equiv ONOO–.

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Interference of BHT with benzopyrylium probe (BZP) activation upon exposure to ONOO–. (a) Mechanism of ONOO–-triggered coumarin release from the BZP probe. (b) Ratiometric fluorescence response of BZP (0.1 mM, 1 equiv) upon exposure to increasing amounts of ONOO– (0–0.5 mM, 0–5 equiv). (c) Ratiometric fluorescence response of BZP to ONOO– in the presence of BHT (0.25 μM). (d) Corresponding comparison of fluorescence ratios (λem 480/λem 730) in the presence (0.25 μM) and absence (0 μM) of BHT, demonstrating an approximately 98% reduction in activation signal at 1 equiv added ONOO–. (e) Time-dependent analysis of coumarin release monitored by fluorescence (λem 480 nm) for BZP samples (0.1 mM) containing 0.25 μM BHT (1:0.0025 equiv BHT:BZP) or 25 μM (1:0.25 eq BHT:BZP); ONOO– (0.1 mM, 1 equiv) was added after 5 min of baseline acquisition. All measurements were conducted in 1:1 H2O/MeCN, using λex 400 nm. Values are reported as mean ± SD (n = 4).

We next introduced BHT (0.25 μM or 0.0025 equiv) as a radical scavenger and evaluated the spectral and colorimetric responses. However, the 480 nm turn-on intensity was markedly reduced while the 730 nm emission persisted, up to higher equivalents of ONOO– (Figure c). Under typical assay conditions, this result could be misinterpreted as reduced probe reactivity, demonstrating how trace antioxidants may mask probe responses and potentially contribute to false-negative outcomes in ONOO– sensing experiments. In these studies, a BHT concentration of 0.25 μM was used, far below the typical 0.9–1.8 mM levels found in 4 L solvent bottles. This lower concentration was chosen to maintain the solubility of both the BZP probe and BHT, but more importantly, it underscores that even trace amounts of BHT can substantially alter ratiometric ONOO– sensing responses. The influence of BHT reactivity on peroxynitrite detection was evaluated as the product-to-precursor fluorescence ratio (F480/F730). Without BHT, full conversion to coumarin was possible with 2 equiv ONOO–; however, in the presence of BHT (0.0025 equiv), 50% more ONOO– (3 equiv) was required to achieve full conversion to coumarin (Figure d). This near-stoichiometric scavenging is consistent with classical antioxidant kinetics, wherein BHT donates a phenolic hydrogen atom to reactive oxidants, yielding a resonance-stabilized phenoxyl radical that terminates further reactivity. Such near-equimolar reactivity, rather than exact 1:1 or 1:2 stoichiometries, is characteristic of phenolic antioxidants and mirrors established behaviors in lipid autoxidation systems.

These results indicate that BHT interferes with the efficiency of peroxynitrite-mediated probe activation. Specifically, the attenuated F480/F730 signal suggests diminished capacity to produce coumarin due either to (i) chemically competitive interactions, in which BHT consumes ONOO– prior to probe activation, or (ii) optical artifacts, including inner filter effects or quenching of the fluorescent species. Spectral controls help to differentiate these possibilities. The presence of 0.0025 equiv of BHT does not alter the emission maxima of either pristine BZP or product coumarin (Figure b,c), confirming that BHT does not interact with pristine BZP or the coumarin product (Figure b,c), arguing against significant ground-state complexation for direct fluorophore perturbation. Furthermore, when excess ONOO– (5 equiv) is added, the same final F480/F730 intensity is achieved (Figure d), indicating that the suppressed signal at lower equivalents arises primarily from reduced effective ONOO– availability rather than irreversible optical quenching. Together, these observations support a predominantly chemical competition mechanism under our experimental conditions, with minor contributions from postreaction photophysical effects.

In the absence of BHT, BZP reacts rapidly with ONOO–, achieving near-complete conversion to coumarin with t 1/2 = 3.7 s (Figure e). This rapid reaction highlights the strong oxidizing potential and nucleophilicity of ONOO– toward the probe. Following the initial activation, a steady decline in fluorescence is observed over the subsequent 10 min. The rate of fluorescence signal decline (8 × 103 ± 2 × 103 a.u./min) was independent of the addition of the amount of BHT (0.25 and 0.0025 equiv) added. Potential causes for this signal decay were hypothesized to be secondary reactions of coumarin with residual reactive species, real-time solubility changes, or fluorophore photobleaching. To evaluate this, we conducted control experiments with pristine coumarin in the presence of ONOO– and BHT (Figure S5). Kinetic measurements show that coumarin alone exhibits a steady decline in emission under the measurement conditions, indicating that this behavior is intrinsic to the fluorophore and independent of chemical transformation (Figure S5a). Upon the addition of only ONOO– or ONOO– and BHT to the coumarin at 5 min, there is a slight increase in fluorescence intensity, likely due to a change in the ionic strength and pH of the solution, which is known to influence the quantum yield of coumarin dyes. , A brief increase in the emission intensity is therefore consistent with the partial disruption of pre-existing coumarin aggregates or the temporary reduction in self-quenching, resulting in enhanced fluorescence before the system re-equilibrates. However, all experiments displayed similar fluorescence decreases over time, confirming that secondary reactions with coumarin are unlikely. To rule out photobleaching effects, we incubated coumarin in the dark and measured fluorescence spectra before and after the same 15 min timeframes, revealing a similar degree of fluorescence decay (Figure S5b). Thus, the most plausible explanation for our signal decay in Figure e is due to coumarin aggregation or solubility-related quenching, consistent with its tendency to form J-aggregates.

In the presence of BHT, fluorescence kinetics are perturbed. The initial time constant for probe activation remains similar (t 1/2 = 3.6–3.3 s), indicating that BHT does not impact the chemical responsiveness of the BZP probe to active ONOO–. However, the maximum signal amplitude was reduced by ∼1.2-fold with 0.25 μM BHT and by ∼1.8-fold with 25 μM of BHT, reflecting competitive scavenging of active ONOO– by BHT. The combination of BHT-mediated competition and minor postreaction quenching emphasizes the sensitivity of the system to trace solvent stabilizers and highlights the importance of controlling such impurities for reproducible ONOO– detection.

Collectively, these results indicate that under the conditions examined here, trace levels of BHT can measurably divert ONOO– from reacting with fluorescent probes. The stoichiometric competition observed underscores the necessity of accounting for stabilizer impurities when assessing probe performance, particularly in biological or formulation contexts where antioxidants are ubiquitous.

Spectroscopic Evidence for BHT–ONOO– Interaction and Mechanistic Considerations

To examine the direct interaction of BHT with ONOO– in solution, we examined their UV–Vis absorption spectra (Figure ). BHT displays a characteristic maximum at approximately 280 nm, whereas ONOO– exhibits a distinct band centered at 302 nm (Figure a). Upon mixing, both features broadened into a merged profile spanning 280–310 nm, indicative of ground-state complexation or early-stage chemical reactivity between the two species. As the amount of BHT increased to 2 equiv, the absorbance increased by 3-fold, while the fluorescence intensity decreased by a similar factor. The absorbance enhancement is consistent with chemical interaction between BHT and ONOO– that alters the electronic structure of BHT and shifts the λmax from 285 to 297 nm, whereas the accompanying fluorescence suppression may reflect a combination of chemical consumption and optical effects (e.g., inner filter attenuation). As fluorescence recovery is observed upon addition of excess ONOO– in probe experiments, purely optical attenuation cannot carefully account for the reduced signal intensity. The intrinsic emission of BHT (λem = 315 nm) progressively diminished upon titration with ONOO–, reaching approximately 77% quenching at a 1:1 molar ratio (Figure b). At this stoichiometry, the effective ONOO– signal is underestimated by nearly 4-fold, highlighting a substantial analytical artifact produced by BHT scavenging.

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Spectroscopic evidence for the interaction between BHT and ONOO– in 50:50 H2O:MeCN. Solutions were incubated for 5 min before measurements. (a) UV–vis absorption spectra of BHT (0.1 mM, 1 equiv) and ONOO– (0.1–0.2 mM, 1–2 equiv) recorded individually and after mixing, showing the emergence of a broadened, intensified absorption profile consistent with chemical interaction between the two species. (b) Fluorescence emission of BHT (0.1 mM, 1 equiv) as a function of ONOO– (0.1–0.2 mM, 1–2 equiv) exposure, demonstrating a progressive decrease in fluorescence intensity upon reaction. (c) 1H NMR spectra of BHT acquired in the absence and presence of ONOO–, revealing chemical shift changes indicative of BHT oxidation.

Through structural elucidation using 1H NMR, we observed that the phenolic proton resonance at 5.20 ppm diminishes with the addition of an equimolar amount of ONOO–, consistent with phenolic proton abstraction and an O-centered radical (Figure c). Concomitant with the loss of the O–H signal, both the methyl protons (2.22–2.40 ppm) and the tert-butyl protons (2.15–2.21 ppm) shift downfield. This deshielding is indicative of decreased electron density on the aromatic ring following oxidation, similar to the 2,4,6-tri-tert-butylphenoxyl radical synthesized by Mayer and co-workers. Removal of the phenolic proton generates a phenoxyl radical or a phenolate-derived oxidized intermediate, each of which renders the ring more electron-withdrawing and alters the local magnetic environment experienced by the alkyl substituents. These electronic changes reduced σ-shielding at the methyl and tert-butyl groups and, in the case of the radical species, introduced additional paramagnetic contributions that further shifted the aliphatic resonances downfield. These collective spectral changes, therefore, support a mechanistic pathway in which ONOO– engages BHT through phenolic proton abstraction, producing an oxidized aromatic framework with a redistributed electron density that manifests as a pronounced hyperchromic effect and slight bathochromic shift in the UV–vis spectrum (Figure a).

To further validate the inhibitory effects of BHT, we performed an oxidation assay with a common reducing agent, reduced nicotinamide adenine dinucleotide (NADH). To continually generate ONOO– in a controllable and concentration-dependent manner, we used 3-morpholinosydnonimine hydrochloride (SIN-1), a synthetic adduct that degrades into ONOO– precursors (Scheme S1). As freshly generated ONOO– comes into contact with fluorescent NADH (λem = 460 nm), it is oxidized to NAD+ (Figure a). , In a 1:1 SIN-1 to NADH solution (0.1 mM), the fluorescence intensity decreases more rapidly than in a sample with BHT, where the oxidation process is slowed down (Figure b). Kinetic fitting reveals that BHT extends the apparent NADH oxidation half-life by approximately 1000-fold, supporting its role as a kinetically competent competing sink for ONOO– or its radical precursors. This suggests that BHT interacts with the radical degradation products of SIN-1, NO· and O2 2–·, thus preventing its ability to generate ONOO– in the first place. This competitive scavenging behavior may extend to other nucleophilic or redox-active fluorescent probes that rely on direct reaction with ONOO–, although the magnitude of the effect will depend on the probe structure and reaction kinetics.

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Oxidative interaction of NADH with ONOO– generated from SIN-1. (a) Schematic representation of NADH oxidation to NAD+ mediated by ONOO– formed in situ from SIN-1 decomposition. (b) Time-dependent fluorescence of NADH recorded in the presence and absence of BHT, illustrating the effect of the antioxidant on the rate and extent of NADH oxidation. Samples containing SIN-1 (0.1 mM, 1 equiv), NADH (0.1 mM, 1 equiv), and BHT (0.1 mM, 1 equiv) were prepared in 50:50 H2O:MeCN immediately prior to fluorescence measurements.

Although the precise mechanism by which BHT scavenges ONOO– is still under investigation, BHT may intercept secondary radicals such as ·OH or NO2· formed during ONOO– degradation, terminating radical chain processes that would otherwise activate the probe; depletion of these degradation products is expected to shift the equilibrium toward more rapid ONOO– decay in accordance with Le Châtelier’s principle (Figure ). Although solvent polarity and microenvironmental effects cannot be completely excluded, a control experiment involving BHT under basic conditions rules out simple acid–base deprotonation as the origin of the observed probe suppression (Figure S6), supporting a predominantly chemical scavenging mechanism.

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Schematic of plausible BHT interference with peroxynitrite. (a) Formation of ONOO– from the diffusion-controlled reaction of nitric oxide and superoxide, followed by its rapid decomposition to yield secondary species. (b) Proposed interaction of BHT with the nitrogen dioxide radical via either hydrogen atom transfer or single-electron oxidation, thereby diverting reactive nitrogen species away from probe activation.

Precedent from lipid autoxidation studies, where BHT efficiently quenches peroxyl and alkoxyl radicals, supports its role as a general radical scavenger in this system. Taken together, the spectroscopic, kinetic, and control experiments indicate that the dominant source of signal suppression in this system arises from the chemical competition between BHT and ONOO–. While minor optical contributions such as inner filter effects or fluorophore aggregation may modulate signal amplitude, the recovery of the probe response upon addition of excess ONOO– and the independent spectroscopic evidence for BHT oxidation support a primarily chemical scavenging mechanism under the conditions examined here.

Conclusion

To the best of our knowledge, this work provides the first systematic evaluation of a common solvent stabilizer on the performance of a model PRP. We demonstrate that BHT, widely present at trace levels in commonly used organic solvents, directly scavenges peroxynitrite and severely compromises the accuracy of fluorescent-based ratiometric assays. Notably, BHT induces measurable interference at concentrations as low as 0.25 μM, more than 3 orders of magnitude lower than the typical concentration found in a 4L solvent bottle, resulting in apparent false-negative responses and unreliable quantitative detection.

These findings identify solvent stabilizer content as a previously overlooked experimental variable that may contribute to the variability in reported optical ONOO– sensing outcomes. Differences in stabilizer content can yield inconsistent or misleading results in laboratory studies, while the routine use of bulk-stabilized solvents introduces reproducibility challenges in industrial settings. By revealing how trace solvent stabilizers can influence probe behavior, this work provides practical guidance for the design, evaluation, and interpretation of optically responsive ONOO– probes and underscores the necessity of routine analytical screening for stabilizers (i.e., NMR, LC-MS, or UV–vis analysis) to ensure quantitative reliability and reproducibility across fundamental and applied studies.

Supplementary Material

ao5c13412_si_001.pdf (431.7KB, pdf)

Acknowledgments

The authors are grateful for financial support from the University of California. A.S.C. acknowledges funding support from the National Science Foundation through a CAREER Award from the Division of Materials Research (DMR-2443975) and a Hellman Family Faculty Fellowship. The authors acknowledge the NSF Major Research Instrumentation Award (MRI-1920299) for magnetic resonance instrumentation. The authors thank Dr. Hongjun Zhou for assistance with NMR experiments. The authors thank AJ Chavez for insightful discussions on possible reaction mechanisms and for suggesting relevant literature. The graphical abstract was created in BioRender. Carlini, A. (2026) https://BioRender.com/k0uydci, and modified in PowerPoint.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c13412.

  • Nuclear magnetic resonance spectra, spectroscopic data, and mechanism of peroxynitrite generation from SIN-1 (PDF)

A.S.C. conceived the idea and secured the funding. E.J.M. synthesized reactive monomers, and S.R.M. identified BHT as a source of sensing interference. A.V.P. designed and carried out experiments, performed data analysis, and wrote the manuscript.

The authors declare no competing financial interest.

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