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Ultrasensitive and versatile hydrogen peroxide sensing via fluorescence quenching†

Jenisha John Peter‡ , Nathaniel Chennattuparambil Roy‡, Flavio Grynszpan and Mindy Levine*
Department of Chemical Sciences, Ariel University, 65 Ramat HaGolan Street, Ariel 40700, Israel. E-mail: mindyl@ariel.ac.il; mindy.levine@gmail.com

First published on 22nd August 2024

Although many detection methods for hydrogen peroxide currently exist, including electrochemical,⁵ optical,⁶ and spectrometric methods,⁷ these methods suffer from noticeable drawbacks, including the use of expensive laboratory instrumentation and/or highly trained personnel. Moreover, more recently-developed methods that eliminate these requirements often use toxic components, including heavy metals, to enable an effective sensor response.⁸ Of the aforementioned methods, fluorescence-based spectrometry has significant potential, due to the high sensitivity and rapid response time of fluorescence-based sensors,⁹ as well as the ability to achieve on-site fluorescence detection using smartphone-based or other portable spectrometers.¹⁰

Our research groups have a long-standing interest in the design and synthesis of novel fluorescent compounds, particularly of bimane-based fluorophores,¹¹ and in the development of fluorescence-based chemical sensors, including for the detection of cobalt(II) cations,¹² fluoride,¹³ iodine,¹⁴ and water.¹⁵ As part of this work, we ascertained that compound 1 (relative quantum yield = 0.17) acts as an effective water sensor, with water-induced hydrolysis of the aryl boronate moieties resulting in the formation of non-fluorescent compound 3 (Fig. 1, bottom path).¹⁶

	Fig. 1 Schematic depiction of the reactions between compound 1 and analytes, resulting in analyte-specific fluorescence quenching.

As part of those investigations, we determined that hydrogen peroxide caused oxidation of the aryl boronate moieties of compound 1 to phenols, leading to the formation of virtually non-fluorescent compound 2 (relative quantum yield = 0.043; Fig. 1, top path) with no trace of compound 3. The formation of this product was confirmed by: (a) ¹H NMR spectroscopy, which showed that the addition of increasing amounts of hydrogen peroxide resulted in a decrease in the ¹H NMR peaks corresponding to compound 1 and an increase in the peaks corresponding to compound 2 (Fig. 2); and (b) high-resolution mass spectrometry (see ESI† for more details), which showed the evolution of a new signal with the mass of compound 2. The lack of formation of compound 3 (the compound reversibly formed from the reaction of compound 1 with water) is particularly noteworthy, given the fact that the hydrogen peroxide analyte was tested as an aqueous solution, and is due to the markedly slower kinetics of the reaction of compound 1 with water compared to its reaction with hydrogen peroxide (vide infra).

	Fig. 2 1H NMR spectra showing the hydrogen peroxide-triggered conversion of compound 1 to compound 2 (A) aromatic region of the 1H NMR spectra; (B) aliphatic region of the 1H NMR spectra (black trace: compound 1; green trace: compound 1 + 20 μL H2O2 (30% w/v); blue trace: compound 1 + 160 μL H2O2 (30% w/v); red trace: compound 2).

The proposed mechanism of hydrogen peroxide-induced oxidation involves the formation of an anionic tetrahedral boronate intermediate B from the initial boronate ester A, followed by a 1,2-metallate rearrangement and O–O bond cleavage to form intermediate C (Fig. 3).¹⁷ Intermediate C reacts further with water to form the phenol product D, with water-induced hydrolysis of the boric ester E forming pinacol and boric acid.¹⁸

	Fig. 3 Proposed mechanism for the hydrogen peroxide-induced oxidation of boronate esters to phenols.

Moreover, the reaction of compound 1 with hydrogen peroxide was accompanied by substantial decreases in the absorbance and emission signals of acetonitrile solutions of compound 1 (Fig. 4), with exposure to 300 μM hydrogen peroxide sufficient to induce a 34% decrease in absorbance and 91% decrease in the fluorescence emission signals.

	Fig. 4 Hydrogen peroxide-induced photophysical changes in the (A) UV-visible absorbance spectra; and (B) steady-state fluorescence emission spectra of acetonitrile solutions of compound 1 ([1] = 10 μM; λex = 450 nm).

To further distinguish compound 1′s responsiveness to hydrogen peroxide from its known responsiveness to water, we directly compared the room-temperature reaction of each analyte with compound 1. This comparison revealed that the reaction of compound 1 with hydrogen peroxide was much faster than its reaction with water, both at the same analyte concentration and when comparing the reaction of compound 1 with a four-fold higher concentration of water (the amount of water present in the H₂O₂ sample). (Fig. 5, Table 1). These results demonstrate that the sensor is effective at detecting H₂O₂ in aqueous solutions even at very low concentrations, where water is present in significantly higher amounts than hydrogen peroxide.

	Fig. 5 Depiction of the differential reaction rates of solutions of compound 1 to the presence of hydrogen peroxide (grey squares), compared to water at the same concentration (red circles) and a four-fold higher concentration (blue triangles). Inset shows images of solutions of compound 1 after analyte exposure, under 365 nm excitation.

The strong responsiveness of compound 1 to hydrogen peroxide suggests its potential as a turn-off fluorescence sensor for this analyte, provided it can demonstrate high sensitivity, practicality, and selectivity. The sensitivity of this system was determined by measuring how the fluorescence emission of compound 1 varies with hydrogen peroxide concentration, resulting in a limit of detection (LOD) of 7.9 pM. This LOD is several orders of magnitude lower than most literature-reported hydrogen peroxide sensors (micromolar to nanomolar),¹⁹ and, to the best of our knowledge (see ESI† for compiled data), is four times lower than the lowest limit of detection for hydrogen peroxide reported to date (32.60 pM).²⁰ Such low detection limits are crucial in many real-world scenarios where hydrogen peroxide is present at picomolar concentrations, such as in intracellular environments²¹ and groundwater samples.²²

The high practicality of a compound 1-based hydrogen peroxide sensor was realized both in solution and by adsorbing compound 1 on Whatman #1 filter paper. Solution-state practicality was evaluated by examining its selectivity toward hydrogen peroxide. We found that the solution-state response of compound 1 to H₂O₂ gradual analyte-induced fluorescence quenching was distinct from its responses to formaldehyde (no quenching) and HCl (an initial decrease in fluorescence due to the strongly acidic pH (1.3), with no further decrease over time).

Further evaluation of the solution-state practicality of this system involved testing the ability of compound 1 to detect H₂O₂ in untreated tap water. Compared to the hydrogen peroxide-induced quenching observed in Milli-Q purified water, a higher quenching efficiency was recorded in tap water (after one hour of exposure to 30 mM hydrogen peroxide: 27% quenching in Milli-Q water; 42% quenching in untreated tap water). This enhanced quenching response in tap water is likely attributable to the different pH of the tap water (7.6, compared to the pH of Milli-Q water: 6.9). Moreover, tap water has a number of additional components, including significant ion concentrations (as determined by conductivity measurements) and other non-ionic species.

Additional practicality was demonstrated through the testing of compound 1 – functionalized filter papers, which exhibited hydrogen peroxide-specific colorimetric changes (Fig. 6). Notably, the same filter papers showed virtually no color change in the presence of pure water, indicating that the differential response of compound 1 to hydrogen peroxide versus water persists on a solid support. Moreover, unlike the reaction of compound 1 with water, which can be readily reversed with the removal of water, the reaction of compound 1 with hydrogen peroxide was irreversible (see ESI† for more details).

	Fig. 6 The quantitative blue values of compound 1 – functionalized filter paper after exposure to hydrogen peroxide or water, photographed under 365 nm excitation. Inset shows photographs of the functionalized papers following exposure to: no analyte (blank), 50 mM H2O2, and 50 mM H2O.

Additional practicality was demonstrated through the use of compound 1 – functionalized filter papers for vaporized hydrogen peroxide (VHP) detection. VHP is highly effective for the bio-decontamination and sterilization of equipment, surfaces, rooms, and airplane cabins due to its bactericidal properties. However, VHP is also toxic to humans, which means that significant efforts are required to reduce its concentration to safe levels (below 1 ppm) after use. Therefore, monitoring low concentrations of VHP is of great importance. Gratifyingly, we observed distinct colorimetric changes in the functionalized filter papers upon exposure to 0.12 ppm (3.5 μM) VHP (Fig. 7). This response was also highly selective, with the addition of sodium hypochlorite vapor, water vapor, formaldehyde vapor, or HCl vapor resulting in minimal colorimetric changes. In contrast, iodine induced markedly different color changes, likely due to its reaction with the filter paper.²³

	Fig. 7 Quantitative colorimetric changes observed in compound 1 – functionalized filter paper when exposed to a variety of analytes in the vapor phase ([analyte]solution = 10 mM; [analyte]vaporphase = 3.5 μM (0.12 ppm)), followed by visualization under 365 nm excitation. Boxes below the graph show photographs of the filter papers after vapor-phase analyte exposure.

In conclusion, our detailed investigations of compound 1 resulted in the development of an ultrasensitive fluorescence-based sensor for hydrogen peroxide. This sensor was readily oxidized with hydrogen peroxide, resulting in the formation of a non-fluorescent product (biphenol 2). A direct comparison of the sensor's previously-reported responsiveness to water with its responsiveness to hydrogen peroxide revealed approximately 23 times greater efficacy in hydrogen peroxide detection, offering a distinct advantage in analyte discrimination. Moreover, the sensor displayed the lowest LOD for hydrogen peroxide reported to date (LOD = 7.9 pM), high practicality (as evidenced by the ability to maintain hydrogen peroxide responsiveness when adsorbed on filter paper), and broad-based applicability (as evidenced by the detection of both solution-state and vapor-phase hydrogen peroxide, at vapor-phase concentrations below 1 ppm). Despite these significant advantages, the sensor has certain limitations, including the instability of compound 1 under highly acidic or highly basic conditions, as well as its susceptibility to strong nucleophiles. Overall, this study contributes to the advancement of H₂O₂ sensing technology by offering a sensitive, and practical fluorescence-based sensor. Its remarkable performance, coupled with its potential for on-site detection and low limits of detection, indicates its potential value in addressing critical needs in hydrogen peroxide detection.

We gratefully acknowledge Ariel University for providing the Levine and the Grynszpan groups with research funding, and for providing N. C. R and J. J. P. with scholarships. F. G. is the incumbent of the Cosman endowment for organic chemistry research. Dr Vered Marks is acknowledged for her role in maintaining the NMR facility at Ariel University, and Dr Rami Krieger is thanked for his assistance in maintaining the departmental instruments. We thank Dr Joy Karmakar for an early synthesis of compound 1 and preliminary results on its reaction with H₂O₂. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

Conflicts of interest

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