Fig. 2 A–C shows brightfield and fluorescence images of microdroplets of 160, 50, and 16 µm in diameter, respectively. Higher fluorescence intensity was observed for microdroplets with smaller diameters, indicating that the yield of H 2 O 2 increased as microdroplet size decreased. A detailed analysis of the relationship between fluorescence intensity and microdroplet size revealed that the fluorescence intensity increased significantly below a diameter of ∼20 µm ( Fig. 2D ).

Fluorescence imaging of spontaneous generation of hydrogen peroxide in aqueous microdroplets: (A) reaction scheme between PF-1 and hydrogen peroxide; (B) schematic of confocal microscope setup for imaging microdroplets; and (C) brightfield and fluorescence images of microdroplets (2 μm to 17 µm in diameter) at Left and bulk water at Right including the flat air-bulk-water interface. Each sample contains 10 µM PF-1. Only microdroplets display fluorescence from fluorescein caused by H 2 O 2 cleavage of PF-1. (Scale bar, 20 μm.)

To examine the production of H 2 O 2 in an aqueous microdroplet, we utilized a H 2 O 2 -sensitive water-soluble fluorescent probe, peroxyfluor-1 (PF-1), originally reported by Chang and coworkers ( 15 , 16 ). The compound PF-1, which is not fluorescent, is known to respond selectively to H 2 O 2 to liberate fluorescein ( Fig. 1A ). In bulk water, fluorescence was observed from a solution of 10 µM PF-1 and 100 µM H 2 O 2 ( SI Appendix, Fig. S1 ), but no fluorescence was observed in the absence of H 2 O 2 ( SI Appendix, Fig. S2 ). An aqueous solution containing 10 µM PF-1 was sprayed onto a hydrophobic silane-treated glass surface. The resulting supported microdroplets were analyzed by confocal microscopy to establish a relationship between microdroplet diameter and observed fluorescence intensity ( Fig. 1B ). Strong fluorescence emission was observed from microdroplets containing 10 µM PF-1, but not in bulk water ( Fig. 1C ). These observations demonstrate that H 2 O 2 was generated in microdroplets, but not in detectable amounts in bulk water or at the air−water interface of bulk water ( Fig. 1 C , Right).

An additional experiment was carried out to assess whether the generation of the phenol 4-HB from 4-CPB was from H 2 O 2 generated in microdroplets and not from another adventitious reaction of an arylboronic acid in microdroplets. In this experiment, D 2 O was sprayed and collected 3 times. The resulting solution was added to a 100-µM D 2 O solution of phenylboronic acid (PB), and this mixture was incubated overnight at room temperature. Analysis of the resulting solution by 1 H NMR revealed that ∼30% of the PB was converted to phenol. This result indicates that hydrogen peroxide is generated in aqueous microdroplets and that the hydrogen peroxide can be collected and utilized for subsequent reactions (see SI Appendix, Fig. S3 and section S2 for further details). This additional experiment also shows that what we have observed by mass spectrometry is not an artifact or a result of microdroplet evaporation in the heated capillary inlet.

Molecular signature of H 2 O 2 production in aqueous microdroplets using boronic acid probe as a function of consecutive sprays. (A) Reaction scheme of H 2 O 2 -promoted deborylation of 4-CPB. (B) Mass spectrum of aqueous microdroplets containing 100 µM 4-CPB and 10 µM sodium benzoate (as internal standard) on the seventh consecutive spray. (C) Normalized ion count of 4-CPB (purple, 165 m/z) starting material, and H 2 O 2 deborylation products, 4-HB acid (red, 137 m/z) and boric acid (blue, 61 m/z), over multiple sprays. Error bars represent 3 replicates for sprays 1 through 4, and 2 replicates for spray 5.

We further confirmed the production of H 2 O 2 in aqueous microdroplets by assaying the cleavage of 4-carboxyphenylboronic acid (4-CPB) by H 2 O 2 , which yields boric acid and 4-hydroxybenzoic acid (4-HB) ( Fig. 3A ). An aqueous solution of 100 µM 4-CPB was sprayed into a mass spectrometer for analysis. In addition to the parent peak centered at 165.0359 mass to charge ratio (m/z) (4-CPB), small peaks at 137.0240 m/z and 61.0103 m/z were observed ( Fig. 3B ), corresponding to 4-HB and boric acid. The solution containing 4-CPB was sprayed into a collection vial, redissolved in water, and then resprayed. This process was repeated up to 7 times, and the relative ion count of both the 4-HB and boric acid increased linearly after each spray ( Fig. 3C ). This result indicates that the observed products of boronic acid cleavage are indeed from a reaction with H 2 O 2 within the sprayed microdroplets and not from trace contaminants or from gas-phase reactions within the mass spectrometer.

The quantitative comparison of H 2 O 2 production yield for microdroplets with different sizes was acquired by controlling microdroplet size with different N 2 nebulization gas pressures. We find that the H 2 O 2 production yield is inversely proportional to microdroplet size ( SI Appendix, Fig. S5 ), which is consistent with the observation of higher fluorescence emission of PF-1 for smaller microdroplets ( Fig. 2D ).

H 2 O 2 concentration as a function of different operating conditions. (A) Absorption spectrum of aqueous PTO solution with added H 2 O 2 . Example microdroplet spectrum in red. (B) Calibration curve at 400 nm from A. The red circle represents the concentration of H 2 O 2 generated from aqueous microdroplets acquired from the spectra in A. (C) The effect of varying the nebulizing gas. (D) The effect of dissolving different gases in water. Both C and D are measured with peroxide test strips. Error bars represent 1 SD from 3 measurements.

Quantitative analysis of H 2 O 2 production from aqueous microdroplets was carried out with potassium titanium oxalate (PTO, K 2 TiO(C 2 O 4 ) 2 ·H 2 O) titration and peroxide test strip assays ( Movie S1 ). The agreement between these 2 quantification methods was confirmed using a standard H 2 O 2 solution ( SI Appendix, Fig. S4 ). Fig. 4A shows the absorption spectra of 0.1 M PTO solution with various concentrations of H 2 O 2 as well as with the microdroplet sample. As shown in Fig. 4B , the H 2 O 2 production yield was ∼30 µM (∼1 part per million [ppm]).

Mechanism of H 2 O 2 Generation in Microdroplets.

Having solidly established that H 2 O 2 is produced in aqueous microdroplets, we investigated possible pathways for its formation. Hydrogen must originate from water, but there are 2 initial sources of oxygen to form H 2 O 2 : water and atmospheric O 2 . First, we measured H 2 O 2 production under different nebulization gases: dry air, N 2 , and O 2 using peroxide test strips (Fig. 4C). Changing the gas from N 2 to air did not change the H 2 O 2 yield significantly. Changing the gas from air to O 2 led to a decrease in the H 2 O 2 yield, suggesting that the reactions that generate H 2 O 2 in microdroplets do not involve atmospheric oxygen as a reactant. In addition, we examined whether the dissolved oxygen is a source by measuring H 2 O 2 yield after bubbling water with O 2 for different durations (Fig. 4D). The amount of H 2 O 2 produced decreased as a function of the time spent bubbling O 2 . These data show that the H 2 O 2 was generated from aqueous microdroplets, not from oxidation by atmospheric or dissolved oxygen. The decrease of H 2 O 2 yield upon dissolving oxygen in water microdroplets may be caused by the trapping of oxygen to form the perhydroxyl radical that interferes with H 2 O 2 formation (17).

Water is not readily oxidized or reduced unless subjected to strong oxidants, reductants, or applied voltage. There are several possible origins for the formation of H 2 O 2 , including triboelectric effect, asymmetric charge separation during microdroplet fission, contact electrification, and the oxidation of water by the intrinsic surface potential of the water microdroplet surface. We have examined each possibility. First, the oxidation of water might be caused by the streaming electrification (18) between water and the capillary. We examined this possibility by measuring the production yield of H 2 O 2 in microdroplets with different capillary lengths. Essentially no difference in the production yield was observed (SI Appendix, Fig. S6). If the phenomenon were caused by streaming electrification, the production yield would be expected to be proportional to the length of capillary. We also examined the production yield using different capillary materials, including silica, polyether ether ketone, and phenyl-methylpolysiloxane−coated fused silica (DB-5, Agilent Technologies). We observed no difference in the production yield (SI Appendix, Fig. S7). We also tested the possibility of electrification between water and the pressurized nebulizing gas being a cause of the water oxidation, by comparing the production yield of H 2 O 2 from microdroplet spray and bulk water blown with the same dry N 2 gas for several hours. There was no H 2 O 2 formation in the bulk water with the contact of a stream of N 2 gas. These data suggest that electrification may not likely be the origin.

Because electrification can occur by charge transfer between the silica capillary and the water inside the capillary, we measured the H 2 O 2 yield after replacing the silica capillary with a stainless steel capillary with and without grounding (0 V). SI Appendix, Fig. S8 clearly shows that there is no difference in the production yield, demonstrating the charge transfer between silica capillary and water inside the capillary was not the origin of the water oxidation.

We also considered whether asymmetric microdroplet fission and imbalanced net charge formation during droplet fission and evaporation (19) could be a cause. Previously, we reported that aqueous microdroplets maintain their sizes with minimum evaporation up to ∼130 µs of microdroplet traveling time (20, 21). Moreover, asymmetric fission has been measured to occur on a longer timescale (22). We did observe the production of H 2 O 2 at a short distance with less than ∼100-µs reaction time. This result shows that droplet fission or evaporation might not be the primary cause of H 2 O 2 formation.

The fourth possibility would be the formation of H 2 O 2 through spontaneous oxidation of water by a strong intrinsic electric field at the water−air interface of microdroplets. Several factors unique to microdroplets may be responsible for our proposed mechanism where an electric field generates hydroxyl radicals from OH−, which recombine into H 2 O 2 (Fig. 5). First, the air−water interface of a microdroplet has a strong electric field, on the order of 109 V/m (23). This electric field strength is enough to ionize hydroxide ions to form hydroxyl radicals. Furthermore, in microdroplets, the hydronium ions and hydroxide ions are separated and heterogeneously distributed (24), which enhances the electric field strength at the microdroplet surface. This line of reasoning is supported by our observation of higher efficiency of H 2 O 2 production for smaller microdroplets that have increased curvature, which induces charge accumulation at the surface, and thereby increases the electric field strength. Second, the redox potential can be shifted by electric field or local pH change (25) in microdroplets (24). In addition, it was shown that the pK a and the redox potential at the water−air interface shifts from that in the bulk, suggesting the microdroplet surface promotes redox reactions by providing an energetically favorable environment (26⇓⇓–29). These changes in redox potential may lower the energetic barrier for the water oxidation at the surface of the microdroplet, as we observed before, as a reduced free-energy barrier for ribose phosporylation in microdroplets (30). Previously, we have shown the spontaneous formation of hydroxyl radicals in water microdroplets using salicylate (31) that forms 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid upon reaction with OH radicals (2). The work of Du et al. (32) shows that OH radicals readily combine to form H 2 O 2 in the presence of water. We do not know the fate of the released electrons, but, possibly, they can be accepted by liquid water or used for the reduction of hydrogen ions in water (33, 34).

Fig. 5. Proposed mechanism to form H 2 O 2 at the air−water interface of microdroplets. First, the autoionization of water into H+ and OH− readily occurs at and near the air−water interface of the microdroplet. Then, due to the pH gradient and electric field, OH radicals are formed, releasing a solvated electron. Finally, 2 OH radicals at and near the water microdroplet interface recombine to form H 2 O 2 .