<p>      Chemical reactions in micron-sized droplets under ambient conditions are often orders of magnitude faster than the equivalent bulk reactions due to the large interfacial effects. The investigation of the underlying mechanisms driving the unique surface chemistry of droplets, as well as their applications and implications in synthesis, has garnered considerable interest. This dissertation delves into three key subtopics: (1) Exploring partial solvation as a mechanism for accelerating reactions in microdroplets, (2) Investigating the spontaneous oxidation and reduction of heteroatom double bonds induced by water radical cations and anions generated from water, and (3) Examining the role of oxazolone intermediates in prebiotic peptide synthesis and the emergence of homochirality in living systems.</p> <p>      Chemical reactions can be accelerated in microdroplets but with previously unclear mechanisms. Here we report a systematic study of organic reactions of common types in microvolumes and compare their rates with those in bulk solution. The observed interfacial area effect, molecularity effect and solvent effect provided experimental evidence for partial solvation at gas/liquid interface as one of the major contributors to the observed more than 10⁴-fold acceleration in microdroplets.</p> <p>      Recent spectroscopic results as well as computations demonstrate the existence of a strong electric field at aqueous droplet surfaces, which can result in microdroplet-specific reactions, especially their intrinsic redox properties. Spontaneous oxidation or reduction without external oxidants or reductants has been reported. One explanation for the existence of active species is dissociation of the radical cation/anion pair (H₂O^+∙/ H₂O^-∙), recently argued to occur in pure bulk water, to provide the free radical cation and radical anion. In this work, we reported spontaneous oxidation of heteroatom double bonds (e.g. sulfone to sulfonic acid, ketone to carboxylic acid) in non-aqueous microdroplets containing traces of water (<1%). Meanwhile, the simultaneous oxidation and reduction of several phosphonates was discovered, supporting the radical pair as the source of reactive species in water microdroplets.</p> <p>      One implication of microdroplet chemistry lies in its connection to prebiotic synthesis. Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here we show that (i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; (ii) addition of water to the oxazolone forms the dipeptide; and (iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.</p>

<p dir="ltr">Scientists across many disciplines have observed a striking phenomenon: chemical reactions that do not appreciably occur in large volumes often proceed readily in microdroplets. At the core of suggested mechanisms is the influence of interfacial chemistry on the overall reaction; when the interfacial area dominates the reactor volume, the measured reaction rate is often accelerated. For instance, microdroplets with a high surface area-to-volume ratio (generally with radii smaller than 10 µm) provide a unique reaction environment and have been observed to accelerate a wide variety of chemical reactions. This is likely surprising to most readers, as much of our chemical intuition comes from experiments performed on benchtops in beakers (large, single-phase systems). However, microdroplets are regularly exploited by nature, from multiphase atmospheric aerosols to biomolecular condensates in cells. Thus, it is vital to have measurement tools capable of studying multiphase, nanoscale reactors. An electrochemical perspective on measuring multiphase chemistry under nanoconfinement is given in Chapters 2-4. To my knowledge, there were no reports of accelerated reactivity in microdroplets from electrochemical studies until the 2021 observation that enzyme turnover rates are inversely-related to the size of the containing nanodroplet (given in Chapter 6). In this dissertation work, we developed new electroanalytical tools to probe chemical transformations/reactions at micro- and nano-interfaces and made use of new reaction schemes that capitalize on multiphase microenvironments.</p><p dir="ltr">Much of the method development was built on the foundation of stochastic nanoelectrochemistry, a technique that is reviewed thoroughly in Chapters 2, 4, and 5. Briefly, stochastic nanoelectrochemistry is the measurement of single nano-entities, one-at-a-time, as the collide with a micron-sized electrode. The nano-entities studied in this dissertation were aqueous droplets, either suspended in an immiscible oil continuous phase or propelled through air. We dove deeply into these studies, from using correlated microscopy to watch how these micro- and nanodroplets collide with other interfaces to building simulations to quantify changes to the chemistry inside. We showed how the surface environment directs water nanodroplet collisions (Chapter 10) and measured the sub-diffraction-limited nanometer contact area that forms between a microdroplet and a metal surface (Chapter 11). Using the nanodroplets as tiny reactors, we measured accelerated rate constants and promoted unfavorable nucleation events in attoliter-femtoliter aqueous droplets (see Chapter 6-7 and Chapter 12, respectively) and in microliter aqueous droplets (see Chapter 8 and Chapter 9, respectively).</p><p dir="ltr">As mentioned above, microdroplets are ubiquitous in air (<i>e.g.,</i> aerosols). However, electrochemistry is not an obvious choice for the measurement of intact aerosols because electrochemistry is traditionally performed in a conductive solution, and electrochemistry in air is difficult. In this dissertation we laid the groundwork for a path forward that allows electrochemical access the air|microdroplet interface. We designed and characterized a novel electrochemical cell, where the working electrode is a microwire traversing a suspended liquid film (Chapters 13-15). The early results were born from pure curiosity: Can we do electrochemistry in a soap bubble wall? Chapter 13 shows that the answer is “Yes!”, and that electrochemistry can report on aerosol contents that are collected from the air into this thin film. However, the soap bubble wall was severely limited by the lifetime of the bubble wall (bubbles pop), so in Chapters 14 and 15, we introduce a suspended ionic liquid film that does not pop from evaporation. With the more robust system, we realized the ability to probe intact single microdroplets, one-at-a-time (Chapter 14), giving electrochemical access to the air|water interface.</p><p dir="ltr">As detection of illicit substances from aerosols has the potential for immediate impact on first responder, user, and bystander safety, we employed the new technology to electroanalyze aerosolized methamphetamine (Chapter 13) and fentanyl (Chapter 15). Electrochemistry is small, simple, and affordable, making it a realistic candidate for an in-field sensor. We overcame selectivity challenges by using our understanding of interfacial microenvironments to leverage local pH changes, as demonstrated by the reliable detection of low purity cocaine in mixed powders (Chapter 16). This patented method provides a highly selective technique for cocaine identification in the presence of adulterants without the need to bring any chemicals to the scene (water is our only reagent!).</p><p dir="ltr">In sum, this body of work contributes to the electrochemical studies in nano- and microdroplets, extending the reach to account for droplet size on measured rates and to include microdroplets with a water|air boundary. Applications of the work were focused on in-field detection of illicit substances.</p>

<p dir="ltr">Microdroplets (typically less than 10 μm in radius) have proven to be unique reaction vessels capable of doing the seemingly impossible: drive the chemistry that could have made life possible. While I am not a biochemist here to explain the intricacies of such a claim, I am a measurement scientist who has worked for the past three and half years to develop new methods which can be used to unveil new chemistries in these droplets. Before electrochemists like me entered the microdroplet realm, mass spectrometrists spent years studying droplets at this scale (typically generated with electrospray methods) and have been able to show that these droplets can promote reaction acceleration by several orders of magnitude, spontaneous generation of reactive species such as water and hydroxide radicals as well as hydrogen peroxide, and other curious chemistries. While these studies have changed the way that scientists view microdroplets, they all require the analysis of thousands of droplets in tandem where values are extrapolated back to the average droplet. The robust correlation of chemistry in an individual droplet of a specific size requires the development of new measurement tools capable of accessing single sub-femtoliter droplets, one at a time. Here, I describe the development of new electrochemical measurement tools which have been used to access this curious chemistry at the single droplet level as well as the implications of the findings from the implementation of these tools. First, stochastic electrochemistry, a method where an electrode effectively “fishes” for droplets suspended in an oil phase, will be outlined and it’s use to probe the spontaneous generation of hydrogen peroxide in such droplets will be presented. Afterwards, a method used for the electroanalysis of droplets in air, or aerosols, where an ionic liquid bubble (suspended by a platinum bubble wand) captures droplets to be analyzed at a carbon fiber wire thread through the middle, will be outlined. The use of these two techniques to correlate enzymatic activity in both droplet types, droplets in oil and aerosols, will then reveal that the gas|liquid interface promotes higher turnover rate acceleration for glucose oxidase. Finally, the fabrication and use of a dual-barrel electrode for the analysis of an acoustically levitated droplet will be presented. These three techniques stand to make electrochemistry a pivotal technique for the analysis of the curious chemistry housed within individual microdroplets. In addition to these methods, methods for extending electrochemistry to the next generation of scientists are presented.</p>

<p>Various organic reactions, including important synthetic reactions involving C–C, C–N, and C–O bond formation as well as reactions of biomolecules, are known to be accelerated when the reagents are present in confined volumes such as sprayed or levitated microdroplets or thin films. This phenomenon of reaction acceleration and the key role of interfaces played in it are of intrinsic interest and potentially of practical value as a simple, rapid method of performing small-scale synthesis. This dissertation has three focusing subtopics in the field of reaction acceleration: (1) application of reaction acceleration in levitated droplets and mass spectrometry to accelerate the reaction-analysis workflow of forced degradation of pharmaceuticals at small scale; (2) fundamental understanding of mechanisms of accelerated reactions at air/solution interfaces; (3) discovery the use of glass particles as a `green' heterogeneous catalysts in solutions and systematical study of solid(glass)/solution interfacial reaction acceleration as a superbase for synthesis and degradation using high-throughput screening.</p><p><br></p><p>Reaction acceleration in confined volumes could enhance analytical methods in industrial chemistry. Forced degradation is critical to probe the stabilities and chemical reactivities of therapeutics. Typically performed in bulk followed by LC-MS analysis, this traditional workflow of reaction/analysis sequence usually requires several days to form and measure desirable amount of degradants. I developed a new method to study chemical degradation in a shorter time frame in order to speed up both drug discovery and the drug development process. Using the Leidenfrost effect, I was able to study, over the course of seconds, degradation in levitated microdroplets over a metal dice. This two-minute reaction/analysis workflow allows major degradation pathways of both small molecules and therapeutic peptides to be studied. The reactions studied include deamidation, disulfide bond cleavage, ether cleavage, dehydration, hydrolysis, and oxidation. The method uses microdroplets as nano-reactors and only require a minimal amount of therapeutics per stress condition and the desirable amount of degradant can be readily generated in seconds by adjusting the droplet levitation time, which is highly advantageous both in the discovery and development phase. Built on my research, microdroplets can potentially be applied in therapeutics discovery and development to rapidly screen stability of therapeutics and to screen the effects of excipients in enhancing formulation stabilities.</p><p><br></p><p>My research also advanced the fundamental understanding of reaction acceleration by disentangles the factors controlling reaction rates in microdroplet reactions using constant-volume levitated droplets and Katritzky transamination as a model. The large surface-to-volume ratios of these systems results in a major contribution from reactions at the air/solution interface where reaction rates are increased. Systems with higher surface-active reactants are subject to greater acceleration, particularly at lower concentrations and higher surface-to-volume ratios. These results highlight the key role that air/solution air/solution interfaces play in Katritzky reaction acceleration. They are also consistent with the view that reaction increased rate constant is at least in part due to limited solvation of reagents at the interface.</p><p><br></p><p><br></p><p>While reaction acceleration at air/solution interfaces has been well known in microdroplets, reaction acceleration at solid/solution interfaces appears to be a new phenomenon. The Katritzky reaction in bulk solution at room temperature is accelerated significantly by the surface of a glass container compared to a plastic container. Remarkably, the reaction rate is increased by more than two orders of magnitude upon the addition of glass particles with the rate increasing linearly with increasing amounts of glass. A similar phenomenon is observed when glass particles are added to levitated droplets, where large acceleration factors are seen. Evidence shows that glass acts as a ‘green’ heterogeneous catalyst: it participates as a base in the deprotonation step and is recovered unchanged from the reaction mixture. </p><p><br></p><p>Subsequent to this study, we have systematically explored the solid/solution interfacial acceleration phenomena using our latest generation of a high-throughput screening system which is capable of screening thousands of organic reactions in a single day. Using desorption electrospray ionization mass spectrometry (DESI-MS) for automated analysis, we have found that glass promotes not only organic reactions without organic catalysts but also reactions of biomolecules without enzymes. Such reactions include Knoevenagel condensation, imine formation, elimination of hydrogen halide, ester hydrolysis and/or transesterification of acetylcholine and phospholipids, as well as oxidation of glutathione. Glass has been used as a general `green' and powerful heterogeneous catalyst.</p>