Robert T. Kennedy

Machine vision camera method for label-free measurement of OD ₆₀₀ of microbes growing in flowing nanoliter droplets at 10 samples per s is presented.

Probing the spatiotemporal release dynamics of multiple neurochemicals simultaneously in vivo is essential for understanding the molecular basis of brain function and behavior. Microdialysis enables multiplexed neurochemical analysis when combined with advanced molecular tools such as mass spectrometry, but existing microdialysis probes typically exhibit low recovery rates and provide temporal resolution of several minutes, which is insufficient to capture rapid neurochemical release events. Moreover, it remains challenging to investigate the release of multiple neurochemicals upon cell type–specific neuromodulation with a single probe. Here, we present an opto-microperfusion platform that integrates time-sequential microfluidics with cell type–specific optogenetic neuromodulation and push–pull microsampling capabilities. We demonstrated subminute temporal resolution in vitro and near-minute temporal resolution in vivo, with in vivo sampling intervals limited by downstream analytical requirements. The platform achieved high recovery rates for both small molecules and neuropeptides, including 76% for serotonin, 69% for dopamine, and approximately 14% for neuropeptide Y, representing a significant improvement over conventional microdialysis probes. In vivo studies in rats demonstrated reliable microsampling of multiple neurochemicals with minimal sample-to-sample variation. Studies in mice demonstrated the simultaneous detection of the release dynamics of up to 23 different neurochemicals following optogenetic stimulation when coupled with mass spectrometry. Together, this multimodal neural probe paves the way for investigating the molecular mechanisms underlying behavior and neurological disorders.

Nanodiscs are tunable and widely used membrane mimetics that can allow for the in vitro study of membrane proteins in a native-like lipid bilayer environment. The lipids incorporated into nanodiscs can be modulated to represent specific biophysical environments, but many nanodisc applications rely on simplified lipid compositions that inadequately represent native membrane complexity. Natural lipid extracts offer more physiological relevance; however, recent work has shown that (1) lipid composition of a nanodisc does not stoichiometrically reflect the initial mixture and that (2) assembly conditions such as temperature and belt size may influence lipid incorporation. Here, we add to this body of work by implementing an untargeted LC-MS/MS lipidomics workflow to profile the incorporation of multiple, combined natural lipid extracts into membrane scaffold protein nanodiscs, examining not only assembly temperature and belt size, but also the effects of detergent and synthetic lipid additives on lipid incorporation. Species level lipidomic analysis, including trends in acyl chain length and degree of unsaturation were evaluated. We observe that assembly temperatures have minimal impact on lipid incorporation, whereas belt size effects vary across extracts, as smaller scaffolds likely enrich lipids that promote a more tightly packed membrane. Furthermore, we found that lipid solubilization and incorporation into nanodiscs is broadly preserved regardless of detergent identity. Conversely, synthetic lipid additives influence incorporated lipid class composition more strongly than detergent, temperature, or MSP belt size, with unsaturated additives favoring glycerolipid enrichment. Taken together, this data demonstrates the interplay of nanodisc assembly conditions in shaping the incorporated lipid environment.

Innate temperament affects vulnerability to substance use disorders (SUDs). Identifying the neurochemistry that differentiates temperaments, alongside the metabolic impact of drugs of abuse, can provide new insights into pathways to SUD and treatments. We show that heritable emotional temperaments associated with SUD vulnerability are encoded by distinct extracellular neurochemical states in the living brain. We apply untargeted metabolomics to microdialysis samples to define the chemical composition of the living brain metabolome with unprecedented depth. Using two phenotypes from a rodent model of diverging temperaments linked to SUDs, dialysate samples were collected before and after acute exposure to cocaine (15 mg/kg, i.p.). Metabolomic analysis revealed previously unknown basal differences in stress-response, bioenergetics, and amino acid neurotransmitters and their related pathways between the two phenotypes. Cocaine administration also had a profound impact on the metabolome, altering amino acid, purine, and arachidonic acid pathways along with cognition-related metabolites. Despite these broad metabolomic changes, cocaine administration narrowed several pre-existing differences between phenotypes, highlighting divergent responses across genetic lines. These findings demonstrate that in vivo metabolomics not only offers novel insights into the neurochemistry underlying temperament and drug response but also serves as framework to advance a systems-level understanding of other brain-related disorders.

Engineered biocatalysts enable highly selective chemical transformations with low environmental impact. Development of biocatalysts by directed evolution requires screening many enzyme variants for improved catalytic properties. The low throughput of commonly used label-free screening methods, e.g., liquid chromatography-mass spectrometry (LC-MS), becomes the rate-limiting step in biocatalyst development, limiting the coverage of protein sequence space explored. Direct MS methods have been applied to biocatalyst screening; however, these methods cannot be used to evaluate isomer selectivity. Ion mobility spectrometry is a separation technique readily combined with MS, facilitating isomer differentiation on the millisecond time scale. Here, we present a droplet microfluidic system coupled to cyclic ion mobility-mass spectrometry (cIM-MS) to enable the screening of isomeric products. The system was applied to the separation of biaryl benzofuran dimers formed by the fungal cytochrome P450 KtnC. The isomeric 5,7'-bibenzofuran and 7,7'-bibenzofuran products from KtnC variants were baseline-resolved within 32 ms by cIM. By infusing biocatalytic reaction mixtures as 5 nL droplets, an analysis throughput of 1.2 s/droplet was achieved using cIM-MS. Droplet cIM-MS was used to quantify standards in the reaction matrix with high agreement to actual concentrations of each isomer (i.e., R² of 0.97 and 0.98 for total bibenzofuran content and fractional 5,7'-bibenzofuran content, respectively). For samples with enzymatically formed product isomers, droplet cIM-MS identified the same active variants and had comparable reproducibility (RSD of 10-15%) to analysis by LC-MS and produced this data 128 times faster. The method is expected to be suitable for improving the rate of biocatalyst development for isomer-selective reactions.

High-throughput screening is important in a diverse array of applications including drug discovery, synthetic reaction development, and enzyme engineering. Well plates are often used for sample preparation and containment in such applications, so analytical methods that are compatible with this format are required. Mass spectrometry (MS) is an attractive analytical technique for high-throughput analysis due to its potential for rapid, sensitive, selective, and label-free multiplexed measurements. Here, we present a method that uses the Venturi effect to withdraw droplet samples from a well plate and infuse them to the electrospray ionization (ESI) source of a mass spectrometer. The Venturi effect is generated by flow of nebulizing gas through a constriction at the outlet of the ESI source. The resulting negative pressure allows sample to be pulled to the ESI source via a sample transfer capillary that is coaxial with the ESI source at the outlet and can be dipped into sample at the inlet. To keep different samples from mixing, 380 nL sample plugs flowing at 330 µL min⁻¹ are sipped into the source and separated by air gaps resulting in segmented flow to the source. The system requires no valves or connections for achieving sample pick-up and analysis. An x,y,z-positioner is used to move the sample inlet for automated sampling from different wells. Increasing capillary inner diameter and nebulizing gas pressure increased the throughput of Venturi droplet-MS by increasing sample flow rate. An interaction of sample plug size and number within the capillary on overall flow rate was observed and affected the possible throughput. When using perfluoroalkoxy alkane (PFA) tubing as the transfer capillary, carryover between samples was 0.88 ± 0.16%. The method is demonstrated by screening 283 whole cell reactions for enzyme engineering at 0.4 samples per s, while showing good agreement (R² = 0.92) with liquid chromatography-mass spectrometry (LC-MS). This work improves upon previous uses of segmented flow for high-throughput MS by integrating sample generation and transfer in one step. Compared to other high-throughput MS methods this approach requires no custom MS sources or specialty sample introduction equipment.

We introduce a rapid (1 min/sample) direct-MS method for analyzing terpene content in fermentation extracts.

Biotechnology is increasingly relying on high-throughput experiments in which large populations of cells are phenotyped and selected based on performance, e.g., for producing a desired product. Droplet microfluidics enables high-throughput screening, and as such, it lends itself ideally to such experiments, but reliance on a fluorescent readout is limiting. Herein, we report a system integrating droplet electrospray ionization-mass spectrometry (ESI-MS) with a microfluidic voltage-mediated droplet printer to enable online analysis and capture of cell-containing aqueous droplets, ultimately offering the possibility for label-free phenotyping and selection of cells. Escherichia coli (E. coli) cell-containing droplets (20 nL) are split into two volume fractions within a microfluidic splitter chip. One fraction is analyzed using a sheath-flow ESI source with MS, while the sibling fraction is printed onto an agar plate using a custom-built voltage-mediated microfluidic printer. Printed droplets can grow into single microbial colonies that map back to their respective droplet ESI-MS signal with 94-99% accuracy and without carryover. Nearly synchronous and stable system operation is shown for infusion rates in the range of 0.4-1.2 droplets/s, while the achieved droplet spacing and printing precision can enable reliable single-colony retrieval for further analyses or gene sequencing. The system is also shown for phenotyping and selection of an E. coli variant engineered to produce l-lysine among control cells. The method enables screening cell colonies for chemical composition and collecting them for further processing with potential application in synthetic biology and enzyme engineering among others.