Room B20

Room B20 | 8:45 a.m.

DEVELOPMENT OF AFFINITY MICRO FREE FLOW ELECTROPHORESIS IMMUNOASSAY FOR LEPTIN

Presented by: Jordan Kunze

Jordan Kunze, Michael Bowser

kunze077@umn.edu, bowser@umn.edu 

While existing immunoassays allow for discrete measurements, there are not yet refined affinity methods for continuous monitoring, leading to an incomplete understanding of biological processes. Micro free flow electrophoresis (µFFE) is a continuous analytical technique for separating, and quantifying analytes. Combining µFFE with affinity assays enables continuous monitoring of antigens. In this approach, sample flows through a planar separation channel while an electric field applied orthogonally separates analytes based on their electrophoretic mobility. This method allows for real-time detection of target molecules, such as signaling peptides like leptin, to evaluate responses to stimuli.

Here, a COC µFFE device was fabricated via hot embossing and bonded using cyclohexane vapor exposure. A competitive µFFE immunoassay for leptin is being developed, in which fluorescently labeled leptin and unlabeled leptin compete for binding sites on anti-leptin antibodies. The bound and unbound fractions are then separated into two streams, and fluorescence intensity of each stream is measured. From the fluorescence intensity of these peaks, the concentration of unlabeled leptin can be determined. Injection of FITC-leptin demonstrates a low-nanomolar limit of detection, enabling the detection of labeled antigen and the generation of a linear calibration curve. Preliminary offline experiments using anti-leptin antibodies confirm small amounts of binding to FITC-Leptin, as proven by the appearance of a second peak. Further experiments are being conducted to establish a competitive immunoassay for leptin before applying the assay to adipose cells to develop the method into an online detection assay.

Room B20 | 9:10 a.m.

DETECTION OF LOW ABUNDANCE SECRETED CELLULAR SENESCENCE MARKERS THROUGH NANOPARTICLE-BASED MASS CYTOMETRY

Presented by: Rose Liyanagunawardana

Rose Liyanagunawardana, Edgar Arriaga*

liyan011@umn.edu, arriaga@umn.edu

Cellular senescence is a primary driver of age-related pathologies, yet characterizing the Senescence-Associated Secretory Phenotype (SASP) is hindered by the limitations of bulk analysis. While Interleukin-6 (IL-6) is a key SASP component readily detected via Enzyme-Linked Immunosorbent Assay (ELISA), these methods fail to report SASP from single cells in mixed senescent populations that characterize aging. This project addresses these constraints by leveraging mass cytometry (CyTOF) using gold nanoparticles (AuNP) to replace traditional metal-chelating polymers as labels in this multiparametric technique. To develop this labeling scheme, Bovine Serum Albumin (BSA) was used to optimize protein conjugation via NHS/EDC chemistry, and conjugation was verified by using magnetic microspheres employing a streptavidin-biotin assay. This nanoparticle-based approach could enhance sensitivity, carrying approximately 30,000 atoms per antibody, theoretically a ~300-fold increase over the <100 lanthanide ions provided by standard polymer labeling. The next phase of this research involves anchoring AuNP-antibody complexes directly to cell surfaces to monitor the SASP (see abstract graphic). The AuNPs will be bound to the exterior of cell membranes using anti-beta-2-microglobulin (B2M) antibodies, which recognize the B2M surface protein. The AuNP also has anti-IL-6 antibodies that capture secreted IL-6, which then is “sandwiched” with a secondary antibody that serves as a reporter. This can be expanded to various cell models and use antibodies against senescent cell markers and phenotypic markers to identify cell types to produce the first comprehensive description of SASP secretion by senescence cells in mixed cell populations, providing a foundation for a deep understanding of age- related pathologies.

Room B20 | 9:35 a.m.

Scalable Native Ion Mobility-Mass Spectrometry with Online Tangential Flow Filtration

Presented by: Gabrielle Blake

Gabrielle Blake¹, Carter Asef², Suraj Dhungana², Mason Chilmonczyck², Varun V. Gadkari*¹

blake654@umn.edu, vgadkari@umn.edu 

1. University of Minnesota, Department of Chemistry, Minneapolis, MN

2. Andson Biotech, Atlanta, GA

Native mass spectrometry requires the elimination of non-volatile salts. Typically, this is done offline using time consuming desalting methods. Tangential flow filtration (TFF) is a rapid and effective desalting technique that can be coupled directly to mass spectrometers – allowing for online cleanup immediately prior to nanoelectrospray ionization. This work evaluates the efficacy of a commercially available TFF device, the DynaChip X1, used for native mass spectrometry measurements. Ion mobility-mass spectrometry (IM-MS) was used to determine whether a  range of protein standards (8.6-465 kDa) maintained native structure through online desalting. These standards were used to evaluate the ability of the TFF system to remove biologically relevant buffer components prior to IM-MS analysis. Collision cross section (CCS) measurements taken of the standards were consistent with native structure literature values. This indicates that the native structures were preserved during the online desalting process. Collision induced unfolding (CIU) experiments were adjusted to the elution timescale of the TFF system, and rapid TFF-CIU measurements were validated for the antibody drug, Infliximab. Typical CIU acquisitions are 20 minutes long. Rapid TFF-CIU workflows were developed to perform a full acquisition in 2 minutes and 4 minutes. The 2-minute and 4-minute CIU fingerprints were both highly reproducible (RMSD <5%) and comparable across both timescales, establishing a 2-minute CIU acquisition as a robust workflow for antibody stability analysis. These results establish online TFF as a rapid and reproducible online desalting technique for native IM-MS and CIU analyses, as well as benchmark a rapid workflow for conducting CIU experiments.

Room B20 | 10:00 a.m.

Development & Investigation of Glutathione Carbon Dots as biosensors for in vitro Oxidative Stress Monitoring in Cells

Presented by: Rhea Caldwell

Rhea Caldwell, Madison Cereno, Dr.Christy L. Haynes* 

caldw316@umn.edu, chaynes@umn.edu 

Redox homeostasis is essential for maintaining physiological function. Its disruption, known as oxidative stress (OS), is related to many diseases and disorders, including neurodegenerative diseases, cancer, and diabetes. Common analytical methods, including mass spectrometry and electrochemistry, can quantify reactive oxygen species, but lack facile spatial detection capabilities. Fluorescence probes are commonly used to monitor OS due to their high sensitivity, spatial detection, and real-time analysis capabilities; however, fluorescent probes can be toxic, expensive, and difficult to synthesize. 

Herein, the use of glutathione-based carbon dots (GSH-CDs) is proposed as a fluorescent sensing modality for OS monitoring. CDs are eco-friendly and biocompatible alternatives to standard fluorescence sensors. Previously, surface-modified GSH-CDs were made via the EDC-NHS coupling reaction. Those GSH CDs exhibited a fluorescence quenching response when in the presence of dopamine. In this work, GSH-CDs are made by embedding GSH into the polymeric backbone of the CDs via a one-step hydrothermal synthesis. The physical properties, fluorescence mechanisms, and binding affinity to OS precursors, including dopamine, is assessed for the GSH-CDs using various characterization strategies. With this data, the localization of GSH CDs in cells will be investigated to observe whether GSH-CDs localize at specific organelles and understand why this occurs. All together, these data will reveal if GSH-CDs are a more biocompatible, eco-friendly, low-cost, and effective strategy for in vitro monitoring of OS in disease systems.

Room B20 | 10:40 a.m.

CONTINUOUS MONITORING OF TUMOR NECROSIS FACTOR ALPHA USING ONLINE AFFINITY MICRO FREE-FLOW ELECTROPHORESIS.

Presented by: Sandra Nwankwo

Sandra Nwankwo, Michael Bowser*

nwank019@umn.edu, bowser@umn.edu

Real-time monitoring of biochemical messengers such as Tumor Necrosis Factor Alpha (TNF-α) is es- sential for understanding dynamic biological processes. However, current methods are limited in their ability to provide continuous data. To address this limitation, we are developing a noncompetitive platform that integrates micro free-flow electrophoresis (μFFE) with aptamer-based affinity interactions to enable selective separation and analysis. μFFE is a separation technique that enables continuous separation of analytes as a sample flows through a planar channel under an electric field applied perpendicular to the direction of flow. In affinity μFFE, binding between an affinity reagent and the target causes electropho- retic mobility shifts, enabling separation of bound and unbound species. Aptamers are used as the affinity reagent due to their small size and high stability, which produce enhanced electrophoretic mobility shifts upon target binding. Significant outcomes included clear separation of bound and unbound aptamer peaks, establishment of a linear calibration curve for TNF-α that confirms the feasibility of the assay, and reproducible signals achieved through optimization of buffer composition and aptamer handling. Ongoing work is focused on enhancing temporal response and improving the assay’s limit of detection. Overall, this work demonstrates a compact, non-competitive platform capable of continuous, real-time monitoring of TNF-α. This approach provides a foundation for improving temporal resolution in biomolecular detec- tion and has strong potential for future diagnostic and analytical applications.

Room B20 | 11:05 a.m.

Characterizing the Higher Order Structure and Efficacy of Stressed Antibodies via Native Ion Mobility-Mass Spectrometry and Gas Phase Unfolding

Presented by: Eledon S Beyene

Eledon S Beyene; Rowan Matney; Varun V Gadkari*

beyen090@umn.edu, vgadkari@umn.edu

Monoclonal antibodies (mAbs) are the fastest growing class of biotherapeutic agents against a wide array of diseases. However, their size and complexity, mAbs are difficult systems to characterize and assess for quality control. Higher-order structure (HOS) of mAbs are influenced by stress conditions encountered during manufacturing, storage, and administration, potentially affecting their efficacy and safety. Stress-induced changes in HOS can lead to reduced biological activity and adverse patient outcomes. While native Ion Mobility-Mass Spectrometry (nIM-MS) and collision-induced unfolding (CIU) have proven effective in differentiating mAbs under stressed conditions, a comprehensive comparison of HOS changes across different stressors is needed to establish the utility of these techniques in the quality control of mAbs.

In this study, immunoglobulin G1κ (IgG1κ), the most functionally abundant antibody subclass in human serum and a common scaffold for biotherapeutic development, was subjected to thermal, pH, and freeze-thaw stress conditions. CIU analysis revealed distinct unfolding pathways between stress conditions, with pH-stressed IgG1κ monomers exhibiting stabilized HOS changes relative to thermally stressed and control samples. Replicate CIU datasets were further used to develop a supervised machine learning classification workflow capable of differentiating stressed IgG1κ samples and distinguishing thermally stressed from pH-stressed species.

Ongoing work extends these workflows to commercially available FDA-approved biotherapeutics to investigate how stress-induced HOS changes correlate with biological function. Specifically, native IM-MS is used to characterize antibody–antigen complex stoichiometry and stability under stressed conditions. Collectively, these results demonstrate the utility of IM-MS and CIU for linking mAb stability, structure, and function in biotherapeutic quality assessment.

Room B20 | 11:30 a.m.

DETECTION AND CHARACTERIZATION OF CATALYTIC DNA USING DROPLET MICROFLUIDICS.

Presented by: Jaya Jakhar

Jaya Jakhar, Michael T. Bowser*

jakha003@umn.edu,  bowser@umn.edu 

Nucleic acids, traditionally recognized for their roles in storing genetic information, have more recently been identified for their catalytic capabilities, offering a readily synthesized and chemically stable alternative to proteins. The discovery of catalytic RNA inspired the identification of single-stranded catalytic DNA molecules, known as DNAzymes. Their integration in high-sensitivity detection assays would reduce technical and logistical requirements compared to protein-based methods, revolutionizing biosensing and providing unique insights into the catalytic potential of nucleic acids, broadening our understanding of early life evolution.  However, bias of selection methods towards single-turnover catalysts has hindered comprehensive characterization of the full catalytic potential of these oligonucleotides in therapeutics and biosensing.

Here, we use a flow-focusing droplet microfluidic platform to generate nL to pL-scale droplets, creating isolated microenvironments to enable real-time study of single enzyme kinetics. By preventing cross-contamination, this offers high-throughput characterization of catalytic oligonucleotides using fluorescence imaging. The 10-23 DNAzyme, a gold standard molecule with high catalytic activity, is used as a positive control, as a single molecule can produce thousands of fluorescent products when confined to a microscale environment, giving a detectable signal. In each experiment, over 10,000 droplets are imaged using a confocal microscope, allowing long-term monitoring of product formation with 3D droplet visualization, yielding rigorous datasets, to assess their folding mechanisms and kinetics. Furthermore, the study of single-enzyme kinetics will provide comprehensive insights into how structural folding and enzymatic activity are affected by diverse microenvironments, advancing the development of catalytic oligonucleotides for biosensing by overcoming limitations in traditional selection methods.

Room B20 | 11:55 a.m.

Fungal Siderophores: Secondary Metabolites Produced by Fungal Endophytes Influence Switchgrass Micronutrient Acquisition

Presented by: Ikumi Ellis

Ikumi Ellis, Rachel Hammer, Christine Hawkes, Rhona Stuart, Rene Boiteau* 

elli0687@umn.edu, rboiteau@umn.edu 

Switchgrass is a promising bioenergy crop, but its sustainability relies on growth on marginal lands with low or no fertilizer inputs. One approach to support growth in these conditions is for plants to rely on mineral-solubilizing fungal partners. Root fungal endophytes are known to aid uptake of macronutrients by switchgrass; however, their effects on host access to micronutrients, specifically Fe, remains poorly understood. Preliminary analyses indicate that diverse endophytic fungal strains can enhance or suppress switchgrass uptake of Fe. We hypothesized that fungal production of siderophores – iron chelators secreted in response to Fe stress – is a key mechanism for fungal-mediated Fe acquisition in switchgrass. To identify siderophores secreted by the fungi in culture, we used untargeted liquid chromatography – high resolution orbitrap mass spectrometry. We identified nine siderophores from at least two known families across 17 fungal strains. Fungi associated with an increase in plant host shoot Fe produced either a full suite of coprogen-B siderophores, a siderophore of an unknown class, or both. This indicates that switchgrass acquisition of Fe solubilized by fungal endophytes is likely to be strain-dependent, as different strains produce distinct siderophore types. Furthermore, the unknown siderophore was associated with the greatest uptake of Fe by the plant, suggesting that specific types of siderophores produced may influence plant Fe acquisition. Ongoing work aims to determine the structure of the unknown siderophore, as well as elucidate the mechanisms for fungal siderophore-mediated Fe dissolution and its influence on plant access to mineral-bound Fe. 

Room B20 | 1:35 p.m.

DRIVERS OF DISSOLVED ORGANIC MATTER COMPOSITION IN PRAIRIE POTHOLE WETLANDS

Presented by: Aiden J. Berndt

Aiden J. Berndt, Dillon Siple, Keith Morrison, Abrar Shahriar, Edward J O’Loughlin, Maxim I. Boyanov, Kenneth M. Kemner, Sheel Bansal, Roser Matamala, Rene Boiteau*

bernd216@umn.edu, rboiteau@umn.edu

Wetlands are global hotspots of carbon cycling, disproportionately storing a larger percent- age of global soil organic matter than the land mass they account for. Within wetlands, dissolved organic matter (DOM) serves as an important substrate that fuels microbial production of carbon dioxide and methane, as well as a precursor for the formation of mineral associated organic matter. Although DOM composition can vary and strongly impact the fate of organic carbon, current models of wetland carbon cycling represent DOM as a single uniform reservoir. To evaluate the major drivers of DOM variability across a wetland, we compared DOM concentrations and molecular composition across spatial, depth, and seasonal scales in a pristine wetland located in the Prairie Pothole Region of North Dakota. Molec- ular characterization was performed using reverse phase liquid chromatography coupled to ultrahigh resolution orbitrap mass spectrometry. We found that permanently saturated sediment porewaters con- sistently contained significantly more DOM than the suboxic porewaters in the transiently inundated tran- sition zone. Molecular analysis revealed that the DOM accumulating in the transiently inundated porewa- ters was significantly depleted in oxidized, high molecular weight species. Thermodynamic modelling suggests the remaining DOM is more energetically favorable for microbial remineralization. Furthermore, the depleted aromatic, oxidized, and high molecular weight DOM adsorbs preferentially to iron oxide minerals through ligand exchange. Our findings indicate wetland DOM is better represented by a two- component model that distinguishes a thermodynamically stable component with high mineral affinity that accumulates in euxinic porewaters from a less stable, lower mineral affinity component that is more widespread. 

Room B20 | 2:00 p.m.

INVESTIGATING ORGANIC INTERFACES FOR EFFICIENT WATER SPLITTING USING VIBRATIONAL SUM FREQUENCY GENERATION SPECTROSCOPY

Presented by: Mia Halliday

Mia Halliday, Aaron Massari*

Halli218@umn.edu

Efficient water splitting has been a persistent goal of energy research due to its potential to cleanly create H₂ gas, a highly energetic fuel. This process typically has a high energetic barrier that must be exceeded to occur. Photocatalytic methods utilizing the photoexcitation of organic semiconduc- tors can increase water splitting efficiency by lowering this barrier. Specifically, a system of 3,4,9,10- perylenetetracarboxylic diimide (PTCDI) and 1,4,5,8-napthalenetetracarboxylic diimide (NTCDI) layers has been shown to fulfill these criteria. The electron deficiency of NTCDI relative to PTCDI spontaneously generates an internal electric field (IEF) at the interface that separates electrons and holes from the interface, leading to increased photocatalytic efficiency. The electric fields have previously only been measured at the surface, rather than by measuring the electric field at the buried interface between semiconductors. Thus vibrational sum frequency generation (VSFG) spectroscopy, an interface specific technique, provides a useful tool for characterizing these IEFs. Each semiconductor has a specific mo- lecular vibrational handle that can be used in VSFG to measure differences in the IEF. As the interface is modified, changes to the resonant and non-resonant components of the VSFG spectrum can be ana- lyzed to determine the factors that lead to the strongest IEF and thus the most efficient water splitting.

Room B20 | 2:25 p.m. 

COMPARISON OF THE CHEMICAL COMPOSITION OF DISSOLVED ORGANIC MATTER FROM GROUNDWATER AND RIVER SOURCES TO THE WEST FLORIDA SHELF

Presented by: Caini Huang

Caini Huang, Ilana Farrell, Kathleen Kouba, Salvatore Caprara, Hannah R. Hunt, Angela Knapp, Joseph Tamborski, Christopher Smith, Tim Conway, Kristen Buck, Phoebe Dreux Chappell, Rene Boiteau*

huan2696@umn.edu, rboiteau@umn.edu

Groundwater and river water are two important components of the coastal carbon and nu- trient budgets, particularly in oligotrophic systems such as the West Florida Shelf (WFS). Along the WFS and adjacent coastal water bodies, inputs of organic nutrients from groundwater and river are thought to play a crucial role in the timing and extent of coastal algal blooms. Significant regional and seasonal variability of riverine sources has been well recognized. However, it remains unknown whether the com- position of dissolved organic matter (DOM) in groundwater and river water discharged into the WFS is also variable. To address this gap, we characterized and compared the molecular composition of solid phase extracted DOM from submarine groundwater discharge (SGD) and river sources on the WFS across four seasons using ultrahigh performance liquid chromatography-Orbitrap mass spectrometry (UHPLC-Orbitrap-MS). SGD-DOM is significantly larger, more aliphatic, and more sulfur-enriched than river-DOM (Wilcoxon rank-sum test p-value < 0.05). Source materials (e.g., vascular plants vs algae and phytoplankton) and processing (e.g., oxic vs anoxic) may lead to the spatial variations. Distinct temporal variation in SGD and riverine DOM was also observed, suggesting that endmembers react differently to environmental changes. Selected marker features have great potential to differentiate DOM composition from different coastal inputs and can be used to track its fate and impact on coastal ecosystems, which enables better prediction of how changing hydrologic processes will impact the organic matter supply.

Room B20 | 2:50 p.m

STABILITY AND COMPETITIVE SORPTION OF LIGNIN-DERIVED ORGANIC ACIDS ON GOETHITE: THE ROLE OF CARBOXYL AND CATECHOL FUNCTIONAL GROUPS

Presented by: Aleksander Rabago

Aleksander Rabago, Rene Boiteau, Lee Penn

rabag005@umn.edu, rboiteau@umn.edu, rleepenn@umn.edu

Soil organic carbon (SOC) dynamics are governed by organic matter stabilization on mineral surfaces. Iron oxides like goethite sequester organic ligands through surface adsorption. Lignin, a heterogeneous biopolymer rich in carboxyl and phenol groups, comprises up to 60% of plant woody tissue and is degraded by soil fungi and bacteria via distinct pathways producing degradation products that form iron-associated SOC (FeOC). The stability of these mineral associations is a governing factor of long-term terrestrial carbon sequestration. This research investigates how specific structural features of model lignin degradation products associated with fungal vs. bacterial pathways dictate adsorption dynamics. Initial experiments evaluated the adsorption of fungal (caffeic acid, p-coumaric acid) and bacterial (catechol) products onto goethite from pH 4-9. The catechol (ortho-dihydroxy) group was confirmed to be a primary driver of mineral association at circumneutral pH. To assess stability, disruption experiments were conducted at pH 7, showing that caffeic acid (containing both carboxyl and catechol groups) displaces pre-adsorbed catechol through ligand exchange. While catechol is slowly released into the aqueous phase, caffeic acid adsorption continues for up to 120 hours. These results imply that while catechol groups bind strongly alone, polyfunctional lignin fragments associated with fungal pathways that also retain carboxyl groups form more thermodynamically stable complexes than simple bacterial metabolites. Thus, fungal pathways of lignin degradation may be greater contributors to the accumulation of mineral associated organic carbon in soils.