Technological development of in-cell NMR: Microfluidic, metabolomic, and fluxomic approaches

Author

Nicholas Sciolino, University at Albany, State University of New YorkFollow

ORCID

https://orcid.org/0000-0001-5575-6209

Date of Award

Spring 2025

Language

English

Embargo Period

3-25-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Program

Chemistry

First Advisor

Alexander Shekhtman

Committee Members

Alexander Shekhtman, Ravichandran Ramasamy, Qiang Zhang, Li Niu, Mehmet Yigit

Keywords

In-Cell NMR, Fluxomics, Metabolomics, Nuclear Magnetic Resonance, NMR

Subject Categories

Biochemistry | Cellular and Molecular Physiology | Medicinal-Pharmaceutical Chemistry | Molecular and Cellular Neuroscience | Molecular Biology | Neuroscience and Neurobiology | Other Neuroscience and Neurobiology | Structural Biology

Abstract

In the fields of structural biology, metabolomics, and fluxomics, there is a driving, technique-based concern when it comes to high-resolution spectrometry and its marriage to biological and physiological relevance. Sample preparation often involves cellular perturbation, lysate separation schemes, combinations with non-biological reagents, and timeline slack. Conceptually, the farther we get from the cell, the greater the potential disparity between what exists in vivo and our in vitro manipulations. This is particularly true when seeking to collect data on transfected targets of interest, and on sensitive metabolic equilibria that occur on the order of minutes. The question remains: if we are insulting cells and decreasing their viability, or concentrating and derivatizing their components in preparation for spectrometric analysis, to what degree does our research truly describe what is going on at a systems level? With such sensitive internal structures and metabolic processes, are our manipulations negatively impacting the protein structure homeostasis and metabolic fluxes which we wish to study, and can we arrive at a supporting methodology to help provide greater confidence beyond the status quo confirmatory assays that already exist? More specifically, can we keep cells alive to study protein structure and metabolic fluxes in real-time? Novel transfection and bioreactor technology developed and advanced by our laboratory, in concert with in-cell NMR, provides us with a new array of potential solutions to these questions.

The integration of the Volume Exchange for Convective Transfer (VECT) methodology with in-cell NMR shows itself to be a reliable, high-throughput method for introduction of isotopically-labeled targets of interest to eukaryotic cells, without the perturbation, high mortality rates, or lengthy optimization timelines of other, common techniques like electroporation. Greater numbers of cells are transfected and kept viable, allowing labeled targets to be observed with atomic-level resolution, in the native cellular environment, over the course of days. The pairing of these technologies has achieved collection of important, and biologically v relevant structural data from inside the cell, serving as proof of concept for future work involving more relevant cell lines and targets of interest.

Observation of metabolite synthesis over time using stable isotope supplementation in media like ¹³C-glucose has been widely adopted for research into metabolic pathways, but lacks true, real-time analysis when cell lysis is required. Bioreactor technology has been used to increase the capacity for analysis of living cells by NMR, an inherently non-destructive technique. The combination of these two methodologies with our recent advances in cell alginate-collagen matrix packaging, media perfusion, and gasification have allowed our group to develop the Real-Time Pulse-Chase NMR (RTPCR-NMR) method, performing fluxomic analysis on living cells that visualized the introduction of stable isotope-labeled media as a “pulse,” quantifying the leading and trailing edges. We used this technique to observe changes to characteristic times and binding constants in three important metabolites indicative of glycolysis, TCA cycle, and alternate energy metabolism across two relevant cell lines. SH-SY5Y cell pulses were modeled and analyzed before and after differentiation. H9C2 cell pulses were modeled and analyzed to record the effects of hypoxia with and without the formin Diaphanous-1. Results for all aspects of this project speak to the capacity of these novel approaches to address the need to obtain real-time, structural and fluxomic data on unperturbed, living cells. They provide greater fidelity on the inner workings of the cell and what can now be achieved with these step-wise advancements. The door is now open to opportunities for even greater discovery.

License

This work is licensed under the University at Albany Standard Author Agreement.

Recommended Citation

Sciolino, Nicholas, "Technological development of in-cell NMR: Microfluidic, metabolomic, and fluxomic approaches" (2025). Electronic Theses & Dissertations (2024 - present). 118.
https://scholarsarchive.library.albany.edu/etd/118