Development of Microfluidic, Immunoassay, Liquid Chromatography, and Mass Spectrometry Methods for Analysis of Islet Cell Secretions

Abstract

Cell secretion is a fundamental and highly regulated process which can inform on health, disease, cellular function, and cell-cell communication when interrogated, though this is analytically challenging. Instrumentation and methods may not be well suited for the numerous chemical classes found in secretions, may not be compatible with conditions like complex matrices required for cell viability, and may be limited by large volume requirements which can increase sample consumption or decrease temporal resolution. To develop methods for monitoring secretions that overcome these challenges, islets of Langerhans were used as a model system. Islets primarily work to maintain blood glucose homeostasis through the hormones insulin and glucagon; though, the small molecules they secrete and their metabolome are also of interest in relation to glucose homeostasis and diabetes.

Approaches to analyze islets have included microfluidic devices to mimic in vivo environments and liquid chromatography (LC), mass spectrometry (MS), and immunoassays to assess secretions. These approaches successfully lay the groundwork for cell studies; however, multiplexed measurements are rare. To enable temporally resolved multimodal measurements of hormones and small molecules from islets-on-chip, a bead-based immunoassay was scaled-down in volume and paired with benzoyl chloride derivatization prior to LC-MS/MS analysis. Derivatization improves ionization and enables better separation of polar analytes on a reversed-phase column. With the multimodal approach, correlation in secretion patterns was identified; however, the biogenic amine concentrations were near the limits of detection. We identified that low recoveries from the device were likely due to loss to the polydimethylsiloxane (PDMS) material commonly used for devices.

Losses of small molecules to PDMS decreases reliability of drug delivery, alters nutrients available to cells, and reduces recovery. This loss is poorly understood. To inform on mechanism of loss, 11 device treatments were screened aimed at changing surface charge and hydrophobicity and device porosity. Results suggest that hydrophobicity is not the main determinant of small molecule retention on-chip and other factors such as surface topology and device porosity need to be further investigated. Results also demonstrate that recovery with treatments varied by analyte and signify a need to test all targeted compounds with a given device material or treatment. Using the best tested treatment to measure islet secretions, more significant differences were observed than without treatment.

To expand on targetable molecules with the benzoyl chloride derivatization step for small molecules, alternative reaction conditions were explored systematically. Higher concentrations of base tended to improve detection of hydroxyl-containing molecules and an alternative order of addition improved detection limits for steroids and nucleosides. Overall, no one combination of conditions enabled complete derivatization of all targeted classes with low limits of detection, but through a combination of described methods, more analyte classes may be targeted with derivatization.

Lastly, integrating temporal and spatial measurements is of interest to provide a more complete picture of cell secretions. Initial steps toward incorporating mass spectrometry imaging with islet-on-chip studies were made, including identifying suitable a matrix deposition method and tissue preparation steps.

Together, these results provide methods for multimodal cell investigations of different chemical classes in a more controlled and accurate on-chip environment. These methods have been applied to study primary cells, cell lines, and neurochemistry, demonstrating applicability in other fields of study. The biological results obtained emphasize the potential for improved understanding of secretions through advances in the analytical approaches used to investigate them.