Development of Microfluidic Droptodes as a New Approach for Ionophore-Based Polyion Sensing
Glenn, Nicholas
2023
Abstract
Negatively or positively charged macromolecular species – alternatively referred to as polyions – are key analytical targets found in several natural and synthetic forms. As the efficacy of these species in chemical and biological reactions is most often linked to their quantitative amount, methods capable of measuring polyion concentrations accurately and efficiently are essential. Herein, this dissertation presents the development of ionophore-based droplet microfluidics as a new analytical technique for polyion sensing. Chapter 1 reviews several methods developed over the past several decades which have incorporated colorimetric and fluorescent molecules for chemical sensing of polyions. Subsequently – microfluidic droptodes derived from both droplet microfluidic and optical ‘optode’ sensing technologies – are detailed as a succeeding technology. Though to this point, a microfluidic droptode chemistry for fluorescent, biphasic sensing of polyanions does not exist, limiting the application of this method. Chapter 2 entails the development, optimization, and application of a novel droptode sensing chemistry for the detection of polyanions using a familiar chromoionophore, ionophore, and ion exchanger format. Heparin – a molecule whose anticoagulative effects in blood are most commonly monitored using unstandardized clotting assays – is pursued as an initial target of interest. This chapter demonstrates polyanion-sensitive droptodes are responsive toward heparin at a wide range of concentrations (3 – 135 µg/mL) which can be fine-tuned based on the concentration of sensing reagents employed. Furthermore, heparin measurements in human plasma demonstrate how the intrinsic features of microfluidic droptodes allow for quantitative polyanion measurements to be made in biologically complex and opaque matrices without optical interference from the sample. Protamine, a positively charged protein, is primarily known as the only FDA-approved antidote for the reversal of heparin. Polycation-sensitive droptodes exist as an alternate method for biphasic protamine sensing, though they require usage of an organic solvent which has low compatibility with polydimethylsiloxane-fabricated microfluidic devices. This limits our ability to study the effects of several key parameters such as aqueous droplet volumes and segment spacing on overall response. Chapter 3 successfully implements a compatible, lipophilic fluorescein derivative-based chemistry to analyze the impact of reaction time, aqueous droplet volume, and segment spacing on response of protamine-sensitive microfluidic droptodes using both bulk (sub-mL volumes) and microfluidic (100s of pL) methods. Chapter 4 advances the utility of microfluidic droptodes beyond biomedical polyion sensing for the measurement of cationic, polymeric quaternary ammonium compounds (PQs). Three PQ species (PQ-2, PQ-6, PQ-10) are detected as targets of interest, each inducing a unique response within our droptode system. Having developed and optimized a polyanion sensing chemistry, microfluidic droptodes are fully demonstrated in their ability to detect electrolytes, polycations, and polyanions. Though to this point, this method has only been demonstrated for single-plex sensing applications. Chapter 5 details the development and application of a droplet microfluidic “H-channel” device for spatial multiplexing of Na+ and K+ in individual samples. Lastly, Chapter 6 concludes this dissertation with an overview of the work presented in each chapter. Microfluidic droptodes are fully demonstrated for biphasic sensing of polyions and other targets in adjacent aqueous droplets. This method stands as a unique and novel variation of traditional droplet microfluidic applications in which optical measurements are made in aqueous droplets. Additionally, suggested applications and alternative strategies for microfluidic droptodes as future work are provided to continue the advancement of this newly introduced category of droplet microfluidics.Deep Blue DOI
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droplet microfluidics fluorescence polyions heparin protamine polyquaterniums
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