title: Electrophoretic separations of small molecules in low diffusion environment
Creators: name: Nanthasurasak, P
Publisher: name: University of Tasmania
Year: 2019

description: Portable analytical devices have been sought after heavily to support clinical diagnostics in lack-of-resource areas where access of advanced medical devices and specialists are restricted. Even though there are many commercially available portable Point-of-care (POC) devices allowing easy and rapid self-diagnostic, these POC were developed for generic infectious diseases such as diabetes or HIV and are not yet made for many common life-threatening disorders. In these cases, portable sample collection devices, such as Guthrie card, are employed for collection of biological samples including blood, urine, or saliva from patients off-hospital then the samples are sent to the centralized laboratory for analysis. However, several post-processing steps of the collected samples (i.e. extraction) upon arrival are required prior to the analysis and these are time-consuming steps that delay the turn-around-time of the result. Electrophoresis, separation of molecules under application of electric field, is a powerful analytical and sample preparation technique mostly utilized in clinical diagnostics to isolate components in samples ready for analysis. Recently, there has been a large number of developments with electrophoresis into miniaturized formats; however, they are not yet fully integrated with a high voltage power supply and liquid electrolytes are needed to perform separation making it unsuitable as portable diagnostic device. In attempt to bring forward a practical platform for clinical sample collection and sample preparation, the main goal of this thesis is to create a portable electrophoretic platform capable of performing solvent-free electrophoresis under relatively low voltage addressing issues found in conventional clinical diagnostics and electrophoresis. In this research, a polymer inclusion membrane (PIM) embedded with a carrier was investigated as a separation medium for electrophoresis for the first time. In chapter 2, PIM casting, dimension design, and electrophoresis apparatus are reported. A thin PIM was employed in this research in a lateral strip and electromigration studies were performed of fluorescent dyes with different charges including Coumarin 334, Fluorescein, and Rhodamine 6G (R6G) as neutral, anionic, and cationic analytes, respectively. The electromigration was monitored and recorded using a portable fluorescence microscope. Three main components of PIM – cellulose triacetate (CTA) as polymer base, 2-nitrophenyl octyl ether (2-NPOE) as plasticizer, and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide ([EMIM][NTf\(_2\)]) as carrier – were optimized as the ratios were found to impact on the migration distance and the spot shape of the dyes. Under a voltage of 2000 V (500 V/cm), only migration of cationic R6G was observed in this PIM leading to further investigation of electrophoresis of several cationic dyes. Successful electrophoretic separation of cationic dyes then allowed the electrophoretic mobilities and diffusion constants to be estimated for each species. Additionally, physical characterization of the PIM and possible mechanism of electromigration were also elucidated. Chapter 3 focuses on the potential of the PIM-based electrophoretic platform being useful in clinical application in the form of a portable electrokinetic device performing separation, as sample preparation, at low voltage while being transited to a central laboratory eliminating additional steps needed upon arrival. A positively charged alkaloid, Berberine chloride (BC), was used as a model analyte resembling small molecule pharmaceuticals spiked into the whole blood sample. Laboratory-based experiments revealed electromigration of BC but not dried drop of blood resulting in successful separation of BC from the blood matrix. To pursue the concept of in-transit separation, the PIM strip was fully assembled into a pocket-sized device with plastic housing equipped with two commercial batteries generating 6 V/cm (24 V) potential for separation. Separation real-time during transit was demonstrated by sending a portable device containing spiked BC in whole blood via internal mail and the analysis was performed once returned. To investigate PIM selectivity towards anions, a different ionic liquid, Aliquat\(^®\)336, was investigated as a carrier in chapter 4. It was found that not only electromigration of anionic species was observed but neutral molecules also migrated presumably from heteroconjugation. The tunability of the PIM was further investigate using several cationic surfactants with a similar chemical structure to Aliquat®336, with different number of carbon units, number of chains, chain length and counter anions, as carriers and their influence on electromigration and selectivity of dyes were investigated. While most of the cationic surfactants were found to have similar selectivity towards anionic molecules to Aliquat\(^®\)336, anionic surfactants investigated revealed selectivity towards cationic species similar to that observed with [EMIM][NTf\(_2\)]. In the last chapter, the possibility of integrating Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for detection of in-transit electrokinetic separations was explored. The experiment was performed to extend the applicability to non-coloured analytes as well as demonstrating for more powerful practical workflow allowing upon-arrival detection without additional labelling or derivatizing of analytes. PIM after in-transit separation was detached and taped on conductive Indium Tin oxide glass slide before being spray-coated with matrix α-Cyano-4-hydroxycinnamic acid (4-HCCA) and scanned using MALDI imaging. Colour intensity profile and distribution of BC molecules after electrophoretic separation from whole blood sample were graphically displayed in such case where the electrophoretic migration distance was unknown. The results showed promising potential for MALDI-TOF to be employed as detection method for in-transit electrokinetic platform concept where non-coloured analytes can be analyzed.