Electrofluidic thread-based analytical devices (eTAD) with ambient ionization mass spectrometry

Thread-based microfluidic analytical devices (˜í¬¿TADs) have attracted growing interest in medical, biological, and environmental analysis, due to their low-cost, biocompatibility, ease of assembly, and low-reagent consumption applications. The hydrophilic properties, mechanical strength, and soft and flexible nature of threads enable the fabrication of ˜í¬¿TADs without hydrophobic wax barriers as required in paper-based microfluidics and are compatible with modification or functionalisation under wet conditions. Additionally, preparation of ˜í¬¿TADs with simple configurations is possible by using threads with different surface chemistries via cutting, knotting, or sewing. Ambient ionization mass spectrometry (AIMS) provides direct ionization of analytes on the surface and subsurface of samples under ambient conditions, with minimal sample manipulation. Over the past few years, a series of ambient ionization techniques have been developed to enhance the capability of AIMS and expand its applications. These techniques have been widely applied in forensics, drug development, and medical diagnostics. However, the lack of simple and rapid sample clean-up and separation procedures typically limits the application of AIMS for the analysis of trace levels of analytes in complex samples like biological fluids, containing high levels of interfering species. This thesis has focused on the combination of ˜í¬¿TADs, for the pretreatment of complex biological samples, and desorption electrospray ionization mass spectrometry (DESI-MS), for the rapid analysis of complex biological samples. The combination of ˜í¬¿TADs with AIMS can address the limitations of singly applying AIMS to the analysis of complex samples. Additionally, ˜í¬¿TADs-AIMS has significant future potential in developing on-site analytical devices by utilising miniature mass spectrometers. Chapter 1 reviews the recent advances and applications of ˜í¬¿TAD in analytical sciences. It gives an overall introduction of the fabrication techniques and detection methods of ˜í¬¿TAD systems, followed by the classification of the applications. The pros and cons of ˜í¬¿TADs and future trends in this area are also discussed. Chapter 2 discusses the advances of AIMS with specific focus on the new generation of ionization techniques (like DESI, direct analysis in real time, DART, extractive electrospray ionization, EESI, paper spray ionization, PSI, etc.), developed to enhance the capabilities of AIMS and to expand its applications. A major focus of this Chapter is on the use and role of nanomaterials in AIMS techniques to further improve their performance and application. In Chapter 3, the research aimed to explore and develop a simple and inexpensive threadbased sample preparation and concentration method and its coupling with DESI-MS for rapid 'on-thread' electrofluidic analysis. However, the detection of water-containing samples is a challenge for DESI-MS. Therefore, herein a platform for thread-based isoelectric focusing (TB-IEF) was 3D-printed, optimised, and applied for the separation and focusing of three model proteins from aqueous media. After the TB-IEF separation and concentration process, the thread with focused analytes was dried through an applied voltage and directly subjected to the DESI-MS source. This combination delivered a novel and low-cost approach for the sample pretreatment and focusing of amphoteric solutes in complex aqueous samples, with direct 'on-thread' ambient MS detection. This setup was applied for the separation and focusing of bovine serum albumin, R-phycoerythrin, myoglobin (MB), and cytochrome c. Successful separation and focusing was achieved within 30 min. A 10-fold increase in the DESI-MS response was achieved following the TB-IEF preconcentration, whilst simultaneously isolating the target solutes from their sample matrix. The TB-IEF-DESI-MS technique is mainly for the analysis of proteins and peptides, therefore, the development of a similar method for small molecules is an area of interest. Hence, in Chapter 4, the focus of the research was to integrate a thread-based isotachophoresis (TBITP) sample treatment approach with DESI-MS for the analysis of small target molecules in complex biological samples. However, to couple the TB-ITP technique with DESI-MS, it was necessary to focus the analytes at a predetermined position, without band diffusion and postfocussing. To achieve this, an on-thread trap was proposed, a simple trapping knot (from a different thread material) tied to the separation thread at a predetermined position. The proposed TB-ITP-DESI-MS setup and methodology was applied for the clean-up, preconcentration, and determination of alkaloids (coptisine, berberine and palmatine) in biological fluids. This system enabled the focusing and rapid analysis of the analytes of interest in complex matrices that were otherwise challenging for direct ambient MS. A single string of Nylon 6 thread was used as the electrophoresis substrate and a cotton knot, tied to the nylon thread, was used as the trapping zone of the ITP focused model analytes. Compared to the direct DESI-MS detection, the signal-to-noise ratio (S/N) for coptisine, berberine and palmatine obtained using the proposed method was increased 11.6-, 5.5- and 5.7-fold, respectively, due to the reduced matrix interference and focusing. Although the TB-ITP was successfully coupled with DESI-MS with the aid of a trapping knot, the applicability of this method was limited by the trapping capability of available thread materials. Accordingly, a sorptive nanomaterial was used to functionalize the trapping knot to improve the efficiency and selectivity. For this purpose, in Chapter 5, a nanomaterial-assisted TB-ITP-DESI-MS setup was developed for the clean-up, focusing, and trapping of target compounds in biological samples. Nylon thread was coated with graphene oxide (GO) by using bovine serum albumin (BSA) as the linker. The GO coated thread was then tied on the electrophoresis substrate (nylon thread) to trap the TB-ITP focused solutes in a predetermined position, followed by exposing the trapped solutes (on the dried knot) to the DESI source. The performance of this setup and strategy was verified by the analysis of model alkaloids in urine samples. Compared with the traditional direct DESI-MS analysis, the sensitivity of this method for coptisine, berberine and palmatine was enhanced 7.8-, 9.2-, 9.0-fold, respectively. Compared to the method developed in Chapter 4, this method delivered higher signal intensities and better selectivity. Most importantly, this research has opened up a new perspective to use a wide range of nanomaterials with different features, to achieve higher selectivity for the TBITP separation and trapping of targeted molecules. Finally, Chapter 6 summarises the results and findings of this project and discusses the future trends in thread-based electrofluidic systems. This project investigated different strategies for sample pretreatment using electrofluidic thread-based systems followed by DESIMS detection for rapid and sensitive detection. The performance and applicability of the proposed systems were demonstrated by analysing biological samples.