# Solid core multi-lumen capillaries for application in (bio)-analytical chemistry

Upadhyay, N ORCID: 0000-0002-5383-5904 2021 , 'Solid core multi-lumen capillaries for application in (bio)-analytical chemistry', PhD thesis, University of Tasmania.

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## Abstract

The following thesis describes research built upon three major aims, such as investigating the evanescent field based optical detection, surface modification, characterisation and non-invasive means of detection of multi-lumen capillaries (MLC).
Where the common overall direction has been to develop practical and novel (bio) analytical applications of surface modified MLC. Within the first Chapter of this thesis, the general background principles and ideas, and the supporting literature behind much of what is discussed in subsequent Chapters is presented. The use of multi-lumen capillaries (MLCs) has expanded over recent years, with numerous applications being reported across many areas of analytical chemistry. Multi-lumen capillaries possess multiple parallel micro-channels, which provide a higher surface-area-to-volume ratio when compared to their single lumen (traditional fused silica capillary) counterparts. MLCs are most well-known for their application as fused silica capillary-based optical fibres otherwise known as photonic crystal fibres (PCFs). PCFs have multiple parallel multi-µm channels within the capillary, often surrounding a solid silica core. MLCs originated within the fields of fibre lasers, fibre sensors, and optical fibre-based communications. These fibres can thus be readily used as waveguides, and have significant potential for novel applications in the fields of sensing and analytical detection. Their unique structure provides an excellent platform within which light and chemical samples can actively interact for quantitative spectroscopic analysis. Additionally, with the enhanced surface area of these capillaries, their potential for applications in areas of in-capillary extraction, reaction and chromatographic separation is also significant.
In the second Chapter we present a complete literature review of the application of MLCs reported to-date, specifically in the area of the separation science. Multi-lumen capillaries of many dimensions have been fabricated and modified, for use within a wide range of applications within separation science. As mentioned above, this extends from sample preparation and sample extraction, to chromatographic separations, and detection sciences. The most commercially available range of MLCs, the so-called photonic crystal fibres (PCFs), emerged in the 1990's, and were specifically designed for optical applications, predominantly as waveguides. Since then, a range of modifications have been reported, with the fibres applied to a wide variety of analytical applications, in liquid and gas phase analysis or detection. This comprehensive review provides an up-to-date and state-of-the-art analysis of such applications of PCFs and related MLCs, detailing the chemical modifications that have been carried out, and the subsequent applications of the developed materials. This Chapter has been published in its entirety as a co-authored review within Trends in Analytical Chemistry.
In the third Chapter, a novel investigation of evanescent field-based optical detection using multi-lumen capillaries (MLCs) is presented. The micro-structured capillaries provide the potential for long effective optical pathways with the additional advantage of high surface area-to-volume ratios. Their use in in-capillary evanescent field-based detection could offer several significant advantages, including on-line sensing, in-capillary reaction monitoring and integration with various chromatographic applications. The Chapter involves comparison of the effective detection pathlength of empty MLCs with MLCs that have gold nanoparticles (AuNPs) immobilised on the surface of the channel. MLCs with 126 parallel channels of either 4 µm or 8 µm inner diameter were used, connecting a light source and optical detector. Experimental measurements were carried out with different concentrations of a brilliant blue dye solution using a flow-injection analysis (FIA) approach. Calibration curves using the brilliant blue dye solution were constructed to demonstrate the potential of the approach, and were linear up to 5.12 mM, with typical R$$^2$$ values of 0.998. The effective pathlengths of the empty MLCs range between 1.205 mm and 0.075 mm, and with AuNPs on the surface the effective pathlengths increased relatively to 1.893 mm and 0.229 mm for the 4-µm and 8-µm MLCs, respectively. In addition, a peak dispersion study was carried out using a scanning capacitively coupled contactless conductivity detector (sC$$^4$$D) and injections of a 10 mM NaCl solution. The 8-µm channel MLC showed lowest dispersion with the difference in width of the peak being 0.004 mm (23.53%) from the beginning to the end of the 12-cm capillary.
In the fourth Chapter, the modification of the internal channel wall of a multi-lumen capillary (MLC) with a polymer layer was investigated. Lauryl methacrylate ethylene dimethacrylate (LMA-co-EDMA)-based monolithic porous layer open tubular (monoPLOT) MLC columns were prepared, using a UV polymerisation approach. The study aimed to rapidly produce a uniform monolith layer within each channel of the MLC using UV light (a 254 nm lamp or a 255 nm LED). The difference in monolithic growth within the MLCs, with and without the acrylate external capillary coating, was noticeable due to the initiating UV light either travelling through the core of the MLC as an evanescent wave or, in the case of the uncoated MLC, directly through the wall. The uniform wall coverage of the monolith was monitored using a scanning capacitively coupled contactless conductivity detector (sC$$^4$$D) and scanning electron microscopy (SEM), with the greatest monolith growth (up to 1000 nm) occurring in the uncoated MLC where the light was able to enter through the wall. Photo-initiation using the UV LED gave the least homogeneous coverage; some channels of the MLC were blocked and some did not show any polymerisation. The application of monolithic MLCs for the retention of series of test proteins was also briefly explored. Under optimised gradient conditions, effective retention of proteins was demonstrated on the MLC column with the greatest amount of immobilised monolith (the uncoated MLC).
In the fifth Chapter of this thesis, the simultaneous homogenous modification of all channels within a multi-lumen capillary (MLC) was investigated. Capillaries were first functionalised with 3-aminopropyltriethoxysilane (APTES), followed by the attachment of gold nanoparticles (AuNPs). A gold layer thickness of ~15 nm was observed, which corresponds well with the 15 nm AuNP particle size and confirmed a mono-layer structure. These attached AuNPs were then used as seeds for a surface reduction reaction, in which a mixture of a reducing agent (hydroxylamine) and chloroauric acid (HAuCl$$^4$$) was slowly passed through the MLC. During the process, the deposited gold particles agglomerated within the capillary when they reached a certain size (100 nm) eventually forming a complete uniform gold layer within each channel. Scanning capacitively coupled contactless conductivity detection (sC$$^4$$D) measurements were applied to determine uniformity of coverage along the length of the capillaries. Under optimal conditions, an AuNP-modified 126 x 4 μm inner diameter (ID) MLC gave an average C$$^4$$D response RSD of 0.8% along the 15 cm length, indicating a uniform axial gold coverage. FE-SEM images of the capillary cross-sections also indicated uniform radial gold coverage in all 126 channels of each MLC. The obtained gold layer was then functionalised with Erythrina cristagalli lectin (ECL) via a bio-linker to demonstrate one potential in-capillary extraction application of the modified MLC. The ECL-modified MLC was applied to the selective extraction and isolation of galactosylated proteins.
In the final Chapter, a novel microreactor prepared using surface modified PCF capillaries was developed. The capillary investigated contained 126 parallel channels, each of 4 μm internal diameter. The modified PCF, along with conventional fused silica capillaries of 25 μm and 50 μm internal diameter, were treated by (3-aminopropyl)triethoxysilane (APTES) and then modified with gold nanoparticles, of ∼20 nm in diameter, to ultimately provide immobilisation sites for the proteolytic enzyme, trypsin. The modified capillaries and PCFs were characterised and again profiled using non-invasive sC$$^4$$D. The sC$$^4$$D profiles confirmed a significantly higher amount of enzyme was immobilised within the PCF when compared to the fused silica capillaries, attributable to the increased surface to volume ratio. The modified PCF was used for dynamic protein digestion, where peptide fragments were collected and subjected to off-line chromatographic evaluation. The digestion was achieved with the PCF reactor, using a residence time of just 1.26 min, following which the HPLC peak associated with the intact protein decreased by >70%. The PCF reactors behaved similarly to the classical in vitro or in-solution approach but provided a reduction in digestion time, and fewer peaks associated with trypsin auto-digestion, which is common using in-solution digestion. This Chapter has been published in its entirety as a co-authored paper within the Analyst.
The thesis is comprised of seven Chapters, first Chapter gives overview of the thesis, second Chapter is literature review of MLC in analytical chemistry. Chapter three to six involved major line of experimental work, with key aims described in the thesis: (i) optical detection using evanescent field-based system, (ii) synthesis of organic polymer layer and AuNPs to provide solid stationary phase for further attachments of legends (lectin and trypsin) in MLC and, (iii) the characterization of MLC using non-invasive techniques

Item Type: Thesis - PhD Upadhyay, N Multi-lumen capillary, Column Chromatography, Sensing, Analytical chemistry, Optical detection, Gold nano-particles, Monolith. 10.25959/100.00038001 Copyright 2021 the author Chapter 2 appears to be the equivalent of a post-print version of an article published as: Currivan, S., Upadhyay, N., Paull, B., 2018. Multi-channel capillaries and photonic crystalfibres for separation sciences, TrAC - Trends in analytical chemistry, 102, 322–331.Chapter 6 appears to be the equivalent of a post-print version of an article published as: Currivan, S. A., Chen, W. Q., Wilson, R., Sanz Rodriguez, E., Upadhyay, N., Connolly, D., Nesterenko, P. N., Paull, B., 2018. Multi-lumen capillary based trypsin micro-reactor for the rapid digestion of proteins, Analyst, 143(200, 4944-4953. View statistics for this item