Phung_whole_thesis_ex_pub_mat.pdf (3.26 MB)
Isotachophoresis of bacterial cells
thesisposted on 2023-05-27, 11:06 authored by Phung, SC
Currently, the standard method for bacterial enumeration is still the plate count method. The issues with this method includes it is time consuming, laborious and it is only able to detect culturable bacteria. While there have been alternative methods reported for bacteria detection, most of the alternative methods either suffer from poor sensitivity or selectivity. This thesis documents research on developing an alternative method for microbial detection using isotachophoresis (ITP) and to transfer this method to a 3D printed microchip with integrated electrodes for the possibility of fabrication of these integrated devices. In the first part, a counter-pressure was applied to extend the duration of the field amplified sample injection (FASI) in ITP to improve the detection of bacterial cells. By using a universal nucleic acid dye, the DNA and RNA in Escherichia coli (E. coli) was stained, and focused into the narrow ITP boundary. The use of the counter-pressure allows more cells to be injected and focused into the stationary ITP boundary. The counter-pressure was then removed to allow the ITP band to move past the detector. To obtain the optimum conditions, negatively charged encapsulated fluorescent beads with a diameter of 0.51 ˜í¬¿m were used as a model. The injection voltages, applied counter-pressure and the injection time was optimised and then transferred to E. coli. Using ‚Äö- 12 kV with a 1.3 psi counter pressure for 4 mins, followed by mobilisation and continued injection at ‚Äö- 6 kV, the limit of detection (LOD) was 78 cells/mL, resulting in an improvement of 4 when compared with FASI-ITP without counter-pressure. The second part of the thesis focussed on an ITP method for rapid hybridisation of the 16S rRNA inside the bacterial cells with a fluorescence in situ hybridisation (FISH) probe. This approach uses a two-stage separation within a single ITP analysis. With the introduction of spacer ions and sieving matrix, the in-line FISH between the probe and E. coli occurs within the first 10% of the total capillary length during ITP. When the ITP band reaches the sieving matrix in second section of the capillary, the spacer ions overtake the stained E. coli forming two discrete ITP bands. The first ITP band is the free FISH probe focussed between the leader and spacer ions and the second ITP band is the stained E. coli focussed between the spacer ions and terminator ions. This method was then studied for selectivity with the use of PseaerA probe on Pseudomonas aeruginosa (P. aeruginosa) and E. coli cells. The analysis results successfully showed that the PseaerA probe does not bind with E. coli using the ITP method. The next part was to study the efficiency of the hybridisation using the ITP method and compared with the off-line FISH labelled cells. Fluorescent microscopy counting and ITP results showed that the in-line FISH ITP method stains 50% of the total cells injected with a 2 mins counter-pressure injection in comparison to the off-line FISH ITP method. When 6.0 x 10\\(^4\\) cells were injected (3 times higher than the LOD reported by Lantz et al. in 2008) for in-line FISH ITP, approximately 3.0 x 10\\(^4\\) cells were stained. This result was confirmed by analysis of off-line staining of 3.0 x 10\\(^4\\) cells/mL. The final body of the thesis is the transfer of the ITP method into microchip for portability. This chapter is divided into two sections. In the first section, a cross PDMS chip with 50 ˜í¬¿m width and 42 ˜í¬¿m depth was used for in-line ITP of intact bacterial cells. 7% PVP was selected as sieving matrix instead of 1.8% HEC due to the high viscosity of the polymer to be mechanically injected into the channel of the PDMS chip. With the optimisation of DMSO concentration to allow the probe to enter the cells for hybridisation during ITP in the PDMS chip, the total analysis time was reduced from 30 min (capillary) to 4 min (PDMS chip). Second part of this chapter was to study the use of a 3D printer to print a multimaterial chip with integrated electrodes for ITP of bacterial cells. A FDM printer (Felix 3.0 dual extruder head) was used to print a microfluidic chip using transparent acrylonitrile butadiene styrene (ABS) base material for the straight channel. The second extruder was used to deposit the conducting PLA electrodes embedded into the devices. The electrical resistance of the 3D integrated electrodes was examined and compared with in-house Pt electrode using the same chip geometry. While joule heating was observed when 600 V was applied onto the 3D printed chip, the 3D printed chip were able to carry a stable current up to 380 ˜í¬¿A that is suitable for ITP. An ITP band of E. coli stained off-line with SYTO 9 was observed in the 3D printed chip. The rapid quantification of the device was examined and the detection limits of the cells using the PMT devices was 4.0 x 10\\(^4\\) cell/mL which is higher than previously reported in capillaries most likely due to the LED fluorescence light used and the small sample volume used in the chip reservoir. While optimisation of the 3D printed chip is required, the 3D printer allows for the print of microchip with integrated electrodes using commercially available thermoplastic materials that can be used for mass productions of the chip when compared to PDMS chip. Moreover, the total time for printing a chip is 2 hours with the cost per chip is AUD $0.50.
Rights statementCopyright 2017 the author Chapter 2 appears to be the equivalent of a post-peer-review, pre-copyedit version of an article published in Analytical and bioanalytical chemistry. The final authenticated version is available online at: http://dx.doi.org/10.1007/s00216-015-8838-4 Chapter 3 appears to be the equivalent of a post-print version of an article published as: Phung, S. C., Cabot, J. M., Macka, M., Powell, S. M., Guijt, R. M., Breadmore, M., 2017. Isotachophoretic fluorescence in situ hybridization of intact bacterial cells, Analytical chemistry, 89(12), 6513-6520