Towards a point-of-care optical waveguide biosensor for enabling judicious use of antibiotics

Dixon, Hazel Jade ORCID: 0000-0002-5045-1788 (2023). Towards a point-of-care optical waveguide biosensor for enabling judicious use of antibiotics. University of Birmingham. Ph.D.

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Abstract

Leaky waveguide (LW) films prepared from cheap and readily available hydrogel materials have the potential to produce affordable, sensitive, and label-free biosensors for use in point-of-care (POC) settings. Specifically, this project focuses on the development of a LW biosensor which, when combined with a 3D printed portable instrument, presents the opportunity to carry out testing for biomarkers at POC.

Overuse of antibiotics is a major health concern and is leading to increased antibiotic resistance and the emergence of new antibiotic-resistant bacteria. The motivation of this project is to work towards a POC biosensor which can distinguish between bacterial and viral infections, thus allowing for more informed treatments and a reduction in overprescription of antibiotics. Consequently, the reduction in antibiotic use should serve to slow down antibiotic resistance. The POC aspect is of vital importance to this as portability and a simplistic design will allow for the device to be used in various settings rather than in dedicated laboratories, leading to quicker results and earlier patient treatment.

A crucial element of POC sensing is the requirement for an internal referencing system. To meet this requirement, two different techniques were studied. These comprised of two-channel systems and stacked systems. The two-channel system allows for direct removal of common mode effects and non-specific adsorption (NSA) via a referencing channel running parallel to the sensing channel, while the stacked approach simplifies the system and reduces the opportunity for channel-to-channel variance by vertically stacking the reference and sensor layers and interrogating via a single channel flow cell.

This thesis is broken down into eight chapters covering the following content:
Chapter 1 – Introduction. This chapter offers a review of current antibiotic usage and an overview of some of the different biosensors currently available.
Chapter 2 – Instrumentation and Techniques. This chapter covers the instrumentation and techniques used throughout the project. Optical biosensing was selected as the focus for this work, allowing for use of LWs to detect analytes via the changes in refractive index (RI) caused when an analyte binds to the LW. The simplistic sensing technique combined with the high sensitivity of LWs offers excellent potential for quick and real-time results.
Chapter 3 – Single Layer Waveguides. To achieve the desired sensitivity and reactivity of the LWs multiple different polymers were investigated. This included assessing the sensitivity, longevity, and cost of the polymers, along with storage requirements and batch-to-batch reproducibility. These polymers included both natural and synthetic polymers. Overall, the material which fulfilled most of the requirements was found to be chitosan (CS), a natural polymer derived from crustacean shells.
Chapter 4 – Stacked Waveguides. This chapter discusses the potential for LWs produced from an inert reference LW and a functionalised sensor LW in a stacked conformation as an internally referenced biosensor. While the inert (reference) LW should only react to environmental changes and NSA, the functionalised (sensor) LW should react to these and the analyte of interest. A differential response can then be taken between the two layers, providing a final response caused only by the analyte. The chapter firstly covers different inert hydrogel films as potential reference layers and tests these in the same way as the single layer LWs. The most suitable reference LW was found to be a synthetic hydrogel produced from poly(ethylene glycol) (PEG)-aldehyde and PEG-hydrazide polymer units. Using these reference layers and the sensor layers developed in Chapter 3, the films are then combined into the stacked conformation and characterised.
Chapter 5 – Protein Detection. After development of the LWs in Chapters 3 and 4, the most promising LWs were tested for their protein detection abilities. As part of this, NSA was investigated and blocking strategies applied to reduce this. Multiple different protein immobilisation strategies were developed and tested on different LWs to assess the reproducibility and sensitivity of the techniques. Finally, protein detection was attempted in different solutions: buffers, synthetic urine, and human serum. Using 1% CS LWs we were able to detect down to 5 nM lactoferrin (LF) in buffer samples which offers an improvement over a surface plasmon resonance (SPR)-based LF sensor which was able to detect down to 10-8 M [1]. We were also able to detect 25 nM anti-biotin antibody (ABA) in 10% (v:v) human serum, which has been detected in this quantity in the literature in buffer but not in human serum [2].
Chapter 6 – Portability. In this chapter a portable waveguiding device, produced and provided by a collaborator, was used to interrogate different LWs and protein immobilisation strategies. The results of these are compared directly with the equivalent results produced on the laboratory-based instrumentation, allowing for assessment of the portable device and comparison of the advantages and limitations of both the laboratory-based and portable devices.
Chapter 7 – Conclusion and Future Outlook. This chapter provides a brief summary of the entire work, compiling information into tables for simplicity and ease of comparison. Future work is also discussed.
Chapter 8 – Experimental. This details the experimental methods used throughout the work.

Overall, this work has developed various LW films and protein detection strategies and proven their ability to detect proteins on both laboratory-based and portable instruments. It has also assessed the advantages and limitations of stacked LWs, providing a basis for future development into a reliable POC biosensor for enabling judicious use of antibiotics.

References
[1] Gupta, R. and N.J. Goddard, A study of diffraction-based chitosan leaky waveguide (LW) biosensors. Analyst, 2021. 146(15): p. 4964-4971.
[2] Tomassetti, M., et al., Lactoferrin determination using flow or batch immunosensor surface plasmon resonance: Comparison with amperometric and screen-printed immunosensor methods. Sensors and Actuators B Chemical, 2013. 179: p. 215-225.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):