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Affordable ligand-based electrochemical detection of bacterial toxins

Principal Supervisor: Dr Paramaconi Rodriguez - School of Chemistry

Co-supervisors: Francisco Fernandez-Trillo - School of Chemistry and School of Pharmacy & Dr Matthew I. Gibson Medical School

PhD project title: Affordable ligand-based electrochemical detection of bacterial toxins

University of Registration: University of Birmingham

Project outline:

In this cross-disciplinary project we will work towards the development of affordable electrochemical sensors to monitor pathogenic concentrations of bacterial toxin in aqueous solutions. Electrochemical sensors are specially suited for this application, as they provide high sensitivity at low cost. Portable electrochemical sensors are commercially available, and offer the possibility of point of care monitoring of analytes. For instance, the current technology to monitor glucose in diabetic patients is based on electrochemical detection.

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Unfortunately, the potential of current electrochemical sensors for bacterial toxins to reach the market has been compromised due to poor experimental design and material selection. Moreover, most reported sensors rely on antibodies to increase affinity towards toxins, a strategy that while highly specific, increases costs significantly. Manufacturing of antibodies is non-trivial and, because of their low stability, cold-chain has to be employed during storage and transport. There is therefore the need to develop a new sensor technology that allows an easy, fast, portable and cheap way to detect bacterial toxins.

This project will target the current bacteria sensor limitations by employing a combination of glycomics, material science, surface chemistry and microbiology. To reach this goal we will combine the expertise of the PI and Co-PIs to optimise the electrode material composition and ligand display.

Research Objectives:

O1. Screening of electrode materials: We will prepare microelectrodes using a laser pulling technique. This method creates reproducible metal disk electrodes inside a glass capillary with diameters from 50 nm to 1 μm. Such microelectrodes provide an accurate quantitative method that can detect trace amounts (10e-6 ng/mL) of toxins. We will screen different materials (i.e.: gold, silver, copper) to optimise electrode sensitivity and selectivity.

O2. Surface Ligand Display: We will explore a range of bioconjugation strategies (i.e.: hydrazine, maleimide) to decorate the surface of the electrode with glycans. N-Acetylneuraminic acid, N-acetylgalactosamine, glucose, mannose or galactose are involved in the binding of bacterial toxins to hosts, and will be investigated.

O3. High-throughput screening: A combinatorial library of electrodes with different material and ligand composition will be evaluated. Bacterial toxins bind with different affinities to glycans, so that electrochemical sensors with high specificity towards the toxin can be developed. Initial experiment will be performed towards the detection of cholera toxin in complex aqueous solution. Once the methodology is optimised, a similar strategy will be employed to develop sensors for other relevant toxins (i.e.: shiga-like toxins or botulinum neurotoxin).

O4. Quantification and validation: We will perform a calibration of optimised sensors against relevant concentration of toxins and we will validate the performance of these sensors against standard methods (i.e.: plating or genomics). These experiments will set up the methodology that will allow us to determine whether pathogenic concentrations of toxin can be produced regardless of the serotype of the bacterial strain.

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

Due to the multidisciplinary approach, the PhD student will develop a wide range of experimental skill described in the following training objectives:

Training 1-Microbiology:

  • Microbial culture: Post-graduate will receive training in standard techniques for microbial culture and identification. This expertise will be key for the completion of Objective 4.
  • Protein expression: Post-graduate will receive training in standard techniques for protein expression. This expertise will be key for the expression of non-commercial toxins needed to complete Objective 3.

Training 2-Statistics and Data analysis:

The skills developed as part of the Foundation Skills Modules (i.e.: Large Dataset Handling, Quantitative Biology) will be supported by training in the following software packages:

  • GraphPad Prism: This software combines scientific graphing, curve fitting, statistics, and data organisation, and is specially designed of experimental biologists. This software will be key for the evaluation and optimisation of electrode performance, Objectives 3-4.
  • Matlab: This software provides an environment for numerical computation, visualisation and programming. Training in this package will allow post-graduate students to perform evaluation of more complex datasets generated as part of Objectives 3-4.

Training 3-Analytical: Postgraduate will receive training in the preparation of nanoelectrodes. It will be also trained in electroanalysis methods as cyclic voltammetry, chronoamperometry and capacitive curves. This expertise will be key for the completion of Objectives 1-3.

Training 4-Synthesis: Postgraduate will receive basic training in material and synthetic chemistry, bioconjugation and polymer science. This expertise will be key for the completion of Objectives 1-2.

Contact: Dr Paramaconi Rodriguez, University of Birmingham