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Bioelectrochemical Sensing of Sexually Transmitted Diseases

Periodic Reporting for period 1 - BSSTD (Bioelectrochemical Sensing of Sexually Transmitted Diseases)

Reporting period: 2016-09-15 to 2018-09-14

"The detection of sexually transmitted diseases (STDs) has become a serious medical concern around the globe, as the number of reported cases increases every year, particularly in the United Kingdom.1 Infection with STDs, such as Gonorrhea and Chlamydia can result in long term health issues, such as infertility, reproductive tract cancer, and poor perinatal outcome. The successful treatment of these diseases depends on a quick and reliable method of diagnosis. The funded project aimed for the development of a detection method for bacteria, associated with STDs, based on bioelectrochemical techniques.
The project’s methodology is based on the identification of a redox couple, functioning as electron donor for bacterial oxidases, the specific binding of bacteria to a macroelectrode and the electrochemical recognition of immobilized bacteria at the macroelectrode. The proposed technique makes use of a biochemical test for aerobic bacteria, the oxidase test, by adapting it for electrochemical detection. In doing so, high selectivity is achieved by targeted binding of living bacteria to immobilized antibodies on an electrode, whereas sensitivity is strongly increased through the localized electrochemical detection of the oxidase test product.
The proposed detection principle has the potential to be transferred to all oxidase-positive bacteria. The detection method can thus find widespread use for a large number of pathogens in health, veterinary care, food, and pharma industries.
This project is structured into 6 main objectives: 1) Identification of a redox couple functioning as electron donor for bacterial oxidases, 2) Specific binding of bacteria to a gold macroelectrode, 3) Proof of concept: Combining objectives #1 and #2, 4) Controls: Testing for false-positive and false-negative results, 5) Varying target cultures, 6) Transfer of the chemical principle onto disposable screen printed electrodes towards the assembly of a biosensing device.
In conclusion, all objectives and work packages outlined in the proposal were achieved, whereby 7 manuscripts were published in scientific high quality peer-reviewed journals, including Chemical Science. Another manuscript is currently in preparation for submission. The established electrochemical concept for the detection of the pathogens E. coli and N gonorrhoea lead to the filing of a patent application and steps are currently underway to bringing the established chemical principle to the commercial market.

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TMPD was characterized electrochemically by cyclic voltammetric measurements using gold electrodes. A rapid oxidation of TMPD by oxygen was observed. The researcher gained knowledge about simulating electrochemical processes with the simulation software DigiSim®. To understand the reaction mechanism and kinetics of TMPD at an electrode, theoretical curves were fitted to experimental data using the DigiSim® simulation software for cyclic voltammetry. Kinetic parameters were deduced from the modelling of cyclic voltammetry in TMPD. An electrocatalytic reaction of ascorbic acid (AA) and TMPD was discovered and explored. To overcome the difficulties associated with the rapid oxidation of TMPD by oxygen, ascorbic acid (Vitamin C) was added to TMPD as reducing agent. An electrocatalytic reaction of AA and TMPD was discovered. Slow electrode kinetics of AA result in irreversible voltammetric behavior and electrode fouling over subsequent voltammetry cycles can be observed. Due to these intrinsic properties, ascorbic acid is difficult to quantify electrochemically directly. However, a suitable redox mediator, such as TMPD, is able to overcome such issues. AA was successfully detected in commercial orange juice. This work was published in Electrochimica Acta 2017, 242, 19.
The compound N,N,N',N'-tetramethyl-para-phenylenediamine (TMPD) is rapidly oxidized by atmospheric oxygen, therefore the radical cation salt TMPD-BF4 was synthesized, to assure solution stability and higher accuracy during bioelectrochemical measurements. As a model organisms, E.coli as well as B. subtilis bacteria were immobilized onto a gold macroelectrode by dropcasting. Cyclic voltammetry and Chronoamperommetry were performed in the presence and the absence of bacteria, showing an increase in reductive current in the presence of bacteria (Figure 1). The expression of cytochrome c oxidase in both organisms was quantified and expressed as a turnover number. For the first time cytochrome c oxidase activity was measured in aerobic E. coli. This work was published in Chemical Science 2017, 8, 7682 and a review was written on recent strategies, advances and challenges in the field of electrochemical detection of pathogenic bacteria in Chemistry – An Asian Journal 2018 (in press.).
To make this detection principle selective for specific pathogens, self-assembled-monolayer modifications were carried out at macro- and screen printed electrodes (SPEs). Three types of SPEs were evaluated for their suitability to the proposed method. E. coli bacteria as well as N. gonorrhoea were successfully detected at SPEs (Figure 2) at medically relevant concentrations, whereby this work is currently in preparation for submission.
- Foundation of the ECS Oxford Student Chapter
As anticipated, in September 2016, an ECS Student chapter was founded at Oxford University and is already attracting attention in the UK and abroad. The chapter is holding monthly meetings, consists currently of about 20 members and is continuously growing. An initial poster session was organized in March 2017, allowing undergraduate and graduate students to present their research to the scientific community. Two student symposia have been taken place in 2017 and 2018, and a third event is currently being organised for 2019.
- Presentation and Judging at Madley Brook Primary School Science Week
An outreach activity was conducted at Madley Brook Community Primary School in Witney, Oxfordshire in the context of the school’s yearly science week. Students were introduced to the principle and impact of biosensing and a simple experiment in the field of materials science was performed. The demonstration involved copper coins that were coated with zinc, which transforms into brass upon heating, making the coin appear golden. Furthermore, we had the opportunity to evaluate the student’s science projects, ask questions and give advice on future ideas. The event was a great success and students enjoyed the demonstration and interaction with real scientists.
- Career development Activities
Oxford University offers a wide range of courses beyond state of the art science and research to facilitate employee’s integration into the job market after their appointment at the University. The course “Academic Application and Interview Skills for Research Staff & DPhils” was completed on November 7th 2016, providing useful information about job applications.
- Supervision
Four graduate students were supervised by the researcher Dr Sabine Kuss, which lead to additional publications.
- Conference attendance
As anticipated, results were presented at the Gordon Research Conference – Electrochemistry in Ventura, California, USA in January 2018.
- Future Employment
The researcher Sabine Kuss secured an academic position as Assistant Professor (tenure-track) in Canada following her appointment in Oxford.
Electrochemical recognition of cytochrome c oxidase at macroelectrodes
Electrochemical detection of various concentrations of E. coli at SPEs