Fluorogenic biosensor immobilisation within surface modified fluoropolymer microdevices for rapid smartphone antibiotic susceptibility testing

Antimicrobial resistance is widely acknowledged to be a global health grand challenge. Limited diagnostic technology contributes to this problem because antibiotics are usually prescribed before the patient sample has been tested to see if it is sensitive or resistant to an antibiotic. This is because very old technology is currently used detect and measure microbes and bacteria, which is effective but very slow and uses large equipment only found in centralised labs.

One important example is urinary tract infection (UTI). Although we are familiar with "water infections" which seem common but minor problems, in fact they are one of the most expensive infections nationally, because in some patients the infection gets worse, either because early infection is not treated with antibiotics, or the infection is resistant to the chosen antibiotic. Ideally, the patient urine sample will be tested to see which antibiotic will effectively treat the infection. But unfortunately it takes several days to receive the test result, so GPs often don't bother testing and either select a ''most likely" antibiotic based on current guidelines, or they don't treat at all.

Miniaturised lab systems- termed "lab-on-a-chip" or microfluidic technology have recently been shown to be ideally suited to smaller, more portable, and potentially faster microbial testing. Whilst these initial proof-of-concept studies (one of which was recently published by PI Edwards' research group) show the potential for miniaturisation to overcome the challenge of rapid microbiology testing, significant technical barriers must now be overcome to make sure this exciting technology fulfils its potential.

Our own EPSRC first grant funded proof-of-concept study used a novel technology invented by applicant Dr Edwards' group (Reading) with collaborator Dr Reis (Bath) that allows very low cost microfluidic devices to be mass-produced from a novel material called "microcapillary film". These are very transparent allowing sensitive biological tests to be performed, and the device geometry is ideal for reading test results using a mobile phone camera, flatbed scanner, or digital camera. This low cost manufacturing method coupled to simple digital recording, opens the door to revolutionary digital microbiology tools that can transport antibiotic resistance testing out of the clinical lab and near to the patient. Our first study showed that it is possible to perform antibiotic resistant tests using these novel low-cost devices.

The focus of this project goes beyond proof-of-principle and refines and studies in great detail several important components of microfluidic devices for clinical microbiology. One major focus is on developing effective chemistry that allows us to add brightly fluorescent dyes that detect bacteria inside microcapillaries. Our fluoropolymer devices benefit from unusual material properties of these "Teflon" plastics, but unfortunately the 'non-stick' nature of this material makes it harder to chemically modify the devices. We will in this project use specific chemical modification methods to react with this non-stick surface and make it more useful for bacterial detection. In parallel, we will study the underlying science of speed and sensitivity of bacterial detection using several different dyes that change colour in the presence of bacteria, and thus work out the best way to very rapidly detect bacteria, even when they are very dilute in patient urine samples. The faster we can detect bacteria, the quicker we can tell if they are killed by antibiotics, and therefore the sooner that the patient can be given effective treatment.

Ultimately, this synthetic chemistry research combined with the engineering science of miniaturised bacterial testing devices, will give us the tools and technology needed to speed up diagnostic clinical microbiology, and ensure patients with bacterial infections are treated faster and more effectively.

Patient and Public Benefit
By developing rapid miniaturised analytical microbiology testing we plan to ultimately transport antibiotic susceptibility testing out of central labs to the patient, offering two major benefits.
Firstly, individual patients will benefit from diagnostic tests that inform accurate antibiotic prescribing. Currently, antibiotics are empirically selected based on the least common resistance profile; but if the wrong antibiotic is given, an infection with a resistant strain will worsen. Because microbiology tests are slow, a frequent alternative to empirical antibiotic selection is simply no treatment- wait and see. Again, delayed treatment can allow infection to worsen, often with serious consequences.
Secondly, the entire public benefit from tackling antibiotic resistance, a major threat to our health system. Proper antibiotic selection by improved diagnostic testing is vital to reduce the selective pressures that drive antibiotic resistance development and spread. Current slow testing puts a high burden on clinical judgement or guesswork, and we rely heavily on extremely precious 'last-line' antibiotics with a low prevalence of resistance, or combination therapy. Inevitably this leads to a march of resistance; and without innovation in diagnostics we will run out of effective antibiotics.
Whilst the technology and science developed has multiple potential applications, we identified three specific clinical applications for development:
1 Faster clinical microbiology lab tests
Our initial target is new rapid tools for the clinical microbiology lab- a MCF device could increase speed and throughput of conventional clinical microbiology with minimal changes. Whilst the clinical benefits may be less "disruptive", the barrier to adoption of new technology into labs is significantly lower than for point-of-care testing, with the potential for faster evaluation and adoption. Public health systems and clinical labs will benefit from reduced costs, and patients will benefit from faster time-to-results.
2 Novel research and microbiology testing tools
Technology developed has the potential to accelerate antibiotic discovery with high-throughput screening. This research application allows fastest route to market with no regulatory barrier and expert end-users. Similar devices are likewise ideal for pharmaceutical and food safety microbiological quality control.
3 Point-of-Care microbiology
Our ultimate goal is decentralised diagnostic microbiology devices that rapidly report antibiotic susceptibility directly from a clinical sample, to inform antibiotic selection at point-of-care. This truly disruptive innovation has the longest path to product, but the dramatic benefits justify this long path. Technical challenges include the complexity and heterogeneity of unprocessed clinical samples, a requirement for full clinical trials to evaluate test accuracy, and high investment costs. The disruptive impact on clinical patient pathways will likewise present a barrier to adoption; however, the clinical 'pull' is extremely strong.
Economic Impact and Building Capability
Whilst public health benefits will follow after a long path of technology adoption, as described in the EPSRC's innovation toolkit, this path brings many economic benefits to the UK. To reach market, significant investment in R&D is required, which will follow from activities described in pathway to impact. This will contribute to economic growth in the UK's vital health tech and innovation sector. The project PDRAs will be trained in multidisciplinary healthcare technology research and also involved closely in impact and research translation activities.
Public Understanding of Science
A further benefit of this project is the research objectives and themes are readily accessible to the public, with potential for engagement activities promoted through the concept of "Using smartphones and Teflon to combat the march of antibiotic resistance.