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

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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.