Team:TU Eindhoven/Device
Device
Device
We focused on decreasing antimicrobial resistance by reducing the misuse of antibiotics, which can be achieved by improving the diagnostics of bacterial infections. After talking with several stakeholders and experts, it was clear to us that the detection method had to be fast, specific and modular. Fast because we want to decrease the current time needed for diagnosis, specific since false positives and negatives should be evaded and modular to be able to diagnose a variety of bacterial infections. These three criteria are met by using the specificity and amplification speed of bacteriophages in combination with the specificity, sensitivity and modularity of one of our two dCas9-NanoLuc-sensor systems. A next step is to make the translation from a detection method to a point-of-care diagnostic device that can be used in a clinical setting. For this purpose, a device has been designed and presented to General Practitioners to validate whether they would use such a device in a clinical setting.
When using our device, the user first needs to make a selection of the different types of bacteria that need to be detected. For example, a urinary tract infection is caused by either E. coli, Klebsiella, Proteus or the Pseudomonas aeruginosa bacteria, so when analyzing a urine sample, one would likely screen for these four bacteria. All bacteria are coupled to a unique phage-type, so after selecting the to be screened bacteria, the to be used phages are automatically selected too. Each phage-bacteria couple also has its own sgRNA needed for detection. In essence, the user chooses the bacteria to screen for in the sample and can then add the corresponding phage and sgRNA to the device. After loading the sample, the user will know within an hour whether any of these bacteria are present, after which specific treatment can be started.
Under the hood
The steps of the detection process inside the device will be discussed below. The user has chosen which bacteria to screen for and the corresponding phages and sgRNAs are loaded to the device (Figure 1). Next, the sample is loaded onto the device and filtered initially. Both phage mixtures and sample are pumped towards the microfluidic chip inside the device. Here, continuous flows of sample and phage are mixed. The sample-phage mixture then flows through incubation loops for thirty minutes to allow the phages to possibly infect bacteria, amplify and lyse the bacteria to release their progeny. Subsequently, the mixture is heated 70 °C to denature the (newly formed) phages and release their DNA after which the mixture cooled again. Simultaneously, dCas9-NanoLuc is incubated with the corresponding sgRNA for ten minutes at 37 °C and is then added to the sample-phage mixture (for dCas9-split-NanoLuc, the dCas9-Sbit and dCas9-Lbit will be incubated separately). This whole mixture is incubated for thirty minutes after which the NanoLuc substrate furimazine is added and mixed. Ultimately, the read-out takes place with a light sensor. An elevated phage DNA concentration in the mixture results in an increased light intensity in the case of dCas9-split-NanoLuc, or an increased ratio in red Cy3-generated light opposed to blue NanoLuc light because of an increase in BRET. In this way, it can be determined which type of bacterium was present in the sample. Finally, the mixture leaves the microfluidic chip towards a waste container and the channels are washed so a next sample can be tested.
Figure 1: Design of the point-of-care device that can be used to detect bacteria in a fast, modular and specific way: Left the sample is loaded and the types of phages,
based on the bacteria one wants to screen. These are pumped towards the microfluidic chip where the whole process takes place so on the right a result is generated.
