Building a Versatile Microfluidic Optical Biosensor: Demonstrated with Antimicrobial Sensitivity Testing

Antimicrobial resistance is a growing global concern. Current antibiotic susceptibility testing methods are slow, often taking over 24 hours, due to the necessity of measuring bacterial growth. This delay leads to a reliance on broad-spectrum antibiotics, exacerbating the AMR threat. More rapidly determining if a culture is growing in the presence of antibiotics would reduce clinical turnaround times for treatment and the reliance on broad spectrum antimicrobials. Many methods have been presented but limited research addresses the feasibility of incorporating the measurement scheme into a clinical workflow. This work presents a novel, compact benchtop device that rapidly determines bacterial colony density using an ATP bioluminescence measurement, evaluating colony growth in 4 hours. The samples are prepared identically to samples for a broth microdilution assay, volumes of less than 10 µL are required for the measurement. This test can be run in parallel without additional samples. This is demonstrated on Escherichia Coli, Pseudomonas Aeruginosa, Staphylococcus Aureus, and Streptococcus Pyogenes as well as clinical urine and joint fluid samples. The measurement is performed within 3D printed cassettes held directly on top of a CMOS image sensor. A limit of detection for ATP was shown to be 0.16 nM for 30 µL of solution. A key advantage of the device is that luminous output can be expressed specifically using a mathematical model and volumes as small as 150nL have been directly compared to volumes of 60µL. The model also enables accurate compensation of brightness loss due to fluid attenuation. With the device suited for microfluidics a novel microfluidic fabrication technique, penultimate layer lithography, is presented which can incorporate microscale features onto 3D printed parts at scale. A two-minute measurement has an LOD below 1000 CFUs the system could feasibly be used for rapid point of care susceptibility testing with minimal additional labour costs.