Machine learning classification of bacterial species using mix-and-match reagents on paper microfluidic chips and smartphone-based capillary flow analysis

Traditionally, specific bioreceptors such as antibodies have rapidly identified bacterial species in environmental water samples. However, this method has the disadvantages of requiring an additional process to conjugate or immobilize bioreceptors on the assay platform, which becomes unstable at room temperature. Here, we demonstrate a novel mix-and-match method to identify bacteria species by loading the bacterial samples with simple bacteria interacting components (not bioreceptors), such as lipopolysaccharides, peptidoglycan, and bovine serum albumin, and carboxylated particles, all separately on multiple channels. Neither covalent conjugation nor surface immobilization was necessary. Interactions between bacteria and the above bacteria interacting components resulted in varied surface tension and viscosity, leading to various flow velocities of capillary action through the paper fibers. The smartphone camera and a custom Python code recorded multiple channel flow velocity, each loaded with different bacteria interacting components. A multi-dimensional data set was obtained for a given bacterial species and concentration and used as a machine learning training model. A support vector machine was applied to classify the six bacterial species: Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecium, and Bacillus subtilis. Under optimized conditions, the training model predicts the bacterial species with an accuracy of > 85% of the six bacteria species.