Multiplex sample-to-answer detection of bacteria using a pipette-actuated capillary array comb with integrated DNA extraction, isothermal amplification, and smartphone detection.

A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a "sample-to-answer" manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 °C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of λ-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. "Sample-to-answer" detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex "sample-to-answer" detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.