Nanoporous hydrogel for direct digital nucleic acid amplification in untreated complex matrices for single bacteria counting

Direct quantification of pathogens in unprocessed complex samples remain challenging due to the severe inhibition of nucleic acid amplification. In this work, we report a nanoporous polyethylene glycol hydrogel with self-cleaning capacity for direct amplification of nucleic acid in complex matrices (human whole blood, animal blood, milky tea, humic acid, and surfactants) without any sample pretreatment or DNA extraction. During isothermal amplification inside the hydrogel, the inhibitors in the assay will be adsorbed and removed by the surrounding nanostructured polymers, and nucleic acid amplification was proceeding successfully, resulting in a series of bright dots for single bacteria counting. Thus, the loop-mediated isothermal amplifications (LAMP) performed inside hydrogel demonstrated a high level of resistance to inhibition in various complex matrices. The underlying anti-inhibition mechanism was also investigated. Digital quantification of Escherichia coli, Salmonella typhi and Listeria monocytogenes in whole blood were achieved within 20 min, with wide dynamic range, high specificity and low detection limit down to single bacterium. Visual counting via naked eye was also successfully established with the help of a conventional LED flashlight. We believe the developed hydrogel nanofluidic system has an enormous potential for on-site direct analysis of complex, crude, and unprocessed samples in clinical, food, agricultural, and environmental fields.