Functional hydrogel for fast, precise and inhibition-free point-of-care bacteria analysis in crude food samples

In this work, instead of performing nucleic acid amplification in the bulk solution, we report a nanoporous hydrogel with controlled release function for rapid, precise, and inhibition-free nucleic acid analysis in crude food samples. The cross-linked PEG hydrogel with nanoporous structures possesses adsorption, release, separation, restriction and self-cleaning abilities. When digital loop-mediated isothermal amplification (LAMP) was performed inside this hydrogel, the surrounding nanostructure act as a temporary reservoir for reagents storage and release them on demand during or after amplification. Meanwhile, the restricted nanoconfined environment of hydrogel also favor the enzymatic amplification process. Thus, an enhanced signal readout, robust anti-inhibition, faster amplification rate, more products yields and specific amplification without primer-dimers were obtained. Moreover, direct amplification in untreated complex food sample was successfully performed inside hydrogel without any sample pretreatment, while conventional droplets digital LAMP failed for detection. Absolute quantification of Escherichia coli and Salmonella typhi directly in fresh fruit and vegetables was achieved within 20 min, with high precision and sensitivity down to single cell. This novel lab-on-hydrogel concept has an enormous potential for future molecular diagnostic assays, and can be also applied for other point-of-care assays.