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.

由于核酸扩增的严重抑制，未处理的复杂样品中病原体的直接定量仍然具有挑战性。在这项工作中，我们报告了一种具有自清洁能力的纳米多孔聚乙二醇水凝胶，可直接在复杂基质（人全血，动物血，乳茶，腐殖酸和表面活性剂）中直接扩增核酸，而无需任何样品预处理或DNA提取。在水凝胶内部进行等温扩增期间，测定中的抑制剂将被周围的纳米结构聚合物吸附和去除，核酸扩增成功进行，从而产生了一系列用于单个细菌计数的亮点。因此，在水凝胶内部进行的环介导的等温扩增（LAMP）显示了在各种复杂基质中对抑制的高水平抵抗力。还研究了潜在的抗抑制机制。数字量化在20分钟内可实现全血中的大肠杆菌，伤寒沙门氏菌和单核细胞增生李斯特菌，其动态范围广，特异性高，检出限低至单个细菌。借助常规的LED手电筒，还可以成功建立肉眼可见的计数。我们相信，开发的水凝胶纳米流体系统具有巨大的潜力，可以在临床，食品，农业和环境领域对复杂，粗制和未处理的样品进行现场直接分析。