Accurate, robust, and rapid diagnostics is the basis for all well-functioning healthcare. There is a large need in point-of-care biosensors to facilitate diagnosis and reduce the need for cumbersome laboratory equipment. Proteases are key virulence factors in periodontitis. Periodontal disease is very common and characterized by inflammation and infection in the tooth-supporting structures and is linked to many systemic diseases such as cardiovascular disease, diabetes, and Alzheimer’s disease. Proteases present in periodontal disease, gingipains, are highly responsible for the disease onset and progression and are therefore a promising biomarker. Here we show a novel nanopore-based biosensor strategy for protease activity monitoring. Solid-state nanopores were modified with a proteolytic substrate, restricting the ionic current through the apertures of the nanopores. Protease can digest the proteolytic substrate thus enlarge the aperture and the ionic current. Trypsin was used as an initial model protease to investigate the performance of the sensor. We show that the solid-state nanopore-biosensor can detect trypsin with a limit of detection (LOD) of 0.005 ng/mL (0.2 pM). The detection system developed for the model enzyme was then applied to the detection of gingipains. The LOD for detection of gingipains was 1 ng/mL (0.02 nM), with a 27% recovery of the signal at 0.1 µg/mL, indicating that the sensitivity and dynamic range are relevant for the clinical diagnosis of periodontitis. The generic detection of protease activity and high sensitivity make this a promising sensor technology for both diagnosis of periodontal disease and monitoring of other disease-related proteases.

准确、稳健和快速的诊断是所有运作良好的医疗保健的基础。对即时生物传感器的需求很大，以促进诊断并减少对笨重的实验室设备的需求。蛋白酶是牙周炎的关键毒力因子。牙周病非常常见，其特征是牙齿支撑结构中的炎症和感染，并且与许多全身性疾病有关，例如心血管疾病、糖尿病和阿尔茨海默病。牙周病中存在的蛋白酶，牙龈蛋白酶，对疾病的发作和进展高度负责，因此是一种有前途的生物标志物。在这里，我们展示了一种用于蛋白酶活性监测的新型基于纳米孔的生物传感器策略。用蛋白水解底物修饰固态纳米孔，限制离子电流通过纳米孔的孔径。蛋白酶可以消化蛋白水解底物，从而扩大孔径和离子电流。胰蛋白酶用作初始模型蛋白酶来研究传感器的性能。我们表明，固态纳米孔生物传感器可以检测胰蛋白酶，检测限 (LOD) 为 0.005 ng/mL (0.2 pM)。然后将针对模型酶开发的检测系统应用于牙龈蛋白酶的检测。牙龈蛋白酶检测的 LOD 为 1 ng/mL (0.02 nM)，在 0.1 µg/mL 信号恢复率为 27%，表明灵敏度和动态范围与牙周炎的临床诊断相关。