A handheld platform for target protein detection and quantification using disposable nanopore strips.

Accessible point-of-care technologies that can provide immunoassay and molecular modalities could dramatically enhance diagnostics, particularly for infectious disease control in low-resource settings. Solid-state nanopores are simple and durable sensors with low-energy instrumentation requirements. While nanopore sensors have demonstrated efficacy for nucleic acid targets, selective detection and quantification of target proteins from sample background has not been demonstrated. We present a simple approach for electronic detection and quantification of target proteins that combines novel biomolecular engineering methods, a portable reader device and disposable nanopore test strips. The target of interest can be varied by swapping the binding domain on our engineered detection reagent, which eficiently binds in the bulk-phase to the target and subsequently generates a unique signature when passing through the pore. We show modularity of the detection reagent for two HIV antibodies, TNFα and tetanus toxin as targets. A saliva swab-to-result is demonstrated for clinically relevant HIV antibody levels (0.4-20 mg/liter) in under 60 seconds. While other strip-like assays are qualitative, the presented method is quantitative and sets the stage for simultaneous immunoassay and molecular diagnostic functionality within a single portable platform.

Amyloid ß peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aß-mediated toxicity in vitro.

The gradual accumulation and assembly of ß-amyloid (Aß) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aß is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aß-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)↓(Y/F)↓(S/A/G/T) or RXΦ(Y/F)↓(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aß (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aß42 in vitro, thereby inhibiting Aß fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aß oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aß42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aß-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aß-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.