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

Abstract

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.

Fast and accurate quantification of insertion-site specific transgene levels from raw seed samples using solid-state nanopore technology

“…The most common laboratory methods for this are gel electrophoresis or capillary electrophoresis, with quantification by using a fluorescent intercalating dye or UV absorbance. Another method is to not separate the PCR products at all, and simply quantify their relative amounts by recording the change in electrical signal when individual DNA molecules translocate through a solid-state nanopore sensor [9].…”

“…When a single charged molecule such as a double-stranded DNA is captured and driven through the pore by electrophoresis, the measured current shifts, and the shift depth and duration properties are used to characterize each single-molecule “event.” After recording 100–1000 events in a few minutes, the event distributions are analyzed to characterize the corresponding molecules present [12]. Nanopore sensing thus offers a simple and high-throughput electrical read-out, with an instrument that can have a small footprint at low cost [9]. Prior research has shown that nanopores can discriminate DNA by length, since longer DNA produce longer duration events [11,13].…”

Flossing DNA in a Dual Nanopore Device

“…When the voltage captures a single DNA and drives it through the pore, the passing DNA produces a transient blockade in the current that contains information about the molecule's chemical, conformational, and topological state. Nanopore sensing offers a simple and high‐throughput electrical read‐out with an instrument that can have a small footprint at low cost …”

Stable fabrication of a large nanopore by controlled dielectric breakdown in a high-pH solution for the detection of various-sized molecules

“…For instance, single-molecule DNA sequencing is currently possible 1–14 by using a biological nanopore with a diameter of 2 nm or less. In addition, the detection of proteins, antigen-antibody complexes and probe-labelled DNA is possible 15–21 by using a solid-state nanopore with a diameter of several to tens of nanometres. Moreover, the detection of various viruses is also possible 22–27 by using a solid-state nanopore with a diameter of several tens to hundreds nanometers.…”