Electrical Sensing of Phosphonates by Functional Coupling of Phosphonate Binding Protein PhnD to Solid-State Nanopores

Abstract

Combining the stability of solid-state nanopores with the unique sensing properties of biological components in a miniaturized electrical hybrid nanopore device is a challenging approach to advance the sensitivity and selectivity of small-molecule detection in healthcare and environment analytics. Here, we demonstrate a simple method to design an electrical hybrid nanosensor comprising a bacterial binding protein tethered to a solid-state nanopore allowing high-affinity detection of phosphonates. The diverse family of bacterial substrate-binding proteins (SBPs) binds specifically and efficiently to various substances and has been implicated as an ideal biorecognition element for analyte detection in the design of hybrid bionanosensors. Here, we demonstrate that the coupling of the purified phosphonate binding protein PhnD via primary amines to the reactive NHS groups of P(DMAA-co-NMAS) polymers inside a single track-etched nanopore in poly(ethylene terephthalate) (PET) foils results in ligand-specific and concentration-dependent changes in the nanopore current. Application of the phosphonate 2-aminoethylphosphonate (2AEP) or ethylphosphonate (EP) induces a large conformational rearrangement in PnhD around the hinge in a venus flytrap mechanism resulting in a concentration depended on increase of the single pore current with binding affinities of 27 and 373 nM, respectively. Thus, the specificity and stability of this simple hybrid sensor concept combine the advantages of both, the diversity of ligand-specific substrate-binding proteins and solid-state nanopores encouraging further options to produce robust devices amenable to medical or environmental high-throughput-based applications in nanotechnology.

Ultrasensitive and Selective Protein Recognition with Nanobody‐Functionalized Synthetic Nanopores

“…Second, taking advantage of the defined 3D structure of proteins, the assembly and immobilization of receptors can be achieved in a directed and controlled fashion. In contrast, conventional immobilization methods based on non‐covalent electrostatic interactions [ 10 ] or reactions between surface accessible lysine residues with activated N ‐hydroxysuccinimide esters [ 11 ] only provide little control how a fusion protein is immobilized. In particular, immobilization may turn out heterogenous with detrimental effects on the performance of a biosensor especially when the binding site of receptor is occluded.…”

“…[ 3,4 ] Key considerations that determine the responsiveness of receptor‐modified nanopores toward a desired analyte concerns their material, size, geometry, and surface properties. [ 3 ] In this regard, several label‐free sensors have been developed exploiting small molecules [ 5–7 ] or DNA aptamers [ 8,9 ] as receptors to detect a range of different analytes including metal ions, [ 5,10 ] low molecular weight ligands, [ 6,8,11 ] nucleic acids, [ 12 ] or proteins. [ 7,9,13,14 ] In contrast, the use of receptor proteins has been limited to a few model interactions, in particular, conventional antibody‐antigen combinations and streptavidin‐biotin.…”

Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

“…SDS-PAGE and western blot analysis was performed as described elsewhere 24 . In brief, proteins were separated using a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).…”

Nanosensor Chemical Cytometry: Advances and Opportunities in Cellular Therapy and Precision Medicine

“…These elements can be integrated onto materials of nano-micro scale, including 2D materials such as graphene and MoS 2 , 1D materials including carbon nanotubes (CNTs) and Si nanowire, and 0D materials including Au nanoparticles (AuNPs). − Optical signal detection through transducers is characterized by a unique interaction between the active site of the transducer and light signals, which arises from distinct physical, chemical, or structural properties, leading to high sensitivity . Optical transducers utilize a range of spectroscopic techniques, including absorption, fluorescence, and Raman scattering. − This approach has been utilized to monitor the differentiation state of stem cells including human neural stem cells (hNSCs) and MSCs by analyzing the presence and quantity of specific molecules in various environments. − Electrical sensors are using conductance or resistance variation between electrodes by adsorption of biochemical molecules on conducting channel materials. − Electrical transducers can detect and quantify a wide range of cellular environmental changes, from pH shifts to the presence of large molecules such as antibodies and proteins. This allows for distinguishing cancer cells, observing stem cell differentiation, and monitoring immune cell activation .…”