Improved Measurement of Proteins Using a Solid-State Nanopore Coupled with a Hydrogel

Abstract

Although resistive pulse sensing using solid-state nanopores is capable of single-molecule sensitivity, previous work has shown that nanoparticles, such as proteins, pass through nanopores too quickly for accurate detection with typical measurement apparatus. As a result, nanopore measurements of these particles significantly deviate from theoretically estimated current amplitudes and detection rates. Here, we show that a hydrogel placed on the distal side of a nanopore can increase the residence time of nanoparticles within the nanopore, significantly increasing the detection rate and allowing improved resolution of blockage currents. The method is simple and inexpensive to implement while being label-free and applicable to a wide range of nanoparticle targets. Using hydrogel-backed nanopores, we detected the protein IgG with event frequencies several orders of magnitude higher than those in the absence of the hydrogel and with larger measured currents that agree well with theoretical models. We also show that the improved measurement also enables discrimination of IgG and bovine serum albumin in a mixed solution. Finally, we show that measurements of IgG with the hydrogel-backed nanopores can also yield current amplitude distributions that can be analyzed to infer its approximate shape.

Quantitative Measurements of Protein Volume and Concentration using Hydrogel-Backed Nanopores

“…We measured IgG in its native state using nanopores 23–31 nm in diameter and 15 nm long, backed with a poly­(ethylene glycol)-dimethacrylate (PEG-DMA) hydrogel of average mesh size ∼3.1 nm, as described previously (Figure S1). To determine the average event frequency ( f ), we divided the number of single-molecule events ( N ) by the duration of observation (Δ t ): f = N /Δ t . , We applied −50 mV to the nanopore and found that the event frequency rapidly increased following the protein addition and plateaued after 5–9 min (Figure S2), indicating the amount of time for protein monomers to homogeneously disperse throughout the measurement chamber.…”

“…As the neutral PEG-DMA hydrogel has an antifouling property and sterically interacts with translocating proteins, the size of the protein relative with respect to the mesh size of the hydrogel is an important factor to determine the efficiency of the hydrogel in facilitating a protein detection. We measured 400 pM poly- l -glutamic acid sodium salt (≤5.5 kDa), which has a hydrodynamic radius of 1.35 nm estimated from Equation S19, smaller than the 3.1 nm average mesh size of the PEG-DMA hydrogel . In the absence of a hydrogel, no events were detected over 45 min at −100 mV of applied voltage.…”

“…Recently, we described an approach to improving nanopore sensitivity by polymerizing a hydrogel on one side of the nanopore . The presence of the hydrogel extended analyte residence time within the nanopore, increasing the detection rate by several orders of magnitude for a given analyte concentration while using a standard amplifier and requiring no significant restriction on nanopore size.…”

Single-Entity Detection With TEM-Fabricated Nanopores

“…Most of the traditional techniques require a large number of identical protein samples, wherein it is challenging to record a singleprotein conformational change in a real-time manner (Ding et al, 2019). The nanopore-based approaches can achieve sensitive and label-free detection of a single protein even at extremely low concentrations as well as provide a dynamic view of protein conformation, confirming their ability for protein characterization (Acharya et al, 2020).…”

“…Fragment sizing, sequencing, and structure identification of biomolecules remain major objectives in the field of nanopore sensing. Likewise, the shape and size of NPs can be identified with these typical detection principles (Acharya et al, 2020;Si et al, 2020). For example, Karmi et al used Si 3 N 4 nanopores to detect gold NPs (AuNP) and differentiate the charged monomers (single particles) and dimers (two particles) based on their different translocation time through the pore (Figure 2J; Karmi et al, 2021).…”