Polymer Coatings to Minimize Protein Adsorption in Solid‐State Nanopores

Abstract

can be classified as biological, [8] solidstate, [9] or a combination of the two. [10] Biological nanopores include channel proteins such as the product of Curli specific gene G (CsgG) from Escherichia coli, [11] α-hemolysin (αHL) from Staphylococcus aureus, [12] Mycobacterium smegmatis porin A (MspA) [13] and Aeromonas hydrophila Aerolysin (AeL), [14] which spontaneously embed themselves in a lipid bilayer. These types of nanopores can be produced in large numbers by protein expression and purification, have well-characterized crystal structures, and have played a critical role in advancing the resistive pulse sensing technique. [15-17] It is, however, difficult to generate protein pores with diameters that exceed 4 nm. Another limitation is the need to reconstitute these protein pores into lipid or block copolymer membranes, which can be mechanically and chemically fragile. [18] Conversely, solid-state nanopores can be fabricated in various materials such as silicon nitride, silicon oxide, aluminum oxide, hafnium oxide, or graphene. [9,19] Their diameters and geometries can be tuned depending on user requirements, and they can be used repeatedly for experiments with considerable experimental flexibility (e.g., temperature, buffer conditions, presence of detergents or solvents, extreme pH values, applied potential differences, etc.). [20-22] Despite these attractive characteristics, solid-state nanopores suffer from at least three drawbacks. First, they usually interact nonspecifically with biomolecules, leading to resistive pulse artifacts such as attenuated rotation, translation and Nanopore-based resistive-pulse recordings represent a promising approach for single-molecule biophysics with applications ranging from rapid DNA and RNA sequencing to "fingerprinting" proteins. Based on advances in fabrication methods, solid-state nanopores are increasingly providing an alternative to proteinaceous nanopores from living organisms; their widespread adoption is, however, slowed by nonspecific interactions between biomolecules and pore walls, which can cause artifacts and pore clogging. Although efforts to minimize these interactions by tailoring surface chemistry using various physisorbed or chemisorbed coatings have made progress, a straightforward, robust, and effective coating method is needed to improve the robustness of nanopore recordings. Here, covalently attached nanopore surface coatings are prepared from three different polymers using a straightforward "dip and rinse" approach and compared to each other regarding their ability to minimize nonspecific interactions with proteins. It is demonstrated that polymer coatings approach the performance of fluid lipid coatings with respect to minimizing these interactions. Moreover, these polymer coatings enable accurate estimates of the volumes and spheroidal shapes of freely translocating proteins; uncoated or inadequately coated solid-state pores do not have this capability. In addition, these polymer coatings impart physical and chemical stability and enable effic...

The NEOtrap – en route with a new single-molecule technique

“…iScience --, 103007, --, 2021 the passivating lipid bilayer coating or by changing the solution pH (Firnkes et al, 2010;Fujinami Tanimoto et al, 2021), but the latter may have undesirable effects on the analyte. Alternative passivation protocols, such as tethered lipid bilayers (Andersson and Knoll, 2019) or grafted polymers (Awasthi et al, 2020;Giamblanco et al, 2018b;Roman et al, 2017), may improve the coating stability and tolerate higher voltages without adding noise.…”

“…This can be achieved by adding lipids with negatively or positively charged head groups to the passivating lipid bilayer coating or by changing the solution pH ( Firnkes et al., 2010 ; Fujinami Tanimoto et al., 2021 ), but the latter may have undesirable effects on the analyte. Alternative passivation protocols, such as tethered lipid bilayers ( Andersson and Knoll, 2019 ) or grafted polymers ( Awasthi et al., 2020 ; Giamblanco et al., 2018b ; Roman et al., 2017 ), may improve the coating stability and tolerate higher voltages without adding noise.

Figure 2 The NEOtrap and envisioned technical extensions (A) New opportunities arise through chemical functionalization of the pore passivation layer and the origami sphere to lock it in place.

…”

Single-Entity Detection With TEM-Fabricated Nanopores

“…For instance, SiN x or glass made nanopores can be modified using silane chemistry, whereas metallic (e.g., Au) nanopores can be modified by applying thiol chemistries via solution or vapor-based depositions (Yin et al, 2017). Likewise, several monolayer agents have been used to functionalize the surface (e.g., chemical groups, roughness, and surface charge) of solid-state nanopores including cetyltrimethylammonium bromide, Tween 20, polyethylene glycol (PEG), organosilanes via non-covalent and covalent bonding interactions (Giamblanco et al, 2018;Eggenberger et al, 2019;Awasthi et al, 2020). For example, Meller group used 3-(aminopropyl)trimethoxysilane (APTMS) to modify the surface of SiN x nanopores (Figures 1C,D; Anderson et al, 2013).…”

Selective target protein detection using a decorated nanopore into a microfluidic device

“…The second strategy involves coating the nanopores with a fluid lipid bilayer (Venkatesan et al, 2011;Yusko et al, 2017Yusko et al, , 2011. Another one is to control the surface chemistry of the solid-state nanopore by membrane surface functionalization, which allows to increase their lifetime, to control the pore size and to passivate the membrane (Arima et al, 2018b(Arima et al, , 2018aAwasthi et al, 2020;Giamblanco et al, 2018;Lepoitevin et al, 2016;Li et al, 2019;Malekian et al, 2018;Roman et al, 2018Roman et al, , 2017Wanunu and Meller, 2007). Nevertheless, specific sensing remains a challenge to design new diagnostic tools (Li et al, 2019).…”