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1.
Int J Mol Sci ; 23(10)2022 May 20.
Article in English | MEDLINE | ID: mdl-35628527

ABSTRACT

Nano- and micrometer-sized compartments composed of synthetic polymers are designed to mimic spatial and temporal divisions found in nature. Self-assembly of polymers into compartments such as polymersomes, giant unilamellar vesicles (GUVs), layer-by-layer (LbL) capsules, capsosomes, or polyion complex vesicles (PICsomes) allows for the separation of defined environments from the exterior. These compartments can be further engineered through the incorporation of (bio)molecules within the lumen or into the membrane, while the membrane can be decorated with functional moieties to produce catalytic compartments with defined structures and functions. Nanometer-sized compartments are used for imaging, theranostic, and therapeutic applications as a more mechanically stable alternative to liposomes, and through the encapsulation of catalytic molecules, i.e., enzymes, catalytic compartments can localize and act in vivo. On the micrometer scale, such biohybrid systems are used to encapsulate model proteins and form multicompartmentalized structures through the combination of multiple compartments, reaching closer to the creation of artificial organelles and cells. Significant progress in therapeutic applications and modeling strategies has been achieved through both the creation of polymers with tailored properties and functionalizations and novel techniques for their assembly.


Subject(s)
Polymers , Unilamellar Liposomes , Catalysis , Ions , Polymers/chemistry , Proteins
2.
Nanoscale ; 11(42): 19636-19657, 2019 Nov 14.
Article in English | MEDLINE | ID: mdl-31603455

ABSTRACT

Since their introduction in 2001, solid-state nanopores have been increasingly exploited for the detection and characterization of biomolecules ranging from single DNA strands to protein complexes. A major factor that enables the application of nanopores to the analysis and characterization of a broad range of macromolecules is the preparation of coatings on the pore wall to either prevent non-specific adhesion of molecules or to facilitate specific interactions of molecules of interest within the pore. Surface coatings can therefore be useful to minimize clogging of nanopores or to increase the residence time of target analytes in the pore. This review article describes various coatings and their utility for changing pore diameters, increasing the stability of nanopores, reducing non-specific interactions, manipulating surface charges, enabling interactions with specific target molecules, and reducing the noise of current recordings through nanopores. We compare the coating methods with respect to the ease of preparing the coating, the stability of the coating and the requirement for specialized equipment to prepare the coating.


Subject(s)
Coated Materials, Biocompatible/chemistry , DNA/analysis , Nanopores , Proteins/analysis , Animals , Humans , Portraits as Topic
3.
Nanotechnology ; 30(32): 325504, 2019 Aug 09.
Article in English | MEDLINE | ID: mdl-30991368

ABSTRACT

In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bilayer coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls by exposing a zwitterionic, fluid surface. Second, they can slow down protein translocation and rotation by the opportunity to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility to impart analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Fourth, they offer a method for tuning nanopore diameters by choice of the length of the lipid's acyl chains. The work presented here compares four properties of various lipid compositions with regard to their suitability as nanopore coatings for protein sensing experiments: (1) electrical noise during current recordings through solid-state nanopores before and after lipid coating, (2) long-term stability of the recorded current baseline and, by inference, of the coating, (3) viscosity of the coating as quantified by the lateral diffusion coefficient of lipids in the coating, and (4) the success rate of generating a suitable coating for quantitative nanopore-based resistive pulse recordings. We surveyed lipid coatings prepared from bolaamphiphilic, monolayer-forming lipids inspired by extremophile archaea and compared them to typical bilayer-forming phosphatidylcholine lipids containing various fractions of curvature-inducing lipids or cholesterol. We found that coatings from archaea-inspired lipids provide several advantages compared to conventional phospholipids; the stable, low noise baseline qualities and high viscosity make these membranes especially suitable for analysis that estimates physical protein parameters such as the net charge of proteins as they enable translocation events with sufficiently long duration to time-resolve dwell time distributions completely. The work presented here reveals that the ease or difficulty of coating a nanopore with lipid membranes did not depend significantly on the composition of the lipid mixture, but rather on the geometry and surface chemistry of the nanopore in the solid state substrate. In particular, annealing substrates containing the nanopore increased the success rate of generating stable lipid coatings.


Subject(s)
Archaea/metabolism , Lipid Bilayers/chemistry , Nanopores , Unilamellar Liposomes/chemistry , Diffusion , Phosphatidylcholines/chemistry , Phospholipids/chemistry , Surface Properties
4.
ACS Nano ; 13(5): 5231-5242, 2019 05 28.
Article in English | MEDLINE | ID: mdl-30995394

ABSTRACT

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.


Subject(s)
Nanopores , Proteins/chemistry , Diffusion , Electric Conductivity , Lipid Bilayers/chemistry , Rotation
5.
ACS Nano ; 12(11): 11458-11470, 2018 11 27.
Article in English | MEDLINE | ID: mdl-30335956

ABSTRACT

Nanopores with diameters from 20 to 50 nm in silicon nitride (SiN x) windows are useful for single-molecule studies of globular macromolecules. While controlled breakdown (CBD) is gaining popularity as a method for fabricating nanopores with reproducible size control and broad accessibility, attempts to fabricate large nanopores with diameters exceeding ∼20 nm via breakdown often result in undesirable formation of multiple nanopores in SiN x membranes. To reduce the probability of producing multiple pores, we combined two strategies: laser-assisted breakdown and controlled pore enlargement by limiting the applied voltage. Based on laser power-dependent increases in nanopore conductance upon illumination and on the absence of an effect of ionic strength on the ratio between the nanopore conductance before and after laser illumination, we suggest that the increased rate of controlled breakdown results from laser-induced heating. Moreover, we demonstrate that conductance values before and after coating the nanopores with a fluid lipid bilayer can indicate fabrication of a single nanopore versus multiple nanopores. Complementary flux measurements of Ca2+ through the nanopore typically confirmed assessments of single or multiple nanopores that we obtained using the coating method. Finally, we show that thermal annealing of CBD pores significantly increased the success rate of coating and reduced the current noise before and after lipid coating. We characterize the geometry of these nanopores by analyzing individual resistive pulses produced by translocations of spherical proteins and demonstrate the usefulness of these nanopores for estimating the approximate molecular shape of IgG proteins.

6.
Nat Nanotechnol ; 12(4): 360-367, 2017 05.
Article in English | MEDLINE | ID: mdl-27992411

ABSTRACT

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.


Subject(s)
Lipid Bilayers/chemistry , Models, Chemical , Multiprotein Complexes/chemistry , Nanopores
7.
Biophys J ; 110(11): 2430-2440, 2016 06 07.
Article in English | MEDLINE | ID: mdl-27276261

ABSTRACT

This work explores the proton/hydroxide permeability (PH+/OH-) of membranes that were made of synthetic extremophile-inspired phospholipids with systematically varied structural elements. A fluorescence-based permeability assay was optimized to determine the effects on the PH+/OH- through liposome membranes with variations in the following lipid attributes: transmembrane tethering, tether length, and the presence of isoprenoid methyl groups on one or both lipid tails. All permeability assays were performed in the presence of a low concentration of valinomycin (10 nM) to prevent buildup of a membrane potential without artificially increasing the measured PH+/OH-. Surprisingly, the presence of a transmembrane tether did not impact PH+/OH- at room temperature. Among tethered lipid monolayers, PH+/OH- increased with increasing tether length if the number of carbons in the untethered acyl tail was constant. Untethered lipids with two isoprenoid methyl tails led to lower PH+/OH- values than lipids with only one or no isoprenoid tails. Molecular dynamics simulations revealed a strong positive correlation between the probability of observing water molecules in the hydrophobic core of these lipid membranes and their proton permeability. We propose that water penetration as revealed by molecular dynamics may provide a general strategy for predicting proton permeability through various lipid membranes without the need for experimentation.


Subject(s)
Hydroxides/chemistry , Liposomes/chemistry , Membrane Lipids/chemistry , Protons , Unilamellar Liposomes/chemistry , Archaea/chemistry , Biomimetic Materials/chemistry , Fluorescent Dyes , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Ionophores/chemistry , Membrane Potentials , Methacrylates , Microscopy, Atomic Force , Molecular Dynamics Simulation , Permeability , Valinomycin/chemistry , Water/chemistry
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