Poly(ethylene glycol) (PEG) nanofilms are used to avert the nonspecific binding of biomolecules on substrate surfaces in biomedicine and bioanalysis including modern fluorescence-based DNA sensing and sequencing chips. A fundamental and coherent understanding of the interactions between fluorophore-tagged DNA, PEG-films, and substrates in terms of molecular and energetic factors is, however, missing. Here we explore a large parameter space to elucidate how PEG layers passivate metal oxide surfaces against Cy3-labeled DNA probes. The driving force for probe adsorption is found to be the affinity of the fluorophore to the substrate, while the high-quality PEG films prevent adsorption to bare ITO surfaces. The amount of nonrepelled, surface-bound DNA strongly depends on oligonucleotide size, PEG chain length, and incubation temperature. To explain these observations, we develop an experimentally validated theory to provide a microscopic picture of the PEG layer and show that adsorbed DNA molecules reside within the film by end-tethering the fluorophore to the ITO surface. To compensate for the local accumulation of negatively charged DNA, counterions condense on the adsorbed probes within the layer. The model furthermore explains that surface passivation is governed by the interdependence of molecular size, conformation, charge, ion condensation, and environmental conditions. We finally report for the first time on the detailed thermodynamic values that show how adsorption results from a balance between large opposing energetic factors. The insight of our study can be applied to rationally engineer PEG nanolayers for improved functional performance in DNA analysis schemes and may be expanded to other polymeric thin films.

DNA strands can be analyzed at the single-molecule level by isolating them inside nanoscale holes. The strategy is used for the label-free and portable sequencing with nanopores. Nanochannels can also be applied to map genomes with high resolution, as shown by Jeffet et al. in this issue of ACS Nano. Here, we compare the two strategies in terms of biophysical similarities and differences and describe that both are complementary and can improve the DNA analysis for genomic research and diagnostics.

Hyperbranched dendrimers are nanocarriers for drugs, imaging agents, and catalysts. Their nanoscale confinement is of fundamental interest and occurs when dendrimers with bioactive payload block or pass biological nanochannels or when catalysts are entrapped in inorganic nanoporous support scaffolds. The molecular process of confinement and its effect on dendrimer conformations are, however, poorly understood. Here, we use single-molecule nanopore measurements and molecular dynamics simulations to establish an atomically detailed model of pore dendrimer interactions. We discover and explain that electrophoretic migration of polycationic PAMAM dendrimers into confined space is not dictated by the diameter of the branched molecules but by their size and generation-dependent compressibility. Differences in structural flexibility also rationalize the apparent anomaly that the experimental nanopore current read-out depends in nonlinear fashion on dendrimer size. Nanoscale confinement is inferred to reduce the protonation of the polycationic structures. Our model can likely be expanded to other dendrimers and be applied to improve the analysis of biophysical experiments, rationally design functional materials such as nanoporous filtration devices or nanoscale drug carriers that effectively pass biological pores.

A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing.

Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.Keywords: bilayer membrane; DNA nanotechnology; nanofluidics; nanopore; PEG; single-molecule;

Biological channels embedded in cell membranes regulate ionic transport by responding to external stimuli such as pH, voltage, and molecular binding. Mimicking the gating properties of these biological structures would be instrumental in the preparation of smart membranes used in biosensing, drug delivery, and ionic circuit construction. Here we present a new concept for building synthetic nanopores that can simultaneously respond to pH and transmembrane potential changes. DNA oligomers containing protonatable A and C bases are attached at the narrow opening of an asymmetric nanopore. Lowering the pH to 5.5 causes the positively charged DNA molecules to bind to other strands with negative backbones, thereby creating an electrostatic mesh that closes the pore to unprecedentedly high resistances of several tens of gigaohms. At neutral pH values, voltage switching causes the isolated DNA strands to undergo nanomechanical movement, as seen by a reversible current modulation. We provide evidence that the pH-dependent reversible closing mechanism is robust and applicable for nanopores with opening diameters of up to 14 nm. The concept of creating an electrostatic mesh may also be applied to different organic polymers.

DNA nanotechnology excels at rationally designing bottom-up structures that can functionally replicate naturally occurring proteins. Here we describe the design and generation of a stable DNA-based nanopore that structurally mimics the amphiphilic nature of protein pores and inserts into bilayers to support a steady transmembrane flow of ions. The pore carries an outer hydrophobic belt comprised of small chemical alkyl groups which mask the negatively charged oligonucleotide backbone. This modification overcomes the otherwise inherent energetic mismatch to the hydrophobic environment of the membrane. By merging the fields of nanopores and DNA nanotechnology, we expect that the small membrane-spanning DNA pore will help open up the design of entirely new molecular devices for a broad range of applications including sensing, electric circuits, catalysis, and research into nanofluidics and controlled transmembrane transport.

The voltage-driven passage of biological polymers through nanoscale pores is an analytically, technologically, and biologically relevant process. Despite various studies on homopolymer translocation there are still several open questions on the fundamental aspects of pore transport. One of the most important unresolved issues revolves around the passage of biopolymers which vary in charge and volume along their sequence. Here we exploit an experimentally tunable system to disentangle and quantify electrostatic and steric factors. This new, fundamental framework facilitates the understanding of how complex biopolymers are transported through confined space and indicates how their translocation can be slowed down to enable future sensing methods.

DNA is a powerful biomaterial for creating rationally designed and functionally enhanced nanostructures. DNA nanoarchitectures positioned at substrate interfaces can offer unique advantages leading to improved surface properties relevant to biosensing, nanotechnology, materials science, and cell biology. This Perspective highlights the benefits and challenges of using assembled DNA as a nanoscale building block for interfacial layers and surveys their applications in three areas: homogeneous dense surface coatings, bottom-up nanopatterning, and 3D nanoparticle lattices. Possible future research developments are discussed at the end of the Perspective.

We present a generic and flexible method to nanopattern biomolecules on surfaces. Carbon-containing nanofeatures are written at variable diameter and spacing by a focused electron beam on a poly(ethylene glycol) (PEG)-coated glass substrate. Proteins physisorb to the nanofeatures with remarkably high contrast factors of more than 1000 compared to the surrounding PEG surfaces. The biological activity of model proteins can be retained as shown by decorating avidin spots with biotinylated DNA, thereby underscoring the universality of the nano-biofunctionalized platform for the binding of other biotinylated ligands. In addition, biomolecule densities can be tuned over several orders of magnitude within the same array, as demonstrated by painting a microscale image with nanoscale pixels. We expect that these unique advantages open up entirely new ways to design biophysical experiments, for instance, on cells that respond to the nanoscale densities of activating molecules.

The creation of synthetic devices that mimic functionality of biological systems is a task of fundamental importance for the future development of bio- and nanotechnology and also an ultimate test of our understanding of the biological systems. Among a plethora of bio-inspired devices, designed nanopores and nanochannels with an embedded functionality are of particular interest because of their potential applications in nanofluidic electronics, biosensing, separation, synthetic biology, and single-molecule manipulation. In this respect, nanopores with built-in stimulus-responsive properties are of special benefit. A transmembrane potential is a particularly useful stimulus as it is non-invasive, tunable, and can act over a short time scale. This critical review considers engineered solid-state and protein nanopores with voltage-responsive properties. The engineered systems show nonlinear current–voltage curves, and/or voltage-dependent switching between discrete conductance states (141 references).

Proteins have been synergistically combined with electrochemistry to develop biosensors and biofuel cells. The focus of this review is to discuss the role of enzymes and highlight how protein engineering and immobilization can enhance device performance.

The labeling of nucleotides and oligonucleotides with reporter groups is an important tool in the sequence-specific sensing of DNA, as exemplified by fluorescence tags. Here we show that chemical tags can encode sequence information for electrical nanopore recordings. In nanopore recordings, individual DNA strands are electrophoretically driven through a nanoscale pore leading to detectable blockades of ionic current. The tags cause characteristic electrical signatures in the current blockades of translocating DNA strands and thereby encode sequence information. The viability of the strategy is demonstrated by discriminating between drug resistance-conferring point mutations. By being independent of pore engineering, the new approach can potentially enhance the sensing repertoire of durable solid-state nanopores for which alternative sensing strategies developed for protein pores are not easily accessible.

We describe microarrays of receptors on gold/glass substrates for the selective capturing of viral particles at high density. Microscale gold squares were surface-modified with alkanethiol derivatives which enabled the immobilization of the His6-tagged virus-binding domain from the very-low density lipoprotein (VLDL) receptor. The free glass areas surrounding the gold squares were passivated with a dense film of poly(ethylene glycol) (PEG). As assessed by atomic force microscopy, human rhinovirus particles were captured onto the VLDL-receptor patches with a high surface coverage but were effectively repelled by the PEG layer, resulting in a 330 000-fold higher density of the particles on the gold as compared to the glass surfaces. The metal chelate-based coupling strategy was found to be superior to two alternative routes, which used the covalent coupling of viral particles or viral receptors to the substrate surface. The high-density receptor arrays were employed for sensing and characterizing viral particles with so far unprecedented selectivity.

We describe a soft thin film which selectively adsorbs DNA but averts the non-specific binding of proteins. Indium tin oxide (ITO) substrates were surface-modified with a poly(L-lysine)-g-poly(ethylene glycol) (PLL-PEG) film which carries an outer protein-repelling PEG layer and an underlying positively charged PLL layer that attracts DNA. Binding of DNA could be tuned by a factor of over 90 by varying the salt concentration. The dependence of DNA binding on ionic strength was described with a physicochemical model which led to the conclusion of an unexpectedly high enrichment of salt inside the PEG layer. In addition, the model led to an expanded definition of the Debye–Hückel type effective screening length parameter z. Our new findings on a film with dual passivation/attraction properties can find applications in biopolymer-specific coatings useful in bioseparation and biosensing. In addition, the physicochemical characterisation provides new insight into the interactions between biopolymers and polymer-coated interfaces.

We describe the synthesis of 2′-deoxyuridine-5′-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep VentR exo− from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2′-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate.

Many biologically relevant structures are formed by the self-assembly of identical protein units. Examples include virus capsids or cytoskeleton components. Synthetic biology can harness these bottom-up assemblies and expand their scope for applications in cell biology and biomedicine. Nanobiotechnology and materials science also stand to gain from assemblies with unique nanoscale periodicity. In these disciplines, the soft scaffolds can serve as templates to produce new metallic or inorganic materialsof predefined dimensions. This review article describes how the structure and function of biological assemblies has inspired researchers to develop engineered systems with designed properties for new biomolecular applications.

Multimeric protein assemblies are essential components in viruses, bacteria, eukaryotic cells, and organisms where they act as cytoskeletal scaffold, storage containers, or for directional transport. The bottom-up structures can be exploited in nanobiotechnology by harnessing their built-in properties and combining them with new functional modules. This review summarizes the design principles of natural protein assemblies, highlights recent progress in their structural elucidation, and shows how rational engineering can create new biomaterials for applications in vaccine development, biocatalysis, materials science, and synthetic biology.Highlights► Protein assemblies occur in every virus or biological cell. ► They act as cytoskeletal scaffold, storage container, or for directional transport. ► Rational engineering can exploit protein assemblies for nanobiotechnology. ► Structural insight can guide the generation of designed biomaterials. ► Applications of engineered systems range from vaccine development, biocatalysis, materials science, to synthetic biology.

Surface layer (S-layer) proteins self-assemble into two-dimensional crystalline lattices that cover the cell wall of all archaea and many bacteria. We have generated assembly-negative protein variants of high solubility that will facilitate high-resolution structure determination. Assembly-negative versions of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 were obtained using an insertion mutagenesis screen. The haemagglutinin epitope tag was inserted at 23 amino acid positions known to be located on the monomer protein surface from a previous cysteine accessibility screen. Limited proteolysis, circular dichroism, and fluorescence were used to probe whether the epitope insertion affected the secondary and tertiary structures of the monomer, while electron microscopy and size-exclusion chromatography were employed to examine proteins' ability to self-assemble. The screen not only identified assembly-compromised mutants with native fold but also yielded correctly folded, self-assembling mutants suitable for displaying epitopes for biomedical and biophysical applications, as well as cryo-electron microscopy imaging. Our study marks an important step in the analysis of the S-layer structure. In addition, the approach of concerted insertion and cysteine mutagenesis can likely be applied for other supramolecular assemblies.

Bacterial surface layer (S-layer) proteins self-assemble into large two-dimensional crystalline lattices that form the outermost cell-wall component of all archaea and many eubacteria. Despite being a large class of self-assembling proteins, little is known about their molecular architecture. We investigated the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 to identify residues located at the subunit–subunit interface and to determine the S-layer's topology. Twenty-three single cysteine mutants, which were previously mapped to the surface of the SbsB monomer, were subjected to a cross-linking screen using the photoactivatable, sulfhydryl-reactive reagent N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide. Gel electrophoretic analysis on the formation of cross-linked dimers indicated that 8 out of the 23 residues were located at the interface. In combination with surface accessibility data for the assembled protein, 10 residues were assigned to positions at the inner, cell-wall-facing lattice surface, while 5 residues were mapped to the outer, ambient-exposed lattice surface. In addition, the cross-linking screen identified six positions of intramolecular cross-linking within the assembled protein but not in the monomeric S-layer protein. Most likely, these intramolecular cross-links result from conformational changes upon self-assembly. The results are an important step toward the further structural elucidation of the S-layer protein via, for example, X-ray crystallography and cryo-electron microscopy. Our approach of identifying the surface location of residues is relevant to other planar supramolecular protein assemblies.

We describe the synthesis of 2′-deoxyuridine-5′-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep VentR exo− from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2′-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate.

The creation of synthetic devices that mimic functionality of biological systems is a task of fundamental importance for the future development of bio- and nanotechnology and also an ultimate test of our understanding of the biological systems. Among a plethora of bio-inspired devices, designed nanopores and nanochannels with an embedded functionality are of particular interest because of their potential applications in nanofluidic electronics, biosensing, separation, synthetic biology, and single-molecule manipulation. In this respect, nanopores with built-in stimulus-responsive properties are of special benefit. A transmembrane potential is a particularly useful stimulus as it is non-invasive, tunable, and can act over a short time scale. This critical review considers engineered solid-state and protein nanopores with voltage-responsive properties. The engineered systems show nonlinear current–voltage curves, and/or voltage-dependent switching between discrete conductance states (141 references).