Sterically Enhanced Control of Enzyme-Assisted DNA Assembly.

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100 bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA that spatially restrict enzymatic activity. This approach, sterically controlled nuclease enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, when using the same starting materials, conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation.

Scale-Up of Room-Temperature Constructive Quantum Interference from Single Molecules to Self-Assembled Molecular-Electronic Films.

The realization of self-assembled molecular-electronic films, whose room-temperature transport properties are controlled by quantum interference (QI), is an essential step in the scale-up of QI effects from single molecules to parallel arrays of molecules. Recently, the effect of destructive QI (DQI) on the electrical conductance of self-assembled monolayers (SAMs) has been investigated. Here, through a combined experimental and theoretical investigation, we demonstrate chemical control of different forms of constructive QI (CQI) in cross-plane transport through SAMs and assess its influence on cross-plane thermoelectricity in SAMs. It is known that the electrical conductance of single molecules can be controlled in a deterministic manner, by chemically varying their connectivity to external electrodes. Here, by employing synthetic methodologies to vary the connectivity of terminal anchor groups around aromatic anthracene cores, and by forming SAMs of the resulting molecules, we clearly demonstrate that this signature of CQI can be translated into SAM-on-gold molecular films. We show that the conductance of vertical molecular junctions formed from anthracene-based molecules with two different connectivities differ by a factor of approximately 16, in agreement with theoretical predictions for their conductance ratio based on CQI effects within the core. We also demonstrate that for molecules with thioether anchor groups, the Seebeck coefficient of such films is connectivity dependent and with an appropriate choice of connectivity can be boosted by ∼50%. This demonstration of QI and its influence on thermoelectricity in SAMs represents a critical step toward functional ultra-thin-film devices for future thermoelectric and molecular-scale electronics applications.

Rapid Fragmentation during Seeded Lysozyme Aggregation Revealed at the Single Molecule Level.

Protein aggregation is associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The poorly understood pathogenic mechanism of amyloid diseases makes early stage diagnostics or therapeutic intervention a challenge. Seeded polymerization that reduces the duration of the lag phase and accelerates fibril growth is a widespread model to study amyloid formation. Seeding effects are hypothesized to be important in the "infectivity" of amyloids and are linked to the development of systemic amyloidosis in vivo. The exact mechanism of seeding is unclear yet critical to illuminating the propagation of amyloids. Here we report on the lateral and axial fragmentation of seed fibrils in the presence of lysozyme monomers at short time scales, followed by the generation of oligomers and growth of fibrils.

Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge.

There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.

A Redox-Activated G-Quadruplex DNA Binder Based on a Platinum(IV)-Salphen Complex.

There has been increasing interest in the development of small molecules that can selectively bind to G-quadruplex DNA structures. The latter have been associated with a number of key biological processes and therefore are proposed to be potential targets for drug development. Herein, we report the first example of a reduction-activated G-quadruplex DNA binder. We show that a new octahedral platinum(IV)-salphen complex does not interact with DNA in aqueous media at pH 7.4; however, upon addition of bioreductants such as ascorbic acid or glutathione, the compound is readily reduced to the corresponding square planar platinum(II) complex. In contrast to the parent platinum(IV) complex, the in situ generated platinum(II) complex has good affinity for G-quadruplex DNA.

Single-Molecule Conductance Studies of Organometallic Complexes Bearing 3-Thienyl Contacting Groups.

The compounds and complexes 1,4-C6 H4 (C≡C-cyclo-3-C4 H3 S)2 (2), trans-[Pt(C≡C-cyclo-3-C4 H3 S)2 (PEt3 )2 ] (3), trans-[Ru(C≡C-cyclo-3-C4 H3 S)2 (dppe)2 ] (4; dppe=1,2-bis(diphenylphosphino)ethane) and trans-[Ru(C≡C-cyclo-3-C4 H3 S)2 {P(OEt)3 }4 ] (5) featuring the 3-thienyl moiety as a surface contacting group for gold electrodes have been prepared, crystallographically characterised in the case of 3-5 and studied in metal|molecule|metal junctions by using both scanning tunnelling microscope break-junction (STM-BJ) and STM-I(s) methods (measuring the tunnelling current (I) as a function of distance (s)). The compounds exhibit similar conductance profiles, with a low conductance feature being more readily identified by STM-I(s) methods, and a higher feature by the STM-BJ method. The lower conductance feature was further characterised by analysis using an unsupervised, automated multi-parameter vector classification (MPVC) of the conductance traces. The combination of similarly structured HOMOs and non-resonant tunnelling mechanism accounts for the remarkably similar conductance values across the chemically distinct members of the family 2-5.

Deep learning for single-molecule science.

Exploring and making predictions based on single-molecule data can be challenging, not only due to the sheer size of the datasets, but also because a priori knowledge about the signal characteristics is typically limited and poor signal-to-noise ratio. For example, hypothesis-driven data exploration, informed by an expectation of the signal characteristics, can lead to interpretation bias or loss of information. Equally, even when the different data categories are known, e.g., the four bases in DNA sequencing, it is often difficult to know how to make best use of the available information content. The latest developments in machine learning (ML), so-called deep learning (DL) offer interesting, new avenues to address such challenges. In some applications, such as speech and image recognition, DL has been able to outperform conventional ML strategies and even human performance. However, to date DL has not been applied much in single-molecule science, presumably in part because relatively little is known about the 'internal workings' of such DL tools within single-molecule science as a field. In this Tutorial, we make an attempt to illustrate in a step-by-step guide how one of those, a convolutional neural network (CNN), may be used for base calling in DNA sequencing applications. We compare it with a SVM as a more conventional ML method, and discuss some of the strengths and weaknesses of the approach. In particular, a 'deep' neural network has many features of a 'black box', which has important implications on how we look at and interpret data.

The role of ion-water interactions in determining the Soret coefficient of LiCl aqueous solutions.

The application of a thermal gradient to an aqueous electrolyte solution induces the Soret effect, and the salt migrates towards hot (thermophilic) or cold regions (thermophobic). Experimental studies of LiCl reported changes in the sign of the Soret coefficient as well as a minimum in this coefficient at specific salt concentrations and temperatures. At the minimum the thermodiffusive response of the solution is enhanced significantly. We have performed non-equilibrium molecular dynamics simulations of LiCl solutions to quantify the dependence of the sign change and minimum of the Soret coefficient with salt concentration and temperature. We find that the ion mass plays a secondary role in determining the magnitude of the Soret coefficient, while the diameter of the cation has a significant impact on the coefficient and on the observation of the minimum. Our simulations show that the ordering of water around Li+ plays a key role in determining the Soret coefficient of LiCl salts.

Ferrocene- and Biferrocene-Containing Macrocycles towards Single-Molecule Electronics.

Cyclic multiredox centered systems are currently of great interest, with new compounds being reported and developments made in understanding their behavior. Efficient, elegant, and high-yielding (for macrocyclic species) synthetic routes to two novel alkynyl-conjugated multiple ferrocene- and biferrocene-containing cyclic compounds are presented. The electronic interactions between the individual ferrocene units have been investigated through electrochemistry, spectroelectrochemistry, density functional theory (DFT), and crystallography to understand the effect of cyclization on the electronic properties and structure.

Trianguleniums as Optical Probes for G-Quadruplexes: A Photophysical, Electrochemical, and Computational Study.

Nucleic acids can adopt non-duplex topologies, such as G-quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA-M2 as a unique fluorescence probe for such studies. This probe's emission lifetime is highly dependent on the topology of the DNA it interacts with opening up the possibility of carrying out live-cell imaging studies. Herein, we describe the origin of its fluorescence selectivity for G-quadruplexes. Cyclic voltammetry predicts that the appended morpholino groups can act as intra- molecular photo-induced electron transfer (PET) quenchers. Photophysical studies show that a delicate balance between this effect and inter-molecular PET with nucleobases is key to the overall fluorescence enhancement observed upon nucleic acid binding. We utilised computational modelling to demonstrate a conformational dependence of intra-molecular PET. Finally, we performed orthogonal studies with a triangulenium compound, in which the morpholino groups were removed, and demonstrated that this change inverts triangulenium fluorescence selectivity from G-quadruplex to duplex DNA, thus highlighting the importance of fine tuning the molecular structure not only for target affinity, but also for fluorescence response.

High-bandwidth detection of short DNA in nanopipettes.

Glass or quartz nanopipettes have found increasing use as tools for studying the biophysical properties of DNA and proteins, and as sensor devices. The ease of fabrication, favourable wetting properties and low capacitance are some of the inherent advantages, for example compared to more conventional, silicon-based nanopore chips. Recently, we have demonstrated high-bandwidth detection of double-stranded (ds) DNA with microsecond time resolution in nanopipettes, using custom-designed electronics. The electronics design has now been refined to include more sophisticated control features, such as integrated bias reversal and other features. Here, we exploit these capabilities and probe the translocation of short dsDNA in the 100 bp range, in different electrolytes. Single-stranded (ss) DNA of similar length are in use as capture probes, so label-free detection of their ds counterparts could therefore be of relevance in disease diagnostics.

New Insights into Single-Molecule Junctions Using a Robust, Unsupervised Approach to Data Collection and Analysis.

We have applied a new, robust and unsupervised approach to data collection, sorting and analysis that provides fresh insights into the nature of single-molecule junctions. Automation of tunneling current-distance (I(s)) spectroscopy facilitates the collection of very large data sets (up to 100,000 traces for a single experiment), enabling comprehensive statistical interrogations with respect to underlying tunneling characteristics, noise and junction formation probability (JFP). We frequently observe unusual low-to-high through-molecule conductance features with increasing electrode separation, in addition to numerous other "plateau" shapes, which may be related to changes in interfacial or molecular bridge structure. Furthermore, for the first time we use the JFP to characterize the homogeneity of functionalized surfaces at the nanoscale.

Challenges of biomolecular detection at the nanoscale: nanopores and microelectrodes.

The interest in analytical devices, which typically rely on the reactivity of a biological component for specificity, is growing rapidly. In this Perspective, we highlight current challenges in all-electrical biosensing as these systems shrink toward the nanoscale and enable the detection of analytes at the single-molecule level. We focus on two sensing principles: nanopores and amperometric microelectrode devices.

Electrodeposition and bipolar effects in metallized nanopores and their use in the detection of insulin.

Solid-state nanopore devices with integrated electrodes are an important class of single-molecule biosensors, with potential applications in DNA, RNA, and protein detection and sequence analysis. Here we investigate solid-state nanopore sensors with an embedded gold film, fabricated using semiconductor processing techniques and focused ion beam milling. We characterize their geometric structure in three dimensions on the basis of experimental conductance studies and modeling as well as transmission electron microscopy imaging and tomography. We used electrodeposition to further shrink the pores to effective diameters below 10 nm and demonstrate how bipolar electrochemical coupling across the membrane can lead to significant contributions to the overall pore current and discuss its implications for nanopore sensing. Finally, we use metallized nanopores modified with homocysteine for the detection of insulin. We show that adsorption of the protein to the chemically modified nanopores slows down the translocation process to tens of milliseconds, which is orders of magnitude slower than expected for conventional electrophoretic transport.

Which way up? Recognition of homologous DNA segments in parallel and antiparallel alignments.

Homologous gene shuffling between DNA molecules promotes genetic diversity and is an important pathway for DNA repair. For this to occur, homologous genes need to find and recognize each other. However, despite its central role in homologous recombination, the mechanism of homology recognition has remained an unsolved puzzle of molecular biology. While specific proteins are known to play a role at later stages of recombination, an initial coarse grained recognition step has, however, been proposed. This relies on the sequence dependence of the DNA structural parameters, such as twist and rise, mediated by intermolecular interactions, in particular, electrostatic ones. In this proposed mechanism, sequences that have the same base pair text, or are homologous, have lower interaction energy than those sequences with uncorrelated base pair texts. The difference between the two energies is termed the "recognition energy." Here, we probe how the recognition energy changes when one DNA fragment slides past another, and consider, for the first time, homologous sequences in antiparallel alignment. This dependence on sliding is termed the "recognition well." We find there is a recognition well for anti-parallel, homologous DNA tracts, but only a very shallow one, so that their interaction will differ little from the interaction between two nonhomologous tracts. This fact may be utilized in single molecule experiments specially targeted to test the theory. As well as this, we test previous theoretical approximations in calculating the recognition well for parallel molecules against MC simulations and consider more rigorously the optimization of the orientations of the fragments about their long axes upon calculating these recognition energies. The more rigorous treatment affects the recognition energy a little, when the molecules are considered rigid. When torsional flexibility of the DNA molecules is introduced, we find excellent agreement between the analytical approximation and simulations.

Single molecule ionic current sensing in segmented flow microfluidics.

Herein, we describe the integration of two glass nanopores into a segmented flow microfluidic device with a view on enhancing the functionality of label free, single molecule nanopore sensors. Within a robust and mechanically stable platform, individual droplet compositions are distinguished before single molecule translocations from the droplet are detected electrochemically via the Coulter principle. This result is highly significant, combining the sensitivity of single molecule methods and their ability to overcome the clouding of the ensemble average with the "isolated microreactor" benefits of droplet microfluidics. Furthermore, devices as presented here provide the platform for the development of systems where the injection and extraction of single molecules allow droplet composition to be controlled at the molecular level.

Label-free Pb(II) whispering gallery mode sensing using self-assembled glutathione-modified gold nanoparticles on an optical microcavity.

An ultrasensitive assay for the detection of Pb(II) has been developed using whispering gallery mode (WGM) sensing. In this technique a photonic microcavity was decorated with glutathione (GSH)-modified gold nanoparticles (Au NPs). The resonator was functionalized using an aminosilane to promote adhesion of the GSH-modified NPs creating a highly sensitive sensor specific to Pb(II). Upon introduction of Pb(II) solutions via a fluidic cell, Pb(II) ions bind to the GSH-Au NP complex and induce a shift of the resonant wavelength. Using this detection strategy we show that we are able to detect Pb(II) concentrations down to 0.05 nM in the presence of alkaline and heavy metal interferences such as Mg(II), Mn(II), Ca(II), Ni(II), Cd(II), Cr(II), Fe(II), and Hg(II). The signal was found to be proportional to the Pb(II) concentration within the range of 2.40-48.26 nM and was found to have an association constant of 2.15 × 10(5) M(-1) s(-1). The sensitivity obtained shows unparalleled advantages over currently available technology and satisfies the exposure thresholds set out by world organizations such as International Agency for Research on Cancer (IARC) and the Environmental Protection Agency (EPA). We believe that this sensor has the potential to be made portable for applications in environmental monitoring and in-field applications.