Preparation of colloidal molecules with temperature-tunable interactions from oppositely charged microgel spheres.

The self-assembly of small colloidal clusters, so-called colloidal molecules, into crystalline materials has proven extremely challenging, the outcome often being glassy, amorphous states where positions and orientations are locked. In this paper, a new type of colloidal molecule is therefore prepared, assembled from poly(N-isopropylacrylamide) (PNIPAM)-based microgels that due to their well documented softness and temperature-response allow for greater defect tolerance compared to hard spheres and for convenient in situ tuning of size, volume fraction and inter-particle interactions with temperature. The microgels (B) are assembled by electrostatic adsorption onto oppositely charged, smaller-sized microgels (A), where the relative size of the two determines the valency (n) of the resulting core-satellite ABn-type colloidal molecules. Following assembly, a microfluidic deterministic lateral displacement (DLD) device is used to effectively isolate AB4-type colloidal molecules of tetrahedral geometry that possess a repulsive-to-attractive transition on crossing the microgels' volume phase transition temperature (VPTT). These soft, temperature-responsive colloidal molecules constitute highly promising building blocks for the preparation of new materials with emergent properties, and their optical wavelength-size makes them especially interesting for optical applications.

Stochastic unfolding of nanoconfined DNA: Experiments, model and Bayesian analysis.

Nanochannels provide a means for detailed experiments on the effect of confinement on biomacromolecules, such as DNA. Here we introduce a model for the complete unfolding of DNA from the circular to linear configuration. Two main ingredients are the entropic unfolding force and the friction coefficient for the unfolding process, and we describe the associated dynamics by a non-linear Langevin equation. By analyzing experimental data where DNA molecules are photo-cut and unfolded inside a nanochannel, our model allows us to extract values for the unfolding force as well as the friction coefficient for the first time. In order to extract numerical values for these physical quantities, we employ a recently introduced Bayesian inference framework. We find that the determined unfolding force is in agreement with estimates from a simple Flory-type argument. The estimated friction coefficient is in agreement with theoretical estimates for motion of a cylinder in a channel. We further validate the estimated friction constant by extracting this parameter from DNA's center-of-mass motion before and after unfolding, yielding decent agreement. We provide publically available software for performing the required image and Bayesian analysis.

Competitive binding-based optical DNA mapping for fast identification of bacteria--multi-ligand transfer matrix theory and experimental applications on Escherichia coli.

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.

How nanochannel confinement affects the DNA melting transition within the Poland-Scheraga model.

When double-stranded DNA molecules are heated, or exposed to denaturing agents, the two strands are separated. The statistical physics of this process has a long history and is commonly described in terms of the Poland-Scheraga (PS) model. Crucial to this model is the configurational entropy for a melted region (compared to the entropy of an intact region of the same size), quantified by the loop factor. In this study, we investigate how confinement affects the DNA melting transition, by using the loop factor for an ideal Gaussian chain. By subsequent numerical solutions of the PS model, we demonstrate that the melting temperature depends on the persistence lengths of single-stranded and double-stranded DNA. For realistic values of the persistence lengths, the melting temperature is predicted to decrease with decreasing channel diameter. We also demonstrate that confinement broadens the melting transition. These general findings hold for the three scenarios investigated: 1. homo-DNA, i.e., identical basepairs along the DNA molecule, 2. random sequence DNA, and 3. "real" DNA, here T4 phage DNA. We show that cases 2 and 3 in general give rise to broader transitions than case 1. Case 3 exhibits a similar phase transition as case 2 provided the random sequence DNA has the same ratio of AT to GC basepairs (A - adenine, T - thymine, G - guanine, C - cytosine). A simple analytical estimate for the shift in melting temperature is provided as a function of nanochannel diameter. For homo-DNA, we also present an analytical prediction of the melting probability as a function of temperature.

Fluorescent nanowire heterostructures as a versatile tool for biology applications.

Nanowires are increasingly used in biology, as sensors, as injection devices, and as model systems for toxicity studies. Currently, in situ visualization of nanowires in biological media is done using organic dyes, which are prone to photobleaching, or using microscopy methods which either yield poor resolution or require a sophisticated setup. Here we show that inherently fluorescent nanowire axial heterostructures can be used to localize and identify nanowires in cells and tissue. By synthesizing GaP-GaInP nanowire heterostructures, with nonfluorescent GaP segments and fluorescent GaInP segments, we created a barcode labeling system enabling the distinction of the nanowire morphological and chemical properties using fluorescence microscopy. The GaInP photoluminescence stability, combined with the fact that the nanowires can be coated with different materials while retaining their fluorescence, make these nanowires promising tools for biological and nanotoxicological studies.

Geometry-Dependent Elastic Flow Dynamics in Micropillar Arrays.

Regular device-scale DNA waves for high DNA concentrations and flow velocities have been shown to emerge in quadratic micropillar arrays with potentially strong relevance for a wide range of microfluidic applications. Hexagonal arrays constitute another geometry that is especially relevant for the microfluidic pulsed-field separation of DNA. Here, we report on the differences at the micro and macroscopic scales between the resulting wave patterns for these two regular array geometries and one disordered array geometry. In contrast to the large-scale regular waves visible in the quadratic array, in the hexagonal arrays, waves occur in a device-scale disordered zig-zag pattern with fluctuations on a much smaller scale. We connect the large-scale pattern to the microscopic flow and observe flow synchronization that switches between two directions for both the quadratic and hexagonal arrays. We show the importance of order using the disordered array, where steady-state stationary and highly fluctuating flow states persist in seemingly random locations across the array. We compare the flow dynamics of the arrays to that in a device with sparsely distributed pillars. Here, we observe similar vortex shedding, which is clearly observable in the quadratic and disordered arrays. However, the shedding of these vortices couples only in the flow direction and not laterally as in the dense, ordered arrays. We believe that our findings will contribute to the understanding of elastic flow dynamics in pillar arrays, helping us elucidate the fundamental principles of non-Newtonian fluid flow in complex environments as well as supporting applications in engineering involving e.g., transport, sorting, and mixing of complex fluids.

Photophysical image analysis: Unsupervised probabilistic thresholding for images from electron-multiplying charge-coupled devices.

We introduce the concept photophysical image analysis (PIA) and an associated pipeline for unsupervised probabilistic image thresholding for images recorded by electron-multiplying charge-coupled device (EMCCD) cameras. We base our approach on a closed-form analytic expression for the characteristic function (Fourier-transform of the probability mass function) for the image counts recorded in an EMCCD camera, which takes into account both stochasticity in the arrival of photons at the imaging camera and subsequent noise induced by the detection system of the camera. The only assumption in our method is that the background photon arrival to the imaging system is described by a stationary Poisson process (we make no assumption about the photon statistics for the signal). We estimate the background photon statistics parameter, λbg, from an image which contains both background and signal pixels by use of a novel truncated fit procedure with an automatically determined image count threshold. Prior to this, the camera noise model parameters are estimated using a calibration step. Utilizing the estimates for the camera parameters and λbg, we then introduce a probabilistic thresholding method, where, for the first time, the fraction of misclassified pixels can be determined a priori for a general image in an unsupervised way. We use synthetic images to validate our a priori estimates and to benchmark against the Otsu method, which is a popular unsupervised non-probabilistic image thresholding method (no a priori estimates for the error rates are provided). For completeness, we lastly present a simple heuristic general-purpose segmentation method based on the thresholding results, which we apply to segmentation of synthetic images and experimental images of fluorescent beads and lung cell nuclei. Our publicly available software opens up for fully automated, unsupervised, probabilistic photophysical image analysis.

Fibroblasts cultured on nanowires exhibit low motility, impaired cell division, and DNA damage.

Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells.

Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51.

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.

Single-molecule denaturation mapping of DNA in nanofluidic channels.

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.

Lipid-based passivation in nanofluidics.

Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.

Short and long-range cyclic patterns in flows of DNA solutions in microfluidic obstacle arrays.

We observe regular patterns emerging across multiple length scales with high-concentration DNA solutions in microfluidic pillar arrays at low Reynolds numbers and high Deborah numbers. Interacting vortices between pillars lead to long-range order in the form of large travelling waves consisting of DNA at high concentration and extension. Waves are formed in quadratic arrays of pillars, while randomizing the position of the pillar in each unit cell of a quadratic array leads to suppression of the long-range patterns. We find that concentrations exceeding the overlap concentration of the DNA enables the waves, and exploring the behavior of the waves as a function of flow rate, buffer composition, concentration and molecular length, we identify elastic effects as central to the origin of the waves. Our work may not only help increase the low throughput that often limits sample processing in microfluidics, it may also provide a platform for further studies of the underlying viscoelastic mechanisms.

Using symmetry to control viscoelastic waves in pillar arrays.

Solutions of macromolecules exhibit viscoelastic properties and unlike Newtonian fluids, they may break time-reversal symmetry at low Reynolds numbers resulting in elastic turbulence. Furthermore, under some conditions, instead of the chaotic turbulence, the result is large-scale waves in the form of cyclic spatial and temporal concentration variations, as has been shown for macromolecular DNA flowing in microfluidic pillar arrays. We here demonstrate how altering the symmetry of the individual pillars can be used to influence the symmetry of these waves. We control the extent of instabilities in viscoelastic flow by leveraging the effects of the symmetry of the pillars on the waves, demonstrating suppressed viscoelastic fluctuations with relevance for transport and sorting applications, or conversely opening up for enhanced viscoelasticity-mediated mixing. The onset of waves, which changes flow resistance, occurs at different Deborah numbers for flow in different directions through the array of triangular pillars, thus breaking the symmetry of the flow resistance along the device, opening up for using the occurrence of the waves to construct a fluidic diode.

Bandpass filtering of DNA elastic modes using confinement and tension.

During a variety of biological and technological processes, biopolymers are simultaneously subject to both confinement and external forces. Although significant efforts have gone into understanding the physics of polymers that are only confined, or only under tension, little work has been done to explore the effects of the interplay of force and confinement. Here, we study the combined effects of stretching and confinement on a polymer's configurational freedom. We measure the elastic response of long double-stranded DNA molecules that are partially confined to thin, nanofabricated slits. We account for the data through a model in which the DNA's short-wavelength transverse elastic modes are cut off by applied force and the DNA's bending stiffness, whereas long-wavelength modes are cut off by confinement. Thus, we show that confinement and stretching combine to permit tunable bandpass filtering of the elastic modes of long polymers.

A single-step competitive binding assay for mapping of single DNA molecules.

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

Directed self-organization of single DNA molecules in a nanoslit via embedded nanopit arrays.

We show that arrays of nanopit structures etched in a nanoslit can control the positioning and conformation of single DNA molecules in nanofluidic devices. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of a device without additional chemical modification and achieve a high degree of control over local DNA conformation. DNA can be extended between two nanopits and in closely spaced arrays will self-assemble into "connect-the-dots" conformations consisting of locally pinned segments joined by fluctuating linkers. These results have broad implications for nanotechnology fields that require methods for the nanoscale positioning and manipulation of DNA.

High-Throughput Separation of Long DNA in Deterministic Lateral Displacement Arrays.

Length-based separation of DNA remains as relevant today as when gel electrophoresis was introduced almost 100 years ago. While new, long-read genomics technologies have revolutionised accessibility to powerful genomic data, the preparation of samples has not proceeded at the same pace, with sample preparation often constituting a considerable bottleneck, both in time and difficulty. Microfluidics holds great potential for automated, sample-to-answer analysis via the integration of preparatory and analytical steps, but for this to be fully realised, more versatile, powerful and integrable unit operations, such as separation, are essential. We demonstrate the displacement and separation of DNA with a throughput that is one to five orders of magnitude greater than other microfluidic techniques. Using a device with a small footprint (23 mm × 0.5 mm), and with feature sizes in the micrometre range, it is considerably easier to fabricate than parallelized nano-array-based approaches. We show the separation of 48.5 kbp and 166 kbp DNA strands achieving a significantly improved throughput of 760 ng/h, compared to previous work and the separation of low concentrations of 48.5 kbp DNA molecules from a massive background of sub 10 kbp fragments. We show that the extension of DNA molecules at high flow velocities, generally believed to make the length-based separation of long DNA difficult, does not place the ultimate limitation on our method. Instead, we explore the effects of polymer rotations and intermolecular interactions at extremely high DNA concentrations and postulate that these may have both negative and positive influences on the separation depending on the detailed experimental conditions.

Use of dielectrophoresis for directing T cells to microwells before nanostraw transfection: modelling and experiments.

Nanostraw substrates have great potential for achieving minimally invasive cell transfection. Cells located on the nanostraw substrate are subjected to mild DC electric pulses applied across the nanostraw substrate, which open pores in the cell membrane on top of the nanostraws and drives charged cargo through these pores via electrophoresis. However, with this method, the current may leak through uncovered nanostraws, thereby decreasing the desired effect in the cell-covered nanostraws. A minimization of the number of uncovered nanostraws could be achieved by high cell coverage, but this is challenging when working with small cell populations. Nanostraw substrates of smaller area could be covered by smaller cell populations but are hard to integrate into fluidics systems. Here, we use simulations and experiments to show that this issue can be addressed by covering the nanostraw substrate with an insulating layer containing pores of similar size to cells. The pores act as traps into which cells can be guided using dielectrophoresis, ensuring a high degree of occupancy while maintaining a high cell viability, even if the total number of cells is low.

DNA in nanochannels--directly visualizing genomic information.

The power of nanofluidic channels to analyze DNA is described along with practical experimental hints. As an introduction, a general overview is given on conventional DNA analysis tools, as well as tools under development towards the $1000 genome. The focus of this tutorial review is the stretching of DNA in nanoscale channels for coarse-grained mapping of DNA. To understand the behavior of the DNA, basic theory is discussed. Experimental details are revealed so that the reader, with the proper equipment, should be able to perform experiments. Basic approaches to the analysis of the data are discussed. Finally, potential future directions are discussed including the application of melting mapping as a simple barcode for the DNA.

Fluorescence enhancement of single DNA molecules confined in Si/SiO2 nanochannels.

We demonstrate that the detected emission intensity from YOYO-labeled DNA molecules confined in 180 nm deep Si/SiO2 nanofunnels changes significantly and not monotonically with the width of the funnel. This effect may be of importance for quantitative fluorescence microscopy and for experiments with a tight photon budget.