Standalone interferometry-based calibration of convex lens-induced confinement microscopy with nanoscale accuracy

Chen C., Li Y., Kerman S., Neutens P., Willems K., Cornelissen S., Lagae L., Stakenborg T., Van Dorpe P.

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS. Direct and real-time identification of nucleobases in DNA strands is still limited by the sensitivity and spatial resolution of the established solid-state nanopore devices. Here, the authors use CMOS compatible, plasmonic nanoslits to locally enable SERS for identifying nucleobases, both individual and incorporated in DNA strands.

Scott S., Xu Z., Kouzine F., Berard D.J., Shaheen C., Gravel B., Saunders L., Hofkirchner A., Leroux C., Laurin J., Levens D., Benham C.J., Leslie S.R.

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Wang J., Hou J., Zhang H., Tian Y., Jiang L.

Ultrasensitive and selective detection of molecules at nano or sub-nanomolar level is very important for many areas such as early diagnosis and drug testing. Herein, we report a high-sensitive cocaine sensor based on a single nanochannel coupled with DNA aptamers. The single nanochannel-aptamer-based biosensor can recognize cocaine molecules with an excellent sensitivity and good selectivity. A linear relationship between target cocaine concentration and output ionic current is obtained in a wide concentration range of cocaine from 1 nM to 10 μM. The cocaine sensor also shows a detection limit down to 1 nM. This study provides a new avenue to develop new nanochannel-aptamer-based biosensors for rapid and ultratrace detection of a variety of illicit drugs.

Zhou C., Segal‐Peretz T., Oruc M.E., Suh H.S., Wu G., Nealey P.F.

Control over nanopore size and 3D structure is necessary to advance membrane performance in ubiquitous separation devices. Here, inorganic nanoporous membranes are fabricated by combining the assembly of cylinder-forming poly(styrene-block-methyl methacrylate) (PS-b-PMMA) block copolymer and sequential infiltration synthesis (SIS). A key advance relates to the use of PMMA majority block copolymer films and the optimization of thermal annealing temperature and substrate chemistry to achieve through-film vertical PS cylinders. The resulting morphology allows for direct fabrication of nanoporous AlOx by selective growth of Al2O3 in the PMMA matrix during the SIS process, followed by polymer removal using oxygen plasma. Control over the pore diameter is achieved by varying the number of Al2O3 growth cycles, leading to pore size reduction from 21 to 16 nm. 3D characterization, using scanning transmission electron microscopy tomography, reveals that the AlOx channels are continuous through the film and have a gradual increase in pore size with depth. Finally, the ultrafiltration performance of the fabricated AlOx membrane for protein separation as a function of protein size and charge is demonstrated.

Leith J.S., Kamanzi A., Sean D., Berard D., Guthrie A.C., McFaul C.M., Slater G.W., de Haan H.W., Leslie S.R.

We directly measure the free energy of confinement for semiflexible polymers from the nanoscale to bulk regimes in slit-like confinement. We use convex lens-induced confinement (CLiC) microscopy of DNA to directly count molecules at equilibrium in a single chamber of smoothly increasing height. Our data, acquired across a continuum of confinement regimes, provide a bridge with which to connect scaling theories established for qualitatively different regimes. We present new experimental data and simulations that connect the Odijk theory describing sub-persistence-length confinement, the interpolation model by Chen and Sullivan extending Odijk to moderate confinement, and the Casassa theory describing the transition from moderate confinement to bulk. Further, this work establishes a robust, quantitative platform for understanding and manipulating biopolymers at the nanoscale, with key applications and insights toward emerging genomic analysis tools.

Sun Z., Liao T., Zhang Y., Shu J., Zhang H., Zhang G.

A very simple sensing device based on biomimetic nanochannels has been developed for label-free, ultrasensitive and highly sequence-specific detection of DNA. Probe DNA was modified on the inner wall of the nanochannel surface by layer-by-layer (LBL) assembly. After probe DNA immobilization, DNA detection was realized by monitoring the rectified ion current when hybridization occurred. Due to three dimensional (3D) nanoscale environment of the nanochannel, this special geometry dramatically increased the surface area of the nanochannel for immobilization of probe molecules on the inner-surface and enlarged contact area between probes and target-molecules. Thus, the unique sensor reached a reliable detection limit of 10 fM for target DNA. In addition, this DNA sensor could discriminate complementary DNA (c-DNA) from non-complementary DNA (nc-DNA), two-base mismatched DNA (2bm-DNA) and one-base mismatched DNA (1bm-DNA) with high specificity. Moreover, the nanochannel-based biosensor was also able to detect target DNA even in an interfering environment and serum samples. This approach will provide a novel biosensing platform for detection and discrimination of disease-related molecular targets and unknown sequence DNA.

Bassu M., Holik P., Schmitz S., Steltenkamp S., Burg T.P.

We describe the fabrication of ultra uniform nanoslit arrays and their application to continuous separation of small molecules by charge.

Ando K., Tanabe M., Morigaki K.

The biological membrane is a natural biosensing platform that can detect specific molecules with extremely high sensitivity. We developed a biosensing methodology by combining a model biological membrane and a nanometer-sized gap structure on a glass substrate. The model membrane comprised lithographically patterned polymeric and fluid lipid bilayers. The polymeric bilayer was bonded to a poly(dimethylsiloxane) (PDMS) sheet by using an adhesion layer with a defined thickness (lipid vesicles). Extruded lipid vesicles having a biotin moiety on the surface were used as the adhesion layer in conjunction with the biotin-streptavidin linkage. A gap structure was formed between the fluid bilayer and PDMS (nanogap junction). The thickness of the gap structure was several tens of nanometers, as determined by the thickness of the adhesion layer. The nanogap junction acted as a sensitive biosensing platform. From a mixture of proteins (cholera toxin and albumin), the target protein (cholera toxin) was selectively transported into the gap by the specific binding to a glycolipid (GM1) in the fluid bilayer and lateral diffusion. The target protein molecules were then detected with an elevated signal-to-noise ratio due to the reduced background noise in the nanometric gap. The combination of selective transport and reduced background noise drastically enhanced the sensitivity toward the target protein. The nanogap junction should have broad biomedical applications by realizing highly selective and sensitive biosensing in samples having diverse coexisting molecules.

Ahamed M.J., Mahshid S., Berard D.J., Michaud F., Sladek R., Reisner W.W., Leslie S.R.

Confining DNA molecules with high throughput and structural integrity is an important challenge in nanochannel-based genomic mapping technology. Here we demonstrate dynamic confinement and linearization of DNA polymers within an embedded nanogroove array with 95% channel occupation. In standard nanofluidic technology, the free energy of confinement experienced by the DNA molecules increases strongly with decreasing device dimensions, leading to a suppression of molecule concentration in nanoconfined spaces. We overcome this limitation by combining “convex lens-induced confinement” (CLiC) geometry with in situ electrophoresis, simultaneously establishing gentle and continuously adjustable nanoconfinement and precise electrokinetic control. Together, these capabilities enable trapping and visualization of extended DNA molecules with high yield over an extended range of conditions. We demonstrate 10-fold increased DNA concentration in a confined region from 10 to 500 nm. Moreover, we develop and validate a p...

Mahshid S., Ahamed M.J., Berard D., Amin S., Sladek R., Leslie S.R., Reisner W.

We demonstrate a lab-on-a-chip that combines micro/nano-fabricated features with a Convex Lens-Induced Confinement (CLIC) device for the in situ analysis of single cells. A complete cycle of single cell analysis was achieved that includes: cell trapping, cell isolation, lysis, protein digestion, genomic DNA extraction and on-chip genomic DNA linearization. The ability to dynamically alter the flow-cell dimensions using the CLIC method was coupled with a flow-control mechanism for achieving efficient cell trapping, buffer exchange, and loading of long DNA molecules into nanofluidic arrays. Finite element simulation of fluid flow gives rise to optimized design parameters for overcoming the high hydraulic resistance present in the micro/nano-confinement region. By tuning design parameters such as the pressure gradient and CLIC confinement, an efficient on-chip single cell analysis protocol can be obtained. We demonstrate that we can extract Mbp long genomic DNA molecules from a single human lybphoblastoid cell and stretch these molecules in the nanochannels for optical interrogation.

de Carvalho S.J., Metzler R., Cherstvy A.G.

What are the fundamental laws for the adsorption of charged polymers onto oppositely charged surfaces, for convex, planar, and concave geometries?

Arsenault A., Leith J.S., Henkin G., McFaul C.M., Tarling M., Talbot R., Berard D., Michaud F., Scott S., Leslie S.R.

We present the design and construction of a versatile, open frame inverted microscope system for wide-field fluorescence and single molecule imaging. The microscope chassis and modular design allow for customization, expansion, and experimental flexibility. We present two components which are included with the microscope which extend its basic capabilities and together create a powerful microscopy system: A Convex Lens-induced Confinement device provides the system with single-molecule imaging capabilities, and a two-color imaging system provides the option of imaging multiple molecular species simultaneously. The flexibility of the open-framed chassis combined with accessible single-molecule, multi-species imaging technology supports a wide range of new measurements in the health, nanotechnology, and materials science research sectors.

Haywood D.G., Saha-Shah A., Baker L.A., Jacobson S.C.

Ion, particle, and fluid transport in nanofluidic devices has received considerable attention over the past two decades due to unique transport properties exhibited at the nanoscale.1,2 Phenomena such as double layer overlap, high surface-to-volume ratios, surface charge, ion-current rectification, and entropic barriers can influence transport in and around nanofluidic structures because the length scales of these forces and the critical dimensions of the device are similar. Advances in micro- and nanofabrication techniques provide the ability to design a variety of well-defined nanofluidic geometries to study these phenomena and their effects on ion and fluid transport. Integration of micro- and nanofluidic structures into lab-on-a-chip devices permits increased functionality that is useful for a range of analytical applications.3,4 This Review focuses on recent advances in nanofabrication techniques as well as studies of fundamental transport in nanofluidic devices. Nanopores, nanochannels, and nanopipets are three common nanofluidic structures that have been influential in studying nanofluidic transport. Because of space limitations, we have limited the scope of this Review to studies with these three structures, and we focus our attention primarily on work published between January 2011 and August 2014. We do not discuss work with carbon nanotubes,5 nanomeshes,6 or nanowires.7 Figure ​Figure11 shows examples of the three nanofluidic geometries discussed here. Nanopores are typically formed perpendicular to the plane of a substrate and are characterized by a critical limiting dimension, which is measured by scanning electron microscopy (SEM), transmission electron microscopy (TEM), or conductance measurements. Pores are fabricated in a variety of materials, e.g., poly(carbonate), poly(ethylene terephthalate), or silicon nitride, and can have an asymmetric (Figure ​(Figure1a)1a) or symmetric (Figure ​(Figure1b)1b) shape, depending on the fabrication technique. Symmetric pores are either cylindrically shaped with a constant critical dimension determined by electron microscopy or hourglass-shaped with a critical dimension at the center of the pore. Although electron microscopy is capable of measuring exterior pore dimensions, the exact inner geometry is often unknown and may contain an asymmetry between two symmetric features, e.g., cigar-shaped pores. Asymmetric nanopores typically have a narrow tip and a wide base with a funnel-shaped geometry along the pore axis. Tip and base dimensions are measured by SEM, but the exact pore geometry is often unknown. Nanochannels often refer to in-plane structures with either symmetric (Figure ​(Figure1c)1c) or asymmetric (Figure ​(Figure1d)1d) geometries. Channels may be confined to the nanoscale in depth, width, or both, depending on the fabrication method. Nanochannels are commonly fabricated in glass and polymer substrates and characterized by SEM and atomic force microscopy (AFM). The in-plane nature of these channels allows the integration of well-defined features into more complex geometries, and any two-dimensional (2D) channel architecture can be designed. Nanopipets are specialized nanopores fabricated from pulled glass or fused-silica capillaries (Figure ​(Figure1e,f). The1e,f). The geometry of a nanopipet is conically shaped with a critical tip diameter of tens to hundreds of nanometers, which can be measured by electron microscopy. Unlike nanopores and nanochannels, nanopipets can be easily coupled with position control, which allows the tip of the nanopipets to be positioned in specific locations or used in scanned probe microscopies. Figure 1 Nanopores, nanochannels, and nanopipets are three common nanofluidic platforms. Nanopores are typically out-of-plane structures and have either an asymmetric or symmetric geometry. Conical nanopores have a wide base as shown in panel a that tapers to ...

Kienle D.F., Kuhl T.L.

This work describes an interferometry data analysis method for determining the optical thickness of thin films or any variation in the refractive index of a fluid or film near a surface. In particular, the method described is applied to the analysis of interferometry data taken with a surface force apparatus (SFA). The technique does not require contacting or confining the fluid or film. By analyzing interferometry data taken at many intersurface separation distances out to at least 300 nm, the properties of a film can be quantitatively determined. The film can consist of material deposited on the surface, like a polymer brush, or variation in a fluid’s refractive index near a surface resulting from, for example, a concentration gradient, depletion in density, or surface roughness. The method is demonstrated with aqueous polyethylenimine (PEI) adsorbed onto mica substrates, which has a large concentration and therefore refractive index gradient near the mica surface. The PEI layer thickness determined by the proposed method is consistent with the thickness measured by conventional SFA methods. Additionally, a thorough investigation of the effects of random and systematic error in SFA data analysis and modeling via simulations of interferometry is described in detail.

Stroeve P., Rahman M., Naidu L.D., Chu G., Mahmoudi M., Ramirez P., Mafe S.

The diffusion of two similar molecular weight proteins, bovine serum albumin (BSA) and bovine haemoglobin (BHb), through nanoporous charged membranes with a wide range of pore radii is studied at low ionic strength. The effects of the solution pH and the membrane pore diameter on the pore permeability allow quantifying the electrostatic interaction between the charged pore and the protein. Because of the large screening Debye length, both surface and bulk diffusion occur simultaneously. By increasing the pore diameter, the permeability tends to the bulk self-diffusion coefficient for each protein. By decreasing the pore diameter, the charges on the pore surface electrostatically hinder the transport even at the isoelectric point of the protein. Surprisingly, even at pore sizes 100 times larger than the protein, the electrostatic hindrance still plays a major role in the transport. The experimental data are qualitatively explained using a two-region model for the membrane pore and approximated equations for the pH dependence of the protein and pore charges. The experimental and theoretical results should be useful for designing protein separation processes based on nanoporous charged membranes.

Roopnarine B.K., Schmidt S.C., Maxwell K.J., Morozova S.

Mahanta S., Vallejo-Ramirez P., Karedla N., Puczkarski P., Krishnan M.

From molecules and particles to macroscopic surfaces immersed in fluids, chemical reactions often endow interfaces with electrical charge which in turn governs surface interactions and interfacial phenomena. The ability to measure the electrical properties of a material immersed in any solvent, as well as to monitor the spatial heterogeneity and temporal variation thereof, has been a long-standing challenge. Here, we describe an optical microscopy-based approach to probe the surface charge distribution of a range of materials, including inorganic oxide, polymer, and polyelectrolyte films, in contact with a fluid. The method relies on optical visualization of the electrical repulsion between diffusing charged probe molecules and the unknown surface to be characterized. Rapid image-based measurements enable us to further determine isoelectric points of the material as well as properties of its ionizable chemical groups. We further demonstrate the ability to optically monitor chemically triggered surface charge changes with millisecond time resolution. Finally, we present a scanning-surface probe technique capable of diffraction-limited imaging of spatial heterogeneities in chemical composition and charge over large areas. This technique will enable facile characterization of the solid–liquid interface with wide-ranging relevance across application areas from biology to engineering.

Thompson C.J., Kienle D.F., Schwartz D.K.

The facilitated surface diffusion of transiently adsorbing molecules in a planar confined microenvironment (i.e., slit-like confinement) is highly relevant to biological phenomena, such as extracellular signaling, as well as numerous biotechnology systems. Here, we studied the surface diffusion of individual proteins confined between two symmetric lipid bilayer membranes, under a continuum of confinement heights, using single-molecule tracking and convex lens-induced confinement as well as hybrid, kinetic Monte Carlo simulations of a generalized continuous time random walk process. Surface diffusion was observed to vary non-monotonically with confinement height, exhibiting a maximum at a height of ∼750 nm, where diffusion was nearly 40% greater than that for a semi-infinite system. This demonstrated that planar confinement can, in fact, increase surface diffusion, qualitatively validating previous theoretical predictions. Simulations reproduced the experimental results and suggested that confinement enhancement of surface diffusion for symmetric systems is limited to cases where the adsorbate exhibits weak surface sticking.

Hitimana E., Roopnarine B.K., Morozova S.

Transport through heterogeneous confined geometries is encountered in many processes and applications such as filtration, drug delivery, and enhanced oil recovery. We have used differential dynamic microscopy (DDM) and particle tracking to investigate dynamics of 36 nm negatively-charged polystyrene particles in convex lens-induced confinement (CLiC). The confinement gap height was controlled from 0.085 μm to 3.6 mm by sandwiching the aqueous particle solution between a glass coverslip and a convex lens using a homemade sample holder. With an inverted fluorescence microscope, sequences of micrographs were taken at various radial positions and gap heights for five particle concentrations (i.e. φ = 0.5 × 10-5, 1 × 10-5, 5 × 10-5, 10 × 10-5, 50 × 10-5) and ionic strengths ranging from 10-3 to 150 mM. The resulting image structure functions were fitted with a simple exponential model to extract the ensemble-averaged diffusive dynamics. It was found that particle diffusion was more hindered as a function of increased confinement. In addition, the ensemble-averaged diffusion coefficient was found to depend on the bulk concentration, and the concentration dependence increased as a function of confinement. Increasing particle and salt concentration led to confinement-dependent adsorption onto the geometry surface. Overall, we show that CLiC devices are simple and effective and can be used to study dynamics in continuous confinement from sub 100 nm to 100's of μm. These findings could lead to better understanding of separations and interactions in confining devices.

Morrin G.T., Kienle D.F., Schwartz D.K.

In many technological applications, DNA is confined within nanoenvironments that are smaller than the size of the unconfined polymer in solution. However, the dependence of the diffusion coefficient on molecular weight and characteristic confinement dimension remains poorly understood in this regime. Here, convex lens-induced confinement (CLiC) was leveraged to examine how the diffusion of short DNA fragments varied as a function of slit height by using single-molecule fluorescence tracking microscopy. The diffusion coefficient followed approximate power law behavior versus confinement height, with exponents of 0.27 ± 0.01, 0.32 ± 0.02, and 0.42 ± 0.06 for 692, 1343, and 2686 base pair chains, respectively. The weak dependence on slit height suggests that shorter semiflexible chains may adopt increasingly rodlike conformations and therefore experience weaker excluded-volume interactions as the confinement dimension is reduced. The diffusion coefficient versus molecular weight also exhibited apparent power law behavior, with exponents that varied slightly (from -0.89 to -0.85) with slit height, consistent with hydrodynamic interactions intermediate between Rouse and Zimm model predictions.

Frost R., Débarre D., Jana S., Bano F., Schünemann J., Görlich D., Richter R.P.

We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane-sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine-glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.

Morrin G.T., Kienle D.F., Weltz J.S., Traeger J.C., Schwartz D.K.

The surface diffusion of poly-l-lysine (PLL) in a planar nanoslit was studied using convex lens-induced confinement (CLiC) single-molecule tracking microscopy. Three surface chemistries were employ...

Leslie S., Berard D., Kamanzi A., Metera K., Scott S., Shaheen C., Shayegan M., Tahvildari R., Zhang Z.

Molecular biology is messy and complex. The future of life sciences research, drug development, and many fields depends on our ability to unravel the complex, biophysical phenomena that underlie cellular function with a finer level of resolution. Using current technologies, it is challenging to conduct quantitative measurements that can reveal the complexity of life at the molecular scale. This article reviews the invention, development, and research applications of convex lens-induced confinement (CLiC) microscopy, which is a method to image molecular interactions one molecule at a time, while emulating “cell-like” conditions, with precision and control. By mechanically confining molecules to the field of view, CLiC can eliminate the complexity and potential biases inherent to “tethering” molecules. Looking forward, CLiC is being applied to emerging areas of exploration where single-molecule resolution can be transformative, including visualizing nanoparticle and protein dynamics, CRISPR-Cas9 targeting dynamics, and therapeutics applications.