DNA Translocation through Vertically Stacked 2D Layers of Graphene and Hexagonal Boron Nitride Heterostructure Nanopore.

Cost-effective, fast, and reliable DNA sequencing can be enabled by advances in nanopore-based methods, such as the use of atomically thin graphene membranes. However, strong interaction of DNA bases with graphene leads to undesirable effects such as sticking of DNA strands to the membrane surface. While surface functionalization is one way to counter this problem, here, we present another solution based on a heterostructure nanopore system, consisting of a monolayer of graphene and hexagonal boron nitride (hBN) each. Molecular dynamics studies of DNA translocation through this heterostructure nanopore revealed a surprising and crucial influence of the heterostructure layer order in controlling the base specific signal variability. Specifically, the heterostructure with graphene on top of hBN had nearly 3-10× lower signal variability than the one with hBN on top of graphene. Simulations point to the role of differential underside sticking of DNA bases as a possible reason for the observed influence of the layer order. Our studies can guide the development of experimental systems to study and exploit DNA translocation through two-dimensional heterostructure nanopores for single molecule sequencing and sensing applications.

Bead-based multiplex detection of dengue biomarkers in a portable imaging device.

Dengue is one of the most rapidly spreading mosquito-borne viral diseases in the world. Differential diagnosis is a crucial step for the management of the disease and its epidemiology. Point-of-care testing of blood-borne dengue biomarkers provides an advantageous approach in many health care settings, and the ability to follow more than one biomarker at once could significantly improve the management of the disease. Bead-based multiplex technologies (suspension array) can measure multiple biomarker targets simultaneously by using recognition molecules immobilized on microsphere beads. The overarching objective of our work is to develop a portable detection device for the simultaneous measurement of multiple biomarkers important in dengue diagnosis, monitoring and treatment. Here, we present a bead-based assay for the detection of one of the four serotypes of dengue virus non-structural protein (DENV-NS1) as well as its cognate human IgG. In this system, the fluorescent microspheres containing the classification fluorophore and detection fluorophore are imaged through a microfluidic chip using an infinity-corrected microscope system. Calibration curves were plotted for median fluorescence intensity against known concentrations of DENV-NS1 protein and anti-NS1 human IgG. The limit of quantitation was 7.8 ng/mL and 15.6 ng/mL, respectively. The results of this study demonstrate the feasibility of the multiplex detection of dengue biomarkers and present its analytical performance parameters. The proposed imaging device holds potential for point-of-care testing of biomarkers on a highly portable system, and it may facilitate the diagnosis and prevention of dengue as well as other infectious diseases.

Active modulation of surfactant-driven flow instabilities by swarming bacteria.

Models based on surfactant-driven instabilities have been employed to describe pattern formation by swarming bacteria. However, by definition, such models cannot account for the effect of bacterial sensing and decision making. Here we present a more complete model for bacterial pattern formation which accounts for these effects by coupling active bacterial motility to the passive fluid dynamics. We experimentally identify behaviors which cannot be captured by previous models based on passive population dispersal and show that a more accurate description is provided by our model. It is seen that the coupling of bacterial motility to the fluid dynamics significantly alters the phase space of surfactant-driven pattern formation. We also show that our formalism is applicable across bacterial species.

Effect of single nanoparticle-nanopore interaction strength on ionic current modulation.

Solid-state nanopores are rapidly emerging as promising platforms for developing various single molecule sensing applications. The modulation of ionic current through the pore due to translocation of the target molecule has been the dominant measurement modality in nanopore sensors. Here, we focus on the dwell time, which is the duration taken by the target molecule or particle to traverse the pore and study its dependence on the strength of interaction of the target with the pore using single gold nanoparticles (NPs) as targets interacting with a silicon nitride (SiN) nanopore. The strength of interaction, which in our case is electrostatic in nature, can be controlled by coating the nanoparticles with charged polymers. We report on an operating regime of this nanopore sensor, characterized by attractive interactions between the nanoparticle and the pore, where the dwell time is exponentially sensitive to the target-pore interaction. We used negatively and positively charged gold nanoparticles to control the strength of their interaction with the Silicon Nitride pore which is negatively charged. Our experiments revealed how this modulation of the electrostatic force greatly affects the dwell time. Positively charged NPs with strong attractive interactions with the pore resulted in increase of dwell times by 2-3 orders of magnitude, from 0.4 ms to 75.3 ms. This extreme sensitivity of the dwell time on the strength of interaction between a target and nanopore can be exploited in emerging nanopore sensor applications.

Impact of Bioinspired Nanotopography on the Antibacterial and Antibiofilm Efficacy of Chitosan.

Chitosan derived from chitin is one of the most abundant naturally occurring biocompatible polymers obtained from fungi and arthropods. In this work, we report the enhancement in the bactericidal efficacy of CHI in the presence of a sharp nanotopography. High-aspect ratio nanostructured surface (NSS) was fabricated using a single-step deep reactive ion etching technique (DRIE). Post fabrication, CHI coating was carried out using a layer-by-layer (LBL) dip coating process on the flat and nanostructured surfaces. Antibacterial efficacy of the flat silicon surface coated with CHI (Si_CHI) and NSS coated with CHI (NSS_CHI) was tested against both Gram-negative (G-ve) bacteria E. coli and Gram-positive (G+ve) bacteria S. aureus. NSS_CHI exhibited superior antibacterial property against G-ve and G+ve microbes as compared with Si_CHI and NSS substrates. Scanning electron microscopy (SEM) and fluorescence microscopy were used to study the morphology and viability of the bacteria on all the surfaces. Also, biofilm quantification was carried out on all the engineered surfaces for both E. coli and S. aureus using crystal violet (CV) staining. NSS_CHI was found to have the minimum biofilm formation on its surface exhibiting its superior antibacterial property. This study shows that the antibacterial and antibiofilm efficiency of CHI can be augmented by combining it with a sharp nanotopography.

Polyelectrolyte multilayers for bio-applications: recent advancements.

The synergistic relationship between structure and the bulk properties of polyelectrolyte multilayer (PEM) films has generated tremendous interest in their application for loading and release of bioactive species. Layer-by-layer assembly is the simplest, cost effective process for fabrication of such PEMs films, leading to one of the most widely accepted platforms for incorporating biological molecules with nanometre precision. The bulk reservoir properties of PEM films render them a potential candidate for applications such as biosensing, drug delivery and tissue engineering. Various biomolecules such as proteins, DNA, RNA or other desired molecules can be incorporated into the PEM stack via electrostatic interactions and various other secondary interactions such as hydrophobic interactions. The location and availability of the biological molecules within the PEM stack mediates its applicability in various fields of biomedical engineering such as programmed drug delivery. The development of advanced technologies for biomedical applications using PEM films has seen rapid progress recently. This review briefly summarises the recent successes of PEM being utilised for diverse bio-applications.

High precision measurement of intensity peak shifts in tunable cascaded microring intensity sensors.

We demonstrate a method to precisely track intensity peak shifts in tunable cascaded double-microring based refractive index sensors. Without modifications, width of the intensity peak of a tunable cascaded microring device limits the precision of peak-shift measurements and thereby the limit of detection of the sensor. We overcome this limitation by using dual harmonic lock-in detection for precisely determining the position of the intensity maximum. Using this modification, we have demonstrated a reduction in the full width at half-maximum (FWHM) of the intensity peak by a factor of over 1300. We show that such a reduction in FWHM of the peak curve can significantly improve the detection limit of a tunable cascaded microring-based sensor.

Subdiffraction-Resolution Optical Measurements of Molecular Transport in Thin Polymer Films.

The measurement of molecular transport within polymer films yields information about the internal structural organization of the films and is useful in applications such as the design of polymeric capsules for drug delivery. Layer-by-layer assembly of polyelectrolyte multilayer films has been widely used in such applications where the multilayer structure often exhibits anisotropic transport resulting in different diffusivities in the lateral (parallel to the film) and transverse (normal to the film) directions. Although lateral transport can be probed using techniques such as fluorescence recovery after photobleaching (FRAP), it cannot be applied to probing transverse diffusivity in polymer films smaller than the diffraction limit of light. Here we present a technique to probe the transport of molecules tagged with fluorphores in polymer films thinner than the optical diffraction limit using the modulation of fluorescence emission depending on the distance of the tagged molecules from a metal surface. We have used this technique to probe the diffusion of proteins biotin and bovine serum albumin (BSA) in polyelectrolyte multilayer films. We also studied the interdiffusion of chains in multilayer films using this technique. We observed a 3 order of magnitude increase in interdiffusion as a function of the ionic strength of the medium. This technique, along with FRAP, will be useful in studying anisotropic transport in polymer films, even those thinner than the diffraction limit, because the signal in this technique arises only from transverse and not lateral transport. Finally, this technique is also applicable to studying the diffusion of chromophore-labeled species within a polymer film. We demonstrate this aspect by measuring the transverse diffusion of methylene blue in the PAH-PAA multilayer system.

Bright-field Nanoscopy: Visualizing Nano-structures with Localized Optical Contrast Using a Conventional Microscope.

Most methods for optical visualization beyond the diffraction limit rely on fluorescence emission by molecular tags. Here, we report a method for visualization of nanostructures down to a few nanometers using a conventional bright-field microscope without requiring additional molecular tags such as fluorophores. The technique, Bright-field Nanoscopy, is based on the strong thickness dependent color of ultra-thin germanium on an optically thick gold film. We demonstrate the visualization of grain boundaries in chemical vapour deposited single layer graphene and the detection of single 40 nm Ag nanoparticles. We estimate a size detection limit of about 2 nm using this technique. In addition to visualizing nano-structures, this technique can be used to probe fluid phenomena at the nanoscale, such as transport through 2D membranes. We estimated the water transport rate through a 1 nm thick polymer film using this technique, as an illustration. Further, the technique can also be extended to study the transport of specific ions in the solution. It is anticipated that this technique will find use in applications ranging from single-nanoparticles resolved sensing to studying nanoscale fluid-solid interface phenomena.

Adaptive interferometry of protein on a BioCD.

Adaptive spinning-disk interferometry is capable of measuring surface profiles of a thin biolayer with subnanometer longitudinal resolution. High-speed phase modulation in the signal beam arises from the moving surface height profile on the spinning disk and is detected as a homodyne signal via dynamic two-wave mixing. A photorefractive quantum-well device performs as an adaptive mixer that compensates disk wobble and vibration while it phase-locks the signal and reference waves in the phase quadrature condition (pi/2 relative phase between the signal and local oscillator). We performed biosensing of immobilized monolayers of antibodies on the disk in both transmission and reflection detection modes. Single- and dual-analyte adaptive spinning-disk immunoassays were demonstrated with good specificity and without observable cross-reactivity. Reflection-mode detection enhances the biosensing sensitivity to one-twentieth of a protein monolayer, creates a topographic map of the protein layer, and can differentiate monolayers of different species by their effective optical thicknesses.

High-speed label-free detection by spinning-disk micro-interferometry.

Spinning-disk interferometers are a new class of analytic sensors to detect immobilized biomolecules with high speed and high sensitivity. The disks are composed of a large number of surface-normal self-referencing interferometers, analogous to an optical CD, but operating on the principle of microdiffraction quadrature that achieves sensitive linear detection of bound molecules. The surface-normal structures have a small footprint of only 20 microm each, allowing potential integration to over a million interferometric elements per disk. We have fabricated interferometric microstructures on silicon and on dielectric mirror disks to demonstrate the basic principles of the BioCD. We have detected the presence of immobilized anti-mouse IgG and the specific binding of 10 femtomol of mouse IgG at a sampling rate of 100 kilo-samples/s, while also demonstrating negligible non-specific binding. This technique provides a label-free method that could potentially screen hundreds to thousands of proteins per disk.