Fluidic operation of a polymer-based nanosensor chip for analysing single molecules

Most medical diagnostic tests are expensive, involve slow turnaround times from centralized laboratories and require highly specialized equipment with seasoned technicians to carry out the assay. To facilitate realization of precision medicine at the point of care, we have developed a mixed-scale nanosensor chip featuring high surface area pillar arrays where solid-phase reactions can be performed to detect and identify nucleic acid targets found in diseased patients. Products formed can be identified and detected using a polymer nanofluidic channel. To guide delivery of this platform, we discuss the operation of various components of the device and simulations (COMSOL) used to guide the design by investigating parameters such as pillar array loading, and hydrodynamic and electrokinetic flows. The fabrication of the nanosensor is discussed, which was performed using a silicon (Si) master patterned with a combination of focused ion beam milling and photolithography with deep reactive ion etching. The mixed-scale patterns were transferred into a thermoplastic via thermal nanoimprint lithography, which facilitated fabrication of the nanosensor chip making it appropriate for in vitro diagnostics. The results from COMSOL were experimentally verified for hydrodynamic flow using Rhodamine B as a fluorescent tracer and electrokinetic flow using single fluorescently labelled oligonucleotides (single-stranded DNAs, ssDNAs)

In-plane Extended Nano-coulter Counter (XnCC) for the Label-free Electrical Detection of Biological Particles

This is the peer reviewed version of the following article: Z. Zhao, S. Vaidyanathan, P. Bhanja, S. Gamage, S. Saha, C. McKinney, J. Choi, S. Park, T. Pahattuge, H. Wijerathne, J. M. Jackson, M. L. Huppert, M. A. Witek, S. A. Soper, Electroanalysis 2022, 34, 1961., which has been published in final form at https://doi.org/10.1002/elan.202200091. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.We report an in-plane extended nanopore Coulter counter (XnCC) chip fabricated in a thermoplastic via imprinting. The fabrication of the sensor utilized both photolithography and focused ion beam milling to make the microfluidic network and the in-plane pore sensor, respectively, in Si from which UV resin stamps were generated followed by thermal imprinting to produce the final device in the appropriate plastic (cyclic olefin polymer, COP). As an example of the utility of this in-plane extended nanopore sensor, we enumerated SARS-CoV-2 viral particles (VPs) affinity-selected from saliva and extracellular vesicles (EVs) affinity-selected from plasma samples secured from mouse models exposed to different ionizing radiation doses

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits

Mixed-scale nano-and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 mu m in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared via HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective T(g) was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: similar to 6% for PMMA and similar to 9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 +/- 0.7 x 10(-4) cm(2) V(-1) s(-1) and 7.6 +/- 0.6 x 10(-4) cm(2) V(-1) s(-1) for PMMA and COC, respectively, at a field strength of 25 V cm(-1). The extension factors for lambda-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2-6% standard deviation).close171

Microfluidic affinity selection of active SARS-CoV-2 virus particles

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus’s S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip’s surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent

Fabrication of polymeric dual-scale nanoimprint molds using a polymer stencil membrane

We report on a simple and effective process that allows fabricating polymeric dual-scale nanoimprinting molds. The key for the process is the use of a thin flexible SU-8 stencil membrane, which was fabricated by either photolithography or thermal nanoimprint lithography (NIL). The stencil membrane with microscale pores was assembled into a nanopatterned substrate, producing a dual-scale structure. The assembled structure was used as a template to produce polymeric imprinting molds via UV-NIL. With this method, we demonstrated dual-scale nanoimprint molds having nano-pillars of 251 nm diameter and 146 nm high on top of microscale square protrusions of 5 μm wide and 3.6 μm high. The resin mold with the dual-scale structure was successfully used to produce a freestanding membrane with dual-scale perforated pores via UV-NIL. After metal coating and integrated into microfluidic devices, this freestanding membrane can potentially be used as a substrate for surface plasmon resonance sensors

Selection of UV-resins for nanostructured molds for thermal-NIL

Nanoimprint molds made of soft polymeric materials have advantages of low demolding force and low fabrication cost over Si or metal-based hard molds. However, such advantages are often sacrificed by their reduced replication fidelity associated with the low mechanical strength. In this paper, we studied replication fidelity of different UV-resin molds copied from a Si master mold via UV nanoimprint lithography (NIL) and their thermal imprinting performance into a thermoplastic polymer. Four different UV-resins were studied: two were high surface energy UV-resins based on tripropyleneglycol diacrylate (TPGDA resin) and polypropyleneglycol diacrylate (PPGDA resin), and the other two were commercially available, low surface energy poly-urethane acrylate (PUA resin) and fluorine-containing (MD 700) UV-resins. The replication fidelity among the four UV-resins during UV nanoimprint lithograph from a Si master with sharp nanostructures was in the increasing order of (poorest) PUA resin \u3c MD 700 \u3c PPGDA resin \u3c TPGDA resin (best). The results show that the high surface energy and small monomer size are keys to achieving good UV-resin filling into sharp nanostructures over the viscosity of the resin solution. When the four UV-resin molds were used for thermal-NIL into a thermoplastic polymer, the replication fidelity was in the increasing order of (poorest) MD 700 \u3c TPGDA resin \u3c PUA resin (best), which follows the same order of their Young\u27s moduli. Our results indicate that the selection of an appropriate UV-resin for NIL molds requires consideration of the replication fidelities in the mold fabrication and the subsequent thermal-NIL into thermoplastic polymers

Surface Charge Density-Dependent DNA Capture through Polymer Planar Nanopores

Surface charge density of nanopore walls plays a critical role in DNA capture in nanopore-based sensing platforms. This paper studied the effect of surface charge density on the capture of double-stranded (ds) DNA molecules into a polymer planar nanopore numerically and experimentally. First, we simulated the effective driving force ( F) for the translocation of a dsDNA through a planar nanopore with different sizes and surface charge densities. Focus was given on the capture stage from the nanopore mouth into the nanopore by placing a rodlike DNA at the nanopore mouth rather than inside the nanopore. For negatively charged DNA and nanopore walls, electrophoretic driving force ( F) under an electric field is opposed by the viscous drag force by electroosmotic flow ( F). As the surface charge density of the nanopore wall becomes more negative, F exceeds F beyond a threshold surface charge density, σ, where DNA molecules cannot be driven through the nanopore via electrophoretic motion. For a 10 nm diameter nanopore filled with 1× TE buffer, σ was determined to be -50 mC/m. The simulation results were verified by performing dsDNA translocation experiments using a planar nanopore with 10 nm equivalent diameter imprinted on three polymer substrates with different surface charge densities. Both fluorescence observation and ionic current measurement confirmed that only nanopore devices with the surface charge density less negative than σ allowed DNA translocation, indicating that the simulated σ value can be used as a parameter to estimate the translocation of biopolymers in the design of nanopore devices

Scalable fabrication of sub-10 nm polymer nanopores for DNA analysis

Nanotechnology: New fabrication technique shrinks nanopores Researchers in the United States have developed a method to create nanopores smaller than 10 nanometers in a simple, controllable, cost-effective way. Sunggook Park’s team at Louisiana State University pressed an array of silicon microneedles into a polymer layer to produce pores of around 10 nm. The polymer is then briefly heated to make it reflow, causing the pores to shrink. The size of the nanopores can be controlled by regulating the duration of the reflow process. Using this technique with and without the reflow step, the team fabricated nanopores of 12 nm and 6 nm diameter, respectively, and used them to detect single DNA molecules in a solution passed through the pores. This new approach enables researchers to produce with high throughput nanopore devices with precisely positioned sub-10 nm pores which can be used to detect and analyze individual biomolecules