High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles.

We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2-3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103 particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(-). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

Modifying surface charge density of thermoplastic nanofluidic biosensors by multivalent cations within the slip plane of the electric double layer.

Thermoplastic nanofluidic devices are promising platforms for sensing single biomolecules due to their mass fabrication capability. When the molecules are driven electrokinetically through nanofluidic networks, surface charges play a significant role in the molecular capture and transportation, especially when the thickness of the electrical double layer is close to the dimensions of the nanostructures in the device. Here, we used multivalent cations to alter the surface charge density of thermoplastic nanofluidic devices. The surface charge alteration was done by filling the device with a multivalent ionic solution, followed by withdrawal of the solution and replacing it with KCl for conductance measurement. A systematic study was performed using ionic solutions containing Mg2+ and Al3+ for nanochannels made of three polymers: poly(ethylene glycol) diacrylate (PEGDA), poly(methyl methacrylate) (PMMA) and cyclic olefin copolymer (COC). Overall, multivalent cations within the slip plane decreased the effective surface charge density of the device surface and the reduction rate increased with the cation valency, cation concentration and the surface charge density of thermoplastic substrates. We demonstrated that a 10-nm diameter in-plane nanopore formed in COC allowed translocation of λ-DNA molecules after Al3+ modification, which is attributed to the deceased viscous drag force in the nanopore by the decreased surface charge density. This work provides a general method to manipulate surface charge density of nanofluidic devices for biomolecule resistive pulse sensing. Additionally, the experimental results support ion-ion correlations as the origin of charge inversion over specific chemical adsorption.

Label-Free Identification of Single Mononucleotides by Nanoscale Electrophoresis.

Nanoscale electrophoresis allows for unique separations of single molecules, such as DNA/RNA nucleobases, and thus has the potential to be used as single molecular sensors for exonuclease sequencing. For this to be envisioned, label-free detection of the nucleotides to determine their electrophoretic mobility (i.e., time-of-flight, TOF) for highly accurate identification must be realized. Here, for the first time a novel nanosensor is shown that allows discriminating four 2-deoxyribonucleoside 5'-monophosphates, dNMPs, molecules in a label-free manner by nanoscale electrophoresis. This is made possible by positioning two sub-10 nm in-plane pores at both ends of a nanochannel column used for nanoscale electrophoresis and measuring the longitudinal transient current during translocation of the molecules. The dual nanopore TOF sensor with 0.5, 1, and 5 µm long nanochannel column lengths discriminates different dNMPs with a mean accuracy of 55, 66, and 94%, respectively. This nanosensor format can broadly be applicable to label-free detection and discrimination of other single molecules, vesicles, and particles by changing the dimensions of the nanochannel column and in-plane nanopores and integrating different pre- and postprocessing units to the nanosensor. This is simple to accomplish because the nanosensor is contained within a fluidic network made in plastic via replication.

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels.

Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 µm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O3 activated and an aqueous buffer mobile phase.

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 < MD 700 < PPGDA resin < 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 < TPGDA resin < PUA resin (best), which follows the same order of their Young's 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.

Accurate, predictable, repeatable micro-assembly technology for polymer, microfluidic modules.

A method for the design, construction, and assembly of modular, polymer-based, microfluidic devices using simple micro-assembly technology was demonstrated to build an integrated fluidic system consisting of vertically stacked modules for carrying out multi-step molecular assays. As an example of the utility of the modular system, point mutation detection using the ligase detection reaction (LDR) following amplification by the polymerase chain reaction (PCR) was carried out. Fluid interconnects and standoffs ensured that temperatures in the vertically stacked reactors were within ± 0.2 C° at the center of the temperature zones and ± 1.1 C° overall. The vertical spacing between modules was confirmed using finite element models (ANSYS, Inc., Canonsburg, PA) to simulate the steady-state temperature distribution for the assembly. Passive alignment structures, including a hemispherical pin-in-hole, a hemispherical pin-in-slot, and a plate-plate lap joint, were developed using screw theory to enable accurate exactly constrained assembly of the microfluidic reactors, cover sheets, and fluid interconnects to facilitate the modular approach. The mean mismatch between the centers of adjacent through holes was 64 ± 7.7 µm, significantly reducing the dead volume necessary to accommodate manufacturing variation. The microfluidic components were easily assembled by hand and the assembly of several different configurations of microfluidic modules for executing the assay was evaluated. Temperatures were measured in the desired range in each reactor. The biochemical performance was comparable to that obtained with benchtop instruments, but took less than 45 min to execute, half the time.

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.

Patterned electromagnetic alignment of magnetic nanowires.

A combination of electromagnetic alignment and topological pattern assisted alignment to position magnetic nanowires, which is referred to as the Patterned Electromagnetic Alignment (PEA), is developed and examined. Electrodeposited, FeNiCo nanowires with different lengths were used as the test nanomaterial, and the microscale grooved surface was formed by UV nanoimprint lithography. The accuracy of the PEA with FeNiCo nanowires was evaluated by measuring the deviation angle from the direction of the magnetic field line for different magnetic field strengths and nanowire lengths, and a statistical alignment distribution was reported for different nanowire length groups. The results were compared with those of the electromagnetic alignment on flat surfaces and in grooved-patterned substrates without electromagnetic alignment. Overall, the deviation angle for the PEA was lower than that for the electromagnetic alignment when all other experimental conditions were identical, indicating that the alignment accuracy along the direction of the magnetic field lines was enhanced in the presence of surface micro grooves. This can be attributed to the fact that, upon attachment of nanowires to the substrate surface, the surface micro grooves in the PEA add additional deterministic characteristics to the otherwise stochastic nature of the nanowire deposition and solvent evaporation processes compared to the sole electromagnetic alignment.

Droplet Impinging Behavior on Surfaces with Wettability Contrasts.

Heterogeneous substrates with moderate and extreme wettability contrasts were fabricated by comprising of superhydrophobic/hydrophilic and superhydrophobic/extremely hydrophilic surfaces, respectively. The interactions of water droplets impinging on the surfaces with sharp wettability contrasts were investigated experimentally. The impinging droplets that slightly touch the hydrophilic or extremely hydrophilic areas on each substrate exhibit a directional rebounding towards the more wetting surfaces, i.e., hydrophilic or extremely hydrophilic surface. The trajectory and landing distance of the rebounded droplets were tailored by controlling the releasing height of the droplet, wetting contrast across the border, and portion of the droplet touching the more wetting surface of the substrates with wettability contrasts. The landing distance of the droplet increases with the increased releasing height and higher wettability contrast across the border. Increasing the portion of the impinging droplet touching the more wetting surface of the heterogeneous substrates leads to the shorter landing distance of rebounded droplets.

Effect of Different Fluids on Rectified Motion of Leidenfrost Droplets on Micro/Sub-Micron Ratchets.

Leidenfrost droplets, liquid droplets placed on a hot flat surface above the Leidenfrost temperature of the liquid, are an interesting model system to understand and achieve frictionless motion of droplets on a surface. Controlled unidirectional motion of otherwise random Leidenfrost droplets can be achieved by replacing the flat surface by a surface with topological ratchets. In this study, we show how an increase in the vapor layer thickness below the Leidenfrost droplet influences the droplet motion for underlying ratchets with various periods ranging from 1.5mm down to 800nm. This was exploited by systematically studying the Leidenfrost droplet motion of various liquids with low boiling points including acetone, isopropanol, and R134a on the aforementioned various ratchets. For all liquids with boiling points lower than water, no unidirectional motion was observed for 800 nm. This indicates that the asymmetric vapor flow beneath the Leidenfrost droplet becomes negligible due to the large vapor layer thickness relative to the ratchet depth. However, unidirectional droplet motion was still observed for the micron and millimeter scale ratchets even when the ratchet surface temperature was increased up to 360°C and 230°C for acetone and isopropanol, respectively. This can be attributed to the insulating property of the thick vapor layer which prevent the droplet from producing more vapor with increasing temperature. We also report the effect of the ratchet period on the droplet motion at room temperature using R134a droplets.

Surface charge, electroosmotic flow and DNA extension in chemically modified thermoplastic nanoslits and nanochannels.

Thermoplastics have become attractive alternatives to glass/quartz for microfluidics, but the realization of thermoplastic nanofluidic devices has been slow in spite of the rather simple fabrication techniques that can be used to produce these devices. This slow transition has in part been attributed to insufficient understanding of surface charge effects on the transport properties of single molecules through thermoplastic nanochannels. We report the surface modification of thermoplastic nanochannels and an assessment of the associated surface charge density, zeta potential and electroosmotic flow (EOF). Mixed-scale fluidic networks were fabricated in poly(methylmethacrylate), PMMA. Oxygen plasma was used to generate surface-confined carboxylic acids with devices assembled using low temperature fusion bonding. Amination of the carboxylated surfaces using ethylenediamine (EDA) was accomplished via EDC coupling. XPS and ATR-FTIR revealed the presence of carboxyl and amine groups on the appropriately prepared surfaces. A modified conductance equation for nanochannels was developed to determine their surface conductance and was found to be in good agreement with our experimental results. The measured surface charge density and zeta potential of these devices were lower than glass nanofluidic devices and dependent on the surface modification adopted, as well as the size of the channel. This property, coupled to an apparent increase in fluid viscosity due to nanoconfinement, contributed to the suppression of the EOF in PMMA nanofluidic devices by an order of magnitude compared to the micro-scale devices. Carboxylated PMMA nanochannels were efficient for the transport and elongation of λ-DNA while these same DNA molecules were unable to translocate through aminated nanochannels.

Polymer Stamps for Imprinting Nanopatterns in Polymer Substrate.

Using a silicone or metallic stamp for imprinting multiscale patterns comprising micro down to nanoscale patterns into polymer substrates often results in significant deformation in the molded substrate and loss of pattern transfer fidelity for nanopatterns. In the worst case, the expensive stamp can also be damaged. One method to reduce the problem is to use polymer as the stamp material, which will reduce both adhesion and thermal stress generated at the stamp/substrate interface. In this paper, stamps made of three different polymer materials, i.e., polydimethylsiloxane (PDMS), PPGDA-based UV resin and TPGDA-based UV-resin, were fabricated from the same master containing nanofluidic structures and the replication fidelity from the master, polymer stamps, to thermal-imprinted poly(methyl methacrylate) substrate (PMMA) was compared. The largest loss of pattern fidelity occurs in the thermal imprinting step. Polymer stamps with higher Young's moduli result in a better fidelity in pattern transfer. With TPGDA-based UV resin stamps, multiscale structures with a nanochannel with minimum width and height of -70 nm can be imprinted onto PMMA substrate together with macro-scale patterns by a single nanoimprinting processes.

Synthesis and Characterization of ZIF-7 Membranes by In Situ Method.

Zeolitic imidazolate frameworks (ZIFs) have been the focus of interest in adsorption, catalysis, and membrane applications due to their superior thermal and chemical stability, tunable microporous channels, and tailorable physical/chemical properties. In this study, ZIF-7 membranes were successfully prepared on macroporous a-alumina substrate by in situ solvothermal method, without the necessity of seeding or surface modification step. Addition of sodium formate during the reaction facilitates continuous well-intergrown crystalline ZIF-7 layer. As-synthesized ZIF-7 membrane was characterized by XRD, FE-SEM and gas permeation test. The H2 permeance through 5 µm ZIF-7 membrane was 1.9 x 10(-7) mol/m2 x s x Pa with ideal selectivity of H2/CO2 = 15.2.

A Simplified Model for the Study of Film-Boiling Droplet Motion on Microscale Ratchets.

In this work, we explore a simplified model based on both analytical and computational methods for the study of film-boiling droplet motion on microscale ratchets. We consider a specific ratchet design with the length periods and depth of ratchets much smaller than the size of the droplet. We conclude based on our modeling that for the ratchet configuration considered in this paper, the conduction within the vapor film is the dominant means of heat transfer in comparison with convection and radiation. Furthermore, we demonstrate a more manageable two-dimensional model in which analytical approaches coupled with computational approaches yield reasonably accurate results in comparison to the actual experiments.

Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 µm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Fabrication of perforated micro/nanopore membranes via a combination of nanoimprint lithography and pressed self-perfection process for size reduction.

Artificial membranes with perforated nanopores in defined locations provide an important biomimicking platform for sensing and analysis of biomolecules. This study presents a simple and flexible method to fabricate a freestanding polymer membrane with perforated micro- and nanopores using a combination of nanoimprint lithography (NIL) and pressed self-perfection (PSP) process. NIL was used to define initial pores of a few micrometers in diameter in the SU-8 membrane layer, which was followed by the PSP process where the patterned SU-8 membrane is pressed with a blank silicon wafer at an elevated temperature. This PSP process results in a lateral flow of the SU-8 resist and consequently reduces the pore size in the membrane. With this method, we have demonstrated fabrication of a SU-8 membrane with perforated pores down to approximately 300 nm in diameter. The results indicate that by employing a pore reduction process nanopore membranes can be manufactured without requirements of having a stamp with nanoscale structures and high aspect ratio imprinting with the stamp.

Engineering inlet structures to enhance DNA capture into nanochannels in a polymer nanofluidic device produced via nanoimprint lithography.

Operating nanofluidic biosensors requires threading single molecules to be analyzed from microfluidic networks into nanostructures, mostly nanochannels or nanopores. Different inlet structures have been employed as a means of enhancing the number of the capture events into nanostructures. Here, we systematically investigated the effects of various engineered inlet structures formed at the micro/nanochannel interface on the capture of single λ-DNA molecules into the nanochannels. Different inlet geometries were evaluated and ranked in order of their effectiveness. Adding an inlet structure prior to a nanochannel effectively improved the DNA capture rate by 190 - 700 % relative to that for the abrupt micro/nanochannel interface. The capture of DNA from the microchannel to various inlets was determined mainly by the capture volumes of the inlet structures and the geometrically modified electric field in the inlet structure. However, as the width of the inlet structure increased, the hydrodynamic flow existing in the microchannel negatively influenced the DNA capture by dragging some DNA molecules deep into the inlet structure back to the microchannel. Our results indicate that engineering inlet structures is an effective means of controlling the capture of DNA molecules into nanostructures, which is important for operation of nanofluidic biosensors.

Nano-injection molding with resin mold inserts for prototyping of nanofluidic devices for single molecular detection.

While injection molding is becoming the fabrication modality of choice for high-scale production of microfluidic devices, especially those used for in vitro diagnostics, its translation into the growing area of nanofluidics (structures with at least one dimension <100 nm) has not been well established. Another prevailing issue with injection molding is the high startup costs and the relatively long time between device iterations making it in many cases impractical for device prototyping. We report, for the first time, functional nanofluidic devices with dimensions of critical structures below 30 nm fabricated by injection molding for the manipulation, identification, and detection of single molecules. UV-resin molds replicated from Si masters served as mold inserts, negating the need for generating Ni-mold inserts via electroplating. Using assembled devices with a cover plate via hybrid thermal fusion bonding, we demonstrated two functional thermoplastic nanofluidic devices. The first device consisted of dual in-plane nanopores placed at either end of a nanochannel and was used to detect and identify single ribonucleotide monophosphate molecules via resistive pulse sensing and obtain the effective mobility of the molecule through nanoscale electrophoresis to allow its identification. The second device demonstrated selective binding of a single RNA molecule to a solid phase bioreactor decorated with a processive exoribonuclease, XRN1. Our results provide a simple path towards the use of injection molding for device prototyping in the development stage of any nanofluidic or even microfluidic application, through which rapid scale-up is made possible by transitioning from prototyping to high throughput production using conventional Ni mold inserts.

A simulation study on the effect of cross-linking agent concentration for defect tolerant demolding in UV nanoimprint lithography.

The chemistry and composition of UV-sensitive resists are key factors determining the stress in the molded resist structure in UV nanoimprint lithography (UV-NIL) and thus the success of the process. The stress in the molded structure is mainly generated due to shrinkage of the resist in the UV curing step and also adhesion and friction at the stamp/resist interface in the subsequent demolding step. Thus, understanding of the stress generated in these steps is critical to the improvement of the process as well as the development of new UV resists. In this paper the effect of resist composition on the stress generation was studied by numerical simulations of the curing and demolding steps in UV-NIL. Parameters required for the simulation, such as resist shrinkage, Young's modulus, fracture strength, friction coefficient, crack initiation stress, and debonding energy, were determined experimentally for different resist compositions. As the cross-linking agent concentration increases the fracture strength also improves. In addition, as more cross-linking agent is added to the resist composition, both shrinkage stress due to the curing and also adhesion at the stamp/resist interface increase resulting in a larger maximum local stress experienced by the resist on demolding. By normalizing the overall maximum local stress by the fracture stress of the resist, we found that there is an optimum for the cross-linking agent concentration that leads to the most successful imprinting. Our finding is also corroborated by qualitative experimentations performed for UV-NIL with various resist compositions.

Replication of a thin polydimethylsiloxane stamp and its application to dual-nanoimprint lithography for 3D hybrid nano/micropatterns.

This paper presents the fabrication of a thin and flexible polydimethylsiloxane (PDMS) stamp with a thickness of a few tens of um and its application to nanoimprint lithography (NIL). The PDMS material generally has a low elastic modulus and high adhesive characteristics. Therefore, after being treated, the thin PDMS stamp is easily deformed and torn, adhering to itself and other materials. This paper introduces the use of a metal ring around the flange of a thin PDMS stamp to assist with the handling of this material. A PDMS stamp with a motheye pattern in nanometer scale was inserted between a substrate and a microstamp with concave patterns in micrometer scale. Subsequently, three-dimensional (3D) hybrid nano/micropatterns were fabricated by pressing these two stamps and curing the resist. The fabricated hybrid patterns were measured and verified in both the microscale and nanoscale. The process, termed "dual NIL," can be applied to the fabrication of optical components or bio-sensors that require repetitive nanopatterns on micropatterns.