Associations between short-term exposure to air pollution and thyroid function in a representative sample of the Korean population.

BACKGROUND: Disruption of thyroid function can profoundly affect various organ systems. However, studies on the association between air pollution and thyroid function are relatively scarce and most studies have focused on the long-term effects of air pollution among pregnant women. OBJECTIVES: This study aimed to explore the associations between short-term exposure to air pollution and thyroid function in the general population. METHODS: Data from the Korea National Health and Nutrition Examination Survey (2013-2015) were analyzed (n = 5,626). Air pollution concentrations in residential addresses were estimated using Community Multiscale Air Quality models. The moving averages of air pollution over 7 days were set as exposure variables through exploratory analyses. Linear regression and quantile g-computation models were constructed to assess the effects of individual air pollutants and air pollution mixture, respectively. RESULTS: A 10-ppb increase in NO2 (18.8-µg/m3 increase) and CO (11.5-µg/m3 increase) was associated with 2.43% [95% confidence interval (CI): 0.42, 4.48] and 0.19% (95% CI: 0.01, 0.36) higher thyroid-stimulating hormone (TSH) levels, respectively. A 10-µg/m3 increase in PM2.5 and a 10-ppb increase in O3 (19.6-µg/m3 increment) were associated with 0.87% (95% CI: 1.47, -0.27) and 0.59% (95% CI: 1.18, -0.001) lower free thyroxine (fT4) levels, respectively. A simultaneous quartile increase in PM2.5, NO2, O3, and CO levels was associated with lower fT4 but not TSH levels. CONCLUSIONS: As the subtle changes in thyroid function can affect various organ systems, the present results may have substantial public health implications despite the relatively modest effect sizes. Because this was a cross-sectional study, it is necessary to conduct further experimental or repeated-measures studies to consolidate the current results.

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.

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.

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.

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.

Low cost fabrication of a superhydrophobic V-grooved polymer surface.

Engineering of polymer surfaces to control their wetting properties has shown a wide range of potential applications. In this paper we show low cost fabrication of a superhydrophobic polymer surface via a hierarchical combination of hot embossing, O2 reactive ion etching (RIE) and deposition of a hydrophobic silane. The hot embossing and O2 RIE were used to produce respective micro and nanoscale surface roughness which is a requirement for obtaining superhydrophobic surfaces, while the deposition of a hydrophobic silane modified surface chemistry. In order to increase the water/air interface in the Cassie-Baxter composite wetting model, a brass mold with microscale V-grooves was used for hot embossing. Images of droplets in both static water contact angles and dynamic impact tests with the surface clearly show that the wetting state follows the Cassie-Baxter wetting model. The results of this study indicate that our design of the dual level surface roughness and the fabrication process allow for low cost and easy production of a highly superhydrophobic surface.

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.

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.

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.

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.

Flexible-templated imprinting for fluorine-free, omniphobic plastics with re-entrant structures.

HYPOTHESIS: Compared to vertical micro-pillars, re-entrant micro-structures exhibited superior omniphobicity for suspending liquids to Cassie-Baxter state. However, the existing re-entrant structures rely on complex multi-step deposition and etching procedures. The conventional, rigid-templated imprinting would instead damage the re-entrant structures. This leads to the question: is it possible to preserve the re-entrant curvatures by a flexible-templated imprinting? EXPERIMENTS: We facilely imprinted the re-entrant structures on a plastic substrate using a flexible nylon-mesh template. The effect of imprinting time (15-35 min), temperature (110-120 °C) and pressure (15-50 Bar) was investigated. To further improve the liquid-repellency and abrasion resistance, the silica nanoparticles (30-650 nm) along with epoxy resin binder (10 mg/mL) were pre-coated. FINDINGS: A one-step imprinting is sufficient to fabricate the re-entrant structures by utilizing flexible nylon-mesh template, without damaging the imprinted structures after the demolding process. The pre-coated silica nanoparticles and epoxy resin (1) improved liquid repellency by introducing hierarchical surface structures (e.g. contact angle hysteresis of olive oil reduced > 10°), and (2) acted as a protective layer against mechanical abrasion (omniphobicity maintained after 25 cycles, ~1.6 kPa sand paper abrasion). Additionally, the fluorine-free post-treatment was sufficient for the omniphobicity on the obtained plastic structures.

Electrokinetic identification of ribonucleotide monophosphates (rNMPs) using thermoplastic nanochannels.

With advances in the design and fabrication of nanofluidic devices during the last decade, there have been a few reports on nucleic acid analysis using nanoscale electrophoresis. The attractive nature of nanofluidics is the unique phenomena associated with this length scale that are not observed using microchip electrophoresis. Many of these effects are surface-related and include electrostatics, surface roughness, van der Waals interactions, hydrogen bonding, and the electric double layer. The majority of reports related to nanoscale electrophoresis have utilized glass-based devices, which are not suitable for broad dissemination into the separation community because of the sophisticated, time consuming, and high-cost fabrication methods required to produce the relevant devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm, depth x width) for the free solution electrokinetic analysis of ribonucleotide monophosphates (rNMPs). Thermoplastic devices with micro- and nanofluidic networks were fabricated using nanoimprint lithography (NIL) with the structures enclosed via thermal fusion bonding of a cover plate to the fluidic substrate. Unique to this report is that we fabricated devices in cyclic olefin copolymer (COC) that was thermally fusion bonded to a COC cover plate. Results using COC/COC devices were compared to poly(methyl methacrylate), PMMA, devices with a COC cover plate. Our results indicated that at pH = 7.9, the electrophoresis in free solution resulted in an average resolution of the rNMPs >4 (COC/COC device range = 1.94 - 8.88; PMMA/COC device range = 1.4 - 7.8) with some of the rNMPs showing field-dependent electrophoretic mobilities. Baseline separation of the rNMPs was not possible using PMMA- or COC-based microchip electrophoresis. We also found that COC/COC devices could be assembled and UV/O3 activated after device assembly with the dose of the UV/O3 affecting the magnitude of the electroosmotic flow, EOF. In addition, the bond strength between the substrate and cover plate of unmodified COC/COC devices was higher compared to PMMA/COC devices. The large differences in the electrophoretic mobilities of the rNMPs afforded by nanoscale electrophoresis will enable a new single-molecule sequencing platform we envision, which uses molecular-dependent electrophoretic mobilities to identify the constituent rNMPs generated from an intact RNA molecule using a processive exonuclease. With optimized nanoscale electrophoresis, the rNMPs could be identified via mobility matching at an accuracy >99% in both COC/COC and PMMA/COC devices.

Thermoplastic nanofluidic devices for identifying abasic sites in single DNA molecules.

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy of many drugs, such as doxorubicin. While there are different assays to search for DNA damage, they are fraught with limitations, such as the need for large amounts of DNA secured from millions of cells. This is challenging due to the growing importance of using liquid biopsies as a source of biomarkers for many in vitro diagnostic assays. To accommodate the mass limits imposed by the use of liquid biopsies, we report a single-molecule DNA damage assay that uses plastic nanofluidic chips to stretch DNA to near its full contour length when the channel dimensions (width and depth) are near the persistence length (∼50 nm) of double-stranded (ds) DNA. The nanofluidic chip consisted of input funnels for high loading efficiency of single DNA molecules, entropic traps to store the DNA and simultaneously load a series of nanochannels for high throughput processing, and an array of stretching nanochannels to read the AP sites. Single dsDNA molecules, which were labeled with an intercalating dye and a biotinylated aldehyde reactive probe (bARP), could be parked in the stretching nanochannels, where the AP sites were read directly using a dual-color fluorescence microscope equipped with an EMCCD camera. One color of the microscope was used to read the DNA length and the second color detected the AP sites. The nanofluidic chip was made from thermoplastics via nanoimprint lithography, which obviated the need for direct writing the devices in glass or quartz using focused ion beam milling. We show that we can read the frequency of AP sites in single dsDNA molecules with the frequency of AP sites determined by associating fluorescently-labeled streptavidin with bARP through a biotin/streptavidin complex.