Multifunctional magnetic nanoparticle cloud assemblies for in situ capture of bacteria and isolation of microbial DNA.

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for in situ bacterial capture and DNA extraction. M-clouds are obtained as a result of magnetic field density variations when magnetizing an array of micropillars coated with a soft ferromagnetic NiP layer. Numerical simulations suggest that the gradient in the magnetic field created by the pillars is four orders of magnitude higher than the gradient generated by the external magnets. The pillars therefore serve as the sole magnetic capture sites for MNPs which accumulate on opposite sides of each pillar facing the magnets. Composed of loosely aggregated MNPs, the M-cloud can serve as a porous capture matrix for target analyte flowing through the array. The concept is demonstrated by using a multifunctional M-cloud comprising immunomagnetic NPs (iMNPs) for capture of Escherichia coli O157:H7 from river water along with silica-coated NPs for subsequent isolation and purification of microbial DNA released upon bacterial lysis. Confocal microscopy imaging of fluorescently labeled iMNPs and E. coli O157:H7 reveals that bacteria are trapped in the M-cloud region between micropillars. Quantitative assessment of in situ bacterial capture, lysis and DNA isolation using real-time polymerase chain reaction shows linear correlation between DNA output and input bacteria concentration, making it possible to confirm E. coli 0157:H7 at 103 cells per mL. The M-cloud method further provides one order of magnitude higher DNA output concentrations than incubation of the sample with iMNPs in a tube for an equivalent period of time (e.g., 10 min). Results from assays performed in the presence of Listeria monocytogenes (at 106 cells per mL each) suggest that non-target organisms do not affect on-chip E. coli capture, DNA extraction efficiency and quality of the eluted sample.

Polymer Micropillar Arrays for Colorimetric DNA Detection.

We describe the use of periodic micropillar arrays, produced from cyclic olefin copolymer using high-fidelity microfabrication, as templates for colorimetric DNA detection. The assay involves PCR-amplified gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) incorporating a detectable digoxigenin label, which is revealed through an immunoenzymatic process following hybridization with target-specific oligonucleotide capture probes. The capacity of micropillar arrays to induce wicking is used to distribute and confine capture probes with spatial control, making it possible to achieve a uniform signal while allowing multiple, independent probes to be arranged in close proximity on the same substrate. The kinetic profile of color pigment formation on the surface was followed using absorbance measurements, showing maximum signal increase between 20 and 60 min of reaction time. The relationship between microstructure and colorimetric signal was investigated through variation of geometric parameters, such as pitch (10-50 µm), pillar diameter (5-40 µm), and height (16-48 µm). Our findings suggest that signal intensity is largely influenced by the edges of the pillars and less by their height such that it deviates from a linear relationship when both aspect ratio and pillar density become very high. A theoretical model used to simulate the changes in surface composition at the molecular level suggests that differences in the temporal and spatial accumulation of assay components account for this observation.

Microfluidic Integration of a Cloth-Based Hybridization Array System (CHAS) for Rapid, Colorimetric Detection of Enterohemorrhagic Escherichia coli (EHEC) Using an Articulated, Centrifugal Platform.

We describe the translation of a cloth-based hybridization array system (CHAS), a colorimetric DNA detection method that is used by food inspection laboratories for colony screening of pathogenic agents, onto a microfluidic chip format. We also introduce an articulated centrifugal platform with a novel fluid manipulation concept based on changes in the orientation of the chip with respect to the centrifugal force field to time the passage of multiple components required for the process. The platform features two movable and motorized carriers that can be reoriented on demand between 0 and 360° during stage rotation. Articulation of the chip can be used to trigger on-the-fly fluid dispensing through independently addressable siphon structures or to relocate solutions against the centrifugal force field, making them newly accessible for downstream transfer. With the microfluidic CHAS, we achieved significant reduction in the size of the cloth substrate as well as the volume of reagents and wash solutions. Both the chip design and the operational protocol were optimized to perform the entire process in a reliable, fully automated fashion. A demonstration with PCR-amplified genomic DNA confirms on-chip detection and identification of Escherichia coli O157:H7 from colony isolates in a colorimetric multiplex assay using rfbO157, fliCH7, vt1, and vt2 genes.

Microfluidic filtration and extraction of pathogens from food samples by hydrodynamic focusing and inertial lateral migration.

Detecting pathogenic bacteria in food or other biological samples with lab-on-a-chip (LOC) devices requires several sample preparation steps prior to analysis which commonly involves cleaning complex sample matrices of large debris. This often underestimated step is important to prevent these larger particles from clogging devices and to preserve initial concentrations when LOC techniques are used to concentrate or isolate smaller target microorganisms for downstream analysis. In this context, we developed a novel microfluidic system for membrane-free cleaning of biological samples from debris particles by combining hydrodynamic focusing and inertial lateral migration effects. The microfluidic device is fabricated using thermoplastic elastomers being compatible with thermoforming fabrication techniques leading to low-cost single-use devices. Microfluidic chip design and pumping protocols are optimized by investigating diffusive losses numerically with coupled Navier-Stokes and convective-diffusion theoretical models. Stability of inertial lateral migration and separation of debris is assessed through fluorescence microscopy measurements with labelled particles serving as a model system. Efficiency of debris cleaning is experimentally investigated by monitoring microchip outlets with in situ optical turbidity sensors, while retention of targeted pathogens (i.e., Listeria monocytogenes) within the sample stream is assessed through bacterial culture techniques. Optimized pumping protocols can remove up to 50 % of debris from ground beef samples while percentage for preserved microorganisms can account for 95 % in relatively clean samples. However, comparison between inoculated turbid and clean samples (i.e., with and without ground beef debris) indicate some degree of interference between debris inertial lateral migration and hydrodynamic focusing of small microorganisms. Although this interference can lead to significant decrease in chip performance through loss of target bacteria, it remains possible to reach 70 % for sample recovery and more than 50 % for debris removal even in the most turbid samples tested. Due to the relatively simple design, the robustness of the inertial migration effect itself, the high operational flow rates and fabrication methods that leverage low-cost materials, the proposed device can have an impact on a wide range of applications where high-throughput separation of particles and biological species is of interest.

Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2.

Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per µL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per µL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results.

Use of Polymer Micropillar Arrays as Templates for Solid-Phase Immunoassays.

We investigate the use of periodic micropillar arrays produced by high-fidelity microfabrication with cyclic olefin polymers for solid-phase immunoassays. These three-dimensional (3D) templates offer higher surface-to-volume ratios than two-dimensional substrates, making it possible to attach more antibodies and so increase the signal obtained by the assay. Micropillar arrays also provide the capacity to induce wicking, which is used to distribute and confine antibodies on the surface with spatial control. Micropillar array substrates are modified by using oxygen plasma treatment, followed by grafting of (3-aminopropyl)triethoxysilane for binding proteins covalently using glutaraldehyde as a cross-linker. The relationship between microstructure and fluorescence signal was investigated through variation of pitch (10-50 µm), pillar diameter (5-40 µm), and pillar height (5-57 µm). Our findings suggest that signal intensity scales proportionally with the 3D surface area available for performing solid-phase immunoassays. A linear relationship between fluorescence intensity and microscale structure can be maintained even when the aspect ratio and pillar density both become very high, opening the possibility of tuning assay response by design such that desired signal intensity is obtained over a wide dynamic range compatible with different assays, analyte concentrations, and readout instruments. We demonstrate the versatility of the approach by performing the most common immunoassay formats-direct, indirect, and sandwich-in a qualitative fashion by using colorimetric and fluorescence-based detection for a number of clinically relevant protein markers, such as tumor necrosis factor alpha, interferon gamma (IFN-γ), and spike protein of severe acute respiratory syndrome coronavirus 2. We also show quantitative detection of IFN-γ in serum using a fluorescence-based sandwich immunoassay and calibrated samples with spike-in concentrations ranging from 50 pg/mL to 5 µg/mL, yielding an estimated limit of detection of ∼1 pg/mL for arrays with high micropillar density (11561 per mm2) and aspect ratio (1:11.35).

Printing of sub-20 nm wide graphene ribbon arrays using nanoimprinted graphite stamps and electrostatic force assisted bonding.

Nano-graphene ribbons are promising in many electronic applications, as their bandgaps can be opened by reducing the widths, e.g. below 20 nm. However, a high-throughput method to pattern large-area nano-graphene features is still not available. Here we report a fabrication method of sub-20 nm ribbons on graphite stamps by nanoimprint lithography and a transfer-printing of the graphene ribbons to a Si wafer using electrostatic force assisted bonding. These methods provide a path for fast and high-throughput nano-graphene device production.

Hydrodynamic metamaterials: microfabricated arrays to steer, refract, and focus streams of biomaterials.

We show that it is possible to direct particles entrained in a fluid along trajectories much like rays of light in classical optics. A microstructured, asymmetric post array forms the core hydrodynamic element and is used as a building block to construct microfluidic metamaterials and to demonstrate refractive, focusing, and dispersive pathways for flowing beads and cells. The core element is based on the concept of deterministic lateral displacement where particles choose different paths through the asymmetric array based on their size: Particles larger than a critical size are displaced laterally at each row by a post and move along the asymmetric axis at an angle to the flow, while smaller particles move along streamline paths. We create compound elements with complex particle handling modes by tiling this core element using multiple transformation operations; we show that particle trajectories can be bent at an interface between two elements and that particles can be focused into hydrodynamic jets by using a single inlet port. Although particles propagate through these elements in a way that strongly resembles light rays propagating through optical elements, there are unique differences in the paths of our particles as compared with photons. The unusual aspects of these modular, microfluidic metamaterials form a rich design toolkit for mixing, separating, and analyzing cells and functional beads on-chip.

Fabrication of a 60-nm-diameter perfectly round metal-dot array over a large area on a plastic substrate using nanoimprint lithography and self-perfection by liquefaction.

Typically, nanopatterning on plastic substrates has poor fidelity, poor adhesion, and low yield. Here the proposal of and the first experiment using a new fabrication method that overcomes the above obstacles and has achieved arrays of 60-nm-diameter, perfectly round metal dots over a large area on a polyethylene terephthalate (PET) substrate with high fidelity and high yield is reported. This new method is based on the use of a thin hydrogen silsesquioxane (HSQ) layer on top of PET, nanoimprint lithography, and self-perfection by liquefaction (SPEL). The HSQ layer offers excellent thermal protection to the PET substrate during SPEL, as well as good surface adhesion and etching resistance. Nanoimprinting plus a lift off created a large-area array of Cr squares (100 nm x 130 nm) on HSQ and SPEL changed each Cr square into a perfectly round Cr dot with a diameter of 60 nm, reducing the Cr footprint area by 78%. Compared to bare PET, the use of HSQ also reduced the variation in the diameter of the Cr dots from 11.3 nm (standard deviation) to 1.7 nm, an improvement of over 660%. This new technology can be scaled to much larger areas (including roll-to-roll web processing) and thus potentially has applications in various fields.

Crossing microfluidic streamlines to lyse, label and wash cells.

We present a versatile method for continuous-flow, on-chip biological processing of cells, large bio-particles, and functional beads. Using an asymmetric post array in pressure-driven microfluidic flow, we can move particles of interest across multiple, independent chemical streams, enabling sequential chemical operations. With this method, we demonstrate on-chip cell treatments such as labeling and washing, and bacterial lysis and chromosomal extraction. The washing capabilities of this method are particularly valuable because they allow many analytical or treatment procedures to be cascaded on a single device while still effectively isolating their reagents from cross-contamination.

Microfluidic device for label-free measurement of platelet activation.

In this work we demonstrate a new microfluidic method for the rapid assessment of platelet size and morphology in whole blood. The device continuously fractionates particles according to size by displacing them perpendicularly to the fluid flow direction in a micro-fabricated post array. Whole blood, labeled with the fluorescent, platelet specific, antibody PE-anti-CD41, was run through the device and the positions of fluorescent objects noted as they exited the array. From this, histograms of platelet size were created which show marked increases in size after exposure to thrombin or a temperature of 4 degrees C. We infer that the well known morphological changes that occur during activation are causing the observed increase in size.

Twin tubular pinch effect in curving confined flows.

Colloidal suspensions of buoyancy neutral particles flowing in circular pipes focus into narrow distributions near the wall due to lateral migration effects associated with fluid inertia. In curving flows, these distributions are altered by Dean currents and the interplay between Reynolds and Dean numbers is used to predict equilibrium positions. Here, we propose a new description of inertial lateral migration in curving flows that expands current understanding of both focusing dynamics and equilibrium distributions. We find that at low Reynolds numbers, the ratio δ between lateral inertial migration and Dean forces scales simply with the particle radius, coil curvature and pipe radius as (Rp(3)R)/a(4). A critical value δc = 0.148 of this parameter is identified along with two related inertial focusing mechanisms. In the regime below δc, coined subcritical, Dean forces generate permanently circulating, twinned annuli, each with intricate equilibrium particle distributions including eyes and trailing arms. At δ > δc (supercritical regime) inertial lateral migration forces are dominant and particles focus to a single stable equilibrium position.

Sub-10 nm self-enclosed self-limited nanofluidic channel arrays.

We report a new method to fabricate self-enclosed optically transparent nanofluidic channel arrays with sub-10 nm channel width over large areas. Our method involves patterning nanoscale Si trenches using nanoimprint lithography (NIL), sealing the trenches into enclosed channels by ultrafast laser pulse melting and shrinking the channel sizes by self-limiting thermal oxidation. We demonstrate that 100 nm wide Si trenches can be sealed and shrunk to 9 nm wide and that lambda-phage DNA molecules can be effectively stretched by the channels.

Wafer-scale patterning of sub-40 nm diameter and high aspect ratio (>50:1) silicon pillar arrays by nanoimprint and etching.

We demonstrate wide-area fabrication of sub-40 nm diameter, 1.5 µm tall, high aspect ratio silicon pillar arrays with straight sidewalls by combining nanoimprint lithography (NIL) and deep reactive ion etching (DRIE). Imprint molds were used to pre-pattern nanopillar positions precisely on a 200 nm square lattice with long range order. The conventional DRIE etching process was modified and optimized with reduced cycle times and gas flows to achieve vertical sidewalls; with such techniques the pillar sidewall roughness can be reduced below 8 nm (peak-to-peak). In some cases, sub-50 nm diameter pillars, 3 µm tall, were fabricated to achieve aspect ratios greater than 60:1.

Single sub-20 nm wide, centimeter-long nanofluidic channel fabricated by novel nanoimprint mold fabrication and direct imprinting.

We report and demonstrate a new method to fabricate single fluidic-channels of uniform channel width (11-50 nm) and over 1.5 cm in length, which are essential to developing innovative bio/chemical sensors but have not been fabricated previously. The method uses unconventional nanofabrication (a combination of crystallographic anisotropic etching, conformal coating, and edge patterning, etc.) to create an imprint mold of a channel pattern and nanoimprint to duplicate such channel. The centimeter-long channel continuity is verified by flowing fluorescent dye-stained water and stretching and transporting DNAs. The 18 by 20 nm channel cross-section was confirmed by measuring the liquid conductance in the channel.

Deterministic hydrodynamics: taking blood apart.

We show the fractionation of whole blood components and isolation of blood plasma with no dilution by using a continuous-flow deterministic array that separates blood components by their hydrodynamic size, independent of their mass. We use the technology we developed of deterministic arrays which separate white blood cells, red blood cells, and platelets from blood plasma at flow velocities of 1,000 microm/sec and volume rates up to 1 microl/min. We verified by flow cytometry that an array using focused injection removed 100% of the lymphocytes and monocytes from the main red blood cell and platelet stream. Using a second design, we demonstrated the separation of blood plasma from the blood cells (white, red, and platelets) with virtually no dilution of the plasma and no cellular contamination of the plasma.

Statics and dynamics of single DNA molecules confined in nanochannels.

The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics.