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.

Evaporation-Driven Water-in-Water Droplet Formation.

We present new observations of aqueous two-phase system (ATPS) thermodynamic and interfacial phenomena that occur inside sessile droplets due to water evaporation. Sessile droplets that contain polymeric solutions, which are initially in equilibrium in a single phase, are observed at their three-phase liquid-solid-air contact line. As evaporation of a sessile droplet proceeds, we find that submicron secondary water-in-water (W/W) droplets emerge spontaneously at the edges of the mother sessile droplet due to the resulting phase separation from water evaporation. To understand this phenomenon, we first study the secondary W/W droplet formation process on different substrate materials, namely, glass, polycarbonate (PC), thermoplastic elastomer (TPE), poly(dimethylsiloxane)-coated glass slide (PDMS substrate), and Teflon-coated glass slide (Teflon substrate), and show that secondary W/W droplet formation arises only in lower-contact-angle substrates near the three-phase contact line. Next, we characterize the size of the emergent secondary W/W droplets as a function of time. We observe that W/W drops are formed, coalesced, aligned, and trapped along the contact line of the mother droplet. We demonstrate that this W/W multiple emulsion system can be used to encapsulate magnetic particles and blood cells, and achieve size-based separation. Finally, we show the applicability of this system for protein sensing. This is the first experimental observation of evaporation-induced secondary W/W droplet generation in a sessile droplet. We anticipate that the phenomena described here may be applicable to some biological assay applications, for example, biomarker detection, protein sensing, and point-of-care diagnostic testing.

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.

Sample-to-answer centrifugal microfluidic droplet PCR platform for quantitation of viral load.

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 µm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per µL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification.

Reversible bonding in thermoplastic elastomer microfluidic platforms for harvestable 3D microvessel networks.

Transplantable ready-made microvessels have therapeutic potential for tissue regeneration and cell replacement therapy. Inspired by the natural rapid angiogenic sprouting of microvessels in vivo, engineered injectable 3D microvessel networks are created using thermoplastic elastomer (TPE) microfluidic devices. The TPE material used here is flexible, optically transparent, and can be robustly yet reversibly bonded to a variety of plastic substrates, making it a versatile choice for microfluidic device fabrication because it overcomes the weak self-adhesion properties and limited manufacturing options of poly(dimethylsiloxane) (PDMS). By leveraging the reversible bonding characteristics of TPE material templates, we present their utility as an organ-on-a-chip platform for forming and handling microvessel networks, and demonstrate their potential for animal-free tissue generation and transplantation in clinical applications. We first show that TPE-based devices have nearly 6-fold higher bonding strength during the cell culture step compared to PDMS-based devices while simultaneously maintaining a full reversible bond to (PS) culture plates, which are widely used for biological cell studies. We also demonstrate the successful generation of perfusable and interconnected 3D microvessel networks using TPE-PS microfluidic devices on both single and multi-vessel loading platforms. Importantly, after removing the TPE slab, microvessel networks remain intact on the PS substrate without any structural damage and can be effectively harvested following gel digestion. The TPE-based organ-on-a-chip platform offers substantial advantages by facilitating the harvesting procedure and maintaining the integrity of microfluidic-engineered microvessels for transplant. To the best of our knowledge, our TPE-based reversible bonding approach marks the first confirmation of successful retrieval of organ-specific vessel segments from the reversibly-bonded TPE microfluidic platform. We anticipate that the method will find applications in organ-on-a-chip and microphysiological system research, particularly in tissue analysis and vessel engraftment, where flexible and reversible bonding can be utilized.

Facile Fabrication of Flexible Polymeric Membranes with Micro and Nano Apertures over Large Areas.

Freestanding, flexible and open through-hole polymeric micro- and nanostructured membranes were successfully fabricated over large areas (>16 cm2) via solvent removal of sacrificial scaffolds filled with polymer resin by spontaneous capillary flow. Most of the polymeric membranes were obtained through a rapid UV curing processes via cationic or free radical UV polymerisation. Free standing microstructured membranes were fabricated across a range of curable polymer materials, including: EBECRYL3708 (radical UV polymerisation), CUVR1534 (cationic UV polymerisation) UV lacquer, fluorinated perfluoropolyether urethane methacrylate UV resin (MD700), optical adhesive UV resin with high refractive index (NOA84) and medical adhesive UV resin (1161-M). The present method was also extended to make a thermal set polydimethylsiloxane (PDMS) membranes. The pore sizes for the as-fabricated membranes ranged from 100 µm down to 200 nm and membrane thickness could be varied from 100 µm down to 10 µm. Aspect ratios as high as 16.7 were achieved for the 100 µm thick membranes for pore diameters of approximately 6 µm. Wide-area and uniform, open through-hole 30 µm thick membranes with 15 µm pore size were fabricated over 44 × 44 mm2 areas. As an application example, arrays of Au nanodots and Pd nanodots, as small as 130 nm, were deposited on Si substrates using a nanoaperture polymer through-hole membrane as a stencil.

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.

An automated centrifugal microfluidic assay for whole blood fractionation and isolation of multiple cell populations using an aqueous two-phase system.

Fractionating whole blood and separating its constituent components one from another is an essential step in many clinical applications. Currently blood sample handling and fractionation processes remain a predominantly manual task that require well-trained operators to produce reliable and reproducible results. Herein, we demonstrate an advanced on-chip whole human blood fractionation and cell isolation process combining (i) an aqueous two-phase system (ATPS) to create complex separation layers with (ii) a centrifugal microfluidic platform (PowerBlade) with active pneumatic pumping to control and automate the assay. We use a polyethylene glycol (PEG) and dextran (DEX) mixture as the two-phase density gradient media and our automated centrifugal microfluidic platform to fractionate blood samples. Different densities of precisely tuned PEG-DEX solutions were tested to match each of the cell types typically targeted during blood fractionation applications. By employing specially designed microfluidic devices, we demonstrate the automation of the following steps: loading of a whole blood sample on-chip, layering of the blood on the ATPS solution, blood fractionation, precise radial repositioning of the fractionated layers, and finally extraction of multiple, selected fractionated components. Fractionation of up to six distinct layers is shown: platelet-rich plasma, buffy coat, PEG, DEX with neutrophils, red blood cells (RBCs) and high density gradient media (HDGM). Furthermore, through controlled dispensing of HDGM to the fractionation chamber, we show that each of the fractionated layers can be repositioned radially, on-the-fly, without disturbing the interfaces, allowing precise transfer of target fractions and cell types into external vials via a chip-to-world interface. Cell counting analysis and cell viability studies showed equivalence to traditional, manual methods. An overall cell viability greater than 90% of extracted cells demonstrates that the proposed approach is suitable for cell isolation applications. This proof-of-principle demonstration highlights the utility of the proposed system for automated whole blood fractionation and isolation for blood cell applications. We anticipate that the proposed approach will be a useful tool for many clinical applications such as standard cell isolation procedures and other bioanalytical assays (e.g., circulating tumor cells, and cell and gene therapy).

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.

Anisotropic permeability in deterministic lateral displacement arrays.

We uncover anisotropic permeability in microfluidic deterministic lateral displacement (DLD) arrays. A DLD array can achieve high-resolution bimodal size-based separation of microparticles, including bioparticles, such as cells. For an application with a given separation size, correct device operation requires that the flow remains at a fixed angle to the obstacle array. We demonstrate via experiments and lattice-Boltzmann simulations that subtle array design features cause anisotropic permeability. Anisotropic permeability indicates the microfluidic array's intrinsic tendency to induce an undesired lateral pressure gradient. This can cause an inclined flow and therefore local changes in the critical separation size. Thus, particle trajectories can become unpredictable and the device useless for the desired separation task. Anisotropy becomes severe for arrays with unequal axial and lateral gaps between obstacle posts and highly asymmetric post shapes. Furthermore, of the two equivalent array layouts employed with the DLD, the rotated-square layout does not display intrinsic anisotropy. We therefore recommend this layout over the easier-to-implement parallelogram layout. We provide additional guidelines for avoiding adverse effects of anisotropy on the DLD.

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.

All-thermoplastic nanoplasmonic microfluidic device for transmission SPR biosensing.

Early and accurate disease diagnosis still remains a major challenge in clinical settings. Biomarkers could potentially provide useful tools for the detection and monitoring of disease progression, treatment safety and efficacy. Recent years have witnessed prodigious advancement in biosensor development with research directed towards rapid, real-time, label-free and sensitive biomarker detection. Among emerging techniques, nanoplasmonic biosensors pose tremendous potential to accelerate clinical diagnosis with real-time multiplexed analysis, rapid and miniaturized assays, low sample consumption and high sensitivity. In order to translate these technologies from the proof-of-principle concept level to point of care clinical diagnosis, integrated, portable devices having small footprint cartridges that house low-cost disposable consumables are sought. Towards this goal, we developed an all-polymeric nanoplasmonic microfluidic (NMF) transmission surface plasmon resonance (SPR) biosensor. The device was fabricated in thermoplastics using a simple, single step and cost-effective hot embossing technique amenable to mass production. The novel 3D hierarchical mold fabrication process enabled monolithic integration of blazed nanogratings within the detection chambers of a multichannel microfluidic system. Consequently, a single hard thermoplastic bottom substrate comprising plasmonic and fluidic features allowed integration of active fluidic elements, such as pneumatic valves, in the top soft thermoplastic cover, increasing device functionality. A simple and compact transmission-based optical setup was employed with multiplexed end-point or dual-channel kinetic detection capability which did not require stringent angular accuracy. The sensitivity, specificity and reproducibility of the transmission SPR biosensor was demonstrated through label-free immunodetection of soluble cell-surface glycoprotein sCD44 at clinically relevant picomolar to nanomolar concentrations.

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.