Reagent integration and controlled release for multiplexed nucleic acid testing in disposable thermoplastic 2D microwell arrays.

The seamless integration of reagents into microfluidic devices can serve to significantly reduce assay complexity and cost for disposable diagnostics. In this work, the integration of multiplexed reagents into thermoplastic 2D microwell arrays is demonstrated using a scalable pin spotting technique. Using a simple and low-cost narrow-bore capillary spotting pin, high resolution deposition of concentrated reagents within the arrays of enclosed nanoliter-scale wells is achieved. The pin spotting method is further employed to encapsulate the deposited reagents with a chemically modified wax layer that serves to prevent disruption of the dried assay components during sample introduction through a shared microchannel, while also enabling temperature-controlled release after sample filling is complete. This approach supports the arbitrary patterning and release of different reagents within individual wells without crosstalk for multiplexed analyses. The performance of the in-well spotting technique is characterized using on-chip rolling circle amplification to evaluate its potential for nucleic acid-based diagnostics.

Enhanced sample filling and discretization in thermoplastic 2D microwell arrays using asymmetric contact angles.

Sample filling and discretization within thermoplastic 2D microwell arrays is investigated toward the development of low cost disposable microfluidics for passive sample discretization. By using a high level of contact angle asymmetry between the filling channel and microwell surfaces, a significant increase in the range of well geometries that can be successfully filled is revealed. The performance of various array designs is characterized numerically and experimentally to assess the impact of contact angle asymmetry and device geometry on sample filling and discretization, resulting in guidelines to ensure robust microwell filling and sample isolation over a wide range of well dimensions. Using the developed design rules, reliable and bubble-free sample filling and discretization is achieved in designs with critical dimensions ranging from 20 µm to 800 µm. The resulting devices are demonstrated for discretized nucleic acid amplification by performing loop-mediated isothermal amplification for the detection of the mecA gene associated with methicillin-resistant Staphylococcus aureus.

A Scalable Random Access Micro-traps Array for Formation, Selective Retrieval and Capturing of Individual Droplets.

Formation, selective retrieval and capturing of individual droplets are key operational capabilities needed for a broad range of droplet microfluidic applications. The membrane displacement trap (MDT) element gives a robust method for uniform discretization and controllable manipulation of aqueous droplets using an enclosed micro-well covered by an elastomer membrane. This capability can be scaled up by combining the modular elements with a system design that requires a minimal number of signal inputs. Incorporation of MDT elements with a pneumatically-controllable multiplexer system can lead to a scalable random access MDT array platform for liquid discretization and selective manipulation. Herein, we report the design and development of a programmable droplet microfluidic platform for liquid sampling and selectively handling up to 32 individual droplets using 10 pneumatic signal inputs. The multiplexer system can logarithmically scale up capacity of the MDT array platform, making it possible to manipulate hundreds droplets.

Flow-through microfluidic immunosensors with refractive index-matched silica monoliths as volumetric optical detection elements.

A sensitive and rapid absorbance based immunosensor that utilizes ex situ functionalized porous silica monoliths as volumetric optical detection elements is demonstrated in this study. The porous monolith structure facilitates high capture probe density and short diffusion length scales, enabling sensitive and rapid assays. Silica monoliths, synthesized and functionalized with immunocapture probes off-chip before integration into a sealed thermoplastic microfluidic device, serve to capture target antigens during perfusion through the porous structure. Gold nanoparticle immunoconjugates are combined with silver enhancement to create microscale silver clusters, followed by perfusion of an aqueous sucrose solution to limit light scattering and enhance optical signal. Using this approach, detection limits as low as 1 ng/mL are achieved for a sandwich assay, with a dynamic range of at least 4 logs. The results confirm that the combination of on-chip index matching with functionalized porous silica monoliths can enables simple and practical flow-through immunoassays for the sensitive and rapid detection of target antigens.

Staggered trap arrays for robust microfluidic sample digitization.

A sample digitization method that exploits the controlled pinning of fluid at geometric discontinuities within an array of staggered microfluidic traps is presented. The staggered trap design enables reliable sample filling within high aspect ratio microwells, even when employing substrate materials such as thermoplastics that are not gas permeable. A simple geometric model is developed to predict the impact of device geometry on sample filling and discretization, and validated experimentally using fabricated cyclic olefin polymer devices. Using the developed design guidelines, a 768-element staggered trap array is demonstrated, with reliable passive loading and discretization achieved within 5 min. The resulting discretization platform offers a simplified workflow with flexible trap design, reliable discretization, and repeatable operation using low-cost thermoplastic substrates.

Controlled droplet discretization and manipulation using membrane displacement traps.

An innovative platform enabling complex discretization and manipulation of aqueous droplets is described. The system uses simple membrane displacement trap elements to perform multiple functions including droplet discretization, release, metering, capture, and merging. Multi-layer PDMS devices with membrane displacement trap arrays are used to discretize sample into nanoliter scale droplet volumes, and reliably manipulate individual droplets within the arrays. Performance is characterized for varying capillary number flows, membrane actuation pressures, trap and membrane geometries, and trapped droplet volumes, with operational domains established for each platform function. The novel approach to sample digitization and droplet manipulation is demonstrated through discretization of a dilute bacteria sample, metering of individual traps to generate droplets containing single bacteria, and merging of the resulting droplets to pair the selected bacteria within a single droplet.

Novel functionalities of hybrid paper-polymer centrifugal devices for assay performance enhancement.

The presented work demonstrates novel functionalities of hybrid paper-polymer centrifugal devices for assay performance enhancement that leverage the advantages of both paper-based and centrifugal microfluidic platforms. The fluid flow is manipulated by balancing the capillary force of paper inserts with the centrifugal force generated by disc rotation to enhance the signal of a colorimetric lateral flow immunoassay for pathogenic E. coli. Low-cost centrifugation for pre-concentration of bacteria was demonstrated by sample sedimentation at high rotational speeds before supernatant removal by a paper insert via capillary force after deceleration. The live bacteria capture efficiency of the device was similar to a commercial centrifuge. This pre-concentrated sample when combined with gold nanoparticle immunoconjugate probes resulted in a detection limit that is 10× lower than a non-concentrated sample for a lateral flow immunoassay. Signal enhancement was also demonstrated through rotational speed variation to prevent the flow for on-device incubation and to reduce the flow rate, thus increasing the sample residence time for the improved capture of gold nanoparticle-bacteria complexes in an integrated paper microfluidic assay. Finally, multiple sequential steps including sample pre-concentration, filtration, incubation, target capture by an integrated paper microfluidic assay, silver enhancement and quenching, and index matching were completed within a single device. The detection limit was 105 colony forming units per ml, a 100× improvement over a similar paper-based lateral flow assay. The techniques utilize the advantages of paper-based microfluidic devices, while facilitating additional functionalities with a centrifugal microfluidic platform for detection performance enhancement in a low-cost, automated platform amenable to point-of-care environments.

Screw-actuated displacement micropumps for thermoplastic microfluidics.

The fabrication of on-chip displacement pumps integrated into thermoplastic chips is explored as a simple and low cost method for achieving precise and programmable flow control for disposable microfluidic systems. The displacement pumps consist of stainless steel screws inserted into threaded ports machined into a thermoplastic substrate which also serve as on-chip reagent storage reservoirs. Three different methods for pump sealing are investigated to enable high pressure flows without leakage, and software-defined control of multiple pumps is demonstrated in a self-contained platform using a compact and self-contained microcontroller for operation. Using this system, flow rates ranging from 0.5-40 µl min-1 are demonstrated. The pumps are combined with on-chip burst valves to fully seal multiple reagents into fabricated chips while providing on-demand fluid distribution in a downstream microfluidic network, and demonstrated for the generation of size-tunable water-in-oil emulsions.

Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon.

Optical detection enhancement in porous volumetric microfluidic capture elements using refractive index matching fluids.

Porous volumetric capture elements in microfluidic sensors are advantageous compared to planar capture surfaces due to higher reaction site density and decreased diffusion lengths that can reduce detection limits and total assay time. However a mismatch in refractive indices between the capture matrix and fluid within the porous interstices results in scattering of incident, reflected, or emitted light, significantly reducing the signal for optical detection. Here we demonstrate that perfusion of an index-matching fluid within a porous matrix minimizes scattering, thus enhancing optical signal by enabling the entire capture element volume to be probed. Signal enhancement is demonstrated for both fluorescence and absorbance detection, using porous polymer monoliths in a silica capillary and packed beds of glass beads within thermoplastic microchannels, respectively. Fluorescence signal was improved by a factor of 3.5× when measuring emission from a fluorescent compound attached directly to the polymer monolith, and up to 2.6× for a rapid 10 min direct immunoassay. When combining index matching with a silver enhancement step, a detection limit of 0.1 ng mL(-1) human IgG and a 5 log dynamic range was achieved. The demonstrated technique provides a simple method for enhancing optical sensitivity for a wide range of assays, enabling the full benefits of porous detection elements in miniaturized analytical systems to be realized.

Microfluidic remote loading for rapid single-step liposomal drug preparation.

Microfluidic-directed formation of liposomes is combined with in-line sample purification and remote drug loading for single step, continuous-flow synthesis of nanoscale vesicles containing high concentrations of stably loaded drug compounds. Using an on-chip microdialysis element, the system enables rapid formation of large transmembrane pH and ion gradients, followed by immediate introduction of amphipathic drug for real-time remote loading into the liposomes. The microfluidic process enables in-line formation of drug-laden liposomes with drug : lipid molar ratios of up to 1.3, and a total on-chip residence time of approximately 3 min, representing a significant improvement over conventional bulk-scale methods which require hours to days for combined liposome synthesis and remote drug loading. The microfluidic platform may be further optimized to support real-time generation of purified liposomal drug formulations with high concentrations of drugs and minimal reagent waste for effective liposomal drug preparation at or near the point of care.

High-pressure needle interface for thermoplastic microfluidics.

A robust and low dead volume world-to-chip interface for thermoplastic microfluidics has been developed. The high pressure fluidic port employs a stainless steel needle inserted into a mating hole aligned to an embedded microchannel, with an interference fit used to increase pressure resistance. Alternately, a self-tapping threaded needle screwed into a mating hole is also demonstrated. In both cases, the flat bottom needle ports seat directly against the microchannel substrate, ensuring low interfacial dead volumes. Low dispersion is observed for dye bands passing the interfaces. The needle ports offer sufficient pull-out forces for applications such as liquid chromatography that require high internal fluid pressures, with the epoxy-free interfaces compatible with internal microchannel pressures above 40 MPa.

Low temperature bonding of PMMA and COC microfluidic substrates using UV/ozone surface treatment.

The use of UV/ozone surface treatments for achieving low temperature bonds between PMMA and COC microfluidic substrates is evaluated. Low temperature bond strengths, approaching those of native polymer substrates bonded above their glass transition temperatures, are demonstrated for both thermoplastics. To evaluate the effects of the UV/O(3) surface treatment on the operation of bonded microfluidic devices, the relationship between UV/O(3) exposure and polymer hydrophilicity and surface chemistry are measured. Post-treatment surface chemistry is evaluated by XPS (X-ray photoelectron spectroscopy) analysis, and the stability of the treated surfaces following solvent exposure is reported. Electroosmotic flow within fabricated microchannels with modified wall surfaces is also characterized. Overall, UV/O(3) treatment is found to enable strong low temperature bonds between thermoplastic microfluidic substrates using a simple, low cost, and high throughput fabrication technology.

Proteomic analysis of steroid-triggered autophagic programmed cell death during Drosophila development.

Two morphological forms of programmed cell death, apoptosis and autophagic cell death, remove unneeded or damaged cells during animal development. Although the mechanisms that regulate apoptosis are well studied, little is known about autophagic cell death. A shotgun proteome analysis of purified dying larval salivary glands in Drosophila was used to identify proteins that are expressed during autophagic programmed cell death. A total of 5661 proteins were identified from stages before and after the onset of cell death. Analyses of these data enabled us to identify proteins from a number of interesting categories including regulators of transcription, the apoptosis, autophagy, lysosomal, and ubiquitin proteasome degradation pathways, and proteins involved in growth control. Several of the identified proteins, including the serine/threonine kinase warts (Wts), were not detected using whole-genome DNA microarrays, providing support for the importance of such high-throughput proteomic technology. Wts regulates cell-cycle arrest and apoptosis, and significantly, mutations in wts prevent destruction of salivary glands.

Integrated microfluidic UV absorbance detector with attomol-level sensitivity for BSA.

An integrated UV absorbance detection system employing a novel silicon-in-plastic technology to seamlessly integrate bare UV photodiode chips into polymer microfluidic systems has been developed. Detection platforms fabricated using this approach exhibit exceptionally low concentration and mass detection limits down to 15 nM and 9.8 amol, respectively, for bovine serum albumin (BSA) as a model protein. In addition to providing high sensitivity, sub-nanoliter detection volumes are enabled by the use of direct photodiode integration. The fabrication methodology is detailed, and system performance metrics including detection limits, detection volume, dynamic range, and linearity are reported.

Integration of polymeric membranes with microfluidic networks for bioanalytical applications.

The concept of microfluidics has significantly influenced the design and the implementation of modern bioanalytical systems due to the fact that these miniaturized devices can handle and manipulate samples in a much more efficient way than conventional instruments. In an analogy to the development of microelectronics, increasingly sophisticated devices with greater functionalities have become one of the major goals being pursued in the area of micrototal analysis systems. The incorporation of polymeric membranes into microfluidic networks has therefore been employed in an effort to enhance the functionalities of these microfabricated devices. These commercially available membranes are porous, flexible, mechanically robust and compatible with plastic microfluidic networks. The large surface area-to-volume ratio of porous membrane media is particularly important for achieving rapid buffer exchange during microdialysis and obtaining ultrahigh concentration of adsorbed enzymes for various biochemical reactions. Furthermore, the membrane pore diameter in the sub-microm range eliminates the constraints of diffusional mass-transfer resistance for performing chiral separation using adsorbed protein as the chiral stationary phase. A review on the recent advancement in the integration of polymeric membranes with microfluidic networks is presented for their widespread applications in bioanalytical chemistry.

Field-effect flow control in a polydimethylsiloxane-based microfluidic system.

The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.