Centrifugal microfluidic system for colorimetric sample-to-answer detection of viral pathogens.

We describe a microfluidic system for conducting thermal lysis, polymerase chain reaction (PCR) amplification, hybridization, and colorimetric detection of foodborne viral organisms in a sample-to-answer format. The on-chip protocol entails 24 steps which are conducted by a centrifugal platform that allows for actuating liquids pneumatically during rotation and so facilitates automation of the workflow. The microfluidic cartridge is fabricated from transparent thermoplastic polymers and accommodates assay components along with an embedded micropillar array for detection and read-out. A panel of oligonucleotide primers and probes has been developed to perform PCR and hybridization assays that allows for identification of five different viruses, including pathogens such as norovirus and hepatitis A virus (HAV) in a multiplexed format using digoxigenin-labelled amplicons and immunoenzymatic conversion of a chromogenic substrate. Using endpoint detection, we demonstrate that the system can accurately and repetitively (n = 3) discriminate positive and negative signals for HAV at 350 genome copies per µL. As part of the characterization and optimization process, we show that the implementation of multiple (e.g., seven) micropillar arrays in a narrow fluidic pathway can lead to variation (up to 50% or more) in the distribution of colorimetric signal deriving from the assay. Numerical modeling of flow behaviour was used to substantiate these findings. The technology-by virtue of automation-can provide a pathway toward rapid detection of viral pathogens, shortening response time in food safety surveillance, compliance, and enforcement as well as outbreak investigations.

Quantitative Feedback Referencing for Improved Kinetic Fitting of Scanning Electrochemical Microscopy Measurements.

Scanning electrochemical microscopy (SECM) has matured as a technique for studying local electrochemical processes. The feedback mode is most commonly used for extracting quantitative kinetic information. However, approaching individual regions of interest, as is commonly done, does not take full advantage of the spatial resolution that SECM has to offer. Moreover, fitting of experimental approach curves remains highly subjective due to the manner of estimating the tip-to-substrate distance. We address these issues using negative or positive feedback currents as a reference to calculate the tip-to-substrate distance directly for quantitative kinetic fitting of approach curves and line profiles. The method was first evaluated by fitting simulated data and then tested experimentally by resolving negative feedback and intermediate kinetics behavior in a spatially controlled fashion using (i) a flat, binary substrate composed of Au and SiO2 segments and (ii) a dual-mediator system for live-cell measurements. The methodology developed herein, named quantitative feedback referencing (QFR), improves fitting accuracy, removes fitting subjectivity, and avoids substrate-microelectrode contact.

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).

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.

Real-time monitoring of bead-based DNA hybridization in a microfluidic system: study of amplicon hybridization behavior on solid supports.

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.

Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Buoyancy-driven step emulsification on pneumatic centrifugal microfluidic platforms.

We present here a new method for controlling the droplet size in step emulsification processes on a centrifugal microfluidic platform, which, in addition to the centrifugal force, uses pneumatic actuation for fluid displacement. We highlight the importance of the interplay between buoyancy effects and the flow rate at the step junction, and provide a simple analytical model relating these two quantities to the size of the droplets. Numerical models as well as experiments with water-in-oil emulsions are performed in support of the proposed model.

Detection of renal biomarkers in chronic kidney disease using microfluidics: progress, challenges and opportunities.

Chronic kidney disease (CKD) typically evolves over many years in a latent period without clinical signs, posing key challenges to detection at relatively early stages of the disease. Accurate and timely diagnosis of CKD enable effective management of the disease and may prevent further progression. However, long turn-around times of current testing methods combined with their relatively high cost due to the need for established laboratory infrastructure, specialized instrumentation and trained personnel are drawbacks for efficient assessment and monitoring of CKD, especially in underserved and resource-poor locations. Among the emerging clinical laboratory approaches, microfluidic technology has gained increasing attention over the last two decades due to the possibility of miniaturizing bioanalytical assays and instrumentation, thus potentially improving diagnostic performance. In this article, we review current developments related to the detection of CKD biomarkers using microfluidics. A general trend in this emerging area is the search for novel, sensitive biomarkers for early detection of CKD using technology that is improved by means of microfluidics. It is anticipated that these innovative approaches will be soon adopted and utilized in both clinical and point-of-care settings, leading to improvements in life quality of patients.

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.

Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR.

Epigenetic markers attract increasing attention for the study of phenotypic variations, which has led to the investigation of cell-lineage DNA methylation patterns that correlate with human leukocyte populations for obtaining counts of white blood cell (WBC) subsets. Current methods of DNA methylation analysis involve genome sequencing or loci-specific quantitative PCR (qPCR). Herein, a multiplexed digital droplet PCR (ddPCR) workflow for determining epigenetic-based WBC differential count is described for the first time. A microfluidic emulsification device fabricated from a commercially available thermoplastic elastomer (e.g., Mediprene) promotes customizability and cost-effectiveness of the methodology, which are prerequisites for translation into clinical and point-of-care diagnostics. Bisulfite-treated DNA from peripheral blood mononuclear cells and whole blood is encapsulated in droplets with ddPCR reagents containing primers and fluorescent hydrolysis probes specific for CpG loci correlated with WBC sub-population types. The method enables multiplexed detection of various methylation sites within a single droplet. Both qPCR and immunofluorescence staining (IF) were conducted to validate the capacity of the ddPCR methodology to accurately determine WBC sub-populations using epigenetic analysis of methylation sites. ddPCR results correlated closely to cell proportions obtained using IF, whereas qPCR significantly underestimated these values for both high and low copy number gene targets.

Extraction of nucleic acids from blood: unveiling the potential of active pneumatic pumping in centrifugal microfluidics for integration and automation of sample preparation processes.

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).

Evaluating the Use of Edge Detection in Extracting Feature Size from Scanning Electrochemical Microscopy Images.

The edge of a reactive or topographical feature is hard to estimate from feedback-based scanning electrochemical microscopy due to diffusional blurring, but is crucial to determining the accurate size and shape of these features. In this work, numerical simulations are used to demonstrate that the inflection point in a 1D line scan corresponds well to the true feature edge. This approach is then applied in 2D using the Canny algorithm to experimental images of two model substrates and a biological sample. This approach circumvents the need for aligning the imaged region between separate microscopy techniques, reveals hidden details embedded in SECM images, and allows individual features to be separated from their background more effectively.

Determination of the Relationship between Expression and Functional Activity of Multidrug Resistance-Associated Protein 1 using Scanning Electrochemical Microscopy.

Cancer cells can develop multidrug resistance (MDR) after prolonged exposure to chemotherapeutic drugs, which is a severe impediment to successful treatment. MDR is typically associated with transmembrane proteins mediating efflux of administered drugs, thereby keeping their intracellular concentration below the threshold required to kill cells. Although expression assays based on flow cytometry and immunostaining have shown that multidrug resistance-associated protein 1 (MRP1) is prevalent in many cancer types, the functional activity of this efflux pump is more difficult to elucidate, especially at the single-cell level. Herein, we report the measurement of MRP1 functional activity in individual cancer cells using scanning electrochemical microscopy (SECM). Cells were cultured onto plastic substrates containing selective adhesion sites. Optical microscopy and SECM revealed that cells adapt to the underlying surface, while MRP1 functional activity increases once the dimensions of the adhesive islands become smaller than those of the cell itself. Time-lapse SECM imaging revealed a suitable window of 30 min to complete each measurement before the cell undergoes blebbing, which is associated with a considerable increase in functional activity. Distinct cell populations were produced by performing a doxorubicin drug challenge on two parental cell lines (e.g., wild-type HeLa cells and MRP1-overexpressing HeLa-R cells). Expression and functional activity of MRP1 were determined using flow cytometry and SECM, and our findings show that these parameters do not directly correlate. This suggests that functional activity may represent a powerful indicator of a cancer cell's response to chemotherapeutic treatment and should improve our understanding of efflux mechanisms based on MRP1.

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.

Active pneumatic control of centrifugal microfluidic flows for lab-on-a-chip applications.

This paper reports a novel method of controlling liquid motion on a centrifugal microfluidic platform based on the integration of a regulated pressure pump and a programmable electromechanical valving system. We demonstrate accurate control over the displacement of liquids within the system by pressurizing simultaneously multiple ports of the microfluidic device while the platform is rotating at high speed. Compared to classical centrifugal microfluidic platforms where liquids are solely driven by centrifugal and capillary forces, the method presented herein adds a new degree of freedom for fluidic manipulation, which represents a paradigm change in centrifugal microfluidics. We first demonstrate how various core microfluidic functions such as valving, switching, and reverse pumping (i.e., against the centrifugal field) can be easily achieved by programming the pressures applied at dedicated access ports of the microfluidic device. We then show, for the first time, that the combination of centrifugal force and active pneumatic pumping offers the possibility of mixing fluids rapidly (~0.1 s) and efficiently based on the creation of air bubbles at the bottom of a microfluidic reservoir. Finally, the suitability of the developed platform for performing complex bioanalytical assays in an automated fashion is demonstrated in a DNA harvesting experiment where recovery rates of about 70% were systematically achieved. The proposed concept offers the interesting prospect to decouple basic microfluidic functions from specific material properties, channel dimensions and fabrication tolerances, surface treatments, or on-chip active components, thus promoting integration of complex assays on simple and low-cost microfluidic cartridges.

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.

Integrated air stream micromixer for performing bioanalytical assays on a plastic chip.

This paper describes the design, functioning and use of an integrated mixer that relies on air flux to agitate microliter entities of fluid in an embedded microfluidic cavity. The system was fabricated from multiple layers of a thermoplastic elastomer and features circuits for both liquid and air supply along with pneumatic valves for process control. Internally-dyed polymer particles have been used to visualize flow within the fluid phase during agitation. Numerical modelling of the micromixer revealed an overall efficacy of 10(-1) to 10(-2) for momentum transfer at the air-water interface. Simulation of air vortex dynamics showed dependency of the flow pattern on the velocity of the flux entering the cavity. Three bioanalytical assays have been performed as proof-of-concept demonstrations. In a first assay, cells of Listeria monocytogenes were combined with magnetic nanoparticles (NPs), resulting in high-density coverage of the bacteria's surface with NPs after 1 min of agitation. This finding is contrasted by a control experiment without agitation for which interaction between bacteria and NPs remains low. In a second one, capture and release of genomic DNA from fungi through adsorption onto magnetic beads was tested and shown to be improved by agitation compared to non-agitated controls. A third assay finally involved fluorescently-labelled target oligonucleotide strands and polystyrene particles modified with DNA capture probes to perform detection of nucleic acids on beads. Excellent selectivity was obtained in a competitive hybridization process using a multiplexed micromixer chip design.

Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy.

The emergence of resistance to multiple unrelated chemotherapeutic drugs impedes the treatment of several cancers. Although the involvement of ATP-binding cassette transporters has long been known, there is no in situ method capable of tracking this transporter-related resistance at the single-cell level without interfering with the cell's environment or metabolism. Here, we demonstrate that scanning electrochemical microscopy (SECM) can quantitatively and noninvasively track multidrug resistance-related protein 1-dependent multidrug resistance in patterned adenocarcinoma cervical cancer cells. Nonresistant human cancer cells and their multidrug resistant variants are arranged in a side-by-side format using a stencil-based patterning scheme, allowing for precise positioning of target cells underneath the SECM sensor. SECM measurements of the patterned cells, performed with ferrocenemethanol and [Ru(NH3)6](3+) serving as electrochemical indicators, are used to establish a kinetic "map" of constant-height SECM scans, free of topography contributions. The concept underlying the work described herein may help evaluate the effectiveness of treatment administration strategies targeting reduced drug efflux.