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Summary We developed a universal method termed OnCELISA to detect cytokine secretion from individual cells by applying a capture technology on the cell membrane. OnCELISA uses fluorescent magnetic nanoparticles as assay reporters that enable detection on a single-cell level in microscopy and flow cytometry and fluorimetry in cell ensembles. This system is flexible and can be modified to detect different cytokines from a broad range of cytokine-secreting cells. Using OnCELISA we have been able to select and sort highly cytokine-secreting cells and identify cytokine-secreting expression profiles of different cell populations in vitro and ex vivo. We show that this system can be used for ultrasensitive monitoring of cytokines in the complex biological environment of atherosclerosis that contains multiple cell types. The ability to identify and select cell populations based on their cytokine expression characteristics is valuable in a host of applications that require the monitoring of disease progression., Graphical Abstract, Highlights • We developed an assay for detecting cytokine secretion from individual cells • This system is universal to detect different cytokines from a broad range of cell types • The assay can select highly cytokine-secreting cells and purify their populations • This study opens a host of applications for monitoring disease progression, Sensor; Biotechnology; Cell; Nanomaterials

Red blood cell (RBC) shape change under static and dynamic shear stress has been a source of interest for at least 50 years. High-speed time-lapse microscopy was used to observe the rate of deformation and relaxation when RBCs are subjected to periodic shear stress and deformation forces as they pass through an obstacle. We show that red blood cells are reversibly deformed and take on characteristic shapes not previously seen in physiological buffers when the maximum shear stress was between 2.2 and 25 Pa (strain rate 2200 to 25,000 s−1). We quantify the rates of RBC deformation and recovery using Kaplan–Meier survival analysis. The time to deformation decreased from 320 to 23 milliseconds with increasing flow rates, but the distance traveled before deformation changed little. Shape recovery, a measure of degree of deformation, takes tens of milliseconds at the lowest flow rates and reached saturation at 2.4 s at a shear stress of 11.2 Pa indicating a maximum degree of deformation was reached. The rates and types of deformation have relevance in red blood cell disorders and in blood cell behavior in microfluidic devices.

Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re

Interferon-gamma (IFN-γ), a proinflammatory cytokine, has been used as an early indicator of multiple infectious diseases or tumors. In order to explore the detection capability of a commonly used anti-IFN-γ aptamer, a simple target induced strand-displacement aptasensing strategy was tested by introducing three different complementary strands and two different signal/quencher pairs. The Texas red/BHQ2-based sensor showed the best affinity constant (Kd) of 21.87 ng mL−1. It was found that the strand-displacement aptasensing strategy was impacted by the complementary position and length of the complementary strands. Additionally, the hybridization chain reaction (HCR) amplification strategy was introduced, yielding a 12-fold improved sensitivity of 0.45 ng mL−1. In order to further explore the sensing platform for spatially localized cytokine detection, the Texas red/BHQ2-based strand-displacement aptasensor was successfully fabricated on the 3D optical fiber surface to achieve a deployable sensing device for monitoring IFN-γ based on the fluorescence spots counting strategy. Finally, the three developed aptasensing strategies (strand-displacement strategy, HCR amplification strategy, 3D optical fiber aptasensor) were applied for detection of IFN-γ secreted by PBMCs with comparable results to those of ELISA. The deployable 3D optical fiber aptasensor with the superior sensitivity is potential to be used for detection of spatially localized IFN-γ in vivo.

A microfabricated needle-like probe has been designed and applied for hydrogen peroxide (H2O2) sampling and detection using a commercial, single-step fluorescent H2O2 assay. In this work, droplets of the assay reagent are generated and sent to the needle tip using a mineral-oil carrier fluid. At the needle tip, the sample is drawn into the device through 100 μm long hydrophilic capillaries by negative pressure. The sampled fluid is immediately merged with the assay droplet and carried away to mix and react, producing a sequence of droplets representing the H2O2 concentration as a function of time. We have characterized the assay fluorescence for small variations in the sample volume. With the calibration, we can calculate the concentration of H2O2 in the sampled liquid from the size and intensity of each merged droplet. This is a microfluidic data-logger system for on-site continuous sampling, controlled reaction, signal storage and on-line quantitative detection. It is a useful tool for monitoring dynamic chemical reactions in analytical chemistry and biological applications.

This paper concerns the design, fabrication and characterization of graphite/PDMS sensors for low-force sensing applications. Exploiting the design flexibility of 3D printing, moulds of specific dimensions were prepared onto which graphite powder and PDMS were cast, to develop sensor patches. The sensor patches were highly flexible with repeatable responses to iterative bending cycles. The patches were tested in terms of stretchability, strain and bending-cycle responses. The sensor patches had interdigitated electrodes operating on capacitive sensing, where the effective capacitance changes with an applied force because of changes in their dimensions. Forces ranging from 3.5 mN to 17.5 mN were applied to determine the capability of these sensor patches for low-force sensing applications. The sensor patches had a quick recovery having a sensitivity and SNR per unit force of 0.2542 pF mN−1 and 10.86 respectively. The patches were capable of differentiating the forces applied on them, when they were attached to different objects in daily use.

Deterministic Lateral Displacement (DLD) is a microfluidic method of separating particles by size. DLD relies on precise flow patterns to deliver highresolutionparticle separation. These patterns determine which particles are displacedlaterally, and which follow the flow direction. Prior research has demonstrated that the lateral array boundaries can be designed to improve the uniformity of the critical size and hence separation performance. A DLD device with an invariant critical size throughout is yet unknown. In this work we propose a 3D design approach. We first represent the flow through the DLD as a 2D lattice of resistors. This is used to determine the relative flow resistances at the boundaries that will deliver the correct flux patterns. We then use the Lattice Boltzmann Method to simulate fluid flow in a 3D unit cell of the DLD and measure the fluidic resistance for a range or typical dimensions. The results of this work are used to create a new equation for fluidic resistance as a function of post size, post height, and post spacing.We use this equation to determine array geometries that should have the appropriate resistances. We then design and simulate (in COMSOL) complete devices and measure fluid fluxes and first flow-lane widths along the boundaries. We find that the first flow-lane widths are much more uniform than in any devices described previously. This work providesthe best method for designing periodic boundaries, and enables narrower, shorter and higher throughput DLD devices.

The advent of microfluidics in the 1990s promised a revolution in multiple industries from healthcare to chemical processing. Deterministic lateral displacement (DLD) is a continuous-flow microfluidic particle separation method discovered in 2004 that has been applied successfully and widely to the separation of blood cells, yeast, spores, bacteria, viruses, DNA, droplets, and more. Deterministic lateral displacement is conceptually simple and can deliver consistent performance over a wide range of flow rates and particle concentrations. Despite wide use and in-depth study, DLD has not yet been fully elucidated or optimized, with different approaches to the same problem yielding varying results. We endeavor here to provide up-to-date expert opinion on the state-of-art and current fundamental, practical, and commercial challenges with DLD as well as describe experimental and modeling opportunities. Because these challenges and opportunities arise from constraints on hydrodynamics, fabrication, and operation at the micro- and nanoscale, we expect this Perspective to serve as a guide for the broader micro- and nanofluidic community to identify and to address open questions in the field.

In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg&minus, 1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.

Gradient-based electrophoretic separations enable simultaneous separation and concentration of molecules. Compared with conventional injection-based separations, they enable enrichment of low-concentration analytes from larger sample volumes that are not limited by an injection volume. We have demonstrated that a nanochannel, connecting two chemically different reservoirs, can maintain a stationary chemical gradient while trapping biomolecules and effectively averaging out many of the complex physicochemical hydrodynamics that would broaden the bands in a meso- or microscale capillary. Here we describe chemical and physical methods that enable this work.

Droplet-based microfluidics are capable of transporting very small amounts of fluid over long distances. This characteristic may be applied to conventional fluid delivery using needles if droplets can be reliably expelled from a microfluidic channel. In this paper, we demonstrate a system for the extraction of water droplets from an oil-phase in a polymer microfluidic device. A hydrophilic membrane with a strong preference for water over oil is integrated into a droplet microfluidic system and observed to allow the passage of the transported aqueous phase droplets while blocking the continuous phase. The oil breakthrough pressure of the membrane was observed to be 250 ± 20 kPa, a much greater pressure than anywhere within the microfluidic channel, thereby eliminating the possibility that oil will leak from the microchannel, a critical parameter if droplet transport is to be used in needle-based drug delivery.

Previous work has demonstrated the simultaneous concentration and separation of proteins via a stable ion concentration gradient established within a nanochannel (Inglis Angew. Chem., Int. Ed. 2001, 50, 7546-7550). To gain a better understanding of how this novel technique works, we here examine experimentally and numerically how the underlying electric potential controlled ion concentration gradients can be formed and controlled. Four nanochannel geometries are considered. Measured fluorescence profiles, a direct indicator of ion concentrations within the Tris-fluorescein buffer solution, closely match depth-averaged fluorescence profiles calculated from the simulations. The simulations include multiple reacting species within the fluid bulk and surface wall charge regulation whereby the deprotonation of silica-bound silanol groups is governed by the local pH. The three-dimensional system is simulated in two dimensions by averaging the governing equations across the (varying) nanochannel width, allowing accurate numerical results to be generated for the computationally challenging high aspect ratio nanochannel geometries. An electrokinetic circuit analysis is incorporated to directly relate the potential drop across the (simulated) nanochannel to that applied across the experimental chip device (which includes serially connected microchannels). The merit of the thick double layer, potential-controlled concentration gradient as a particle focusing and separation tool is discussed, linking this work to the previously presented protein trapping experiments. We explain why stable traps are formed when the flow is in the opposite direction to the concentration gradient, allowing particle separation near the low concentration end of the nanochannel. We predict that tapered, rather than straight nanochannels are better at separating particles of different electrophoretic mobilities.

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., 13 pages, 10 figures, 1 table, DLD, particle separation, microfluidics, anisotropic permeability

Glass bonding is often necessary in microfluidic, nanofluidic and MEMS applications. Fusion bonding is a popular method in which glass substrates are permanently bonded using chemical treatment, low pressure and annealing. This project investigates a range of bonding methods and measures the bond strength. Float glass substrates were chemically cleaned and prepared using four different methods (Ethanol Rub, Ethanol Rub + NH4OH, piranha + NH4OH, and piranha + HCl + NH4OH), pressed together by hand, and placed in the oven at 400 °C for 10 h. Bond strength was assessed using compressive shear stress. It was determined that the piranha + HCl +NH4OH produced the strongest bond. All methods produced satisfactory bonds and the simplicity and safety of a simple Ethanol rub means it should be considered whenever possible.

We have developed a microfluidic needle-like device that can extract and deliver nanoliter samples. The device consists of a T-junction to form segmented flows, parallel channels to and from the needle tip, and seven hydrophilic capillaries at the tip that form a phase-extraction region. The main microchannel is hydrophobic and carries segmented flows of water-in-oil. The hydrophilic capillaries transport the aqueous phase with a nearly zero pressure gradient but require a pressure gradient of 19 kPa for mineral oil to invade and flow through. Using this device, we demonstrate the delivery of nanoliter droplets and demonstrate sampling through the formation of droplets at the tip of our device. During sampling, we recorded the fluorescence intensities of the droplets formed at the tip while varying the concentration of dye outside the tip. We measured a chemical signal response time of approximately 3 s. The linear relationship between the recorded fluorescence intensity of samples and the external dye co...

Isoelectric focusing of proteins in a silica nanofluidic channel filled with citric acid and disodium phosphate buffers is investigated via numerical simulation. Ions in the channel migrate in response to (i) the electric field acting on their charge and (ii) the bulk electroosmotic flow (which is directed toward the cathode). Proteins are focused near the low pH (anode) end when the electromigration effect is more significant and closer to the high pH (cathode) end when the electroosmotic effect dominates. We simulate the focusing behavior of Dylight labeled streptavidin (Dyl-Strep) proteins in the channel, using a relationship between the protein's charge and pH measured in a previous experiment. Protein focusing results compare well to previous experimental measurements. The effect of some key parameters, such as applied voltage, isoelectric point (pI), bulk pH, and bulk conductivity, on the protein trapping behavior in a nanofluidic channel is examined.

We present an improvement to deterministic lateral displacement arrays, which allows higher particle concentration enhancement. We correct and extend previous equations to a mirror-symmetric boundary. This approach allows particles to be concentrated into a central channel, no wider than the surrounding gaps, thereby maximizing the particle enrichment. The resulting flow patterns were, for the first time, experimentally measured. The performance of the device with hard micro-spheres and cells was investigated. The observed flow patterns show important differences from our model and from an ideal pattern. The 18 μm gap device showed 11-fold enrichment of 7 μm particles and nearly perfect enrichment—of more than 50-fold—for 10 μm particles and Jurkat cells. This work shows a clear path to achieve higher-than-ever particle concentration enhancement in a deterministic microfluidic separation system.