Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Enrichment of bovine milk-derived extracellular vesicles using surface-functionalized cellulose nanofibers.

The isolation of extracellular vesicles (EVs) from milk, a complex mixture of colloidal structures having a comparable size to EVs, is challenging. Although ultracentrifugation (UC) has been widely used for EV isolation, this has significant limitations, including a long processing time at high g-force conditions and large sample volume requirements. We introduced a new approach based on nature nanoentities cellulose nanofibers (CNFs) and short time and low g-force centrifugation to isolate EVs from various milk fractions. The flexible and entangled network of CNFs forms nanoporous, which entraps the EVs. Further, positively charged CNFs interact with anionic EVs through an electrostatic attraction, promoting their isolation with efficiency comparable with UC. The functionality and toxicity of isolated milk EVs were tested in Caco2 cells. Overall, the newly developed approach provides straightforward isolation and biocompatibility and preserves the natural properties of the isolated EVs, enabling further applications.

Xenogenic Neural Stem Cell-Derived Extracellular Nanovesicles Modulate Human Mesenchymal Stem Cell Fate and Reconstruct Metabolomic Structure.

Extracellular nanovesicles, particularly exosomes, can deliver their diverse bioactive biomolecular content, including miRNAs, proteins, and lipids, thus providing a context for investigating the capability of exosomes to induce stem cells toward lineage-specific cells and tissue regeneration. In this study, it is demonstrated that rat subventricular zone neural stem cell-derived exosomes (rSVZ-NSCExo) can control neural-lineage specification of human mesenchymal stem cells (hMSCs). Microarray analysis shows that the miRNA content of rSVZ-NSCExo is a faithful representation of rSVZ tissue. Through immunocytochemistry, gene expression, and multi-omics analyses, the capability to use rSVZ-NSCExo to induce hMSCs into a neuroglial or neural stem cell phenotype and genotype in a temporal and dose-dependent manner via multiple signaling pathways is demonstrated. The current study presents a new and innovative strategy to modulate hMSCs fate by harnessing the molecular content of exosomes, thus suggesting future opportunities for rSVZ-NSCExo in nerve tissue regeneration.

Rose petal topography mimicked poly(dimethylsiloxane) substrates for enhanced corneal endothelial cell behavior.

Low proliferation capacity of corneal endothelial cells (CECs) and worldwide limitations in transplantable donor tissues reveal the critical need of a robust approach for in vitro CEC growth. However, preservation of CEC-specific phenotype with increased proliferation has been a great challenge. Here we offer a biomimetic cell substrate design, by optimizing mechanical, topographical and biochemical characteristics of materials with CEC microenvironment. We showed the surprising similarity between topographical features of white rose petals and corneal endothelium due to hexagonal cell shapes and physiologically relevant cell density (≈ 2000 cells/mm2). Polydimethylsiloxane (PDMS) substrates with replica of white rose petal topography and cornea-friendly Young's modulus (211.85 ± 74.9 kPa) were functionalized with two of the important corneal extracellular matrix (ECM) components, collagen IV (COL 4) and hyaluronic acid (HA). White rose petal patterned and COL 4 modified PDMS with optimized stiffness provided enhanced bovine CEC response with higher density monolayers and increased phenotypic marker expression. This biomimetic approach demonstrates a successful platform to improve in vitro cell substrate properties of PDMS for corneal applications, suggesting an alternative environment for CEC-based therapies, drug toxicity investigations, microfluidics and organ-on-chip applications.

Bioinspired hydrogel surfaces to augment corneal endothelial cell monolayer formation.

Corneal endothelial cells (CECs) have limited proliferation ability leading to corneal endothelium (CE) dysfunction and eventually vision loss when cell number decreases below a critical level. Although transplantation is the main treatment method, donor shortage problem is a major bottleneck. The transplantation of in vitro developed endothelial cells with desirable density is a promising idea. Designing cell substrates that mimic the native CE microenvironment is a substantial step to achieve this goal. In the presented study, we prepared polyacrylamide (PA) cell substrates that have a microfabricated topography inspired by the dimensions of CECs. Hydrogel surfaces were prepared via two different designs with small and large patterns. Small patterned hydrogels have physiologically relevant hexagon densities (∼2000 hexagons/mm2 ), whereas large patterned hydrogels have sparsely populated hexagons (∼400 hexagons/mm2 ). These substrates have similar elastic modulus of native Descemet's membrane (DM; ∼50 kPa) and were modified with Collagen IV (Col IV) to have biochemical content similar to native DM. The behavior of bovine corneal endothelial cells on these substrates was investigated and results show that cell proliferation on small patterned substrates was significantly (p = 0.0004) higher than the large patterned substrates. Small patterned substrates enabled a more densely populated cell monolayer compared to other groups (p = 0.001 vs. flat and p < 0.0001 vs. large patterned substrates). These results suggest that generating bioinspired surface topographies augments the formation of CE monolayers with the desired cell density, addressing the in vitro development of CE layers.

SERS-active linear barcodes by microfluidic-assisted patterning.

Simple, low-cost, robust, and scalable fabrication of microscopic linear barcodes with high levels of complexity and multiple authentication layers is critical for emerging applications in information security and anti-counterfeiting. This manuscript presents a novel approach for fabrication of microscopic linear barcodes that can be visualized under Raman microscopy. Microfluidic channels are used as molds to generate linear patterns of end-grafted polymers on a substrate. These patterns serve as templates for area-selective binding of colloidal gold nanoparticles resulting in plasmonic arrays. The deposition of multiple taggant molecules on the plasmonic arrays via a second microfluidic mold results in a linear barcode with unique Raman fingerprints that are enhanced by the underlying plasmonic nanoparticles. The width of the bars is as small as 10 µm, with a total barcode length on the order of 100 µm. The simultaneous use of geometric and chemical security layers provides a high level of complexity challenging the counterfeiting of the barcodes. The additive, scalable, and inexpensive nature of the presented approach can be easily adapted to different colloidal nanomaterials and applications.

In vitro analysis of multiple blood flow determinants using red blood cell dynamics under oscillatory flow.

The flow behavior of blood is determined mainly by red blood cell (RBC) deformation and aggregation as well as blood viscoelasticity. These intricately interdependent parameters should be monitored by healthcare providers to understand all aspects of circulatory flow dynamics under numerous cases including cardiovascular and infectious diseases. Current medical instruments and microfluidic systems lack the ability to quantify these parameters all at once and in physiologically relevant flow conditions. This work presents a handheld platform and a measurement method for quantitative analysis of multiple of these parameters from 50 µl undiluted blood inside a miniaturized channel. The assay is based on an optical transmission analysis of collective RBC deformation and aggregation under near-infrared illumination during a 1 s damped oscillatory flow and at stasis, respectively. Measurements with blood of different hemo-rheological properties demonstrate that the presented approach holds a potential for initiating simultaneous and routine on-chip blood flow analysis even in resource-poor settings.

Electro-Viscoelastic Migration under Simultaneously Applied Microfluidic Pressure-Driven Flow and Electric Field.

Under the simultaneous use of pressure-driven flow and DC electric field, migration of particles inside microfluidic channels exhibits intricate focusing dynamics. Available experimental and analytical studies fall short in giving a thorough explanation to particle equilibrium states. Also, the understanding is so far limited to the results based on Newtonian and neutral viscoelastic carrier fluids. Hence, a holistic approach is taken in this study to elaborate the interplay of governing electrophoretic and slip-induced/elastic/shear gradient lift forces. First, we carried out experimental studies on particle migration in Newtonian, neutral viscoelastic, and polyelectrolyte viscoelastic media to provide a comprehensive understanding of particle migration. The experiments with the viscoelastic media led to contradictory results with the existing explanations. Then, we introduced the Electro-Viscoelastic Migration (EVM) theory to give a unifying explanation to particle migration in Newtonian and viscoelastic solutions. Confocal imaging with fluorescent-labeled polymer solutions was used to explore the underlying migration behavior. A surprising outcome of our results is the formation of cross-sectionally nonuniform viscoelasticity that may have unique applications in microfluidic particle focusing.

Real-time impedimetric droplet measurement (iDM).

Droplet-based microfluidic systems require a precise control of droplet physical properties; hence, measuring the morphological properties of droplets is critical to obtain high sensitivity analysis. The ability to perform such measurements in real-time is another demand which has not been addressed yet. In this study, we used coplanar electrodes configured in the differential measurement mode for impedimetric measurement of size and velocity. To obtain the size of the droplets, detailed 3D finite element simulations of the system were performed. The interaction of the non-uniform electric field and the droplet was investigated. Electrode geometry optimization steps were described and design guideline rules were laid out. User-friendly software was developed for real-time observation of droplet length and velocity together with in situ statistical analysis results. A comparison between impedimetric and optical measurement tools is given. Finally, to illustrate the benefit of having real-time analysis, iDM was used to synthesize particles with a predefined monodispersity limit and to study the response times of syringe pump and pressure pump driven droplet generation devices. This analysis allows one to evaluate the 'warm-up' time for a droplet generator system, after which droplets reach the desired steady-state size required by the application of interest.

Reversible decryption of covert nanometer-thick patterns in modular metamaterials.

Continuous development of security features is mandatory for the fight against forgery of valuable documents and products, the most notable example being banknotes. Such features demonstrate specific properties under certain stimuli such as fluorescent patterns glowing under ultraviolet light. These security features should also be hard to copy by unlicensed people and be interrogated by anyone using easily accessible tools. To this end, this Letter demonstrates the development of an ideal security feature enabled by the realization of modular metamaterials based on metal-dielectric-metal cavities that consist of two separate parts: metal nanoparticles on an elastomeric substrate and a bottom mirror coated with a thin dielectric. Patterns generated by creating nanometer-thick changes in the dielectric layer are invisible (encrypted) and can only be detected (decrypted) by sticking the elastomeric patch on. The observed optical effects such as visibility and colors can only be produced with the correct combination of materials and film thicknesses, making the proposed structures a strong alternative to compromised security features.

Real-Time In Vivo Control of Neural Membrane Potential by Electro-Ionic Modulation.

Theoretically, by controlling neural membrane potential (Vm) in vivo, motion, sensation, and behavior can be controlled. Until now, there was no available technique that can increase or decrease ion concentration in vivo in real time to change neural membrane potential. We introduce a method that we coin electro-ionic modulation (EIM), wherein ionic concentration around a nerve can be controlled in real time and in vivo. We used an interface to regulate the Ca2+ ion concentration around the sciatic nerve of a frog and thus achieved stimulation and blocking with higher resolution and lower current compared with electrical stimulation. As EIM achieves higher controllability of Vm, it has potential to replace conventional methods used for the treatment of neurological disorders and may bring a new perspective to neuromodulation techniques.

Impedance-based viscoelastic flow cytometry.

Elastic nature of the viscoelastic fluids induces lateral migration of particles into a single streamline and can be used by microfluidic based flow cytometry devices. In this study, we investigated focusing efficiency of polyethylene oxide based viscoelastic solutions at varying ionic concentration to demonstrate their use in impedimetric particle characterization systems. Rheological properties of the viscoelastic fluid and particle focusing performance are not affected by ionic concentration. We investigated the viscoelastic focusing dynamics using polystyrene (PS) beads and human red blood cells (RBCs) suspended in the viscoelastic fluid. Elasto-inertial focusing of PS beads was achieved with the combination of inertial and viscoelastic effects. RBCs were aligned along the channel centerline in parachute shape which yielded consistent impedimetric signals. We compared our impedance-based microfluidic flow cytometry results for RBCs and PS beads by analyzing particle transit time and peak amplitude at varying viscoelastic focusing conditions obtained at different flow rates. We showed that single orientation, single train focusing of nonspherical RBCs can be achieved with polyethylene oxide based viscoelastic solution that has been shown to be a good candidate as a carrier fluid for impedance cytometry.

Evaporation-Induced Biomolecule Detection on Versatile Superhydrophilic Patterned Surfaces: Glucose and DNA Assay.

We introduce a droplet-based biomolecular detection platform using robust, versatile, and low-cost superhydrophilic patterned superhydrophobic surfaces. Benefitting from confinement and evaporation-induced shrinkage of droplets on wetted patterns, we show enrichment-based biomolecular detection using very low sample volumes. First, we developed a glucose assay using fluorescent polydopamine (PDA) based on enhancement of PDA emission by hydrogen peroxide (H2O2) produced in enzyme-mediated glucose oxidation reaction. Incubation in evaporating droplets resulted in brighter fluorescence compared to that in bulk solutions. Droplet assay was highly sensitive toward increasing glucose concentration while that in milliliter-volume solutions resulted in no fluorescence enhancement at similar time scales. This is due to droplet evaporation that increased the reaction rate by causing enrichment of PDA and glucose/glucose oxidase as well as increased concentration of H2O2 generated in shrinking droplet. Second, we chemically functionalized wetted patterns with single-stranded DNA and developed fluorescence-based DNA detection to demonstrate the adaptability of the patterned surfaces for a different class of assay. We achieved detection of glucose and DNA with concentration down to 130 µM and 200 fM, respectively. Patterned superhydrophobic surfaces with their simple production, sensitive response, and versatility present potential for bioanalysis from low sample volumes.

An integrated microfluidic device for the sorting of yeast cells using image processing.

The process of detection and separation of yeast cells based on their morphological characteristics is critical to the understanding of cell division cycles, which is of vital importance to the understanding of some diseases such as cancer. The traditional process of manual detection is usually tedious and inconsistent. This paper presents a microfluidic device integrated with microvalves for fluid control for the sorting of yeast cells using image processing algorithms and confirmation based on their fluorescent tag. The proposed device is completely automated, low cost and easy to implement in an academic research setting. Design details of the integrated microfluidic system are highlighted in this paper, along with experimental validation. Real time cell sorting was demonstrated with a cell detection rate of 12 cells per minute.

Sheathless Microflow Cytometry Using Viscoelastic Fluids.

Microflow cytometry is a powerful technique for characterization of particles suspended in a solution. In this work, we present a microflow cytometer based on viscoelastic focusing. 3D single-line focusing of microparticles was achieved in a straight capillary using viscoelastic focusing which alleviated the need for sheath flow or any other actuation mechanism. Optical detection was performed by fiber coupled light source and photodetectors. Using this system, we present the detection of microparticles suspended in three different viscoelastic solutions. The rheological properties of the solutions were measured and used to assess the focusing performance both analytically and numerically. The results were verified experimentally, and it has been shown that polyethlyene oxide (PEO) and hyaluronic acid (HA) based sheathless microflow cytometer demonstrates similar performance to state-of-the art flow cytometers. The sheathless microflow cytometer was shown to present 780 particles/s throughput and 5.8% CV for the forward scatter signal for HA-based focusing. The presented system is composed of a single capillary to accommodate the fluid and optical fibers to couple the light to the fluid of interest. Thanks to its simplicity, the system has the potential to widen the applicability of microflow cytometers.

A portable microfluidic system for rapid measurement of the erythrocyte sedimentation rate.

The erythrocyte sedimentation rate (ESR) is a frequently used 30 min or 60 min clinical test for screening of several inflammatory conditions, infections, trauma, and malignant diseases, as well as non-inflammatory conditions including prostate cancer and stroke. Erythrocyte aggregation (EA) is a physiological process where erythrocytes form face-to-face linear structures, called rouleaux, at stasis or low shear rates. In this work, we proposed a method for ESR measurement from EA. We developed a microfluidic opto-electro-mechanical system, using which we experimentally showed a significant correlation (R2 = 0.86) between ESR and EA. The microfluidic system was shown to measure ESR from EA using fingerprick blood in 2 min. 40 µl of whole blood is filled in a disposable polycarbonate cartridge which is illuminated with a near infrared emitting diode. Erythrocytes were disaggregated under the effect of a mechanical shear force using a solenoid pinch valve. Following complete disaggregation, transmitted light through the cartridge was measured using a photodetector for 1.5 min. The intensity level is at its lowest at complete disaggregation and highest at complete aggregation. We calculated ESR from the transmitted signal profile. We also developed another microfluidic cartridge specifically for monitoring the EA process in real-time during ESR measurement. The presented system is suitable for ultrafast, low-cost, and low-sample volume measurement of ESR at the point-of-care.

Impedimetric detection and lumped element modelling of a hemagglutination assay in microdroplets.

Droplet-based microfluidic systems offer tremendous benefits for high throughput biochemical assays. Despite the wide use of electrical detection for microfluidic systems, application of impedimetric sensing for droplet systems is very limited. This is mainly due to the insulating oil-based continuous phase used for most aqueous samples of interest. We present modelling and experimental verification of impedimetric detection of hemagglutination in microdroplets. We have detected agglutinated red blood cells in microdroplets and screened whole blood samples for multiple antibody sera using conventional microelectrodes. We were able to form antibody and whole blood microdroplets in PDMS microchannels without any tedious chemical surface treatment. Following the injection of a blood sample into antibody droplets, we have detected the agglutination-positive and negative droplets in an automated manner. In order to understand the characteristics of impedimetric detection inside microdroplets, we have developed the lumped electrical circuit equivalent of an impedimetric droplet content detection system. The empirical lumped element values are in accordance with similar models developed for single phase electrical impedance spectroscopy systems. The presented approach is of interest for label-free, quantitative analysis of droplets. In addition, the standard electronic equipment used for detection allows miniaturized detection circuitries that can be integrated with a fluidic system for a quantitative microdroplet-based hemagglutination assay that is conventionally performed in well plates.

Microcantilever based disposable viscosity sensor for serum and blood plasma measurements.

This paper proposes a novel method for measuring blood plasma and serum viscosity with a microcantilever-based MEMS sensor. MEMS cantilevers are made of electroplated nickel and actuated remotely with magnetic field using an electro-coil. Real-time monitoring of cantilever resonant frequency is performed remotely using diffraction gratings fabricated at the tip of the dynamic cantilevers. Only few nanometer cantilever deflection is sufficient due to interferometric sensitivity of the readout. The resonant frequency of the cantilever is tracked with a phase lock loop (PLL) control circuit. The viscosities of liquid samples are obtained through the measurement of the cantilever's frequency change with respect to a reference measurement taken within a liquid of known viscosity. We performed measurements with glycerol solutions at different temperatures and validated the repeatability of the system by comparing with a reference commercial viscometer. Experimental results are compared with the theoretical predictions based on Sader's theory and agreed reasonably well. Afterwards viscosities of different Fetal Bovine Serum and Bovine Serum Albumin mixtures are measured both at 23°C and 37°C, body temperature. Finally the viscosities of human blood plasma samples taken from healthy donors are measured. The proposed method is capable of measuring viscosities from 0.86 cP to 3.02 cP, which covers human blood plasma viscosity range, with a resolution better than 0.04 cP. The sample volume requirement is less than 150 µl and can be reduced significantly with optimized cartridge design. Both the actuation and sensing are carried out remotely, which allows for disposable sensor cartridges.

Droplet formation in microfluidic T-junction generators operating in the transitional regime. II. Modeling.

This is the second part of a two-part study on the generation of droplets at a microfluidic T-junction operating in the transition regime. In the preceding paper [Phys. Rev. E 85, 016322 (2012)], we presented our experimental observations of droplet formation and decomposed the process into three sequential stages defined as the lag, filling, and necking stages. Here we develop a model that describes the performance of microfluidic T-junction generators working in the squeezing to transition regimes where confinement of the droplet dominates the formation process. The model incorporates a detailed geometric description of the drop shape during the formation process combined with a force balance and necking criteria to define the droplet size, production rate, and spacing. The model inherently captures the influence of the intersection geometry, including the channel width ratio and height-to-width ratio, capillary number, and flow ratio, on the performance of the generator. The model is validated by comparing it to speed videos of the formation process for several T-junction geometries across a range of capillary numbers and viscosity ratios.