We hereby present a circulating tumor cell (CTC) characterization tool that is more informative than the counting of cells. CTCs are heterogeneous and only a subpopulation may be able to disseminate and metastasize. We hypothesize that (1) the metastatic and tumorigenic CTCs are the less differentiated, more stem-cell-like, and mechanically less stiff population; and (2) one can simultaneously capture CTCs and profile their stiffness, the physical property associated with tumorigenicity, for better prediction of cancer progression, recurrence, and metastasis, compared with simple enumeration. Our hypotheses are strongly supported by clinical investigations of CTCs and disseminating tumor cells, the epithelial to mesenchymal transition (EMT) and cancer stem cell theory, and earlier mechanical measurements of tumorigenic cells. We report a simultaneous microfluidics CTC capture and mechanical profiling chip to study cancer metastasis, with a clinical focus on breast cancer, based on our recently published microfluidics mechanical cell separation chip.

The presence and number of Circulating Tumor Cells (CTCs) in bloodstream of patients with epithelial cancers is an important intermediate step in cancer metastasis and is often associated with disease stage. As compared to obtaining tissue biopsy which is often challenging and invasive, “liquid biopsy” for CTCs detection can be carried out in patients due to accessibility and ease of collection during a routine blood draw. These CTCs in peripheral blood are showing their potential uses for early detection, diagnosis, prognosis and personalized treatment. Here, we demonstrate several effective methods of isolating CTCs by utilizing the unique differences in size and deformability of cancer cells from that of blood cells. By exploiting the fluid dynamics in specially designed microfiltration and spiral inertial microfluidics chips, CTCs which are generally stiffer and larger are physically separated from the more deformable blood constituents. Using this approach, we are able to retrieve intact and viable CTCs. With blood specimens from cancer patients, we demonstrated successful detection, isolation and retrieval of viable CTCs. These CTCs will aid in detecting the malignancy as well as determining their phenotypic and genotypic expressions.

Diabetes mellitus is a chronic metabolic disorder that affects over 347 million people worldwide. Diabetics have to use a blood glucose meter (BGM) several times a day for good control of blood glucose levels. The detection limit of commercial BGMs is ~2.8 mM (50 mg/dL), which prevents their use in fluids such as tears or saliva (i.e., do not require invasive extraction) whose glucose concentrations are typically 50 minus; 100× lower than what is found in blood. Over the past decade, extensive efforts have been exerted toward non-invasive glucose detection.
In this work, the synergistic advantage of combining plasmonic interferometry with an enzyme-driven dye assay yields an optical sensor capable of detecting glucose in saliva with high sensitivity and selectivity. The sensor, coined a “Plasmonic Cuvette,” is built around a groove-slit-groove (GSG) plasmonic interferometer coupled to the Amplex Red/Glucose Oxidase/Glucose (AR/GOx/Glucose) assay. In this implementation, GOx is added in solution to rapidly convert glucose into gluconolactone and H2O2 in a 1:1 stoichiometry. The H2O2 reacts with horseradish peroxidase (HRP) to oxidize Amplex Red into resorufin, a dye molecule which is characterized by a strong optical absorption coefficient at ~571nm. The reaction is monitored by simply measuring changes in the light intensity transmitted through the slit of each interferometer.
A lab-on-a-chip system is set up to perform real-time spectral measurements, using a polydimethylsilozane (PDMS) micro-fluidic channel to guide the analytes in and out of the system. The resulting device offers real-time sensitivity as high as 1.7 × 105 % / M (about one order of magnitude more sensitive than without assay) toward glucose in extremely small sensing volumes (i.e., le; 12 pL), and exhibits glucose selectivity in complex mixtures such as a 50 mM sodium phosphate buffer solution and “artificial” saliva over the physiological range of glucose concentration in saliva (20 minus; 240 mu;M). These results demonstrate the viability of measuring the concentration of glucose in saliva, which is a complex mixture of proteins, salts, and urea.
In addition, to make the device feasible for real-time glucose monitoring in saliva, the underlying reactions of the assay are studied in detail. The effective rate constants are determined and used to tune the reaction time and greatly expand the detection range of the assay. By varying the dye assay used, the integrated “Plasmonic Cuvette” can lead to point-of-care diagnostic tools for biomedical sensing of clinically relevant analytes (such as glucose and insulin) within a very small volume (sub-picoliter) of biological fluid.

Nanoscale electronic devices have the potential to achieve exquisite sensitivity as sensors for the direct detection of molecular interactions, thereby decreasing diagnostics costs and enabling previously impossible sensing in disparate field environments. Semiconducting nanowire-field effect transistors (NW-FETs) hold particular promise, though contemporary NW approaches are inadequate for realistic applications and integrated assays. We present here an integrated nanodevice biosensor approach [1] that is compatible with CMOS technology, has achieved unprecedented sensitivity, and simultaneously facilitates system-scale integration of nanosensors. These approaches enable a wide range of label-free biochemical and macromolecule sensing applications, such as specific protein and complementary DNA recognition assays, and specific macromolecule interactions at femtomolar concentrations.
Critical limitations of nanowire sensors are the Debye screening limitation [3], and the lack of internal calibration for analyte quantification, which has prevented their use in clinical applications and physiologically relevant solutions. We will present approaches that solve these longstanding problems, which demonstrates the detection at clinically important concentrations of cancer biomarkers from whole blood samples [4], integrated assays of cancer biomarkers [5], sensor reversibility [6], and the use of these as a quantitative tool for drug design and discovery [7].

A sensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients&’ health and their response to therapeutics. Such technology would enable truly personalized medicine, wherein therapeutic agents could be tailored with optimal doses for each patient to maximize efficacy and minimize side effects. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets, such as glucose, lactose, and oxygen, and the few existing platforms for continuous measurement are not generalizable for the monitoring of other analytes, such as small-molecule therapeutics. In response, we have developed a real-time biosensor capable of continuously tracking a wide range of circulating drugs in living subjects. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system&’s versatility, we measured therapeutic in vivo concentrations of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats and in human whole blood for several hours with high sensitivity and specificity at sub-minute temporal resolution. Importantly, we show that MEDIC can also obtain pharmacokinetic parameters for individual animals in real-time. Accordingly, just as continuous glucose monitoring technology is currently revolutionizing diabetes care, we believe MEDIC could be a powerful enabler for personalized medicine by ensuring delivery of optimal drug doses for individual patients based on direct detection of physiological parameters.

Domain wall (DW) conduits are powerful tools for the manipulation of biological entities linked to magnetic nano and microparticles. [1] For zig-zag shaped structures a micron sized stepper motor can be implemented allowing to transport magnetic particles over large distances. In case of curved structure, high precision in the positioning of a single nanoparticle can be achieved (down to 100 nm) so as the same platform can be used to implement magnetic tweezers to be employed in biology.
In this work we present a novel functionality recently added to our platform: the on-chip electrical detection of the particle passage at a fixed point. This has been achieved by adding a couple of nano-electrodes to a prototypical zig-zag shaped structure, flanking a particular corner. When a domain wall bound to a magnetic particle pass through said corner, the micromagnetic configuration changes and the resistance between the two electrodes falls down due to the anisotropic magnetoresistance (AMR) effect. The AMR signal can then be monitored during the DW passage, so as to detect at which external magnetic field H the DW jump in and out of the corner. [2] As a matter of fact the H field needed to depin the DW-particle complex from the corner (to make the particle to jump out) is higher than that of a single DW not carrying any particle: this provides the way to distinguish the complex from a simple DW. In this contribution we demonstrate the contemporary actuation and detection of the motion of a magnetic particle along a zig-zag conduit. This paves the way to the development of a closed-loop microfluidic platform where biological entities bound to magnetic particles can be remotely manipulated on-chip, via external magnetic fields, and their position checked electrically, without need of using an external microscope. Applications to high throughput fundamental biological investigations, as well as to sample preparation for diagnostics are foreseen.
[1] M. Donolato et al., Adv. Mater, 22, 2706 (2010)
[2] M. Donolato, M. Gobbi, P. Vavassori, M. Leone, M. Cantoni, V. Metlushko, B. Ilic, M. Zhang, S. X. Wang, and R. Bertacco, Nanotechnology 20, 385501 (2009)

The fabrication of micro and nanofluidic devices has been an emerging field of research in recent years. A growing number of applications are found in the areas of nano-science and biological science such as biosensors, drug delivery systems and DNA analytical systems [1]. Single molecule techniques are nowadays used for the investigation of bio-macromolecules like double strand DNA.
In our experiments, proton beam writing (PBW) is used to fabricate two sets of nanochannels in a perpendicular configuration. A very thin layer of teflon is coated on this master mold to promote the demolding process [2], allowing accurate PDMS replication for single DNA molecule manipulation. By flushing different types of salt buffer or protein in the smaller set of channels, the in-situ response in conformation of single DNA molecules to a change in environmental solution conditions can be observed [3]. We have also shown that DNA can be stretched up to 85% of its contour length by polypeptide bottlebrush coating in an array of nanochannels fabricated by soft lithography in elastomer. This stretch is shown sufficient for sequencing of large-scale genomic information [4].
A ratchet structure is also fabricated by PBW with thickness of 20 µm for magnetic particle separation. Two micro electrodes are incorporated just beside this ratchet microchannel in order to drive magnetic beads. By applying a current up to 0.2 A, we can manipulate the magnetic beads inside the ratchet channel. Different size of beads can be separated by our special ratchet structure because of the different diffusion coefficient.
PBW is a relative new 3D direct write technique. MeV proton beams can be focused down to sub 30 nm spot sizes [5]. A proton beam shows practically no spread while penetrating thick resist layers, resulting in vertical and straight sidewalls, facilitating proximity free production of nanostructures. Since PBW is a direct write technique the throughput can be amplified if combined with NIL like PDMS casting. OrmoStamp [6] will be evaluated in combination with PMDS casting and nano imprinting on PBW fabricated masters.
[1] Patrick Abgrall, Nam Trung Nguyen, Nanofluidics. 2009.p.1-7
[2] [3]C. Zhang, K. Jiang, F. Liu, P. S. Doyle, J.A. van Kan and J.R. C. van der Maarel, Lab Chip. 2013 13(14) 2821-6
[4]C. Zhang, A. H.Garcia, K. Jiang, Z.Y. Gong, D. Guttula, S.U.Ng, P. P. Malar, J.A. van Kan, L. Dai, P. S. Doyle, R. D. Vries and J.R.C. van der Maarel Nucleic Acids Research, 2013, 1-8
[5] J. A. van Kan, P. Malar, and A.B. Vera, Review of Scientific Instrumnets 83, 02B902 (2012)
[6]I.F. Cuesta, A. L. Palmarelli, X.G. Liang; J.Y. Zhang; S. Dhuey, D. Olynick; and S. Cabrini Journal of Vacuum Science & Technology B, 29, 6 (2011)

An array of cracks can form in multilayered materials under an applied tensile strain when either the toughness mismatch or the modulus mismatch is sufficiently large between the layers. This phenomenon has been extensively studied as a fundamental problem in fracture mechanics and for its practical application in micro/nanofabrication. While energy-based theoretical analysis of the mechanics of cracking suggest that an inverse relationship exists between the applied strain and crack spacing, achieved spacing is often better represented by a broad statistical distribution due to the presence of intrinsic flaws in the materials used. This distribution makes it challenging to use the crack-engineering paradigm for precision fabrication purposes. Here, we investigate a robust and versatile method to precisely guide crack formation in multi-layered materials with controlled periodicity. This approach, incorporating stress focusing V-notch microstructures in the elastomer, enables the production of adjustable and well-controlled cracks at user-defined locations on both flat and curved surfaces, by simply stretching the multilayered materials. We also suggest mechanical regimes about the appropriate balanced conditions between the applied strain and the spacing of stress-concentrating V-notch structures, independent from a density of intrinsic flaws in the multi-layered soft materials.

Imaging sub-cellular processes in bacteria is complicated by the small size and motile life-style of these organisms. Currently, many research groups immobilize bacteria for individual live-cell imaging by spreading them on an agar pad and then sealing the culture with a microscope coverslip. This method limits imaging only to few hours before the bacteria form colonies on agar. In colonies, the cells begin overlapping and cannot be individually imaged. Moreover, cells in such colonies have different micro-environments across the colony, growing at different speeds. In recent years, several research groups have begun using microfluidic devices for single-cell imaging that partially alleviate the aforementioned problems. All devices reported so far consist of various passive channels in elastomer or hydrogel materials, which guide bacterial growth and remove extra cells by fluid flow. While these devices have considerably increased the time available for microscopic imaging, they still have some limitations. The shortcomings include cell retention in the channels, uniform nutrient availability in the channels and the ability to quickly change the chemical environment. In this presentation, we first characterize bacterial growth in a promising device reported earlier. The device, referred to as the bacterial mother machine, consists of large number of dead-end microchannels in PDMS elastomer. We show that bacteria in these channels experience nutrient limitation. Moreover, loading cells to these channels is complicated and they tend to grow in overlapping lines, as opposed to single-file lines. To overcome these limitations we implement a continuous flow of cell culture media past the cells in the channel. To prevent the cells from drifting, we use micron-scale pressure-actuated microvalves to partially close off the channels after the cells are loaded. The presentation describes the fabrication process of microchannels with these ultra-small microvalves and preliminary measurements using these novel structures.

Patients with chronic conditions, such as diabetes and hypertension, have higher risk of kidney failure. Approximately 40% of patients with type 1 and 20% with type 2 diabetes develop nephropathy and eventually need artificial dialysis or kidney transplant. When it is possible to identify kidney disease in the early stages, patients and doctors can adjust treatment to have better control of diabetes and hypertension by maintaining tight glycemic control and reducing blood pressure; as a result, the progression of kidney disease can be slowed or prevented.
In this work, we describe an injection moulded plastic bio-cartridge and reader that can be used to measure the concentration of albumin and creatinine in a single urine sample. The system uses the Jaffe reaction to detect creatinine and a new detection method using anti-albumin and highly reactive polymer particles to detect albumin. The plastic cartridge has a sample gathering pipette, pneumatic inlet, needle transferring pneumatic pressure to the sample gathering pipette, and two measurement modules to measure the albumin and creatinine levels in the urine sample. Each measurement module has a reaction chamber with an optical window, micro-sized channel to transfer the urine sample from the sample gathering pipette, venting hole to exhale the excess air in the reaction chamber, and urethane coated iron balls to mix the sample solution and the measurement reagents.
To demonstrate the clinical reliability of the cartridge-reader system, we performed CV (coefficient of variation) testing for both creatinine and albumin.

A recent report presents novel three dimensional (3D) structure of graphene transformed from two dimensional (2D) graphene oxide (GO) sheets due to their enhanced specific surface area, high compression/aggregation resistance and superior electrochemical performance. Thus, the 3D structure and the intrinsic excellent properties of graphene synergistically conbine, so a variety of applications of 3D graphene including energy storage and as a catalyst support seem promising.
Regarding the synthetic method for the 3D graphene particles and their nanohybrids, Huang et al. reported the use of an ultrasonic atomizer to form aerosol droplets and a horizontal tube furnace to induce the shrinkage, resulting in crumpled balls at submicrometer scale. Wang et al. explained the crumpling phenomenon by correlating the confinement force and the evaporation rate. However, to diversify the applications of 3D graphene, there is still room for improvement in generating the 3D graphene structure. Firstly, the aerosol based method has limitations in terms of controlling the size of the droplets. Secondly, the concentration of the encapsulated GO in the aerosol droplet was randomly distributed, rendering the precise tenability of the morphology difficult. Thus, a novel synthetic method for producing a 3D graphene structure with a wide range of controllability for particle size and morphology is required.
Recently, droplet-based microfluidic technology has garnered huge attention due to its unique characteristics such as operational simplicity, high homogeneity of the droplets, facile breakage and convergence of droplets, and high-throughput generation capability. Typically the droplet can serve as a nanoliter-scale reactor, and various chemical and biochemical applications can be performed with high speeds and high uniformity. Not only can the droplet size be tuned from micron to nano by simply changing the flow rate of the aqueous phase or the oil phase, but also the composition of the reagents in the droplet can be varied depending on the input solution. Utilizing those advantages of the microfluidic droplets, many efforts have been dedicated to the development of chemical/biomolecular.
In this study, we have demonstrated the synthesis of 3D graphene microballs (3D GMs) by using a microfluidic droplet generator. The GO solution was encapsulated in the droplets and underwent capillary compression to form the 3D structure. The controllability of the flow rate and the concentration of GO allows us to generate different sizes and morphologies of the 3D GMs. In addition, we loaded iron oxide nanoparticles (Fe3O4 NPs) together with the GO solution, resulting in 3D nanoparticle-graphene hybrids. Finally, we applied the as-synthesized spherical 3D graphene as a polymer composite support, and have shown polymer-graphene microball composites with a core-shell structure for the first time.

Many efforts have focused on the fabrication of three-dimensional (3D) microfluidic channels to provide an artificial microvascular system. The most widely accepted fabrication method is based on soft-lithography or molding techniques. However, SU-8 negative photoresist template and metal mold based stamping methods typically generate square, rectangular or trapezoidal cross-sectional shapes in the microchannels or require an expensive metal molder for the stamping and casting processes. Considering that the cross-sectional shape of a microchannel determines fluidic dynamics such as shear stress on the endothelium layer, the fabrication of a perfect circular microfluidic channel is crucial to mimic the microvacular model. Moreover, the non-circular microchannel causes difficulties in stable cell seeding at the corners, preventing uniform formation of a confluent endothelial cell layer.
In this study, we described a simple and efficient fabrication method for generating microfluidic channels with a circular-cross sectional geometry by exploiting the reflow phenomenon of a thick positive photoresist. Initial rectangular shaped positive photoresist micropatterns on a silicon wafer, which were fabricated by a conventional photolithography process, were converted into a half-circular shape by tuning the temperature to around 105 °C. Through optimization of the reflow conditions, we could obtain a perfect circular micropattern of the positive photoresist, and control the diameter in a range from 100 to 400 mu;m. The resultant convex half-circular photoresist was used as a template for fabricating a concave polydimethylsiloxane (PDMS) through a replica molding process, and a circular PDMS microchannel was produced by bonding two half-circular PDMS layers. A variety of channel dimensions and patterns can be easily prepared, including straight, S-curve, X-, Y-, and T-shapes to mimic an in vivo vascular network. To form an endothelial cell layer, we cultured primary human umbilical vein endothelial cells (HUVECs) inside circular PDMS microchannels, and demonstrated successful cell adhesion, proliferation, and alignment along the channel.

One of the main issues in tissue engineering is mimicking the well-defined three dimensional microvascular architecture found in native tissues in the human body. For fully understanding the functional tissue engineered vascular remodelling, it will be essential to mimic the in vivo environment with in vitro model. Smooth muscle cells (SMCs) in the native blood vessel are three dimensionally and circumferentially aligned and elongated. Moreover, vascular SMCs have contractile and spindle-shaped morphology. Since the mechanical properties of SMCs such as strength, elasticity and contractility mainly rely on the unique 3D structure and multiple layers of vascular SMCs, it is necessary to model the in vivo-like in vitro blood vessel system.
The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40 % stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.

Control of cell-substratum interaction is essential to construct functional engineered-tissues. To this end, a variety of topographical patterns such as grooves, pillars, holes and fibers have been developed, and their effect on the cellular responses including cell morphology, alignment, migration, differentiation, and nuclear deformation have been investigated. The surface topography pattern ranges from micrometer- to nanometer-scale, and both of the micro and nanopatterns lead to the cellular and nuclear morphology change. In a vasculature system, endothelial cells are oriented longitudinally in the intima, while the SMCs (Smooth Muscle Cells) are circumferentially aligned in a media layer. In order to construct a pattern SMC layer, an appropriate nano or microwrinkle structure would be necessary to guide the cellular alignment mimicking the in vivo SMC morphology.
In this study, we fabricated a variety of the nano and microwrinkle structures on the PDMS surfaces by using a non-lithographical strain responsive wrinkling method. By modulating the UV/O exposure time (10, 20, 30, 40, 50, and 60 min) and the stretching rate (10, 20, 30, and 40%), wrinkle-free, nanowrinkle, and hierarchical nano/microwrinkle patterns could be produced, and the wavelength and the amplitude of the resultant wrinkle patterns could be controlled. As the UV/O irradiation time was prolonged, the thickness of the oxide layer was gradually augmented, but the increment was reduced as the exposure time became longer. The wavelength and amplitude of the nano or microwrinkles increased in proportional to the thickness of the oxide layer. The topographical effect of the PDMS surface on the SMC alignment was investigated, and the filamentous actins of the cells on the wrinkle-free PDMS substrate revealed the random orientation and elongation in all directions. In a similar way, the nanowrinkled surface shows the irregular cellular alignment. On the other hand, the filamentous actins of the cells cultured on the nano/microwrinkled substrate were elongated parallel to the wrinkle direction. Our simple methodology could provide the wrinkle structure from nano- to micro-scale, and the produced wrinkle surface could be utilized for constructing a biomimetic cellular organization.

Here we report a readily accessible microfluidic method for generation of micrometer to sub-millimeter scale polymer brush gradients. The lateral gradient in polymer brush chemistry was formed through surface-initiated atom transfer radical polymerization (ATRP) via co-flow of two different monomer solutions in a poly(dimethylsiloxane) (PDMS) microfluidic channel above an initiator-coated substrate. The laminar flow of the two monomer solutions in the channel formed a smooth gradient perpendicular to the flow direction and parallel to the substrate in monomer concentration at the substrate-fluid interface, resulting a laterally varying chemistry of polymer brushes on the substrate. Two monomers, 2-(hydroxyl)ethyl methacrylate (HEMA) and 2-methacryloyloxy ethyl trimethylammonium chloride (METAC), were chosen to form a gradient between neutral and cationic polymers, respectively. We found the optimized condition of chemical compositions for the reliable growth of PHEMA and PMETAC via ATRP in the microfluidic channel under ambient conditions. The density gradients of the two polymer brushes were quantitatively characterized with confocal Raman spectroscopy. The length and the slope of the resulting polymer brush gradients can be controlled by varying the flow rate, and the channel dimension. Brush gradients as narrow as 50 micrometer were formed by this method. This facile microfluidic method is quite general, and enables precise manipulation of narrow polymer brush gradients on surfaces.

Asymmetric structures, as inspired from the Mother Nature, have extensively received attention because of their unique directional physical properties and potential uses in microfluidics and biomedical devices. Although many researchers have so far developed different versions of fabrication methods to realize such asymmetric structures, there are still strong needs for improved fabrication techniques to directly integrate the asymmetric structures within microchannels in simple and economic ways because the integration of patterned structures within microfluidic channels is typically complicated. In this presentation, we propose a direct and facile method to integrate “programmed” asymmetric ratchet structures within microchannels by combining photo-polymerization with guided light transmission through optically asymmetric prism arrays. We employed the selected light refraction into one direction through the optical prism array to crosslink liquid prepolymers within a microchannel in an asymmetric way. The asymmetric ratchet structures, directly realized within microchannels, show the unidirectional liquid flow and can even control the fluid speed in a specific region predefined within a microchannel. To show the proof of concepts, we demonstrate examples of the direct realization of asymmetric ratchet structures within microfluidic channels to control the fluid speed as well as the rate-dependent channel filling showing different dwell times in a split microchannel.

Recently there has been great interests to apply inkjet printing technology in diverse fields of biology, chemistry, medicine and nano-technologies. Two popular techniques are widely employed to make reliable atomized liquid droplets: piezo inkjet and thermal inkjet techniques. In this presentation, we describe our design and construction for a drop-on demand (DOD) droplet dispenser using the piezo inkjet technique that is simple to construct and operate and makes use of readily available components. The droplet dispenser can be easily fitted with cost effective glass nozzles and can be reliably tuned to produce consistent droplet sizes in the micron range. Also, we describe a camera imaging system that is constructed to determine the ejected droplet velocities. Using Stoke's law for fluid drag force, this imaging system further facilitates independent measurement for the dispensed drop diameters through the terminal velocities reached by the droplets.

Previously at the MRS Spring Meeting 2013, we presented the fabrication and characterization of third generation of electrical nano-biosensors array embedded in a microfluidic channel, which we referred to as Nanoneedle biosensors. Here, we present a modified generation of devices, which are made of more biocompatible materials with higher sensitivity compare to the previous generations. The nanoneedle biosensor is a real-time, label-free, direct electrical detection platform, which uses ionic current and impedance modulation to detect biomolecules such as proteins or nucleic acids with high sensitivity. The miniature nanoneedle tip can allow for the accurate electrical detection of a small numbers of molecules binding to the tip. A nanoneedle biosensor structure consists of four thin-film layers, two conductive layers with an insulator layer in between. A protective oxide layer is fabricated above the top electrode and underneath of the bottom electrode to prevent the exposure of conductive electrodes to the solution. The utility of this sensor for label-free bio sensing was demonstrated and the electrical response of nanoneedle for various types of biological agents was studied. We demonstrate the utility of this sensor in affinity biosensing and using biotinilated BSA and Streptavidin as a model system. As a practical example with clinical relevance, we also demonstrated the detection of Vascular Endothelial Growth Factor (VEGF) for cancer diagnosis. Anti-VEGF was immobilized on the sensor surface using ODTS salinization chemistry and impedance changes of the sensor for VEGF proteins as a function of time was measured. As a control experiment Streptavidin was injected to the microfluidic channel. As expected no binding occurred between Streptavidin and Anti-VEGF molecules and the measured impedance level came back to its previous level after a wash step. Our demonstration of label-free and real-time detection of VEGF with this sensor can be envisioned to allow for point-of-care cancer diagnosis.

Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse BBB and infiltrate into the CNS. Cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in CSF may represent important sources to investigate immune-to-brain interaction, or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire (SiNW) array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody-coated 3D nanostructured material such as a silicon nanowire array exhibits vastly improved rare cell capture efficiency. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively capture a specific leukocyte phenotype. Comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer&’s disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool potentially for diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective hematological analysis of CSF from patients.

Microfluidic devices, consisting of networks of channels with micrometer dimensions, have received increased attention in a wide range of applications including analytical systems, biomedical devices, and tools for chemistry and biochemistry. They can produce monodisperse droplets with exceptional precision, which are useful as individual compartments for chemical reactions and templates for preparation of monodisperse functional particles. To date, several types of microfluidic devices such as glass capillary microfluidic devices and polydimethylsiloxane (PDMS) devices by soft lithography have been developed. Although using soft lithography facilitates accurate control of the positions and sizes of the channels in the device through the design of mask patterns, it is difficult to fabricate flow channels in three dimensions, which would limit the utility of the device. We have studied fabrication of three-dimensional microfluidic devices by stereolithography. Stereolithography is a method to build a three-dimensional object layer by layer through the photopolymerization of a liquid monomer resin on the basis of computer-aided design (CAD) data. Therefore, fine-tuning of the three-dimensional channels can be easily and efficiently performed. In this presentation, we report that microfluidic devices with three-dimensional flow channels can be fabricated by stereolithography. We also show that by hydrophilic and hydrophobic surface treatment of the flow channels, the device can produce monodisperse oil-in-water (O/W) and water-in-oil (W/O) emulsions, respectively. In addition, we demonstrate that monodisperse water-in-oil-in-water (W/O/W) double emulsions can be prepared by combing the devices for preparation of monodisperse O/W and W/O emulsions.

The mitochondria function as the powerhouse of the cell by enabling aerobic respiration and play key roles in apoptosis, metabolism, and various signaling pathways. Measuring the mitochondrial oxygen consumption rate not just reflects the functional status of the organelles (e.g. capacity to produce ATP) but can also reveal any perturbing cellular damages in diseases, cancer, and aging. However, available technology requiring significant sample size, namely a large number of cells or a significant amount of mitochondrial protein, prevents the investigations of single cell or individual mitochondrial respiration, which is an unexplored territory and could hold biological significance. The present work demonstrates 10 um x 10 um x 10 um micro-chambers as effective technology to monitor mitochondrial respiration. Specifically, the chambers are etched out of borofloat glass substrate using standard lithography and HF wet etching. Phosphoresent dye Pt-coproporphyrin (PtCP) mixed with polystyrene is deposited the bottom of the chambers as the oxygen sensing layer. Oxygen levels at 0%, 20%, and 100% are used to establish a 3 point calibration curve for each of the micro chambers. Single cells or isolated mitochondria can be deposited randomly into individual micro-chambers for further assessment. After biological sample is placed and confirmed with an optical microscope, a sealing flexible layer made out of PDMS coated with oxygen impermeable Viton rubber is used to prevent diffusion of oxygen from the environment to the chambers. As a result, fluorescence intensity measurements of the oxygen concentration in the chambers (red channel) directly correlate to the rate of respiration of the biological sample trapped inside. Experiments with coupled vs. uncoupled mitochondria i.e. normal vs. FCCP-treated confirms the validity of the micro-chambers in sensing mitochondrial respiration. This is the first demonstration in high-throughput sensing mitochondrial respiration using a low-cost device with without the problem of oxygen diffusion which has limited the use of micro devices. It is also the first demonstration that single mitochondrial respiration measurement is possible.

Single-cell functional proteomics assays can connect genomic information to biological function. through quantitative and multiplex protein measurements. A single-cell functional proteomics assay is one that measures the quantity and functional state (such as phosphorylation) of a given protein or panel of proteins across many otherwise identical cells. A measurement of
the average level of a protein requires many single-cell measurements. Such measurements, if compiled as a histogram of the frequency of observation versus the measured levels, reflect the fluctuations of that protein. If many proteins are simultaneously measured from the same single cells, then it becomes possible to directly quantify protein-protein interactions. The experimental determination of fluctuations and interactions provides a conduit between 'complex' biological problems and the simplifying nature of the physico-chemical laws. In this talk, I will discuss technologies we have developed for such measurements, and how those platforms are being applied towards fundamental and clinical problems in oncology.

Rapid, accurate, and quantitative characterization of immune status of patients is of utmost importance for disease diagnosis and prognosis, evaluating efficacy of immunotherapeutics and tailoring drug treatments. Immune status of patients is often dynamic and patient-specific, and such complex heterogeneity has made accurate, real-time measurements of patient immune status challenging in the clinical setting. Recent advances in microfluidics have demonstrated promising applications of the technology for immune monitoring with minimum sample requirements and rapid functional immunophenotyping capability. In this talk, I will discuss our recent efforts in developing integrated microfluidic immunomonitoring platforms that can perform rapid, accurate, and sensitive cellular functional assays at the single-cell level on different types or subpopulations of immune cells, to provide an unprecedented level of information depth on the distribution of immune cell functionalities. Such innovative tool will allow comprehensive and systems-level immunomonitoring in the clinical setting, unlocking the potential to transform experimental clinical immunology into an information-rich science. Our microfluidics-based technology can serve as a comprehensive and standardized immune monitoring platform to define and characterize the “immunotype” of healthy individuals and patients before, during, and after targeted immunomodulation.

We developed a highly sensitive waveguide-mode sensor with a size of 5cm*10cm*15cm, where a SiO2 waveguide layer was formed by thermal oxidation of the surface of a single crystalline Si layer of a silicon-on-quartz substrate. The final goal of this research is to develop a palmtop sensor to detect hepatitis B virus and hepatitis C virus and to determine the blood type before surgery. Patterns of micro-channel were drawn on a silicon wafer with a mask-less lithography system followed by etching with inductively coupled plasma reactive ion etching system. The patterned silicon wafer was used for mold for poly-dimethylsiloxane (PDMS). The patterned PDMS was fixed on a sensor chip for waveguide mode sensor with illumination of excimer lamp. Thus, micro-channel was created on a sensor chip. Optical microscope was employed to observe the flow during each experiment. A syringe of 1-mL of blood was loaded for each experiment. The blood plasma separation experiment was conducted while changing input flow rate. The blood was diluted with phosphate buffered saline with different dilution levels. The principle of the blood plasma separation from blood cells was supported by the Zweifach-Fung effect and was experimentally demonstrated using microchannels. Human blood plasma separation has direct obvious applications in disease diagnostic and blood typing. A blood sample was mixed with antibodies against type A and B bloods, and the sample was checked whether the blood cells were agglutinated or not. Agglutination for type A and B bloods with respective antibodies can be clearly observed with the developed waveguide mode sensor. This work was financially supported by JST.

Ability of label-free biomolecule detection at an ultra-low concentration has made silicon nanowire field-effect-transistor (SiNW FET) based biosensor technology as one of the most suitable alternatives to the widely used fluorophore labeled approach. In addition, well-matured CMOS technology offers large-scale, high-density integration and possibility to interface with conventional electronic systems to realize low-cost portable sensing devices for instant readout of complex biomolecular binding events. Other advantages of Si FET biosensor include short detection time allowing rapid delivery of test results, multiplexing i.e., simultaneous detection of multiple analytes and high dynamic range. However, the high detection sensitivity of such NW FET achieved through higher surface-to-volume ratio also brings additional challenges associated with strong signal arising from non-specific binding as well as coupling with surrounding electronic disturbances which are often very difficult to distinguish from the signal arising due to the real biomolecular binding. A suitable solution to such problems is to design a delivery system so that the reference signal can be collected in absence of analyte.
We present a multichannel microfluidic platform integrated with Si NW FET sensor arrays, which offer possibilities for multiple sample injection as well as for performing control experiments for reliable detection of biomolecular binding. Microfluidic channels are defined in a polymer layer covered by a PDMS lid. The channels pass the nanowires at right angles and functionalization can be done individually using a spotting approach. Sample delivery is performed with an automated multi-sample injection system to reduce the erroneous sensor responses arising due to switching and fluctuation in flow rate. The efficiency of the design is demonstrated by performing experiment aimed to detect binding of streptavidin molecule to biotin, which is functionalized on NW surface. In addition, chemical sensing was performed by measuring different pH under sequentially injected pH buffers. The analysis of response from the reference sensor shows occasional appearance of different noises and instability, which was then subtracted from the sensor response to get the true signal. Our results demonstrate that the multichannel microfluidic system can be used for simultaneous detection of noises and other undesired response of the sensor and thus can be used to improve the reliability of the biosensor design. In addition, the system also allows simultaneous detection of multiple analytes by using separate delivery channels addressing appropriate sensor arrays.

We are developing general strategies to passively manipulate fluids using simple geometric modifications within microchannels. Our approaches make use of fluid inertia, generally neglected in microfluidic systems, to create well-defined directional forces and fluid deformations that can be combined in a sequential and hierarchical manner to program complex particle and fluid motions. We apply these fundamental techniques to a variety of applications in materials fabrication, cell separation and analysis. I will present one example in which we generate pre-defined complex 3D polymer particles from precursor materials using a combination of flow sculpting and photopolymerization. Pre-programmed shapes can be achieved following a quick design phase with simple to use software that does not require case-by-case fluid dynamic simulation. Low complexity modular components to manipulate cells, particles, and fluid streams in which inertial fluid physics is abstracted from the designer can transform biological, chemical, and materials automation in a similar fashion to how modular control of electrons and abstraction of semiconductor physics transformed computation.

Microfluidic synthesis has received much attention as the advantages of microreactors such as increased safety, the better thermal and mixing control, which leads to higher reproducibility and better efficiency than traditional batch process. Versatile microreactor techniques have been adapted for continuous synthesis of a wide spectrum of materials via polymerization, precipitation, and sol-gel techniques.
This talk covers synthesis of inorganic nanomaterials with the uniform structure such as zeolite, metal organic framework and N-doped graphene in a fast and continuous way and multiple transformations of fine chemicals. At first, a droplet- and ionic liquid-assisted microfluidic process was used for an ultrafast, mild, and continuous synthesis of various inorganic nanomaterials that are difficult to produce. Nanoporous ZSM-5, γ-AlOOH, and β-FeOOH nanorods were synthesized in only “20 min” of reaction time even with simple instrument. Secondly, metal organic framework (MOF) structures with homo- and hetero-compositions were synthesized in confined microdroplets under ultra-fast and continuous solvothermal or hydrothermal conditions. Representative MOF of HKUST-1, MOF-5, IRMOF-3, and UiO-66 were synthesized within a few minutes at solvothermal condition. The preparation of Ru3BTC2 crystals at high-pressure hydrothermal conditions, heterostructured core-shell MOF with enhanced hydrostability or magnetic-core-MOF-shell composites with improved catalytic properties were synthesized by a novel microfluidic approach. Finally, the microsonochemical process was very useful to produce highly N-doped graphene oxide for excellent selectivity in catalytic oxidation of the aliphatic C-H bond containing an active functional group in adjacent position. In addition, several integrated continuous microsystem for synthesis of organic chemicals will be presented. Au, Pd and Ag nanometals were deposited in a site-selective manner on the vertically aligned mesoporous silicate thin film derived from block copolymer. The heterogeneous catalysts in the microfluidic chemical reactions were demonstrated even after thermal and chemical exposures at harsh conditions. A serial process for in-situ generation, separation, and reaction of malodour isocyanides was conducted in an integrated microreactor system.
Key words: Nanomaterial, Microreactor, Continuous flow, ultrafast, Synthesis

Current microfluidic device fabrication is generally based on high cost and low throughput techniques such as photo-lithography, imprinting, micromolding, and CO2 laser writing, which hinders the widespread use of the microfluidic devices. In addition, it is very challenging to design three-dimensional microfluidic devices using the common fabrication techniques. Therefore, simple and high throughput fabrication techniques with design flexibility are needed for next generation microfluidic devices (e. g. for biomolecular analysis, patient home test systems and chemical synthesis).
In this research, we developed a simple method to manipulate fluid behavior in both vertical and lateral directions and to transport a fluid stream crossover another without mixing using hierarchically structured polymer fibers. Utilized rice leaf-like micro/nano structured polyetherimide (PEI) fibers, were produced in two steps. In the first step, star-shaped very long polymer microfibers (typically tens of meters in length and hundreds of microns in diameter) were produced by thermal drawing of a surface-structured PEI preform. Resulting fibers have micro-scale (typically 10 to 50 µm in both width and depth) ordered grooves on their surfaces continuing throughout the fibers. In the second step, an additional hierarchy level was introduced on star-shaped fibers by coating with polydopamine (PDA) or methyl-modified silica (MMS) nanoparticles. The PDA coated star-shaped fibers exhibited anisotropic superhydrophilic behavior, which enables spreading of small liquid portions (1 µL) through distances in centimeter range on the fibers. On the other hand, MMS coated fibers demonstrated anisotropic superhydrophobicity, in other words directional water repellency, which allows droplet transport from one point to another. We simply constructed complex three-dimensional microfluidic architectures by using the fibers cut in proper lengths and adhesive tape as building blocks, and demonstrated water transport and spreading on these prototypes. We believe that this high throughput, low cost and simple method can be used in designing disposable microfluidic devices for early stage diagnosis and point-of-care analysis, as well as proving to be a novel fabrication scheme for further possibilities that require tuning of liquid behavior on large-areas and flexible systems.

Identifying, collecting and analyzing rare malignant cells (ideally free-flowing) in bio-fluids could be crucial to detect patients response to therapy, to monitor drug resistance effects, as well as to early identification of disease recurrence, and ultimately for a personalized medicine approach. We previously developed a bright, spectrally rich, multiplexing SERS platform dubbed SERS Biotags (SBTs), composed of a silver nanoparticle dimer core, that we used to detect the unique neuropilin-1 biomarker expression pattern of prostate cancer cell, contrasting them to healthy prostate cells. The SERS spectrum of an individual SBT acts as a unique barcode that is easily differentiable in a composite SERS spectrum originating from many tags. The SERS intensities achieved are comparable to fluorescence. We have now developed a combined microfluidic and SERS platform for the identification of individual mammalian cells on the fly. In a flow-focused microfluidic channel we injected a mixture of cancer and normal cells, pre-labeled with SBTs. We demonstrate the identification of individual cells by spectral unmixing, of the Raman signature of each cell passing single-file through the Raman laser.

Deterministic lateral displacement (DLD) arrays have been used to concentrate circulating tumor cells (CTCs) in diluted whole blood at flow rates as high as 10 mL/min with capture efficiencies exceeding 85% (K. Loutherback et al., AIP Advances, 2012). However, the equivalent volume of undiluted whole blood that can be processed is limited to 0.3 mL per DLD array due to clogging of the array. Since the concentration of CTCs can be as low as 1 - 10 cells/mL in clinical samples, increasing the volume of blood that can be processed with DLD arrays is important in order to allow collection of sufficient numbers of CTCs for biological experiments. Furthermore, by bumping large cells, such as CTCs, into a buffer stream, DLD arrays can be used to harvest CTCs free of the background of smaller components, such as leukocytes, erythrocytes, platelets, and plasma, present in blood, resulting in a highly enriched product (J.A. Davis, et al., PNAS, 2006).
In this talk, we (i) identify the two dominant biological mechanisms causing clogging of the array, (ii) demonstrate a method for inhibiting these two mechanisms, and (iii) show that shear-induced platelet aggregation is only a minor contributor to clogging of the array. By comparing the reduction in clogging achieved by the calcium-chelating anticoagulants EDTA and ACD to that achieved by the indirect thrombin inhibitor heparin as well as by measuring the EDTA concentration-dependent reduction in clogging, we identify the activity of calcium-dependent integrins as a dominant contributor to clogging. Combining EDTA with the direct thrombin inhibitor PPACK, we identify thrombin-induced platelet activation as the second dominant mechanism contributing to clogging. Using a combination of EDTA and PPACK, we demonstrate a 40-fold decrease in clogging of the array, which allows a commensurate increase in the volume of blood processed. Based on data in a single-channel device (2mm wide), we expect a complete chip to be able to process >100 mL quantities of whole blood in 30 minutes without significant clogging. Finally, we inhibit the glycoprotein IIb/IIIa integrin complex, which is activated by shear forces, using the glycoprotein IIb/IIIa inhibitor tirofiban to show that shear-induced platelet aggregation plays only a minor role in clogging of the array.

All correspondence should be addressed to KMJ (kathryn.miller-jensen@yale.edu) and RF (rong.fan@yale.edu)
Recent evidence indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. Non-genetic heterogeneity is emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies, suggesting the need for practical tools for single cell analysis of proteomic signatures. Herein we describe a microchip platform, which combines spatial multiplexing and spectral multiplexing for simultaneous detection of 42 secreted proteins from a thousand single cells in parallel. This platform was used to study the production of effector function proteins in human macrophages in response to pathogenic ligands that bind to Toll-like receptor (TLR) - 2, 3, and 4, respectively. These cells showed previously unobserved heterogeneous responses and three functional cell subsets within the same cell population were always identified with differential responses to stimulation and such responses are correlated to their initial states. Our results demonstrated the capability of both high throughput and high multiplexing of Proteoplex platform and the assay can be executed in a simple assay “kit” with no need of sophisticated fluid control or bulky equipment. This platform represents an informative tool for comprehensive monitoring of the immune effector functions of single cells and has great value for quantifying cellular heterogeneity at the functional level in the complex biological systems such as the immune system or tumor microenvironment. It also has great potential for both preclinical and clinical evaluation of cellular responses.

Glioblastoma Multiforme (GBM) is the most common malignant primary brain cancer of adults. No early detection, widespread tumor cell infiltration and remarkable resistance to radiation and chemotherapy render GBM the most lethal of all cancer. The therapeutic resistance that arises to treatment may originate in certain genetic mutations or from the signaling network adaptation through pathway crosstalk and/or network rewiring. The great diversity of resistance mechanisms and the profound non-genetic cell-to-cell variability in drug responses and resistance development require a high throughput and robust tool at single cell resolution to reveal the structure and the coordination of the protein signaling networks that is related to a set of druggable core pathways identified by genomic surveys. Microfluidic based Single Cell Barcode Chip (SCBC) developed by our group becomes an ideal choice in this context due to its capacity of connecting genomic information to biological function through quantitatively analyzing a panel of functional proteins associated with growth factor signaling networks across hundreds to thousands of single cells. Protein fluctuations and correlations are unique information of single cell proteomic measurements and can be directly recorded by SCBC to infer the changes in cellular activity and the interactions between signaling nodes. The surface chemistry involved in SCBC has been subtly designed and progressively improved to allow simultaneously detection of secreted, membrane and intracellular proteins with great sensitivity even for single primary cell from patient biopsy samples. With increased multiplexing, such measurements increasingly resolve the structure of the signaling networks. Various theoretical and computational methods, such as unsupervised data-driven modeling, hypothesis-driven network inference and maximum entropy formalism, have been employed to integrate the unique information disclosed by single cell measurements of GBM cancer with functional studies to understand and anticipate the processes of cancer drug response and resistance within a framework that is grounded in the physico-chemical principles, and eventually to translate the molecular catalog into efficacious therapeutic strategies in the clinic.

In this study, we explore the use of microfluidic devices for reproducible wounding and dissection of Stentor Coeruleus, a canonical single-cell regeneration model organism. Stentor Coeruleus is a single-celled ciliate protozoan with fascinating ability to heal wounds and to regenerate after being dissected into small cell fragments. Traditionally, single cell wounding is performed manually using glass needles under a stereoscope. This process requires high dexterity and is time-consuming. Here, we show that the use of microfluidic techniques allows us to wound large numbers of cell reproducibly. It is also possible to encapsulate individual cells and cell fragments into droplets to control the cellular microenvironment, and to track the regeneration process. This work is an important step towards understanding wound healing and regeneration processes at the single cell level.

In 2010, we developed DropLab, an automated platform for performing chemical and biological reactions and screenings in nanoliter-scale droplet array [1, 2]. This system was applied in enzyme inhibition assays, protein crystallization screening and single cell analysis.
Recently, on the basis of DropLab, we developed a sequential operation droplet array (SODA) system for performing fully-automated and flexible droplet manipulation, analysis and screening in the picoliter to nanoliter range using a tapered capillary-syringe pump module and a two-dimensional (2D) oil-covered droplet array [3]. With the SODA system, we developed a novel automated droplet manipulation method with picoliter precision using the programmable combination of the capillary-based liquid aspirating-depositing and the moving of oil-covered 2D droplet array, so-called “aspirating-depositing-moving” method. Flexible droplet manipulations including droplet assembling, generation, indexing, transferring, splitting and fusion, could be automatically achieved, which endows the system with ultralow sample/reagent consumptions and substantial versatility in analysis and screening for multiple different samples. We applied the SODA system in multiple experiments required in drug screening, including the screening of inhibitors for capases-1 from a chemical library, the measurement of IC50 values for the identified inhibitors, and the screening of synergistic effect of multiple inhibitors. In these experiments, the consumptions of samples and reagents are only 60-180 pL for each droplet microreactor, which are commonly 3-5 orders of magnitude lower than those of conventional multi-well plate systems, and 1-2 orders of magnitude lower than other droplet-based microfluidic systems for multiple sample screening.
Most recently, we applied the SODA system in cell-based schedule dependent drug combination screening. Complex multi-step operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel in the semi-open droplet array. The system was applied in parallel schedule-dependent drug combination screening for A549 non-small lung cancer cells with drugs of flavopiridol, paclitaxel and 5-fluorouracil.

References
[1] W. B. Du, M. Sun, S. Q. Gu, Y. Zhu, Q. Fang, Anal. Chem., 2010, 82, 9941.
[2] S-Q. Gu, Y-X. Zhang, Y. Zhu, W-B. Du, B. Yao, Q. Fang, Anal. Chem. 2011, 83, 7570.
[3] Y. Zhu, Y-X. Zhang, L-F. Cai, Q. Fang, Anal. Chem., 2013, 85, 6723.
[4] G-S. Du, J-Z. Pan, S-P. Zhao, Y. Zhu, J. M. J. den Toonder, Q. Fang, Anal. Chem. 2013, 85, 6740.

We present a microfluidic method to continuously produce microfibers containing droplets regularly arranged in a single column along the length of the fiber. These droplet necklaces are multi-compartment material structures that provide heterogeneous microenvironments that are distinct with respect to their hydrophobic/hydrophilic nature and physical state: solid/liquid. The fibers do not only serve as a stable carrier of droplets but can also retain functional structures within the different compartments, which enable more advanced applications as material storage and release systems than conventional microparticles or fibers. In this experiment, we use calcium alginate as a hydrogel material for fibers, which has useful properties such as fast and reversible gelation and biocompatibility. In a hydrophobic microfluidic channel selectively grafted with hydrophilic polymer, we first generate water-in-oil double emulsion droplets in a stream of sodium alginate solution that is then cross-linked with calcium ions to fabricate the structure of droplets-in-fiber. The resulting multiphase necklace can encapsulate different hydrophobic and hydrophilic materials within a single fiber. We explore diverse potential uses of these fiber structures to engineer the release profiles from each compartment, grow cells and study their behaviors inside the functionalized fiber, and manipulate the fiber with magnetic particles encapsulated in one of the compartments.

Droplet-based microfluidic systems have been used in a wide range of applications, including DNA amplification, biochemical diagnostics, high-throughput screening and polymer capsule production. However, the development of these systems for such varied applications remains challenging due to the requirement to identify unique oil/surfactant combinations that are compatible with the intended application and that support stable droplet formation and storage. In this work, we describe how a combinatorial approach based on genetic algorithm-guided screening can be used rapidly identify surfactant/ oil combinations that generate stable emulsion droplets. Further, this approach can then be extended to efficiently optimise oil-water interfaces such that they support mineralization reactions. The mineral shells mechanically stabilize the droplets such that they are stable off-chip and are resistant to merging and leakage. Finally, we demonstrate how this strategy can be harnessed to generate robust “artificial cells” that support in vitro protein expression and are amenable to facile analysis using widely available flow cytometers.

While the beneficial impact of modifying and/or targeting T lymphocytes is becoming increasingly accepted in the treatment of different diseases, the road towards adoptive cell-based immunotherapy is still long and winding. Major challenges that remain include, amongst others, the guidance and exquisite regulation of immune processes ex vivo. In part, this is due to the lack of technical means to synthesize suitable 3D extracellular systems to imitate ex vivo the cellular interactions between T cells and antigen-presenting cells (APCs). Picoliter-sized droplets of a water-in-oil emulsion created in droplet-based microfluidic devices have been tested and recently used as 3D scaffolds for in vitro screening, translation, encapsulation and incubation of different cell types.
The presentation will cover a development and characterization of novel nano-structured and specifically biofunctionalized droplets of water-in-oil emulsions as 3-D APC analogues. To create the droplets, we have synthesized a new type of gold-linked surfactants and used a drop-based microfluidic device. The efficiency of the gold nanoparticles in the nanostructured droplets to provide the required chemical and biological key functions of the APC will be presented. T-cells stored in gold nanostructured droplets, functionalized with cRGD linkers, were found in contact with the gold-linked biomolecules in the pe