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 periphery, whereas T-cells stored in non-functionalized gold nanostructured droplets remained randomly distributed.
Combining flexible biofunctionalization with the pliable physical properties of the nanostructured droplets provided this system with superior properties in comparison with previously reported synthetic APC analogues and can play a crucial role as it results in a flexible and modular system that closely models in situ APC-T cell interactions. The ability of T cells to exert forces in all three dimensions on the biomolecules held by the drop may be important in evaluating the affinity and function of antigen receptors. Like in the natural T-cell APC interaction for creation of the immunological synapse and supramolecular activation clusters, T-cells in this new type of functionalized droplets can rearrange the linked biomolecules in the droplet periphery for optimal activation. Moreover, the ability to create a well-defined picoliter environment for T cell stimulation is preeminent for long-term monitoring of individual T cells over the course of their activation and differentiation and provides this system with superior properties in comparison to previously reported synthetic APC analogs.
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Since virus cannot metabolize or replicate themselves, they need to be cultured inside host cells for further studies. This requirement complicates detection, isolation and characterization of viruses and therefore leaves most of viruses unknown. Droplet microfluidics provides a convenient way for encapsulating single viral particles, and isolating homogenous population of viruses by fluorescent activated sorting. Here, we show that we can isolate SV40 genome out of waste water that contains highly heterogeneous viral species and amplify them for deep sequencing to reconstruct the whole genome by de novo assembly. This technique can be easily implemented to identify the sequence of whole genome of novel viruses.

In this talk, I will discuss (1) What is creativity? (2) Impact of the convergence of integrative art, culture, technology, and science (iACTS), and (3) Effective collaboration for radical convergence in iACTS to find the truth of life; improve our quality of life and interaction with the environment. The transformative convergence of iACTS is crucial to tackle this pursuit. A major challenge of the convergence of iACTS is the communication problems in interdisciplinary research methodologies and values. However, overcoming the barrier of convergence of iACTS by creative transdisciplinary collaboration will enhance our wisdom, science, and capability to establish healthy ecosystem. I will also examine the implications of Pasteur&’s quadrant in science and technology, and the role of transformative convergence of ACTS for solving ill-defined real-world problems. Finally, I will present my vision for the convergence of iACTS to transform medical science, and finding the solutions for preventive personalized medicine and low-cost healthcare systems. The creative convergence of iACTS with collaborative hearts will impact not only on medicine, but also water, food, energy, and environment. As an example of convergence of iACTS, the creation of bionanoscience for innovative global healthcare research and technology (BIGHEART) will be discussed. The BIGHEART via the collaboration of artists, scientists, and engineers can cultivate pioneering interdisciplinary work that has an encouraging impact on society and culture, such as solving both developed and developing world healthcare challenges.

Microparticles find use in a broad range of settings ranging from biosensing to consumer products to fundamental colloid studies. Advanced applications drive the demand for more complex particles with enhanced functionality. This talk will discuss our efforts in developing encoded microparticles using Stop Flow Lithography (SFL). Our SFL synthesis process couples the precise control of flow afforded by microfluidics and the sculpting of light by UV lithographic patterning. The method is general to any free radical polymerization and leverages inhibition layers created by oxygen near the microfluidic channel walls. I will first describe the fundamental transport processes at play in SFL and give demonstrative examples of particles which can be synthesized. Applications in the multiplexed detection of microRNA and object authentication will be discussed.

The National Cancer Institute (NCI) Center for Strategic Scientific Initiatives (CSSI) is a component of the NCI&’s Office of the Director focused on the application of advanced technologies across the full spectrum of cancer basic and clinical research. The Center is tasked with planning, developing, executing, and implementing rapid strategic scientific and technology initiatives that keep the Institute ahead of the scientific curve with respect to potential new exciting areas and discoveries. With an emphasis on complementing the scientific efforts of other NCI divisions, CSSI&’s efforts seek to enable the translation of discoveries into new interventions, both domestically and in the international arena, to detect, prevent and treat cancer more effectively.
The Center&’s mission includes a focus on directly supporting the development of advanced technologies with the potential for transformative impact. The Innovative Molecular Analysis Technologies (IMAT) program provides up to $10.5 million in new awards each year through unique funding mechanisms (3-year phase I and phase II awards) to support investigators from anywhere in the world through both the early and advanced stages of technology development. A variety of IMAT-supported research projects will be highlighted to demonstrate the variety and high level of innovation evident in the IMAT portfolio of supported research and the strong alignment with MRS fields of research. Potential projects of interest and identified technology gaps will also be discussed. The IMAT program continues to represent a unique resource for highly innovative technology development, but runs alongside several active programs at the NCI for supporting cancer-relevant technologies.

Encoded particles at the micron scale bear unique identifiers, or codes, that may be decoded to convey information. While attractive in this role due to small size and an ability to serve as scaffolds for functional payloads like biomolecular sensors, industrial adoption has been limited by a tradeoff between the number of achievable codes and the robustness with which different codes can be distinguished in operationally realistic scenarios (e.g. complex environments, obscurants, noise). Use on an industrial scale requires large numbers of codes (>10^6), compatibility with high-throughput synthesis methods and rapid, straightforward readout. Application-specific needs add yet another layer of complexity, introducing stringent and often opposing constraints on particle properties such as biocompatibility and temperature and solvent resistance. Meeting these exacting requirements for high-value applications such as covert anti-counterfeiting and medical diagnostics presents a significant challenge and remains largely unaddressed by existing particle encoding methodologies.
We report a novel particle encoding strategy by utilizing spatial patterning of rare-earth-doped upconverting nanocrystals (UCNs) via flow lithography and demonstrate applications in anti-counterfeiting and biosensing. A series of multiple colored UCNs were rationally synthesized by changing the dopant ratio with high reproducibility, and then successfully integrated into hydrophilic/hydrophobic polymeric particles to give a distinctive encoding capability. Our coding motif scales exponentially as C^P where C is the ‘number of UCNs colors&’ and P “number of positions” or stripes on the particle. We demonstrate thus far >10^6 codes and a very low false decoding rate (<10^-9). We generated encoded particles in a predictive manner with high reproducibility and provided robust encoding/decoding capability in biological approaches as well as in a variety of harsh industrial processing conditions. We provided a wide range of practical applications including multiplexed bioassays, covert anti-counterfeit encoding on a variety of substrates, and durable labeling of high temperature cast objects (>250 oC). Furthermore, encoded particles were read out using a portable iPhone decoder. UCNs absorb low energy near infrared (NIR) light (980 nm) and emit in the visible spectrum - this is crucial for imaging them when embedded covertly in complex media. Lastly, the rare-earth dopant of UCNs permitted manipulation of encoded particles via applied magnetic fields. Our novel encoding architecture paves the path to virtually unlimited coding capability that can integrated into industrial processes and later scanned on site with an iPhone.

Tissue engineering has the potential to tackle the major problem of organ availability for transplantation. However, the biggest challenge currently faced by the entire tissue-engineering field is how to fabricate fully vascularized functional tissues over sufficiently large scales (several to tens of centimeters). Here, we report a highly promising approach to generate large-scale, defect-free, interconnected, perfusable networks of endothelialized microvessels via microfluidics. This vasculature showed tight cell-cell junction, as indicated by VE-cadherin staining, which is a characteristics of good barrier function. Upon shear flow stimulation, the endothelial layer showed cytoskeleton reorganization along the flow direction, and elevated synthesis of Nitric Oxide. We further demonstrated that this vessel network could switch from nature nonthrombotic state to a prothrombotic state via inflammatory stimulation. All these observation confirms the micro-vessels are structurally correct and biologically functional. To our best knowledge, this is the first report of de novo fabrication of perfusable functional microvessel networks in a gel-free microfluidic system over centimeter scale. The approach is highly repeatable, robust, and represents a significant advance towards building three-dimensional micro-organism.

Many separation techniques, from circulating tumor cell separation to biofuel algae dewatering, require the processing of large volumes of water (from ml to many liters) in short times (seconds to minutes) with the non-clogging separation of particles. I'll describe our development of a home-made 300 mm photo-lithography system which can create ultra-large scale arrays of separation devices and preliminary results.

Technology advances have driven a genomics revolution with sweeping impact on our understanding of life processes. Nevertheless, the arguably more important “proteomics revolution” remains unrealized. Proteins are complex; meaning that multiple physicochemical properties must be assayed. Consequently, proteomic studies are resource intensive and ‘data limited&’. To drive a bold transformation of biomedicine, engineering innovation in proteomics instrumentation is needed.
While microfluidic technology has advanced separations science, progress lags in the multi-stage separations that are a hallmark of proteomics. This talk will summarize new microengineering design strategies for critical multi-stage protein assays. Specifically, I will introduce our tunable photopatterned materials for switchable function, microfluidic architectures for seamless integration of discrete stages, and multiplexed readouts for quantitation. In a translational example, I will detail assay and design advances from our two highly integrated Western blotting platforms. Focus will center on next-generation confirmatory HIV diagnostics. In a life sciences example, I will highlight our recent contributions to protein isoform measurements, here for new prognostic cancer biomarkers and biospecimen repository monitoring. Performance and operational gains will be discussed, including quantitation capability, total assay automation, integration of sample preparation, and workflows that require minutes not days. Ultimately, we aim to infuse engineering advances into the biological and biomedical sciences - collaboration that promises to address a range of unmet scientific, biomedical, & societal needs.

We have developed a controlled synthesis of fluorinated silica nanoparticles (F-SiO2 NPs) for stabilization of water-fluorinated oil interfaces in droplet microfluidics. Numerous industrial processes including those in petroleum and food industry use nanoparticles to stabilize emulsions. These emulsions, also called “Pickering emulsions”, are ideal complements for traditional surfactant based emulsion systems, as they can be engineered to demonstrate high stability, synthetic versatility and environmental compatibility. New functionalities such as electrical conductivity and photocatalytic activity can also be incorporated. In this work, we synthesized F-SiO2 NPs by the fluorination of pre-synthesized pristine SiO2 NPs. This two-step synthetic protocol offers precise control of both particle size and particle hydrophobicity by tuning the surface fluorine content. This approach allows us to produce a wide range of particles for stabilizing emulsions containing different combinations of polar solvents and fluorinated oils. Furthermore, we were able to generate stable monodisperse picoliter aqueous drops from microfluidic flow-focusing devices using these particles as emulsifiers. The high stability and biocompatibility of the particle-stabilized drops allow us to encapsulate and culture micro-organisms and to perform rapid detection of waterborne pathogens.

We report here a novel microfludic approach to enable to investigate the continuous process of heterogeneous nucleation and growth of calcium carbonate. Calcium carbonate was chosen here as a model system to mimic fascinating biomineralization process. Crystallization occurred in an array of spatially defined 48 individual chambers with tuneable sizes, from 50 um to 400 um in diameter and from 2.5 um to 150 um in height. Each room provided independent environment facilitating multiple reactions at once.
Initially Ca solutions with or without soluble additives (organic molecules, synthetic polyelectrolytes or proteins) were preoccupied in the total 48 rooms, where carbonate gas was introduced to initiate precipitation of calcium carbonate. Importantly gradient in supersaturation level of crystallizing solution through the rooms was obtained in progressive manner by controlling the flow rate of carbonate gas. The influence of surface chemistry was also studied by modifying a substrate with self-assembled monolayers with various functional groups. the crystals grown directly on a substrate in the array were examined using SEM and raman microscope, and revealed a number of remarkable features. Each room showed progressive stages of the crystallization due to the gradient level of supersaturation introduced. That is, the room exposed with carbonate gas earlier corresponded to later stage of crystallization, and one occupied later represented earlier stage of crystallizing process. CaCO3 crystallization proceeded significantly more slowly in the smaller room, allowing the mechanism of crystallization to be readily observed. In addition, due to the limited ingredient, the precipitation reaction terminated at an earlier stage than in the bulk solution, easily showing interesting sequences of morphological evolution influenced by the additives with time, which is extremely difficult to capture in bulk crystallization system.
This approach could provide a unique opportunity to study heterogeneous nucleation and crystal growth in environments where the volume and reservoir was restricted. This microfludic design also overcame typical nano/micro reactor issues, such as surfactant contamination and easy solution evaporation. The results are also significant for biomineralization processes, where mineral formation occurs both within compartments and in association with organic matrices, showing that the environment in which a crystal forms can have a significant effect not only on its morphology and orientation but also on the rate of crystallization.

Microfluidic encapsulation of active ingredients or materials has very important applications for biological delivery of drugs, nutrients and cosmetics and pixellation of display pigments. In all cases, capsule membranes enclose and isolate encapsulants from the continuous phase, thereby protecting them from contamination and maintaining their concentration. Microfluidic drop makers have enabled the controlled preparation of double-emulsion drops or drops-in-drop which have been used as template for the production of such capsules due to their core-shell geometry. However, double emulsion often lacks stability, leading to rupture before the consolidation of the shell is fully complete. Moreover, most previous approaches are not available for either control or processing of capsules once formed, which can be essential to fully achieve their potential, particularly for the encapsulation of colloidal crystals where the photonic properties are adjusted through the colloid concentration.
We develop a versatile and pragmatic microfluidic approach to produce microcapsules with three distinct types of membranes: liquid, solid, and rubber shells. Each of these provides high structural stability and unprecedented controllability of the encapsulant concentration. We use a single-step method to produce double-emulsion drops with ultra-thin shells; lubrication resistance along the full interface of the inner drops prevents contact between the inner and outer interfaces, thereby yielding very stable structures. Moreover, the ultra-thin membrane facilitates transport of water through the membrane when subjected to an osmotic pressure difference, enabling the encapsulant concentration within the drops to be precisely and rapidly controlled by an external osmotic pressure. This is particularly valuable for encapsulation of colloidal photonic materials, where the concentration of the colloids can be controlled through transport of water, thereby providing a means to adjust the photonic properties. To demonstrate this, we encapsulate colloidal particles in liquid capsules and concentrate them; this produces photonic effects due to the structural ordering of the particles. These effects can be dramatic when colloidal crystals are formed, resulting in pronounced opalescence or sparkling colors. The photonic liquid capsules can be further stabilized by polymerization of monomers in the membrane, producing either rigid solid capsules or flexible rubber capsules. The rigid photonic capsules are very stable against osmotic pressure. The rubber photonic capsules exhibit reversible change of their volume depending on osmotic pressure, providing osmochromatic properties. We also employ the rubber capsules to demonstrate that formation of colloidal crystals requires a relatively slow increase in concentration whereas a disordered colloidal glass results from a more rapid increase in concentration.

Materials that autonomously manipulate the diffusive transport of molecules through built-in chemical potential gradients offer new opportunities in miniaturized device technologies. Molecular processors based on this new concept enable directed molecular transport and separation of molecules, without the need of external forces or inputs (e.g. electric fields as found in gel electrophoresis, or flowing carrier phases as found in microfluidics). To drive the anisotropic mobility of molecules, chemically specific chemical potential gradients are required. As model systems, we have investigated hydrogel films containing built-in chemical potential gradients. The gradients have been chemically designed to drive the anisotropic movements of molecules. Using these systems, the directed molecular transport, concentration and separation of molecular species on a surface has been achieved. In one example, a molecule was concentrated 20-fold by a radially attractive gradient, and in another example, a mixture of Rhodamine B base (positively charged), and Pyranine (negatively charged) were separated by a gradient which interacted differently with differently charged compounds. Computational analysis has provided a quantitative understanding of such systems and revealed the underpinning physics. This concept is quite general, and may enable the autonomous processing of many chemicals.

Atomically monodisperse silver nanoclusters (also known as nanomolecules) have received particular interest due to their rare and attractive optical properties: multiple strong absorption peaks that cover a broad range of wavelengths (350-950 nm). Silver nanocubes have also received particular interest due to their high potential as catalysts for epoxidation reactions and building blocks for self-assembly. Concerning the silver nanospheres, they have a high potential to boost efficiency the solar cells performance (surface plasmon enhancement). We report a biphasic-liquid segmented continuous flow method for the synthesis of atomically monodisperse thiol-protected silver nanoclusters, high-quality plasmonic single crystal silver nanocubes and nanospheres. The product nanocluster is highly stable in contrast to previous preparation methods. This method is scalable, and produces nanoclusters that are stable in aqueous solution for at least 9 months at room temperature under ambient conditions, with very little degradation to their unique UV-Vis optical absorption spectrum. The nanocubes were synthesized with controllable edge lengths from 20 to 48 nm. Single crystal nanospheres with a mean size of 29 nm were obtained by in-line continuous-flow etching of as-produced 39 nm nanocubes with an aqueous solution of FeNO3. In comparison to batch synthesis, the demonstrated processes represent highly scalable reactions, in terms of both production rate and endurance. The reactions were conducted in a commercially available flow-reactor system that is easily adaptable to industrial-scale production, facilitating widespread utilization of the procedure and the resulting nanoparticles.

The technological and commercial success of printed electronic devices on plastic substrates depends on the development of inexpensive, flexible and solution-processable electrodes. To date the most promising candidates for opaque and transparent electrodes are noble metal nanoparticles and silver nanowires, respectively, both of which offer a good balance of high (opto)electronic performance, environmental stability and processability.
Conventional batch production techniques scale poorly (due to slow mixing and heat transfer) resulting in an undesirable trade off between product quality and production rate which makes them prohibitively expensive.
Here we report the synthesis of metal nanoparticles and nanowires in droplet-based flow reactors, demonstrating a level of control and reproducibility unattainable in batch. The sub microliter volume of the droplets ensures rapid equilibration of temperature and composition, providing a highly uniform environment for nucleation and growth. Most importantly, we demonstrate that by running many identical channels in parallel, greatly increased production rates can be achieved without diminishing the quality of the product, thus avoiding the principal limitation of batch reactors.

Stimuli-responsive hydrogels and microgels are useful for numerous biological applications, where encapsulated therapeutics and cells can be released in an on-demand fashion. Microfluidic systems enable the assembly of uniform populations of microgels through the mixing of various gel precursors. Here, we designed a self-assembly process for microgel formation through the controlled mixing of polymers with complimentary guest or host molecules that bind in a non-covalent manner when mixed due to hydrophobic interactions. This binding is susceptible to disruption through various means, including shear force or with heating. We first synthesized two hyaluronic acid (HA) derivatives: HA modified with β-cyclodextrin (CD-HA) and HA modified with adamantane (Ad-HA). β-cyclodextrin (the host) and adamantane (guest) form a guest-host complex when mixed; thus, CD-HA and Ad-HA polymer solutions form a hydrogel when combined. Rheologically, the shear-modulus is observed to increase by several orders of magnitude as compared to individual polymer solutions. Gold nanorods were synthesized by a seed-mediated process and encapsulated into the gels during mixing. When the samples were exposed to near-infrared (NIR) light they heated through the photothermal effect between the nanorods and NIR light and the gels underwent a disassembly process. Rheological temperature sweeps indicate this disassembly is the direct result of an increase in temperature, as a decrease in modulus of a bulk gel (4 wt% polymer) was observed during heating from 20°C to 80°C. To form microgels, we designed a microfluidic system in which the gel components—CD-HA, Ad-HA, and the gold nanorods—are combined in a droplet and mixed to generate uniform microgels. Microgels were formed with uniform size and distribution, depending on the microfluidic dimensions and flow parameters. Depending on the experimental setup, monodisperse droplets could be generated ranging in size from 30 µm to over 100 µm. Exposure to NIR light resulted in the disruption of the guest-host bonds within the microgels, enabling the triggered release of their contents. Because NIR light penetrates tissue more deeply than shorter wavelengths, this system might ultimately be used to deliver payloads in vivo whose release is triggered using an endoscopic, or even transdermal, light source.

Patterned sheet materials and assembled sheet systems with micro- and nano-scale features are used in a wide range of industries from biotechnology to organic electronics. Here we describe a new methodology for sheet material patterning and sheet system assembly using a hundred-fold widened microfluidic channel, which not only fabricates versatile patterned planar materials but also manufacture assembled sheet systems, both in a one step and high throughput manner. This widened slit microfluidic channel is made by simply placing a supporting glass plate above a PDMS channel to prevent channel deformation, and by combining with stop flow lithography (SFL), it is able to pattern and texture sheet materials continuously.
By simply extending microfluidic channel dimensions, we greatly expand its application scope, from current micro-scale fabrication to centimeter sheet synthesis and assembly. By taking advantage of arbitrary yet high resolution pattern transference of photolithography, along with its flow production fashion, this technique can synthesize sheet materials with any pattern in a high throughput manner. The controllable polymerization in a micro-scale slit channel offers a unique nano-scale surface texturing method. Moreover, the laminar co-flow properties associated with microfluidics allow this technique to pattern sheets with tunable chemical anisotropy. Further, by manipulating the fluid dynamics in a microfluidic channel, this technique is able to position a micro-object in the channel and then in-situ connect it to a patterned sheet via a one step UV projection, readily constructing an assembled functional sheet system.
In this work, we demonstrated its versatile potential from single layer sheet patterning to multi-layer or multi-parts assembly, revealing its powerful processing ability as a tool for sheet material synthesis and assembly. We showed that this technique can pattern functional sheet materials bearing geometrical and chemical anisotropy with controllable surface textures in a one-step fashion. With designed patterned features, we were also able to demonstrate the field assisted self-assembly of layered 3D structures. In addition, the flow focusing feature of microfluidics enabled us to perform single-step connection of RFID dies onto an in-situ synthesized antenna for RFID tag fabrication. With its versatile while one step polymer sheet synthesis and assembly capability, we believe slit channel lithography will encourage more innovations in a wide range of applications where the patterned sheet material is a key feature, such as membrane-based sensors, advanced membrane synthesis, organic flexible electronics and layered structure assembly. Importantly, its flow production strategy and agile manufacturing ability will significantly simplify sheet material patterning and assembly processes, highly demanded in many fast-evolving industries such as biotechnology and material chemistry industry.

The design of novel micro- and nanostructures which allows the study, control and sensing of single cell behaviours is of great importance in cell biology, biophysics, cancer therapy and tissue engineering. Recently, the fabrication of 3D bioanalytic systems for single cell growth and analysis by using rolled-up technology was proposed¹. The systems were made of highly transparent nanomembranes self-folding into microtubes that can be employed for diverse biological applications².
The highly transparent nanomembranes of the scaffold enable the scrutinization of the cellular behaviour inside with the state-of-the-art optical microscopy, while the tubular cavity confines light within to form whispering gallery modes which is highly sensitive to molecular adsorption³ and can be employed as a label-free detection tool for individual cells⁴. Furthermore, the integration of microelectrodes into their thin walls allows the detection of solution and cells with electrochemical methods.
The tubular system can also be used as 3D cell culture scaffold with the inner surface functionalized with various biomolecules to mimic the in vivo micro-environment which offers the advantage of a good directionality and a strict 2D confinement for cell growth⁵. During the cell division, we find that increased spatial confinement results in multiple mitotic errors highlighting both conserved and novel phenomena compared to the effects previously reported in 2D cultured mammalian cells. Thus, our method uncovers a remarkable correlation between the extent of confinement and the fidelity of mitotic chromosome segregation in 3D cultured mammalian cells. Moreover, we detect bipolar distribution of cortex and extensive membrane blebbing for most of the duration of mitosis, a process that in 2D contexts has only been observed in later mitotic stages and to a limited extent.
Collectively, this novel approach represents a multifunctional device which enables the detection and scrutinization of single cell inside the 3D space of cavity of microtubes which would capture more of the complexity present in tissue scaffold.
References:
(1) Mei, Y.; Huang, G.; Solovev, A. A.; Ureña, E. B.; Mönch, I.; Ding, F.; Reindl, T.; Fu, R. K. Y.; Chu, P. K.; Schmidt, O. G. Adv. Mater. 2008, 20, 4085-4090.
(2) Harazim, S. M.; Xi, W.; Schmidt, C. K.; Sanchez, S.; Schmidt, O. G. J. Mater. Chem. 2012, 22, 2878-2884.
(3) Ma, L.; Li, S.; Quiñones, V. A. B.; Yang, L.; Xi, W.; Jorgensen, M.; Baunack, S.; Mei, Y.; Kiravittaya, S.; Schmidt, O. G. Adv. Mater. 2013, 25, 2357-2361.
(4) Smith, E. J.; Schulze, S.; Kiravittaya, S.; Mei, Y.; Sanchez, S.; Schmidt, O. G. Nano Lett. 2010, 11, 4037-4042.
(5) Huang, G.; Mei, Y.; Thurmer, D. J.; Coric, E.; Schmidt, O. G. Lab Chip 2009, 9, 263-268.

Commercially available materials such as glass, polystyrene, and poly(dimethylsiloxane) dominate research into microfluidic devices. While convenient to use, chemical or physical modification of these materials is not necessarily simple.
Here, we demonstrate microchannel fabrication using an alternative polymer network, poly(vinylmethylsiloxane) (PVMS). First, we synthesize PVMS homopolymers with hydroxyl end groups via anionic ring-opening polymerization. Elastomeric networks form by crosslinking the PVMS chains with methoxy crosslinker molecules in the presence of a tin catalyst. PVMS networks behave similarly to PDMS networks, exhibiting low surface energy, low modulus, optical transparency, and hydrophobicity. Replica molding techniques used for PDMS also work with PVMS networks, allowing the fabrication of microchannels through bonding of the channel replica to either glass or PVMS networks after oxygen plasma exposure. The vinyl side groups remain available for modification by thiol-ene chemistry, free radical reactions, or cross metathesis chemistry after formation of the microchannel, enabling the local modification of chemical and mechanical properties of the channel.
This work reveals a simple method to impart local organic solvent resistance to microchannels made from PVMS. A photoinitiator (2-hydroxy-2-methyl-1-phenyl-1-propanone or HMPP) is delivered to the walls of the microchannel by flowing a solution of ethanol and HMPP through the channel for 30-60 minutes. Exposing the channel to UV light initiates the decomposition of the HMPP, starting a free radical reaction which locally crosslinks the PVMS network through the vinyl side groups. This additional crosslinking takes place only in the walls of the channel where there is significant HMPP concentration, hardening the PVMS matrix and preventing swelling upon exposure to organic solvents.
To characterize solvent resistance, we form microchannels from pure PDMS (material which contains no silica particles), commercial Sylgard-184, pure PVMS, and UV-modified PVMS. A solution of toluene and red dye flows through the 60 mu;m tall channel at a flow rate of 1 mL/h. Observation of the channels with optical microscopy for 1-2 h reveals the leaching of red dye into the walls of the channels for pure PDMS, Sylgard-184, and pure PVMS. UV-treated PVMS channels demonstrate remarkable resistance to the leaching of red dye in the presence of toluene. We also observe the behavior of UV-treated PVMS channels in the presence of other organic solvents such as acetone, tetrahydrofuran, and chloroform.
Characterization of the modification as determined by dynamic mechanical analysis, contact angle analysis, and FTIR spectroscopy confirms a chemical and mechanical change in the PVMS networks after UV treatment, explaining the improved resistance to organic solvents.

Polydimethylsiloxane (PDMS) based micro fabricated device has been increasingly employed as a noble platform for cell culture studies as well as for many areas of biomedical applications. It possesses unparallel physicochemical properties over other materials, e.g. easy fabrication process, optical transparency, tunable elasticity, non-toxic, permeability, biological inertness. In addition, PDMS can easily be modified with wide range of functional molecules and proteins which can be further finely tuned to allow specific molecular interactions. Herein, we demonstrate a set of work to understand how the surface functionalization and physicochemical properties of PDMS affects the cell adhesion mechanism. In this regards, phytopathogen bacteria (Xylella Fastidiosa) and human cervical cancer cells (HeLa) have been used for the present study.
The PDMS microfluidic devices with channel width vary form 15-100 µm were developed using conventional photolithographic fabrication methods and functionalized further to enable cell adhesion. The microchannels were activated by oxygen plasma, then aminized with (3-Aminopropyl)triethoxysilane, followed by grafting with carboxylmethyl cellulose (CMC). Different cell adhesive promoters such as gelatin, bovine serum albumin, fibronectin, and collagen were further tethered separately to the CMC grafted channels for comparisons. The influences of different functional groups towards the adhesion of cells were studied in detail. The experiment was performed via a syringe pump to maintain a continuous flow of cells over the functionalized surfaces and monitored in-situ via a fluorescence microscope. This study provides a potential approach to understand the early cell adhesion dynamics on PDMS-based microdevices, and thereby facilitate its potential use in on-chip bioanalysis studies.

Micro and Nanofluidics and Lab-on-Chip can be very beneficial to realize practical applications in detection of disease markers, counting of specific cells from whole blood, and for identification of pathogens, at point-of-care. The use of small sample size and electrical methods for sensitive analysis of target entities can result in easy to use, one-time-use assays that can be used at point-of-care. In this talk, we will present our work on detection of T cells for diagnostics of HIV AIDs for global health, development of a CBC (Complete Blood Cell) analysis on a chip, electrical detection of multiplexed nucleic acid amplification reactions, and detection of epigenetic markers on DNA at the single molecule level. These technologies are all electrically based and use silicon and polymer based micro and nano-fluidics devices.
We will present our work on counting of white blood cells for detection and monitoring of HIV in resource limited settings [1,2]. Credit card sized devices were developed that use a drop of blood and can count the total WBC count, along with specific CD4+ T cells. The devices use a dual coulter counter approach coupled with immuno-capture of target cells and integrate a red blood cell lysing module for preparing of the samples for counting of the white blood cells. This technology can be scaled to now count neutrophils and other cells of interest for realization of a CBC on a chip. We will also present the development of a static digital droplet based approach to electrically measure the amplification of nucleic acid in droplets using silicon based nano-bio FET sensors [3]. This technology can especially bring closer to reality the goal of developing a PCR cartridge that truly brings nucleic acid amplification to point-of-care. And finally, we will also present a nanopore molecular count of approach where we used the binding of a methyl-binding domain protein as a physical label for detection of CpG dinucleotides within a dsDNA molecule [4]. Such detection of epigenetic markers on dsDNA without having to go through the expensive bisulfite conversion of the DNA molecule for detection of methylation.
In short, the projects presented will provide a glimpse on the exciting possibilities that can be brought about by the use of micro and nanotechnology in the application domains of biology and medicine.

REFERENCES
[1]. G. Damhorst, N. N. Watkins, R. Bashir, “Micro and nanotechnology for HIV/AIDS diagnostics in resource-limited settings”, IEEE Transaction of Biomedical Engineering, 60 (3), 715 - 726, 2013.
[2]. N. N. Watkins, U. Hassan, G. L. Damhorst, H. Ni, W. R. Rodriguez, R. Bashir, “Microfluidic CD4+ and CD8+ T Lymphocyte Counters for Point-of-Care HIV/AIDS Diagnostics from Whole Blood”, Science Translational Medicine, Vol. 5, Issue 214, p. 214ra170, 2013.
[3]. E. Salm, C. Duarte, P. Dak, B. Dorvel, B. Reddy Jr., A. Alam, and R. Bashir, "Ultra-localized Thermal Reactions in Sub-Nanoliter Droplets-in-Air," PNAS, DOI:10.1073/pnas.1219639110, 2013.
[4]. J. Shim, G. Humphrey, B. M. Venkatesan, J. M. Munz, X. Zou, S. Chaitanya, K. Schulten, F. Kosari, A. Nardulli, G. Vasmatzis, and R. Bashir, “Detection and Quantification of Methylation in DNA using Solid-State Nanopores,” Sci. Rep. 3, 1389; DOI:10.1038/srep01389, 2013.

Metastatic tumors can spread via release of circulating tumor cells (CTC&’s) into the bloodstream. Early detection of these CTC&’s could greatly improve cancer survival rates by enabling diagnosis, and therefore treatment, before secondary tumors arise. However, tumor cells are typically present in very low concentrations, making them difficult to detect in a fluid dominated by red blood cells (RBCs), leukocytes and serum proteins. Separation of CTCs from blood plasma, leukocytes and RBCs is predicted to improve cell capture via antibody-based methods and reduce interference in capture/detection assays. Previously, members of our team have demonstrated microfluidic, size-based separation of blood components, but have yet to integrate this sorting capability with an affinity-based detection technology. To this end, we have developed a microfluidic platform to separate CTC&’s from mouse whole blood and detect them using grating coupled surface plasmon resonance (GCSPR). We have implemented a size-based sorting array which separates objects based upon their diameter, within a microfluidic channel. Separation of beads (2 µm, 6 µm, 10 µm) has been demonstrated, as well as separation of white blood cells and CTC&’s from whole blood. The resulting stream of large blood cells (including CTCs) is then directed onto an integrated SPR grating for affinity based capture and detection. Using GCSPR vs. conventional SPR enables detection of multiple cell types across the grating, in an array-based format. We have demonstrated differential capture and detection of cells on GCSPR gratings, following size-based separation of whole blood. By using capture antibodies specific to unique CTC surface proteins further enables identification of cell types and may provide prognostic capability, beyond the diagnostic capacity of this system.

DNA sequencing has been known as the key technology to provide human information such as disease, inheritance and individuality. Since the idea was proposed that it might be possible to detect each nucleotides of DNA by using nano-scale pores in 1996, the nanopore detection method has widely researched as a new solution of DNA sensing method as well as a single molecule sensing. Among several nanopores, it is indicated that solid-state nanopore has better feasibility and stability than biological nanopore. Especially, 2-D material nanopore such as graphene nanopore is highlighted that it offers the highest sensing resolution, which is similar to the spacing of each nucleotide (3~5Å). But, it was reported that graphene nanopore has high flicker noise and high hydrophobicity and native pinholes are suspected as the reason. So, in order to obtain both high sensing resolution and low noise characteristics, we developed h-Boron Nitride nanopore including Pyrex glass substrate.
First, we investigated the Pyrex glass-compatible process to decrease high noise in solid-state nanopore. Because Pyrex has better dielectric properties (low dielectric constant & loss) than Si-substrate, Pyrex-based nanopore shows lower dielectric noise than Si-based nanopore. Then, h-BN films are prepared by CVD (chemical vapor deposition) growth on copper foil and h-BN membrane is transferred on 50nm-thick SiN-Pyrex sample with ~100nm round-shape hole. And the nanopore was drilled by focused electron beam of TEM . Actually, pristine h-BN nanopore also shows high flicker noise like graphene nanopore. To reduce flicker noise of solid-state nanopore, O2 plasma or SPM solution treatment is conducted before measurement. Graphene film is known that it can be damaged by even weak O2 plasma but h-BN nanopore has no damage by O2 plasma. Consequently, the plasma-treated BN nanopore has lower rms noise than pristine BN nanopore at the same voltage. And also, the former shows higher signal to noise ratio(ΔI / IRMS) of DNA translocation than the latter when 10nM lambda;-DNA solution is inserted in cis-chamber.
In conclusion, we have investigated the sensitive nanopore device composed of Pyrex substrate and h-BN membrane. Unlike graphene membrane, h-BN is not damaged by flicker noise reduction process, so the treated h-BN nanopore shows higher signal to ratio. This nanopore device will be able to widen the application of solid-state nanopore toward single molecule sensing.

Microcapsules have been widely used in various applications such as pixellation of display pigments and encapsulation of self-healing materials because the impermeable membrane of microcapsules enables long-term storage of materials. The membrane of the microcapsules has been functionalized to use them in various biological applications. For example, the membrane can be tailored to provide triggered release of encapsulated drugs and isolation of cells from immune system. The immuno-isolation is promising and important for treatment of incurable disease. To successfully achieve the immune-isolation, the capsule membrane should be semipermeable and its cut-off value should be precisely controlled; the nanopores on the membrane should allow diffusion of nutrients and stimuli for long-term survival of encapsulated cell and secretion of therapeutic agent while excluding antibodies and immune. However, conventional capsules lack mechanical stability and controllability of the penetration cut-off value. Therefore, development of practical capsule platform for immune-isolation still remains important challenge.
Here, we report a new microfluidic strategy to create microcapsules with selective permeability. With microfluidic glass capillary devices, we prepare water-in-oil-in-water (W/O/W) double-emulsion drops with ultra-thin shell, which serve a template to create microcapsules. The ultra-thin oil shell yields high lubrication resistance along whole shell layer, thereby dramatically enhancing stability of the core-shell structure. As middle oil layer of the double-emulsion drops, we use a homogeneous mixture of photocurable monomers and porogen. Upon UV illumination on the drops, the monomers are polymerized, which leads to phase separation between the polymerized resin and porogen within the shell. Subsequent dissolution of porogen leaves behind regular pores in the polymerized capsule membrane. The pore size is determined by degree of phase separation which is controllable with interaction parameter between the polymer and porogen. With porogen which has relatively high interaction parameter, macropores are generated in the membrane. By contrast, a porogen with small interaction parameter results in micropores. Both macro- and micropores nicely interconnect inner volume of the microcapsules with surrounding fluid. The cut-off value of permeation is evaluated by diffusion of fluorescent dye molecules with various Stokes radius. For preliminary demonstration of immuno-isolation, we encapsulate yeast cells using the semipermeable microcapsules. The cells grow within the microcapsules as the surrounding fluids continuously supply the nutrients. At the same time, detrimental polymers to the cell in the fluids do not influence the survival of cells as the membrane prevents direct contact between the cells and the polymers. This proves great potential of our semipermeable capsules for further biological applications, especially for immune-isolation.

Ionic current modulation is one of main interests in nanofluidic area owing to its potential ability to analyze and control bio-molecules such as DNA, RNA and proteins because the ionic current control is regarded as the way of delivery of chemical species as we want and nanofluidic logic circuit can be utilized like solid-state semiconductor technology by using ionic current rectification behavior. Thus, various approaches for rectifying ionic currents are being extensively researched such as conical, asymmetric bath concentration, bi-polar surface charged nanopores and gate electrode embedded structure. Difference point of those applications is merely the fabrication methods and major principles of the rectification behavior are based on same understanding that the charge accumulation/depletion caused by broken symmetries of nanofluidic system such as geometries, surface charge and mole concentration, and comparable size of nanopores to the Electric Double Layer is the key factor to explain the ionic current rectification. In this work, we suggest a fabrication method for effective ion control device and investigate the ionic rectifying effect caused by all around gate structure according to mole fraction and gate voltage. Our nanopore device, unlike the existing gated nanopores, has relatively thick membrane but thin gate electrode which is localized in small region to make highly broken symmetry and enhance the current controllability. Even with the single nanopore, this pore can show the outstanding ion manipulation property. We have fabricated the nanopore device with 30nm diameter and 800nm thickness. The 30nm thick gate electrode was inserted around 200nm position from the bottom of the pore so that the electrode can be positioned asymmetrically. Silicon nitride, chrome and Al2O3 was chosen as respectively, membrane, gate metal and passivation layer. Our work will be able to provide the possibility of diverse biological and chemical applications which may need ionic current control. The very flexible and tunable nanofluidic device can open new ways to analyze, control, separate and transport bio-molecules like DNA, RNA and proteins. Furthermore, for the lab-on-a chip application, our device can be an important contribution as a building block of the integrated chip.

Intracellular calcium plays a role in a host of cellular functions ranging from synaptic transmission to gene expression. As a transient, short-range signaling molecule, calcium is often presented in complex signal patterns, including oscillations and bursts, which are tightly regulated by the concerted action of pumps, channels, and buffers in conjunction with the ion-impermeable cell membrane. Many techniques measure changes in intracellular calcium, providing insights into the specificity of these signal patterns, but fewer are capable of controlling calcium levels in cells. Patch clamp pipette access can be used to modulate calcium in small numbers of cells, but larger scale control often requires disruption of normal calcium signaling using drugs, in order to couple intracellular calcium to an external calcium signal. Here we present a new technology to rapidly modulate intracellular calcium. We fabricate a platform of supported nanotubes called “nanostraws,” to demonstrate direct ionic delivery into cells. The dimensions of the nanostraws (~100 nm x 1 mu;m tall) allow them to spontaneously penetrate the cell, thereby allowing calcium to bypass the lipid membrane and its constituent calcium channels through the nanostraws. Calcium delivery is controlled by microfluidics, and intracellular calcium oscillations can be induced and modulated in amplitude and frequency. While previous studies have perturbed long-term cell behavior using nanostraws and similar systems, here we apply the technique to transient signaling by rapid intracellular calcium modulation and characterize the spatiotemporal delivery using calcium indicators (e.g. Fluo-4, GCaMP6). By circumventing the intrinsic calcium regulatory mechanisms, nanostraws provide direct access and the ability to mimic biological calcium oscillations, adding a new method for decoding the role of calcium signal patterns and their effects on downstream signaling.

Superhydrophobic surfaces which show high water contact angles (150 deg) and slip angles (10 deg) have been explored for various applications due to their excellent self cleaning and low adhesion properties.1,2 Several superhydrophobic surfaces with hierarchical roughness showed improved resistance to protein adsorption compared to control flat surfaces.3,4 On a superhydrophobic surface, due to interactions between the protein and the hydrophobic surface, attachment of proteins or cells could occur at the contact surface between liquid and solid which fouls the surface. As a result of this change in surface wetting, the superhydrophobic properties of the surfaces are lost. Hydrophilic coatings on hydrophobic surfaces reduce the interactions between the protein and the surface and thus reduce the adsorption of protein on the contact surface; modification of flat surfaces with polyethylene glycol or zwiterionic groups has been shown to be especially effective at preventing protein adsorption5. Making superhydrophobic surfaces with a hydrophilic material is a challenging task. But by introducing surface roughness with reentrant surface structures coated with a hydrophilic material, we have achieved superhydrophobic surfaces which resist protein adsorption. Here, we have studied the effect of surface roughness and chemistry on the biocompatibility of superhydrophobic surfaces with the goal of designing biocompatible super repellent surfaces for biomedical applications.
Superhydrophobic surfaces with reentrant hierarchical roughness were fabricated using a 3-D printing method by dispensing hydrophobic PDMS pre-polymer on a glass substrate; secondary roughness and reentrant structures were introduced by incorporating nanoparticles into the 3D-printed surface. Stable superhydrophobic properties were observed (> five days) even when the surfaces were challenged with bovine serum albumin (BSA, 0.1 and 1 % solution in PBS) protein solutions as indicated by slip angle and contact angle measurements. Protein adsorption studies on flat silica surfaces modified with the same chemistries also were investigated by confocal fluorescence microscopy [BSA labeled with Alexa 488)] and XPS. The results showed almost no protein adsorption on the modified surfaces compare to bare silica surfaces which substantiated our results from slip angle and contact angle measurement study. Additional studies using other proteins, such as fibrinogen and Lysozyme, will also be discussed.
1. Aussillous,P.; Quere, D. Nature 2001,411, 924-927.
2. Xu, QF; Liu, Y.; Lin, FJ.; Mondal, B.; Lyons, A.M., ACS Appl. Mater. Interfaces, 2013, 5 (18), pp 8915-8924.
3. Chen, L.; Han, D.; Jiang, L. Colloids and Surfaces B-Biointerfaces 2011, 85, 2.
4. Webb, H. K.; Hasan, J.; Truong, V. K.; Crawford, R. J.; Ivanova, E. P. Current Medicinal Chemistry 2011, 18, 3367
5. Prime, K. L.; Whitesides, G. M. Journal of the American Chemical Society 1993, 115, 10714.

Production of cytokines by immune cells in blood may be used for diagnosis of infectious disease exposure, for example tuberculosis. Traditional antibody-based immunoassays deployed for cytokine detection require multiple labeling and washing steps and are therefore laborious and time consuming. Aptamers may be used in immunoassays instead of antibodies. The key advantage of such a strategy is the ability to engineer aptamers into beacons that produce signal directly upon binding of target analyte, without the need for labeling and washing steps. The simplicity of getting areadout makes aptamer-based biosensors particularly suitable for point of care testing.
Monitoring a single analyte is often insufficient and detection of multiple cytokines in parallel may be more informative. We sought to develop multiplexed cytokine aptasensors while balancing the need for simplicity of the sensor design. The biosensor consists of an electrode functionalized with redox-labeled aptamers. Appearance of the target cytokine causes the change in the redox current of the electrode and may be converted to cytokine concentration. To enable multiplexing, we designed an approach termed electrochemical redox spectroscopy where specific aptamers are labeled with unique redox reporters. To prove this concept, thiolated aptamers against interferon gamma (IFN)-g, tissue necrosis factor (TNF)-a and transforming growth factor (TGF)-b were labeled with anthraquinone (AQ), methylene blue (MB) and ferrocene (Fc) redox reporters respectively. Electrodes functionalized with a random mixture of these three aptamers showed redox peaks at -0.45V(AQ), -0.3 V(MB) and 0.2V(Fc) vs Ag/AgCl reference. When challenged with individual cytokines, the appropriate redox peak decreased in concentration-dependent fashion.
After confirming responsiveness to recombinant cytokines, the electrodes were miniaturized and integrated into microfluidic devices for detection of endogenous T-cell secreted cytokines. These were two channel devices that contained electrodes and were functionalized with anti-CD antibodies for T-cell capture. A suspension of peripheral blood mononuclear cells was infused into the device resulting in capture of CD4 T-cells. The cells in the first channel were activated and the cytokine molecules released by the cells were detected at the nearby miniature electrodes. The cells in the second channel were not activated and were used for control measurements. The aptasensor multiplexing strategy based on electrochemical redox spectroscopy allowed us to concurrently monitor three cell-secreted cytokines at the same electrode. Because the electrical signal was changing directly as a function of cytokine binding, cell secretions could be monitored dynamically over the course of several hours. In conclusion, the use of electrochemical redox spectroscopy allows to multiplex aptamer-based biosensors for profiling cellular secretions and for diagnostic applications.

MRS Abstract
We have demonstrated a microfluidic system for the concentration and collection of oxygen using the temperature dependent solubility of O2 in perfluoalkanes. The system utilizes countercurrent flow to increase the extraction efficiency, and output concentration of the oxygen. Examples of this type of system, called countercurrent multiplication, can be found throughout nature due to the simplicity and efficiency of the system. These systems utilize a trigger to form a concentration gradient across two adjacent, connected channels. This gradient forms a feedback loop, wherein the concentration at the junction of the two channels rises above the input concentration, and the solute is collected through diffusion at the junction. Previous work has been done in the field of microfluidics to mimic the biological architecture and function of the loop of Henle. The loop of Henle, found in the kidney, utilizes countercurrent multiplication in order minimize water lost during urine excretion. This work however has been limited to two dimensional representations of the countercurrent systems, and the mechanism of solute release has not been generalizable. A general solution to countercurrent multiplication could have applications in flow chemistry and signal amplification in analytical chemistry. By using fabrication methods pioneered in our lab, we show a three dimensional countercurrent multiplication system with a generally applicable release system. We have based our device off of the oxygen concentrating rete mirabile found in deep sea fish.

This project presents the study of fabrication methodologies and the characterization of nanotextured ZnO thin films for use in flexible biosensors. The goal of this project is to develop highly sensitive and selective biosensors which can be worn much like a bandage on the skin for the continual, real time monitoring of target analytes. The continued downscaling and advancement of microelectronic devices has shown the potential for the use of these devices as biosensors for continuous monitoring of physiological changes. However, for these devices to be fully integrated into biosensors, they must be placed in a package which is non-invasive and does not intrude on the movements of the wearer. This area is where being able to fabricate flexible biosensors, which can be worn, will advance such monitoring devices and improve on the current state of the art. For the initial substrate testing, RF magnetron sputtering of ZnO has been performed on nylon membranes to create rapid prototypes for feasibility testing and preliminary analyte detection studies. ZnO was also deposited on SiO2 for comparative analysis and characterization of the ZnO depositions. The ZnO thin films deposited were measured to be between 50-100nm thick with columns that have a diameter of 10-20nm. Porous membranes, with pores that are 200nm in diameter, are used as the flexible substrate based on their ability to filter and aid in the selection of target analytes through size exclusion when placed over the sensing surface. AFM characterization of the ZnO deposition on SiO2 test substrates show the nanocolumnar structure of the ZnO created by the deposition parameters. SEM analysis of the flexible substrates showed the same patterning of the ZnO was possible in the porous membranes. These nano structures of ZnO will be leveraged to improve the sensitivity of the biosensor for analyte detection. Future work for this project will include characterization of various membranes to find the optimum pore configuration and flexibility properties. In addition, continued characterization of the ZnO surface and the device design will be performed to optimize the analyte detection.

Conventional flow cytometry using fluorescent detection method has been a basic tool of biological and physiological experiments. Recently, many research groups have focused highly integrated microfluidic system toward a hand-held flow cytometer because a microfluidic flow cytometer enable a high-throughput, portable, and cheap device.
Generally, white blood cells (WBCs) are protecting cells in the blood that are involved in defending the body against infective organisms and foreign substances. Counting of WBCs has been shown as an indicator of disease. We fabricated devices with microfluidic patterns using polydimethylsiloxane (PDMS) mold. CD45 antibody with avidin as a bridging protein was labeled with magnetic beads for the high detecting efficiency of WBCs. Also, we developed high-throughput lensless complementary metal-oxide-semiconductor (CMOS) image sensor that was able to perform imaging at a high speed about 60 frames/s and high resolution (1280 x 1024 pixels). This lens-free on-chip imaging modality is based on uniform illumination from organic light emitting diodes (OLEDs). OLEDs have several potentials as a light source in medical devices, compared with other light sources such as a light emitting diode (LED), ultraviolet (UV), and halogen lamp.
In this work, we demonstrate lens-less on chip cell imaging platform for medical diagnostic devices which is detecting of immunological labeled WBCs and counting of labeled cells.

In this work, we demonstrate the label-free and ultrasensitive detection of troponin-T, cardiac biomarker using nanoporous nylon membrane integrated on a microelectrode sensor platform. The nanoporous membrane comprises of highly dense nanowells of 200nm in diameter , that allow spatial confinement of protein molecules. The antibodies were immobilized onto the gold electrode surface through thiol linker chemistry. The antibody-antigen complex formation in the nanowells results in charge pertubation within the electrical double layer which is read out using the principle of Electrochemical Impedance Spectroscopy (EIS). The impedance change measured at the low frequency is used to quantitatively determine the concentration of Troponin-T in both pure form as well as in the patient blood samples. The accuracy and reliability of detection using this device was comparable to that of the conventional protein detection technique, Enzyme Linked Immunosorbent Assay (ELISA). Using EIS, we have demonstrated the sensitivity of the sensor for protein biomarker detection to be 1pg/mL. We performed precision study for a period of 5 days and we observed covariance of less than 10% over the dynamic range of detection. Charge pertubations occuring due to specific binding are higher in magnitude than those due to non-specific binding. Using this technique we have success in detecting protein biomarkers in the whole blood, human serum and ionic buffers.This technology provides robust analytical platform for rapid and sensitive detection of protein biomarkers thus making this platform as an ideal candidate for biomarker screening in clinical settings.

Abstract: This presentation will describe the fabrication and use of elastomeric nanochannels with size-tunable cross-sectional dimensions. In this nanofabrication procedure, a poly(dimethylsiloxane) (PDMS) slab with microchannel features are bonded to a thin PDMS film using plasma oxidation. When the resulting PDMS microchannel device is stretched in a direction parallel to that of the microchannels, an array of tunneling cracks are formed. While cracking typically occurs randomly, with the combined use of stress shielding features and control of the degree of stretch to an extent that matches the spacing of the features, crack-based nanostructures can be formed with precisely defined locations and spacing. These cracks serve as tunable nanochannels where the cross-sectional area can be sufficiently widened to allow the efficient loading of large-radius coiled DNA and chromatin into the channels. When the strain is subsequently relaxed, the channel cross-sectional dimensions decrease significantly. This narrowing linearizes and traps biopolymers in an extended state without the application of an electric field by the generation of a mixed shear and elongational flow as well as through the effects of nanoconfinement. This nanoscale squeezing technique enables stretching of lambda DNA to its full contour length in physiological buffers. Additionally, the system enables differentiation of stretchability of chromatin reconstituted with and without histone H1, as well as multi-color mapping of histone acetylation and methylation of single strands of native chromatin isolated from mammalian cells.

We have been exploring a variety of micro/nanofluidic devices for biomolecular analysis, using both lithographic and non-lithographic methods to form nanofluidic structures most often integrated with optical analysis and electrokinetic drive. Some information can be derived from the electrophoretic mobility of individual molecules in the nanofluidics, but the primary analytical method we have used is single molecule fluorescence correlation to detect binding events. We use this to recognize and quantify individual molecules such as chromosomal fragments bearing epigenetic modifications of interest. We are now using this approach, combined with automated switching of electrokinetic drive, to select and recover DNA fragments for epigenetic analysis. We are also using physical properties of microfluidic structures to capture and purify genomic DNA from individual cells and deliver this for on-chip analysis in nanofluidic devices. Larger scale devices have been used to perform multiplexed SELEX to more rapidly obtain and synthesize nucleic acid aptamers as highly specific binding molecules for bioanalysis and other uses.

Rotary nanomotors, a critical but less developed type of NEMS devices, is extremely important for further advancing NEMS technology. In this work, we report innovative design, assembly, and rotation of ordered arrays of nanomotors. The nanomotors were bottom-up assembled from nanoscale building blocks with nanowires as rotors, patterned nanomagnets as bearings, and quadrupole microelectrodes as stators. Arrays of nanomotors can be synchronously rotated with controlled angle, speed (to at least 18,000 rpm), and chirality by electric fields. The fundamental nanoscale electrical, mechanical, and magnetic interactions in the nanomotor system were investigated, which adds new knowledge and understanding for design of various metallic NEMS devices. The innovations from concept, design, to actuation are on multi-levels and may bring transformative impact to NEMS, microfluidics, and lab-on-chip architectures.

Rapid and low-cost method for DNA sequencing is one of the most emerging subjects to analyze genetic information. Solid-state nanopore detectors have been demonstrated as promising device to obtain specific signal from each nucleotides. Here, we describe a method to detect using glass nanopore functionalized with probe DNA molecules. The longer translocation times and the larger conductance drops are observed for DNA molecules with the complementary sequence to probe DNA molecules than for DNA molecules with the non-complementary sequence. Moreover, it is possible to distinguish DNA molecules with different sequences using the glass nanopore modified with probe DNA molecules. These results demonstrate the possibility that the functionalized glass nanopore may be applied for DNA sequencing.

The recently-reported exceptionally fast fluid transport rates in carbon nanotube (CNT) pores of a few nanometer diameters spurred great interest for their application as nanofluidic channels in several areas ranging from desalination and carbon capture, to drug delivery, protein separation, DNA sequencing, and breathable fabrics.
Here, we highlight experimental work performed in our laboratory directed toward: a) a fundamental understanding of the selectivity of these pores for electrolyte solutions under a pressure driving force; b) elucidating ionic conductance behavior in a single CNT nanopore; c) the development of breathable and protective fabrics based on CNT arrays. For our studies, we used ceramic or polymeric membranes with well-aligned, a-few-nm wide CNTs as only through-pores. We demonstrate that ion selectivity for single and mixed electrolytes in narrow CNT pores is dominated by electrostatic interactions between carboxylic groups at the CNT tips and the ions in solution. Ion selectivity can be tuned by acting on solution pH and by employing a controlled surface functionalization with atomic layer deposition (ALD) without loss of their unique ultrafast fluid transport properties. Single-pore ionic conductance measurements revealed a usual power law concentration dependence ~c^n, n<1. Finally, flexible CNT membranes under development for the breathable and protective fabric application provide water vapor transport rates that are comparable to or exceeding state-of-art breathable fabrics at all relative humidities even if the moisture conductive pores are only a-few-nm wide and the membrane porosity <1%. Moreover, these tiny pores enable simultaneous passive protection from biological threats by size exclusion.
This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

Nanopore ionic transistors are electrofluidic elements conceived to manipulate the transport of molecular species through nanopores using electric fields. In this work, we report on a newly discovered diode circuit element existing in the electrolyte-oxide interface, and its importance for electrofluidic gating in ionic transistors. We built sub-20 nm nanopore ionic transistor devices and characterized ionic transport of KCl electrolytes. Simultaneous monitoring of electric currents of source (Is), drain (Id) and gate probes (Ig), allowed us to characterize both ionic and interfacial transports, as a function of gate voltage (Vg). Ionic transport through the nanopore was modulated such that negative (-) gate voltage bias induced an increase of ionic current, representing p-type transport, suggesting that the majority carrier contributing to ionic transport is positively charged potassium ion (K+) which screens the surface charges of pore wall. Interestingly, a characterization of Ig showed the presence of a diode circuit element in the electrolyte-oxide interface: The electric field created by negative (-) gate voltage bias reaches the electrolyte more effectively than that created by a positive (+) gate voltage bias, thus leading more efficient electrical-control over ionic transport. This diode functionality in electrolyte-oxide interface results in the unipolar transport of ionic transistor. Based on the analysis of secondary ion mass spectrometry (SIMS) of the gate oxide layer, we suggest that the diode functionality is attributed to the diffusion of permeable potassium ions into the gate oxide, driven by negative (-) gate voltage biases. Our interpretation of the electrolyte-oxide interfacial effect clarifies electrofluidic gating behavior in ionic transistors.

Assessing the membrane potential of individual mitochondria is critical for understanding functional and behavioral heterogeneity within mitochondrial populations. We have developed cost effective, easy to fabricate arrays of nanochannels with a cross section of 500 nm x 2 µm. The channels are made with soft lithography of PDMS. The mold used to cast the PDMS is fabricated with a simple photolithography step. Isolated mitochondria are passed through the channel