A novel reflectance-based aptasensor using gold nanoparticles for the detection of oxytetracycline.

We present a novel reflectance-based colorimetric aptasensor using gold nanoparticles for the detection of oxytetracycline for the first time. It was found that the reflectance-based measurement at two wavelengths (650 and 520 nm) can generate more stable and sensitive signals than absorbance-based sensors to determine the aggregation of AuNPs, even at high AuNP concentrations. One of the most common antibacterial agents, oxytetracycline (OTC), was detected at concentrations as low as 1 nM in both buffer solution and tap water, which was 25-fold more sensitive, compared to the previous absorbance-based colorimetric aptasensors. This reflectance-based colorimetric aptasensor using gold nanoparticles is considered to be a better platform for portable sensing of small molecules using aptamers.

A novel microneedle array for the treatment of hydrocephalus.

We present a microfabricated 10 by 10 array of microneedles for the treatment of a neurological disease called communicating hydrocephalus. Together with the previously reported microvalve array, the current implantable microneedle array completes the microfabricated arachnoid granulations (MAGs) that mimic the function of normal arachnoid granulations (AGs). The microneedle array was designed to enable the fixation of the MAGs through dura mater membrane in the brain and thus provide a conduit for the flow of cerebrospinal fluid (CSF). Cone-shaped microneedles with hollow channels were fabricated using a series of microfabrication techniques: SU-8 photolithography for tapered geometry, reactive ion etching for sharpening the microneedles, 248 nm deep UV excimer laser machining for creating through-hole inside the microneedles, and metal sputtering for improved rigidity. Puncture tests were conducted using porcine dura mater and the results showed that the fabricated microneedle array is strong enough to pierce the dura mater. The in-vitro biocompatibility test result showed that none of the 100 outlets of the microneedles exposed to the bloodstream were clogged significantly by blood cells. We believe that these test results demonstrate the potential use of the microneedle array as a new treatment of hydrocephalus.

Design, fabrication, and characterization of archaeal tetraether free-standing planar membranes in a PDMS- and PCB-based fluidic platform.

The polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius contains exclusively bipolar tetraether lipids, which are able to form extraordinarily stable vesicular membranes against a number of chemical, physical, and mechanical stressors. PLFE liposomes have thus been considered appealing biomaterials holding great promise for biotechnology applications such as drug delivery and biosensing. Here we demonstrated that PLFE can also form free-standing "planar" membranes on micropores (∼100 µm) of polydimethylsiloxane (PDMS) thin films embedded in printed circuit board (PCB)-based fluidics. To build this device, two novel approaches were employed: (i) an S1813 sacrificial layer was used to facilitate the fabrication of the PDMS thin film, and (ii) oxygen plasma treatment was utilized to conveniently bond the PDMS thin film to the PCB board and the PDMS fluidic chamber. Using electrochemical impedance spectroscopy, we found that the dielectric properties of PLFE planar membranes suspended on the PDMS films are distinctly different from those obtained from diester lipid and triblock copolymer membranes. In addition to resistance (R) and capacitance (C) that were commonly seen in all the membranes examined, PLFE planar membranes showed an inductance (L) component. Furthermore, PLFE planar membranes displayed a relatively large membrane resistance, suggesting that, among the membranes examined, PLFE planar membrane would be a better matrix for studying channel proteins and transmembrane events. PLFE planar membranes also exhibited a sharp decrease in phase angle with the frequency of the input AC signal at ∼1 MHz, which could be utilized to develop sensors for monitoring PLFE membrane integrity in fluidics. Since the stability of free-standing planar lipid membranes increases with increasing membrane packing tightness and PLFE lipid membranes are more tightly packed than those made of diester lipids, PLFE free-standing planar membranes are expected to be considerably stable. All these salient features make PLFE planar membranes particularly attractive for model studies of channel proteins and transmembrane events and for high-throughput drug screening and artificial photosynthesis. This work can be extended to nanopores of PDMS thin films in microfluidics and eventually aid in membrane-based new lab-on-a-chip applications.

Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid.

This paper presents a novel liver model that mimics the liver sinusoid where most liver activities occur. A key aspect of our current liver model is a layered co-culture of primary rat hepatocytes (PRHs) and primary rat liver sinusoidal endothelial cells (LSECs) or bovine aortic endothelial cells (BAECs) on a transwell membrane. When a layered co-culture was attempted with a thin Matrigel layer placed between hepatocytes and endothelial cells to mimic the space of Disse, the cells did not form completely separated monolayers. However, when hepatocytes and endothelial cells were cultured on the opposite sides of a transwell membrane, PRHs co-cultured with LSECs or BAECs maintained their viability and normal morphology for 39 and 57 days, respectively. We assessed the presence of hepatocyte-specific differentiation markers to verify that PRHs remained differentiated in the long-term co-culture and analyzed hepatocyte function by monitoring urea synthesis. We also noted that the expression of cytochrome P-450 remained similar in the co-cultured system from day 1 to day 48. Thus, our novel liver model system demonstrated that primary hepatocytes can be cultured for extended times and retain their hepatocyte-specific functions when layered with endothelial cells.

Flow-induced voltage generation over monolayer graphene in the presence of herringbone grooves.

While flow-induced voltage over a graphene layer has been reported, its origin remains unclear. In our previous study, we suggested different mechanisms for different experimental configurations: phonon dragging effect for the parallel alignment and an enhanced out-of-plane phonon mode for the perpendicular alignment (Appl. Phys. Lett. 102:063116, 2011). In order to further examine the origin of flow-induced voltage, we introduced a transverse flow component by integrating staggered herringbone grooves in the microchannel. We found that the flow-induced voltage decreased significantly in the presence of herringbone grooves in both parallel and perpendicular alignments. These results support our previous interpretation.

The physical foundation of vasoocclusion in sickle cell disease.

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.

Micro-fabricated shunt to mimic arachnoid granulations for the treatment of communicating hydrocephalus.

Hydrocephalus is the abnormal accumulation of cerebrospinal fluid (CSF) within the confines of the skull that if left untreated results in significant morbidity and mortality. The treatment for hydrocephalus has remained essentially unchanged for over 50 years. It was a technological advance in materials that allowed John Holter, in conjunction with neurosurgeons Spitzer and Nulsen, to devise a valve and shunt system that diverted excess CSF from the ventricular space to the peritoneum. This ventriculo-peritoneal (VP) shunt is far from ideal, with problems associated with under/over shunting, mechanical mismatch, infection, high failure rates, disconnection and erosion. With the advances in the field of micro-fabrication and micro-machines we propose an innovative shunt system that would mimic the function of arachnoid granulations. This micro-fabricated shunting device, or micro-mechanical arachnoid granulation (MAG), consists of a multiplicity of micro-valves each 210 µm in diameter that each adhere to individual micro-needles. This work demonstrates the design and initial test results of the micro-valve with parameters for low cracking pressure, optimal flow rate, and reflux that would mimic the function of the native arachnoid granulations.

Improved protein detection on an AC electrokinetic quartz crystal microbalance (EKQCM).

Microscale electrodes supplied with an AC field can generate rotational fluid patterns known as AC electroosmosis. In the present study, this effect was used to improve antibody binding on a biosensor surface. Antibodies, like many other large, slow moving biomolecules, tend to suffer from transport limitations during a reaction with a surface-bound receptor. Stirring such reactions with AC electroosmosis can alleviate this transport limitation by bringing fresh reagent to the surface. For the first time, the use of this phenomenon was used to improve the capture of protein on a sensor. Directly adsorbed antibodies were bound to the surface of specially modified quartz crystal microbalances, known as electrokinetic QCMs (EKQCMs) and the signal was enhanced by about 5.6 times. Modification of the QCM resulted in little reduction of quality factor (from ∼ 5.3 k to ∼ 4.6k) and an increased sensitivity to viscosity changes (151%). Full immunoassays performed on electrodes fabricated on glass surfaces were used to ensure antibody function was not significantly degraded by the enhancement technique.

Chaotic mixing in microchannels via low frequency switching transverse electroosmotic flow generated on integrated microelectrodes.

In this paper we present a numerical and experimental investigation of a chaotic mixer in a microchannel via low frequency switching transverse electroosmotic flow. By applying a low frequency, square-wave electric field to a pair of parallel electrodes placed at the bottom of the channel, a complex 3D spatial and time-dependence flow was generated to stretch and fold the fluid. This significantly enhanced the mixing effect. The mixing mechanism was first investigated by numerical and experimental analysis. The effects of operational parameters such as flow rate, frequency, and amplitude of the applied voltage have also been investigated. It is found that the best mixing performance is achieved when the frequency is around 1 Hz, and the required mixing length is about 1.5 mm for the case of applied electric potential 5 V peak-to-peak and flow rate 75 microL h(-1). The mixing performance was significantly enhanced when the applied electric potential increased or the flow rate of fluids decreased.

Comprehensive analysis of particle motion under non-uniform AC electric fields in a microchannel.

AC electrokinetics is rapidly becoming a foundational tool for lab-on-a-chip systems due to its versatility and the simplicity of the components capable of generating them. Predicting the behavior of fluids and particles under non-uniform AC electric fields is important for the design of next generation devices. Though there are several important phenomena that contribute to the overall behavior of particles and fluids, current predictive techniques consider special conditions where only a single phenomenon may be considered. We report a 2D numerical simulation, using COMSOL Multiphysics, which incorporates the three major AC electrokinetic phenomena (dielectrophoresis, AC electroosmosis and electrothermal effect) and is valid for a wide range of operational conditions. Corroboration has been performed using experimental conditions that mimic those of the simulation and shows good qualitative agreement. Furthermore, a broad range of experiments has been performed using four of the most widely reported devices under varying conditions in order to show their behavior as it relates to the simulation. The large number of experimental conditions reported, together with the comprehensive numerical simulation, will help provide guidelines for scientists and engineers interested in incorporating AC electrokinetics into their lab-on-a-chip systems.

AC electrokinetic phenomena generated by microelectrode structures.

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics, environmental monitoring and homeland security. In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Microfluidic platform for hepatitis B viral replication study.

Hepatocytes, the cells responsible for the metabolic and detoxification processes in the liver, are the predominant target of hepatitis B virus (HBV) infections, a major cause of liver cancer. The limited availability of normal human hepatocytes for cell-culture based studies is a significant challenge in HBV-associated liver cancer research. Therefore, there is a need for miniaturized cell-culture systems that can serve as a platform for studying the effect of HBV infections on hepatocyte physiology. Here, we present a microfluidic platform that can be used to study HBV replication in both rat and human hepatocytes. Polydimethylsiloxane (PDMS) microchannels fabricated using soft lithography techniques served as a culture vessel for both primary rat hepatocytes (PRH) and a human hepatoblastoma cell line, HepG2. The micro cell-culture chamber was then used as a model for HBV replication studies. Cells were grown in static culture conditions and either transfected with an HBV-genome cDNA or infected with the viral genome expressed from a recombinant adenovirus. Supernatants collected from the microchannels were assayed for secreted HBV using polymerase chain reaction (PCR). We achieved approximately 40 and 10% transfection efficiencies in HepG2 cells and PRH respectively, and 80-100% adenoviral infection efficiency in PRH comparable to standard tissue culture methods. Moreover, we successfully detected replicated HBV using our novel platform. This platform can be easily extended to studies involving DNA transfection or HBV infection of primary human hepatocytes since only a small number of cells are required for studies in microfluidic chambers.

Micropatterns of Matrigel for three-dimensional epithelial cultures.

Three-dimensional (3D) epithelial culture models are widely used to promote a physiologically relevant microenvironment for the study of normal and aberrant epithelial organization. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hindered by the lack of existing platforms for large-scale 3D cellular studies. Current 3D standard culture does not allow for single spheroid or 'acinus' analysis required for high-throughput systems. Here, we present general strategies for creating bulk micropatterns of Matrigel that can be used as a platform for 3D epithelial culture and cell-based assays at the single acinus level. Both buried and free-standing micropatterns of Matrigel were created using modified soft lithography techniques such as microtransfer molding (microTM) and dry lift-off technique. Surface modification of poly(dimethylsiloxane) (PDMS) with oxygen plasma followed by treatment with poly(2-hydroxy-ethylmethacrylate) (poly-HEMA) was sufficient to promote deformation-free release of Matrigel patterns. In addition, a novel dual-layer dry lift-off technique was developed to simultaneously generate patterns of Matrigel and poly-HEMA on a single substrate. We also demonstrate that the micropatterned Matrigel can support 3D culture originating from a single normal human mammary epithelial (MCF-10A) cell or a human breast cancer cell (MDA-MB-231) with comparable phenotypes to standard 3D culture techniques. Culture of normal MCF-10A cells on micropatterned Matrigel resulted in formation of structures with the characteristic apoptosis of centrally located cells and formation of hollow lumens. Moreover, the carcinoma cell line showed their characteristic formation of disorganized invasive cellular clusters, lacking the normal epithelial architecture on micropatterned Matrigel. Hence, micropatterned Matrigel can be used as a 3D epithelial cell-based platform for a wide variety of applications in epithelial and cancer biology, tissue engineering, as well as gene/drug screening technology.