High resolution monitoring of chemotherapeutic agent potency in cancer cells using a CMOS capacitance biosensor.

Monitoring cell viability and proliferation in real-time provides a more comprehensive picture of the changes cells undergo during their lifecycle than can be achieved using traditional end-point assays. Particularly for drug screening applications, high-temporal resolution cell viability data could inform decisions on drug application protocols that might lead to better treatment outcomes. We describe a CMOS biosensor that monitors cell viability through high-resolution capacitance measurements of cell adhesion quality. The system consists of a 3 × 3 mm2 chip with an array of 16 sensors, on-chip digitization, and serial data output that can be interfaced with inexpensive off-the-shelf components. An imaging system was developed to provide ground-truth data of cell coverage concurrently with data recordings. Results showed the sensor's ability to detect single-cell binding events, track cell morphology changes, and monitor cell motility. A chemotherapeutic assay was conducted to examine dose-dependent cytotoxic effects on drug-resistant and drug-sensitive cancer cell lines. Concentrations higher than 5 µM elicited cytotoxic effects on both cell lines, while a dose of 1 µM allowed discrimination of the two cell types. The system demonstrates the use of real-time capacitance measurements as a proof-of-concept tool that has potential to hasten the drug development process.

Correlation of Capacitance and Microscopy Measurements Using Image Processing for a Lab-on-CMOS Microsystem.

We present a capacitance sensor chip developed in a 0.35-µm complementary metal-oxide-semiconductor process for monitoring biological cell viability and proliferation. The chip measures the cell-to-substrate binding through capacitance-to-frequency conversion with a sensitivity of 590 kHz/fF. In vitro experiments with two human ovarian cancer cell lines (CP70 and A2780) were performed and showed the ability to track cell viability in realtime over three days. An imaging platform was developed to provide time-lapse images of the sensor surface, which allowed for concurrent visual and capacitance observation of the cells. The results showed the ability to detect single-cell binding events and changes in cell morphology. Image processing was performed to estimate the cell coverage of sensor electrodes, showing good linear correlation and providing a sensor gain of 1.28 ± 0.29 aF/µm2, which agrees with values reported in the literature. The device is designed for unsupervised operation with minimal packaging requirements. Only a microcontroller is required for readout, making it suitable for applications outside the traditional laboratory setting.

Recognizing Hemiparetic Ankle Deficits Using Wearable Pressure Sensors.

OBJECTIVE: To provide proof-of-concept for a novel method to recognize impaired push-off and foot-drop deficits in hemiparetic gait using analog pressure sensors. These data may enhance feedback from a modular ankle exoskeleton (such as Anklebot) for stroke rehabilitation, which now employs on/off foot switches under the foot. METHODS: A pressure sensor was positioned on the posterior side of the calcaneus. Experiments were conducted on two healthy subjects with normal walking and with hip circumduction and foot drop, the latter to mimic hemiparetic gait post-stroke. RESULTS: Unlike the foot switches, the pressure sensor yielded data during swing. The initial swing and terminal stance readings followed local foot-shoe dynamics and were thus able to detect foot drop swing deficits while also providing push-off information during stance. DISCUSSION: The analog pressure sensors provided more information than foot switches, even during stance. This system may provide clinicians with a tool to monitor foot drop and push-off.

LTCC Packaged Ring Oscillator Based Sensor for Evaluation of Cell Proliferation.

A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic (LTCC) module with a flip chip bonding technique. A microcontroller operates the chip, while the whole measurement system was controlled by PC. The developed biosensor was applied for measurement of the proliferation stage of adherent cells where the sensor response depends on the ratio between healthy, viable and multiplying cells, which adhere onto the chip surface, and necrotic or apoptotic cells, which detach from the chip surface. This change in cellular adhesion caused a change in the effective permittivity in the vicinity of the sensor element, which was sensed as a change in oscillation frequency of the ring oscillator. The sensor was tested with human lung epithelial cells (BEAS-2B) during cell addition, proliferation and migration, and finally detachment induced by trypsin protease treatment. The difference in sensor response with and without cells was measured as a frequency shift in the scale of 1.1 MHz from the base frequency of 57.2 MHz. Moreover, the number of cells in the sensor vicinity was directly proportional to the frequency shift.

Real-Time Measurements of Cell Proliferation Using a Lab-on-CMOS Capacitance Sensor Array.

We describe a capacitance sensor array that has been incorporated into a lab-on-CMOS system for applications in monitoring cell viability. This paper presents analytical models, calibration results, and measured experimental results of the biosensor. The sensor has been characterized and exhibits a sensitivity of 590 kHz/fF. We report results from benchtop tests and in vitro experiments demonstrating on-chip tracking of cell adhesion as well as monitoring of cell viability. Human ovarian cancer cells were cultured on chip, and measured capacitance responses were validated by comparison with images from photomicrographs of the chip surface. Analysis was performed to quantify cell proliferation and adhesion, and responses to live cells were estimated to be 100 aF/cell.

Targeted Feature Recognition Using Mechanical Spatial Filtering with a Low-Cost Compliant Strain Sensor.

A tactile sensing architecture is presented for detection of surface features that have a particular target size, and the concept is demonstrated with a braille pattern. The approach is akin to an inverse of mechanical profilometry. The sensing structure is constructed by suspending a stretchable strain-sensing membrane over a cavity. The structure is moved over the surface, and a signal is generated through mechanical spatial filtering if a feature is small enough to penetrate into the cavity. This simple design is tailorable and can be realized by standard machining or 3D printing. Images of target features can be produced with even a low-cost compliant sensor. In this work a disposable elastomeric piezoresistive strain sensor was used over a cylindrical "finger" part with a groove having a width corresponding to the braille dot size. A model was developed to help understand the working principle and guide finger design, revealing amplification when the cavity matches the feature size. The new sensing concept has the advantages of being easily reconfigured for a variety of sensing problems and retrofitted to a wide range of robotic hands, as well as compatibility with many compliant sensor types.

Challenges in realizing a self-contained hydraulically-driven contractile fiber actuator.

The field of soft robots would benefit from electrically controlled contractile actuators in the form of fibers that achieve a strain of 20% in less than a second while exerting high force. This work explores possible designs for achieving this goal using self-contained electroosmotic fluid pumping within a tube-shaped structure. The most promising configuration is a combination of a bellows and a McKibben-type muscle, since pumping fluid from the former to the latter results in contraction of both portions. Realizing such a device entails challenges in fabrication and electrokinetic fluid pumping in closed systems. Further studies of electroosmotic flow in salt-free organic solvents are needed.

System-on-Chip Considerations for Heterogeneous Integration of CMOS and Fluidic Bio-Interfaces.

CMOS chips are increasingly used for direct sensing and interfacing with fluidic and biological systems. While many biosensing systems have successfully combined CMOS chips for readout and signal processing with passive sensing arrays, systems that co-locate sensing with active circuits on a single chip offer significant advantages in size and performance but increase the complexity of multi-domain design and heterogeneous integration. This emerging class of lab-on-CMOS systems also poses distinct and vexing technical challenges that arise from the disparate requirements of biosensors and integrated circuits (ICs). Modeling these systems must address not only circuit design, but also the behavior of biological components on the surface of the IC and any physical structures. Existing tools do not support the cross-domain simulation of heterogeneous lab-on-CMOS systems, so we recommend a two-step modeling approach: using circuit simulation to inform physics-based simulation, and vice versa. We review the primary lab-on-CMOS implementation challenges and discuss practical approaches to overcome them. Issues include new versions of classical challenges in system-on-chip integration, such as thermal effects, floor-planning, and signal coupling, as well as new challenges that are specifically attributable to biological and fluidic domains, such as electrochemical effects, non-standard packaging, surface treatments, sterilization, microfabrication of surface structures, and microfluidic integration. We describe these concerns as they arise in lab-on-CMOS systems and discuss solutions that have been experimentally demonstrated.

Fabrication of a Miniature Paper-Based Electroosmotic Actuator.

A voltage-controlled hydraulic actuator is presented that employs electroosmotic fluid flow (EOF) in paper microchannels within an elastomeric structure. The microfluidic device was fabricated using a new benchtop lamination process. Flexible embedded electrodes were formed from a conductive carbon-silicone composite. The pores in the layer of paper placed between the electrodes served as the microchannels for EOF, and the pumping fluid was propylene carbonate. A sealed fluid-filled chamber was formed by film-casting silicone to lay an actuating membrane over the pumping liquid. Hydraulic force generated by EOF caused the membrane to bulge by hundreds of micrometers within fractions of a second. Potential applications of these actuators include soft robots and biomedical devices.

Bubble-free electrokinetic flow with propylene carbonate.

For electroosmotic pumping, a large direct-current (DC) electric field (10+ V/cm) is applied across a liquid, typically an aqueous electrolyte. At these high voltages, water undergoes electrolysis to form hydrogen and oxygen, generating bubbles that can block the electrodes, cause pressure fluctuations, and lead to pump failure. The requirement to manage these gases constrains system designs. This article presents an alternative polar liquid for DC electrokinetic pumping, propylene carbonate (PC), which remains free of bubbles up to at least 10 kV/cm. This offers the opportunity to create electrokinetic devices in closed configurations, which we demonstrate with a fully sealed microfluidic hydraulic actuator. Furthermore, the electroosmotic velocity of PC is similar to that of water in PDMS microchannels. Thus, water could be substituted by PC in existing electroosmotic pumps.

Multiplex quantitative measurement of mRNAs from fixed tissue microarray sections.

The development of prognostic and diagnostic biomarkers, such as those from gene expression studies, requires independent validation in clinical specimens. Immunohistochemical analysis on tissue microarrays (TMAs) of formalin-fixed paraffin-embedded tissue is often used to increase the statistical power, and it is used more often than in situ hybridization, which can be technically limiting. Herein, we introduce a method for performing quantitative gene expression analysis across a TMA using an adaptation of 2D-RT-qPCR, a recently developed technology for measuring transcript levels in a histologic section while maintaining 2-dimensional positional information of the tissue sample. As a demonstration of utility, a TMA with tumor and normal human prostate samples was used to validate expression profiles from previous array-based gene discovery studies of prostate cancer. The results show that 2D-RT-qPCR expands the utility of TMAs to include sensitive and accurate gene expression measurements.

Packaging commercial CMOS chips for lab on a chip integration.

Combining integrated circuitry with microfluidics enables lab-on-a-chip (LOC) devices to perform sensing, freeing them from benchtop equipment. However, this integration is challenging with small chips, as is briefly reviewed with reference to key metrics for package comparison. In this paper we present a simple packaging method for including mm-sized, foundry-fabricated dies containing complementary metal oxide semiconductor (CMOS) circuits within LOCs. The chip is embedded in an epoxy handle wafer to yield a level, large-area surface, allowing subsequent photolithographic post-processing and microfluidic integration. Electrical connection off-chip is provided by thin film metal traces passivated with parylene-C. The parylene is patterned to selectively expose the active sensing area of the chip, allowing direct interaction with a fluidic environment. The method accommodates any die size and automatically levels the die and handle wafer surfaces. Functionality was demonstrated by packaging two different types of CMOS sensor ICs, a bioamplifier chip with an array of surface electrodes connected to internal amplifiers for recording extracellular electrical signals and a capacitance sensor chip for monitoring cell adhesion and viability. Cells were cultured on the surface of both types of chips, and data were acquired using a PC. Long term culture (weeks) showed the packaging materials to be biocompatible. Package lifetime was demonstrated by exposure to fluids over a longer duration (months), and the package was robust enough to allow repeated sterilization and re-use. The ease of fabrication and good performance of this packaging method should allow wide adoption, thereby spurring advances in miniaturized sensing systems.

High-throughput particle separation and concentration using spiral inertial filtration.

A spiral inertial filtration (SIFT) device that is capable of high-throughput (1 ml/min), high-purity particle separation while concentrating recovered target particles by more than an order of magnitude is reported. This device is able to remove large fractions of sample fluid from a microchannel without disruption of concentrated particle streams by taking advantage of particle focusing in inertial spiral microfluidics, which is achieved by balancing inertial lift forces and Dean drag forces. To enable the calculation of channel geometries in the SIFT microsystem for specific concentration factors, an equivalent circuit model was developed and experimentally validated. Large particle concentration factors were then achieved by maintaining either the average fluid velocity or the Dean number throughout the entire length of the channel during the incremental removal of sample fluid. The SIFT device was able to separate MCF7 cells spiked into whole blood from the non-target white blood cells (WBC) with a recovery of nearly 100% while removing 93% of the sample volume, which resulted in a concentration enhancement of the MCF7 cancer cells by a factor of 14.

Characterization of conjugated polymer actuation under cerebral physiological conditions.

Conjugated polymer actuators have potential use in implantable neural interface devices for modulating the position of electrode sites within brain tissue or guiding insertion of neural probes along curved trajectories. The actuation of polypyrrole (PPy) doped with dodecylbenzenesulfonate (DBS) is characterized to ascertain whether it can be employed in the cerebral environment. Microfabricated bilayer beams are electrochemically cycled at either 22 or 37 °C in aqueous NaDBS or in artificial cerebrospinal fluid (aCSF). Nearly all the ions in aCSF are exchanged into the PPy-the cations Na(+) , K(+) , Mg(2+) , Ca(2+) , as well as the anion PO4 (3-) ; Cl(-) is not present. Nevertheless, deflections in aCSF are comparable to those in NaDBS and they are monotonic with oxidation level: strain increases upon reduction, with no reversal of motion despite the mixture of ionic charges and valences being exchanged. Actuation depends on temperature. Upon warming, the cyclic voltammograms show additional peaks and an increase of 70% in the consumed charge. Bending is, however, much less affected: strain increases somewhat (6%-13%) but remains monotonic, and deflections shift (up to 20%). These results show how the actuation environment must be taken into account, and demonstrate proof of concept for actuated implantable neural interfaces.

A novel surface modification technique for forming porous polymer monoliths in poly(dimethylsiloxane).

A new method of surface modification is described for enabling the in situ formation of homogenous porous polymer monoliths (PPMs) within poly(dimethylsiloxane) (PDMS) microfluidic channels that uses 365 nm UV illumination for polymerization. Porous polymer monolith formation in PDMS can be challenging because PDMS readily absorbs the monomers and solvents, changing the final monolith morphology, and because PDMS absorbs oxygen, which inhibits free-radical polymerization. The new approach is based on sequentially absorbing a non-hydrogen-abstracting photoinitiator and the monomers methyl methacrylate and ethylene diacrylate within the walls of the microchannel, and then polymerizing the surface treatment polymer within the PDMS, entangled with it but not covalently bound. Four different monolith compositions were tested, all of which yielded monoliths that were securely anchored and could withstand pressures exceeding the bonding strength of PDMS (40 psi) without dislodging. One was a recipe that was optimized to give a larger average pore size, required for low back pressure. This monolith was used to concentrate and subsequently mechanical lyse B lymphocytes.

Quantifying mRNA levels across tissue sections with 2D-RT-qPCR.

Measurement of mRNA levels across tissue samples facilitates an understanding of how genes function and what their roles are in disease. Quantifying low-abundance mRNA requires a workflow that preserves spatial information, isolates RNA, and performs reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This is complex because these steps are typically performed in three separate platforms. In the present study, we describe two-dimensional RT-qPCR (2D-RT-qPCR), a method that quantifies RNA across tissues sections in a single integrated platform. The method uses the grid format of a multi-well plate to macrodissect tissue sections and preserve the spatial location of the RNA; this also eliminates the need for physical homogenization of the tissue. A new lysis and nucleic acid purification protocol is performed in the same multi-well plate, followed by RT-qPCR. The feasibility 2D-RT-qPCR was demonstrated on a variety of tissue types. Potential applications of the technology as a high-throughput tissue analysis platform are discussed.

Edge effects determine the direction of bilayer bending.

We elucidate the reason for preferential bending along the long edge in thin rectangular bilayers in which one of the layers is isotropically strained. While this preference has been observed previously, the physical basis for this preference has not been understood. We find that the bending direction is determined by the existence of doubly curved regions at the curled edges, which lower the energy. This energy difference between "spiral" and "cigar" shapes increases with aspect ratio.

2D-PCR: a method of mapping DNA in tissue sections.

A novel approach was developed for mapping the location of target DNA in tissue sections. The method combines a high-density, multi-well plate with an innovative single-tube procedure to directly extract, amplify, and detect the DNA in parallel while maintaining the two-dimensional (2D) architecture of the tissue. A 2D map of the gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was created from a tissue section and shown to correlate with the spatial area of the sample. It is anticipated that this approach may be easily adapted to assess the status of multiple genes within tissue sections, yielding a molecular map that directly correlates with the histology of the sample. This will provide investigators with a new tool to interrogate the molecular heterogeneity of tissue specimens.