Full-wafer in-situ fabrication and packaging of microfluidic flow cytometer with photo-patternable adhesive polymers.

Integration of microelectronics with microfluidics enables sophisticated lab-on-a-chip devices for sensing and actuation. In this paper, we investigate a novel method for in-situ microfluidics fabrication and packaging on wafer level. Two novel photo-patternable adhesive polymers were tested and compared, PA-S500H and DXL-009. The microfluidics fabrication method employs photo lithographical patterning of spin coated polymer films of PA or DXL and direct bonding of formed microfluidics to a top glass cover using die-to-wafer level bonding. These new adhesive materials remove the need for additional gluing layers. With this approach, we fabricated disposable microfluidic flow cytometers and evaluated the performance of those materials in the context of this application. DXL-009 exhibits lower autofluorescence compared to PA-S500H which improves detection sensitivity of fluorescently stained cells. Results obtained from the cytotoxicity test reveals that both materials are biocompatible. The functionality of these materials was demonstrated by detection of immunostained monocytes in microfluidic flow cytometers. The flexible, fully CMOS compatible fabrication process of these photo-patternable adhesive materials will simplify prototyping and mass manufacturing of sophisticated microfluidic devices with integrated microelectronics.

Micro vapor bubble jet flow for safe and high-rate fluorescence-activated cell sorting.

Safe, high-rate and cost-effective cell sorting is important for clinical cell isolation. However, commercial fluorescence-activated cell sorters (FACS) are expensive and prone to aerosol-induced sample contamination. Here we report a microfluidic cell sorter allowing high rate and fully enclosed cell sorting. The sorter chip consists of an array of micro heating hotspots. Pulsed resistive heating in the hotspots produces numerous micro vapor bubbles with short duration, which gives rise to a rapid jet flow for cell sorting. With this method, we demonstrated high sorting rate comparable to commercial FACS and the significant enrichment of rare cancer cells. This vapor bubble based cell sorting method can be a powerful tool for contamination-free and affordable clinical cell sorting such as circulating tumor cell isolation and cancer cell therapy.

Bottom-up SiO2 embedded carbon nanotube electrodes with superior performance for integration in implantable neural microsystems.

The reliable integration of carbon nanotube (CNT) electrodes in future neural probes requires a proper embedding of the CNTs to prevent damage and toxic contamination during fabrication and also to preserve their mechanical integrity during implantation. Here we describe a novel bottom-up embedding approach where the CNT microelectrodes are encased in SiO(2) and Parylene C with lithographically defined electrode openings. Vertically aligned CNTs are grown on microelectrode arrays using low-temperature plasma-enhanced chemical vapor deposition compatible with wafer-scale CMOS processing. Electrodes with 5, 10, and 25 µm diameter are realized. The CNT electrodes are characterized by electrochemical impedance spectroscopy and cyclic voltammetry and compared against cofabricated Pt and TiN electrodes. The superior performance of the CNTs in terms of impedance (≤4.8 ± 0.3 kΩ at 1 kHz) and charge-storage capacity (≥513.9 ± 61.6 mC/cm(2)) is attributed to an increased wettability caused by the removal of the SiO(2) embedding in buffered hydrofluoric acid. Infrared spectroscopy reveals an unaltered chemical fingerprint of the CNTs after fabrication. Impedance monitoring during biphasic current pulsing with increasing amplitudes provides clear evidence of the onset of gas evolution at CNT electrodes. Stimulation is accordingly considered safe for charge densities ≤40.7 mC/cm(2). In addition, prolonged stimulation with 5000 biphasic current pulses at 8.1, 40.7, and 81.5 mC/cm(2) increases the CNT electrode impedance at 1 kHz only by 5.5, 1.2, and 12.1%, respectively. Finally, insertion of CNT electrodes with and without embedding into rat brains demonstrates that embedded CNTs are mechanically more stable than non-embedded CNTs.

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 µm CMOS chip.

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 µm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.

Micro-sized syringes for single-cell fluidic access integrated on a micro-electrode array CMOS chip.

Very-large scale integration and micro-machining have enabled the development of novel platforms for advanced and automated examination of cells and tissues in vitro. In this paper, we present a CMOS chip designed in a commercial 0.18 µm technology with integrated micro-syringes combined with micro-nail shaped electrodes and readout electronics. The micro-syringes could be individually addressed by a through-wafer micro-fluidic channel with an inner diameter of 1 µm. We demonstrated the functionality of the micro-fluidic access by diffusion of fluorescent species through the channels. Further, hippocampal neurons were cultured on top of an array of micro-syringes, and focused ion beam-scanning electron microscopy cross-sections revealed protrusion of the cells inside the channels, creating a strong interface between the membrane and the chip surface. This principle demonstrates a first step towards a novel type of automated in vitro platforms, allowing local delivery of substances to cells or advanced planar patch clamping.

Single-cell stimulation and electroporation using a novel 0.18 µ CMOS chip with subcellular-sized electrodes.

In drug screening and pharmaceutical research, high-throughput systems that are able to perform single-cell measurements are highly desired. Micro-electrode arrays try to answer this need but still suffer from significant drawbacks such as a small amount of electrodes and the inability to address single cells. Here, we present a novel multi-transistor array chip with 16,384 subcellular-sized electrodes based on 0.18 µm CMOS technology. We show that single-cell stimulation is possible by applying voltage pulses on the electrode to stimulate the cells lying on top. Electroporation of the cell membrane is observed using the whole-cell patch clamp technique and fluorescent dye-based live imaging. This technology could be used for high-throughput, single-cell manipulations for the purpose of large-scale drug screening and the investigation of fundamental cell processes.

Using reciprocal derivative chronopotentiometry as a technique to determine safe charge injection limits of electrodes used for neural stimulation.

We used reciprocal derivative chronopotentiometry (RDC) with platinum electrodes of 50 microm diameter in 0.15 M phosphate buffered saline solution to identify the various electrochemical processes occurring at the electrode during biphasic current pulsing. RDC allowed to determine the limits of water hydrolysis based on the specific (dt/dE)-E data representation employed in this technique resulting in curves similar to the voltammetric i-E response. Current stimulation was performed by either varying the pulse amplitude or pulse width. We found that the limits for H(2) and O(2) evolution for constant-amplitude pulses lied at 0.51 mC/cm(2) and 0.67 mC/cm(2), respectively, while for constant-width pulses they occurred at slightly lower values of 0.49 mC/cm(2) and 0.61 mC/cm(2), respectively. We could also extract values for the anodic and cathodic overvoltages associated with gas evolution. The cathodic overvoltage for H(2) evolution was 1.43 V for both constant-amplitude and constant-width pulses, while the anodic overpotentials for O(2) evolution were 2.45 V in the first and 2.24 V in the latter case. These values are clearly larger than the gas evolution limits generally found with steady-state voltammetry.

A novel 16k micro-nail CMOS-chip for in-vitro single-cell recording, stimulation and impedance measurements.

In neurophysiological and pharmaceutical research, parallel and individual access to a dense population of in-vitro cultured neurons is a key feature for analyzing networks of neurons. This paper presents a 0.18µm CMOS chip containing a dense array of micro-nail electrodes, a 128×128 sensor/actuator matrix with in-situ differential amplification circuits, pico-Ampere current stimulation, and impedance measurement circuits. Measurements on packaged chips show successful impedance measurements matching the simulation model and electrical recordings of in-vitro cultured cardiomyocytes, correlated with recorded changes in intra-cellular calcium concentrations. This system is a first step towards a high-throughput neuron/chip interface.

Localized electrical stimulation of in vitro neurons using an array of sub-cellular sized electrodes.

The investigation of single-neuron parameters is of great interest because many aspects in the behavior and communication of neuronal networks still remain unidentified. However, the present available techniques for single-cell measurements are slow and do not allow for a high-throughput approach. We present here a CMOS compatible microelectrode array with 84 electrodes (with diameters ranging from 1.2 to 4.2 µm) that are smaller than the size of cell, thereby supporting single-cell addressability. We show controllable electroporation of a single cell by an underlying electrode while monitoring changes in the intracellular membrane potential. Further, by applying a localized electrical field between two electrodes close to a neuron while recording changes in the intracellular calcium concentration, we demonstrate activation of a single cell (∼270%, DF/F(0)), followed by a network response of the neighboring cells. The technology can be easily scaled up to larger electrode arrays (theoretically up to 137,000 electrodes/mm(2)) with active CMOS electronics integration able to perform high-throughput measurements on single cells.

Local electrical stimulation of single adherent cells using three-dimensional electrode arrays with small interelectrode distances.

In this paper, we describe the localized and selective electrical stimulation of single cells using a three-dimensional electrode array. The chip consisted of 84 nail-like electrodes with a stimulation surface of 0.8 microm(2) and interelectrode distances as small as 3 microm. N2A cells were used to compare bipolar stimulation between one electrode in- and one outside the cell on the one hand, and two electrodes in the same cell on the other hand. Selective and localized stimulation of primary embryonic cardiomyocytes showed the possibility to use this chip with excitable cells. The response of the cells to applied electrical fields was monitored using calcium imaging whereas assessment of electroporation was determined following influx of propidium iodide. Arrays of these three-dimensional electrodes could eventually be used as a tool to selectively electroporate the membrane of single cells for genetic manipulation or to obtain electrical access to the inner compartment of the cell.

Local electrical stimulation of cultured embryonic cardiomyocytes with sub-micrometer nail structures.

In this paper, we demonstrate the feasibility of selective extracellular electrical stimulation at the (sub)cellular level in dissociated cultured cells. Using a CMOS-compatible process, we have fabricated an electrode array with sub-micrometer nail probes. Due to their particular configuration, the nails are strongly engulfed by the cellular membrane. By measuring the calcium signals, we found that electrical stimulation via the micronails activates the cell locally, in a dose-dependent manner, with very low applied currents. The results suggest the applicability of the device in pharmacological or signal propagation studies.