Nanoporous elements in microfluidics for multiscale manipulation of bioparticles

Solid materials, such as silicon, glass, and polymers, dominate as structural elements in microsystems including microfluidics. Porous elements have been limited to membranes sandwiched between microchannel layers or polymer monoliths. This paper reports the use of micropatterned carbon-nanotube forests confined inside microfluidic channels for mechanically and/or chemically capturing particles ranging over three orders of magnitude in size. Nanoparticles below the internanotube spacing (80 nm) of the forest can penetrate inside the forest and interact with the large surface area created by individual nanotubes. For larger particles (>80 nm), the ultrahigh porosity of the nanotube elements reduces the fluid boundary layer and enhances particle-structure interactions on the outer surface of the patterned nanoporous elements. Specific biomolecular recognition is demonstrated using cells (≈10 μm), bacteria (≈1 μm), and viral-sized particles (≈40 nm) using both effects. This technology can provide unprecedented control of bioseparation processes to access bioparticles of interest, opening new pathways for both research and point-of-care diagnostics.     

Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation

Dielectrophoretic/gravitational field-flow fractionation (DEP/G-FFF) was used to separate cultured human breast cancer MDA-435 cells from normal blood cells mixed together in a sucrose/dextrose medium. An array of microfabricated, interdigitated electrodes of 50 microns widths and spacings, and lining the bottom surface of a thin chamber (0.42 mm H x 25 mm W x 300 mm L), was used to generate DEP forces that levitated the cells. A 10-microL cell mixture sample containing approximately 50,000 cells was introduced into the chamber, and cancerous and normal blood cells were levitated to different heights according to the balance of DEP and gravitational forces. The cells at different heights were transported at different velocities under the influence of a parabolic flow profile that was established in the chamber and were thereby separated. Separation performance depended on the frequency and voltage of the applied DEP field and the fluid-flow rate. It took as little as 5 min to achieve cell separation. An analysis of the DEP/G-FFF results revealed that the separation exploited the difference in dielectric and density properties between cell populations. The DEP/G-FFF technique is potentially applicable to many biological and biomedical problems, especially those related to microfluidic systems.     

Controlled dielectrophoretic nanowire self-assembly using atomic layer deposition and suspended microfabricated electrodes

Effects of design and materials on the dielectrophoretic self-assembly of individual gallium nitride nanowires (GaN NWs) onto microfabricated electrodes have been experimentally investigated. The use of TiO(2) surface coating generated by atomic layer deposition (ALD) improves dielectrophoretic assembly yield of individual GaN nanowires on microfabricated structures by as much as 67%. With a titanium dioxide coating, individual nanowires were placed across suspended electrode pairs in 46% of tests (147 out of 320 total), versus 28% of tests (88 out of 320 total tests) that used uncoated GaN NWs. An additional result from these tests was that suspending the electrodes 2.75 μm above the substrate corresponded with up to 15.8% improvement in overall assembly yield over that of electrodes fabricated directly on the substrate.     

Development and characterization of microfabricated disposable gold working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection

We have developed a new type of microfabricated thin-film electrode on polymeric substrates. The microfabrication process allows for inexpensive and reproducible mass production of disposable working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection (IPAD). These microfabricated electrodes are disposable and have been optimized for use in flow-through low-dead-volume electrochemical cells. The analytical performance of microfabricated gold electrodes was characterized with the help of the IPAD method for amino acid detection under alkaline conditions required for anion-exchange separations. When used with a new optimized six-potential IPAD waveform, the electrodes functioned properly for weeks. Compared to nondisposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration, and reproducibility. Disposable electrodes make it possible to avoid polishing and reconditioning, which are required with nondisposable electrodes.     

Electrophoretic and dielectrophoretic field gradient technique for separating bioparticles

We describe a new device for separation of complex biological particles and structures exploiting many physical properties of the biolytes. The device adds a new longitudinal gradient feature to insulator dielectrophoresis, extending the technique to separation of complex mixtures in a single channel. The production of stronger local field gradients along a global gradient allows particles to enter, initially transported through the channel by electrophoresis and electroosmosis, and to be isolated according to their characteristic physical properties, including charge, polarizability, deformability, surface charge mobility, dielectric features, and local capacitance. In this work, the separation mechanism is described in terms of the relevant electromechanical principles, and proof-of-principle is demonstrated using various bacteria cells as model systems. The results demonstrate the selectivity of the technique and suggest that it may form the foundation for a versatile and useful tool for separating mixtures of complex biological particles and structures.     

The hydrodynamics of liquid-solid and gas-liquid-solid inverse fluidized beds with bioparticles

The hydrodynamic characteristics, such as minimum fluidization velocity (U_lmf for liquid-solid (LS) system and U_g,if for gas-liquid-solid (GLS) system) and bed expansion ratio (BER), of liquid-solid and gas-liquid-solid inverse fluidized beds (LSIFB and GLSIFB) with bare particles and particles with biofilm were investigated. In the LSIFB system, U_lmf and BER of the bare particles were independent of the solids loading. For bioparticles, the increase of the biofilm thickness reduced U_lmf and increased BER, suggesting that the fluidizability increases with the presence of the biofilm. In the GLSIFB system, the initial fluidization gas velocity (U_g,if) and the complete fluidization gas velocity (U_g,cf) both increased with increasing particle diameter and decreasing particle density under fixed superficial liquid velocities. Biofilm attachment led to a decrease of both U_g,if and U_g,cf, and an increase of bed expansion, again suggesting increased fluidizability with the presence of biofilm.     

Chemical sensor based on microfabricated wristwatch tuning forks

We report here a chemical sensor based on detecting the mechanical response of a thin (approximately 10-microm) polymer wire stretched across the two prongs of a wristwatch quartz tuning fork (QTF). When the fork is set to oscillate, the wire is stretched and compressed by the two prongs. The stretching/compression force changes upon adsorption of analyte molecules onto/into the polymer wire, which is detected by the QTF with pico-Newton force sensitivity. An array of such sensors with different polymer wires is used for simultaneous detection of several analytes and for improvement of pattern recognition. The low cost (approximately 10 cent) of the QTF, together with that an array of QTFs can be driven to oscillate simultaneously and their resonance frequencies detected with the same circuit, promises a high performance, low cost, and portable sensor for detecting various chemical vapors. We demonstrate here detection of parts-per-billion-level water, ethylnitrobenzene, and ethanol vapors using the QTF arrays.     

Silicon microfabricated column with microfabricated differential mobility spectrometer for GC analysis of volatile organic compounds

A 3.0-m-long, 150-microm-wide, 240-microm-deep channel etched in a 3.2-cm-square silicon chip, covered with a Pyrex wafer, and coated with a dimethyl polysiloxane stationary phase is used for the GC separation of volatile organic compounds. The column, which generates approximately 5500 theoretical plates, is temperature-programmed in a conventional convection oven. The column is connected through a heated transfer line to a microfabricated differential mobility spectrometer. The spectrometer incorporates a 63Ni source for atmospheric-pressure chemical ionization of the analytes. Nitrogen or air transport gas (flow 300 cm(3)/min) drives the analyte ions through the cell. The spectrometer operates with an asymmetric radio frequency (RF) electric field between a pair of electrodes in the detector cell. During each radio frequency cycle, the ion mobility alternates between a high-field and a low-field value (differential mobility). Ions oscillate between the electrodes, and only ions with an appropriate differential mobility reach a pair of biased collectors at the downstream end of the cell. A compensation voltage applied to one of the RF electrodes is scanned to allow ions with different differential mobilities to pass through the cell without being annihilated at the RF electrodes. A unique feature of the device is that both positive and negative ions are detected from a single experiment. The combined microfabricated column and detector is evaluated for the analysis of volatile organic compounds with a variety of functionalities.     

Manipulation of bioparticles using traveling wave dielectrophoresis: numerical approach

A typical microelectrode structure, interdigitated bar electrodes, has been developed for the selective manipulation and separation of bioparticles using traveling field dielectrophoresis effects under AC electrokinetics. This paper presents meshless numerical solutions of traveling wave dielectrophoresis for such interdigitated electrode array energized with 4-phase signal. This meshless method is based on a truncated 2D Taylor series expansion with the first three orders together with a weighted least-square approximation procedure. Dielectrophoretic forces and traveling wave forces are calculated and their applications for bioparticle manipulation and separation are discussed.     

Interpreting the charge state assignment in electrospray mass spectra of bioparticles

In electrospray ionization mass spectra of heterogeneous protein complexes and other bioparticles, accurate mass determination is often hampered by the inaccuracy in determination of the charge states for individual signals. Here, we describe an algorithm that automatically minimizes the standard deviation in a series of related ion peaks with varying numbers of charges. The algorithm assumes that the mass is invariant and allows the determination of the correct charge state in a peak series. The analysis results in a periodic pattern, which can be interpreted as a harmonic oscillator, when the minimum standard deviation of a charge state series is found. We observed that a mass resolution of much less than 1000 in the acquired mass spectra is sufficient to achieve a correct charge state assignment. Moreover, the boundaries of mixed species can be identified by examining the loss of periodicity in the pattern of the analysis. We tested our algorithm successfully on novel spectra and on spectra reported in the literature with sample masses up to several million Dalton, e.g., viral particles, polyethylene glycol polymers, and polystyrene nanoparticles.     

Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices

We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.     

Chondrocyte spheroids on microfabricated PEG hydrogel surface and their noninvasive functional monitoring

Abstract

A two-dimensional microarray of 10 000 (100 × 100) chondrocyte spheroids was constructed with a 100 μm spacing on a micropatterned gold electrode that was coated with poly(ethylene glycol) (PEG) hydrogels. The PEGylated surface as a cytophobic region was regulated by controlling the gel structure through photolithography. In this way, a PEG hydrogel was modulated enough to inhibit outgrowth of chondrocytes from a cell adhering region in the horizontal direction, which is critical for inducing formation of three-dimensional chondrocyte aggregations (spheroids) within 24 h. We further report noninvasive monitoring of the cellular functional change at the cell membrane using a chondrocyte-based field effect transistor. This measurement is based on detection of extracellular potential change induced as a result of the interaction between extracellular matrix protein secreted from spheroid and substrate at the cell membrane. The interface potential change at the cell membrane/gate interface can be monitored during the differentiation of spheroids without any labeling materials. Our measurements of the time evolution of the interface potential provide important information for understanding the uptake kinetics for cellular differentiation.     

Electric field induced anomalous relaxation in a liquid not contacting with potential electrodes

It is experimentally demonstrated that a contactless exposure of alcohols to a high-strength electric field gives rise to an optical anisotropy varying with time both during the exposure and upon switching off the electric field. The effect is related to a field-induced structurization of the liquid and the formation of quasielectret layers with anomalous relaxation properties.     

Stopped-flow enzyme assays on a chip using a microfabricated mixer

This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in < 1 s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. Beta-galactosidase (beta-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-beta-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 +/- 13 microM and 44 +/- 3 s(-1) were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 +/- 6 microM and kcat was 47 +/- 4 s(-1). Enzyme inhibition assays with phenylethyl-beta-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.     

Electrophoretic transport in surfactant nanotube networks wired on microfabricated substrates

Nanofluidic devices are rapidly emerging as tools uniquely suited to transport and interrogate single molecules. We present a simple method to rapidly obtain compact surfactant nanotube networks of controlled geometry and length. The nanotubes, 100-300 nm in diameter, are pulled from lipid vesicles using a micropipet technique, with multilamellar vesicles serving as reservoirs of surfactant material. In a second step, the nanotubes are wired around microfabricated SU-8 pillars. In contrast to unrestrained surfactant networks that minimize their surface free energy by minimizing nanotube path length, the technique presented here can produce nanotube networks of arbitrary geometries. For example, nanotubes can be mounted directly on support pillars, and long stretches of nanotubes can be arranged in zigzag patterns with turn angles of 180 degrees. The system is demonstrated to support electrophoretic transport of colloidal particles contained in the nanotubes down to the limit of single particles. We show that electrophoretic migration velocity is linearly dependent on the applied field strength and that a local narrowing of the nanotube diameter results from adhesion and bending around SU-8 pillars. The method presented here can aid in the fabrication of fully integrated and multiplexed nanofluidic devices that can operate with single molecules.     

Three-dimensional control of protein patterning in microfabricated devices

We present a new procedure that allows for the controlled patterning of functional proteins on the sidewalls of three-dimensional microfabricated chambers. We build microchambers with walls containing gold areas of a defined submicrometer size, to which proteins can be bound specifically, via thiol chemistry. We tested our system by observing the gliding of microtubules along motor-coated microchamber sidewalls and find that introducing an intermediate multilayer of proteins greatly enhances motor protein activity. This combination of spatial confinement and three-dimensional patterning of protein activity opens up new possibilities for the in vitro study of cellular processes, as well as for the development of guided transport mechanisms in nanodevices.     

Morphological and chemical characterization of microfabricated fibres for biological applications

Monodisperse fibres and particulates of different materials with controllable three-dimensional shape, size and chemical composition are of interest in research on toxic respirable fibres as well as wear debris around orthopaedic implants. We have previously demonstrated the production of well-controlled, metal and oxide microfabricated fibres having dimensions 0.1 to 10 m. While our previous results focused on how controlled fibres can be prepared by microfabrication methods, this paper evaluates property–production relationships for microfabricated fibres. Here we have briefly reviewed the production of 0.1 m×0.5 m×10 m microfabricated fibres made by electron beam lithography from evaporated titanium or silicon oxide films using a double lift-off method. We have also analysed the properties of these fibres with respect to morphology and chemical composition, and how they are affected by variations in the production process. Two different solution types have been used to place fibres into liquid suspension and to clean and sterilize them for biological testing. One method involves the use of organic solvents; the other a hydroxide solution and water. While fibre dimensions appear to be material-specific, differences can be corrected for by compensation of the size of the lithographic pattern. Similarly the crystallinity of fibres is material-specific, as is to be expected of evaporated thin films, but should be possible to modify by varying deposition parameters or heat treating, for example. Of the cleaning methods used, the one using an aqueous hydroxide solution is preferred over solvent cleaning, as it is easier to perform and appears to be more effective at removing resist from the fibre suspension.     

Optimization of dielectrophoretic DNA stretching in microfabricated devices

We have found that the surface and bulk solution properties in a microfabricated device affect the degree and probability of electrostretching of DNA molecules. Using lambda phage DNA, we found that significantly hydrophilic surfaces between the electrodes decrease the efficiency of stretching. Surfaces treated with higher silane (trimethylchlorosilane) concentrations performed better presumably due to the decreased nonspecific adsorption of DNA on these surfaces compared to their more hydrophilic counterparts. The shape and dimensions of the electrodes also affected the efficiency of stretching. Both liftoff and metal etching methods produced electrodes with random microscopic peaks along the electrode's edge and were poorly suited for stretching. Annealing the electrodes (450 degrees C for 10 min) removed most of these peaks and allowed for more controlled stretching to be obtained. We also found that thin electrodes (65 nm) gave close to a 90% success rate of DNA stretching but stretching with thick electrodes (350 nm) produced only a 20% success rate.