Carbon nanotube-based separation columns for microchip electrochromatography.

Fabrication of the stationary phase for microchip chromatography is most often done by packing of the individual separation channel after fabrication of the microfluidic chip, which is a very time-consuming and costly process (Kutter. J Chromatogr A 1221:72-82, 2012). Here, we describe in detail the fabrication and operation protocols for devices with microfabricated carbon nanotube stationary phases for reverse-phase chromatography. In this protocol, the lithographically defined stationary phase is fabricated in the channel before bonding of a lid, thereby circumventing the difficult packaging procedures used in more conventional protocols.

Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip.

Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low applied voltages. This will allow small biomolecules with diameters less than 30 A to rapidly diffuse from the permeabilized cells to the surrounding solution. By controlling the applied voltage, partial and transient to complete cell opening can be obtained. By using DC voltages below 0.5 V, cell lysis can be avoided and the transiently formed pores can be closed again and the cells survive. This method has been used to extract RNA and GFP molecules under conditions of electropermeability. Plasmid DNA could be recovered when the applied voltage was increased to 2 V, thus causing complete cell lysis.

Validation of a fully autonomous phosphate analyser based on a microfluidic lab-on-a-chip.

This work describes the design of a phosphate analyser that utilises a microfluidic lab-on-a-chip. The analyser contains all the required chemical storage, pumping and electronic components to carry out a complete phosphate assay. The system is self-calibrating and self-cleaning, thus capable of long-term operation. This was proven by a bench top calibration of the analyser using standard solutions and also by comparing the analyser's performance to a commercially available phosphate monitor installed at a waste water treatment plant. The output of the microfluidic lab-on-a-chip analyser was shown to have sensitivity and linear range equivalent to the commercially available monitor and also the ability to operate over an extended period of time.

Carbon nanotubes integrated in electrically insulated channels for lab-on-a-chip applications.

A fabrication process for monolithic integration of vertically aligned carbon nanotubes in electrically insulated microfluidic channels is presented. A 150 nm thick amorphous silicon layer could be used both for anodic bonding of a glass lid to hermetically seal the microfluidic glass channels and for de-charging of the wafer during plasma enhanced chemical vapor deposition of the carbon nanotubes. The possibility of operating the device with electroosmotic flow was shown by performing standard electrophoretic separations of 50 microM fluorescein and 50 microM 5-carboxyfluorescein in a 25 mm long column containing vertical aligned carbon nanotubes. This is the first demonstration of electroosmotic pumping and electrokinetic separations in microfluidic channels with a monolithically integrated carbon nanotube forest.

Photonic crystal resonator integrated in a microfluidic system.

We report on a novel optofluidic system consisting of a silica-based 1D photonic crystal, integrated planar waveguides, and electrically insulated fluidic channels. An array of pillars in a microfluidic channel designed for electrochromatography is used as a resonator for on-column label-free refractive index detection. The resonator was fabricated in a silicon oxynitride platform, to support electro-osmotic flow, and operated at lambda=1.55 microm. Different aqueous solutions of ethanol with refractive indices ranging from n=1.3330 to 1.3616 were pumped into the column/resonator, and the transmission spectra were recorded. Linear shifts of the resonant wavelengths yielded a maximum sensitivity of Deltalambda/Deltan=480 nm/RIU (refractive index unit), and a minimum difference of Deltan=0.007 RIU was measured.

Acoustic resonances in straight micro channels: beyond the 1D-approximation.

Acoustic actuation can be used to perform several tasks in microfluidic systems. In this paper, we investigate an acoustic separator through micro-PIV analysis in stop-flow mode and numerical simulations, and a good agreement between the two is found. Moreover, we demonstrate that it is not sufficient only to characterize devices in flow-through mode, since in these systems much different resonant patterns can result in similarly looking band formations. Furthermore, we conclude that extended 1D approximations of the acoustic radiation force are inadvisable, and instead, a 2D model is preferred. The results presented here provide valuable insight into the nature and functionality of acoustic microdevices, and should be useful in the interpretation and understanding of the same.

Acoustic resonances in microfluidic chips: full-image micro-PIV experiments and numerical simulations.

We show that full-image micro-PIV analysis in combination with images of transient particle motion is a powerful tool for experimental studies of acoustic radiation forces and acoustic streaming in microfluidic chambers under piezo-actuation in the MHz range. The measured steady-state motion of both large 5 microm and small 1 microm particles can be understood in terms of the acoustic eigenmodes or standing ultra-sound waves in the given experimental microsystems. This interpretation is supported by numerical solutions of the corresponding acoustic wave equation.

Electrophoretic partitioning of proteins in two-phase microflows.

This work reports on protein transport phenomena discovered in partitioning experiments with a novel setup for continuous-flow two-phase electrophoresis consisting of a microchannel in which a phase boundary is formed in flow direction. Proteins can be partitioned exploiting their affinity to different aqueous phases in two-phase systems. This separation process may be enhanced or extended by applying an electric field perpendicular to the phase boundary. In this context, microsystems offer new possibilities, as interfacial forces usually dominate over volume forces, thus allowing a superior control of the formation and arrangement of liquid/liquid phase boundaries. The two immiscible phases which are injected separately into the microchannel are taken from a polyethylene glycol (PEG)-dextran system. The side walls of the channel are partially made of gel material which serves as an ion conductor and decouples the channel from the electrodes, thus preventing bubble generation inside the separation channel. The experiments show that the electrophoretic transport of proteins between the laminated liquid phases is characterized by a strong asymmetry. When bovine serum albumin (BSA) is introduced into the PEG-rich phase, it can easily be transferred into the dextran-rich phase via an applied electric field of low strength or just by diffusion. In the reverse case, up to a certain field strength the transfer to the opposing phase is strongly inhibited. Only if the field strength is further increased will the BSA molecules leave the dextran-rich phase almost completely.

Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces.

There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more conventional methods to determine biocompatibility such as cellular growth rate, morphology and the hydrophobicity of the surfaces. HeLa cells grown on polymethylmethacrylate (PMMA) or a SU-8 surface treated with HNO3-ceric ammonium nitrate (HNO3-CAN) and ethanolamine showed no differences in growth rate, morphology or gene expression profiles as compared to HeLa cells grown in cell culture flasks. Cells grown on SU-8 treated with only HNO3-CAN showed almost the same growth rate (36 +/- 1 h) and similar morphology as cells grown in cell culture flasks (32 +/- 1 h), indicating good biocompatibility. However, more than 200 genes showed different expression levels in cells grown on SU-8 treated with HNO3-CAN compared to cells grown in cell culture flasks. This shows that gene expression profiling is a simple and precise method for determining differences in cells grown on different surfaces that are otherwise difficult to find using conventional methods. It is particularly noteworthy that no correlation was found between surface hydrophobicity and biocompatibility.

Lab-on-a-chip with integrated optical transducers.

Taking the next step from individual functional components to higher integrated devices, we present a feasibility study of a lab-on-a-chip system with five different components monolithically integrated on one substrate. These five components represent three main domains of microchip technology: optics, fluidics and electronics. In particular, this device includes an on-chip optically pumped liquid dye laser, waveguides and fluidic channels with passive diffusive mixers, all defined in one layer of SU-8 polymer, as well as embedded photodiodes in the silicon substrate. The dye laser emits light at 576 nm, which is directly coupled into five waveguides that bring the light to five different locations along a fluidic channel for absorbance measurements. The transmitted portion of the light is collected at the other side of this cuvette, again by waveguides, and finally detected by the photodiodes. Electrical read-out is accomplished by integrated metal connectors. To our knowledge, this is the first time that integration of all these components has been demonstrated.

Measurements of scattered light on a microchip flow cytometer with integrated polymer based optical elements.

Flow cytometry is widely used for analyzing microparticles, such as cells and bacteria. In this paper, we report an innovative microsystem, in which several different optical elements (waveguides, lens and fiber-to-waveguide couplers) are integrated with microfluidic channels to form a complete microchip flow cytometer. All the optical elements, the microfluidic system, and the fiber-to-waveguide couplers were defined in one layer of polymer (SU-8, negative photoresist) by standard photolithography. With only a single mask procedure required, all the fabrication and packaging processes can be finished in one day. Polystyrene beads were measured in the microchip flow cytometer, and three signals (forward scattering, large angle scattering and extinction) were measured simultaneously for each bead. To our knowledge this is the first time forward scattered light and incident light extinction were measured in a microsystem using integrated optics. The microsystem can be applied for analyzing different kinds of particles and cells, and can easily be integrated with other microfluidic components.

Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter.

The integration of complete analyses systems "on chip" is one of the great potentials of microfabricated devices. In this study we present a new pressure-driven microfabricated fluorescent-activated cell sorter chip with advanced functional integration. Using this sorter, fluorescent latex beads are sorted from chicken red blood cells, achieving substantial enrichments at a sample throughput of 12000 cells s(-1). As a part of the sorter chip, we have developed a monolithically integrated single step coaxial flow compound for hydrodynamic focusing of samples in flow cytometry and cell sorting. The structure is simple, and can easily be microfabricated and integrated with other microfluidic components. We have designed an integrated chamber on the chip for holding and culturing of the sorted cells. By integrating this chamber, the risk of losing cells during cell handling processes is eliminated. Furthermore, we have also developed integrated optics for cell detection. Our new design contributes to the ongoing efforts for building a fully integrated micro cell sorting and analysing system.

Monolithic integration of microfluidic channels and optical waveguides in silica on silicon.

Sealing of the flow channel is an important aspect during integration of microfluidic channels and optical waveguides. The uneven topography of many waveguide-fabrication techniques will lead to leakage of the fluid channels. Planarization methods such as chemical mechanical polishing or the etch-back technique are possible, but troublesome. We present a simple but efficient alternative: By means of changing the waveguide layout, bonding pads are formed along the microfluidic channels. With the same height as the waveguide, they effectively prevent leakage and hermetically seal the channels during bonding. Negligible influence on light propagation is found when 10-mum-wide bonding pads are used. Fabricated microsystems with application in absorbance measurements and flow cytometry are presented.

Ultraviolet transparent silicon oxynitride waveguides for biochemical microsystems.

The UV wavelength region is of great interest in absorption spectroscopy, which is employed for chemical analysis, since many organic compounds absorb in only this region. Germanium-doped silica, which is often preferred as the waveguide core material in optical devices for telecommunication, cannot accommodate guidance below 400 nm, owing to the presence of UV-absorbing centers. We show that silicon oxynitride (SiO(x) N(y)) waveguides exhibit very good UV performance. The propagation loss for 24-microm -wide SiO(x)N (y) waveguides was found to be ~1.0dB/cm in the wavelength range 220-550 nm. The applicability of these waveguides was demonstrated in a biochemical microsystem consisting of multimode buried-channel SiO(x)N (y) waveguides that were monolithically integrated with microfluidic channels. Absorption measurements of a beta -blocking agent, propranolol, at 212-215 nm were performed. The detection limit was reached at a concentration of 13microM , with an optical path length of 500microm (signal/noise ratio, 2).

Solvent-programmed microchip open-channel electrochromatography.

Open-channel electrochromatography in combination with solvent programming is demonstrated using a microchip device. Channel walls were coated with octadecylsilanes at ambient temperatures, yielding stationary phases for chromatographic separations of neutral dyes. The electroosmotic flow after coating was sufficient to ensure transport of all species and on-chip mixing of isocratic and gradient elution conditions with acetonitrile-buffer mixtures. Chips having different channel depths between 10.2 and 2.9 µm were evaluated for performance, and van Deemter plots were established. Channel depths of about 5 µm were found to be a good compromise between efficiency and ease of operation. Isocratic and gradient elution conditions were easily established and manipulated by computer-controlled application of voltages to the terminals of the microchip. Linear gradients with different slopes, start times, duration times, and start percentages of organic modifier proved to be powerful tools to tune selectivity and analysis time for the separation of a test mixture. Even very steep gradients still produced excellent efficiencies. Together with fast reconditioning times, complete runs could be finished in under 60 s.