Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring.

The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data.

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.

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.

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.

Monolithic integration of optical waveguides for absorbance detection in microfabricated electrophoresis devices.

The fabrication and performance of an electrophoretic separation chip with integrated optical waveguides for absorption detection is presented. The device was fabricated on a silicon substrate by standard microfabrication techniques with the use of two photolithographic mask steps. The waveguides on the device were connected to optical fibers, which enabled alignment free operation due to the absence of free-space optics. A 750 microm long U-shaped detection cell was used to facilitate longitudinal absorption detection. To minimize geometrically induced band broadening at the turn in the U-cell, tapering of the separation channel from a width of 120 down to 30 microm was employed. Electrical insulation was achieved by a 13 microm thermally grown silicon dioxide between the silicon substrate and the channels. The breakdown voltage during operation of the chip was measured to 10.6 kV. A separation of 3.2 microM rhodamine 110, 8 microM 2,7-dichlorofluorescein, 10 microM fluorescein and 18 microM 5-carboxyfluorescein was demonstrated on the device using the detection cell for absorption measurements at 488 nm.

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).