Lars Wallman

Design and development of a silicon microfabricated flow-through dispenser for on-line picolitre sample handling

The development of a piezoactuated flow-through microdispenser is described. The dispenser can be used for on-line sampling in a continuous-liquid-flow system. A major application area is rapid sample handling in chemical microsystems, e.g. automated high-throughput analysis or screening systems. The microdispenser is constructed of two joined silicon structures forming a flow-through channel. One channel wall couples the impulse movement of a piezoceramic element into the flow channel, generating a pressure pulse. Droplets (of typically 100 picolitres) are ejected from an orifice in the opposite channel wall. The sequential improvements of the dispenser over three generations are presented. The actuation voltage could be lowered from 150 V to approximately 50 V. The final version of the dispenser had an internal volume of 2.6 microlitres. The impact on droplet stability and directivity of introducing a protruding p-n etch-stop-defined nozzle and a means of matching surface properties to the dispensed liquid for improved performance are discussed. The maximum frequency for stable droplet formation was found to be approximately 500 Hz.

Rat sciatic nerve regeneration through a micromachined silicon chip.

The capacity of regenerating nerve fibres to grow through a perforated silicon chip was tested using the silicone chamber model for nerve regeneration. The chips were fabricated as circular membranes, 4 mm in diameter, thickness 60 microns, with a perforated area, 2 mm in diameter, in the centre. Three types of chips were fabricated utilizing anisotropic etching. The chips were glued with silicone adhesive between two halves of silicone rubber tubing (total length 8 mm, inner diameter 1.8 mm, outer diameter 3.0 mm) which was used to bridge a 4 mm gap between the proximal and distal nerve stumps of a transected rat sciatic nerve. The capacity of regenerating nerve fibres to grow through the holes of the chip was analysed by light and scanning electron microscopy after 4 or 16 weeks of regeneration. Furthermore, the muscle contractility force of the gastrocnemius muscle was measured after 16 weeks of regeneration and compared as a percentage of the contralateral uninjured side. Nerves generated through chips with hole diameters of 10 or 50 microns were morphological and functional failures. The nerve structures distal to chips with hole diameters of 100 microns contained many myelinated nerve fibres in a minifascicular pattern after both 4 and 16 weeks of regeneration. The muscle contractility force was 56% of that of contralateral control muscles.

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) there is often a need to purify and concentrate the sample solution, especially after in‐gel digestion of proteins separated by two‐dimensional gel electrophoresis (2‐DE). A silicon microextraction chip (SMEC) for sample clean‐up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed‐phase chromatography media (POROS™ R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI‐TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 M urea in 0.1 M phosphate‐buffered saline with MALDI‐TOF MS. It is demonstrated that the microchip sample clean‐up and enrichment of peptides can facilitate identification of proteins from 2‐DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.

The geometric design of micromachined silicon sieve electrodes influences functional nerve regeneration

A neural interface could be used to control a limb prosthesis. Such an interface can be created by facilitating axonal regeneration through a sieve electrode and then register nerve signals intended to control the prosthesis. A key question is how to design the electrodes to ensure the best possible regeneration. Our previous studies have indicated that regeneration can be achieved using electrodes with square-shaped, 100 x 100 microm, via holes (holes that axons will regenerate through). Other reports have indicated a suitable range of these holes between 40 and 65 microm. In the present study we used silicon sieve electrodes with via holes of either 30 or 90 microm. The transparency, i.e. the percentage of the total via hole area, of these electrodes was either 20 or 30%. The electrodes were inserted into a silicone chamber which was used to bridge a gap in a rat sciatic nerve. After 12 weeks of nerve regeneration electrodes with a hole size of 30 microm and a 30% transparency had the most favourable result as judged by the regained gastrocnemius muscle force and the formation of reactive tissue inside the chamber. The sieve electrode transparency is crucial for ensuring regeneration.

Nanowire-Based Electrode for Acute In Vivo Neural Recordings in the Brain

We present an electrode, based on structurally controlled nanowires, as a first step towards developing a useful nanostructured device for neurophysiological measurements in vivo. The sensing part of the electrode is made of a metal film deposited on top of an array of epitaxially grown gallium phosphide nanowires. We achieved the first functional testing of the nanowire-based electrode by performing acute in vivo recordings in the rat cerebral cortex and withstanding multiple brain implantations. Due to the controllable geometry of the nanowires, this type of electrode can be used as a model system for further analysis of the functional properties of nanostructured neuronal interfaces in vivo.

Sample enrichment in a single levitated droplet for capillary electrophoresis

This paper describes sample enrichment in a single levitated droplet for capillary electrophoresis (CE) analysis. The droplet was trapped in an acoustical field. The minute sample volumes needed for the enrichment procedure were precisely handled using a piezoelectric flow-through liquid microdispenser. Droplets with a volume of 65 pl were ejected from the device at a repetition rate ranging from one single droplet up to several hundreds per second. By counting the number of droplets ejected and accumulated in the levitated drop the sample volume was controlled. Through solvent evaporation the analytes were enriched in the diminishing droplet. The droplet was then injected into a CE capillary and the analytes, dansyl-Gly and dansyl-Val dissolved in ethanol, were separated in a 100 mM borate buffer (pH 9.0) utilising UV-absorption detection at 200 nm near the capillary outlet. Enrichment of 36000 sample droplets (2.3 microl) through solvent evaporation in the levitated drop resulted in a concentration limit of detection (CLOD) of 15 nM for the dansylated amino acids as compared to a CLOD of 2.5 microM which was achieved using standard hydrodynamic injection without preconcentration.

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation.

A recently introduced silicon microextraction chip (SMEC), used for on‐line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid‐SMEC design improves the operating characteristics for solid‐phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics‐module FLOTRAN in the ANSYS® software package. The modeling and analysis of the previously reported weir‐SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid‐SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix‐assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir‐SMEC and the new grid‐SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

Tissue reactions evoked by porous and plane surfaces made out of silicon and titanium

Square-shaped silicon or titanium implants with plane or porous surfaces surrounded by a rim of silicone were implanted in the rat abdominal wall for evaluation of the tissue response after one, six, or 12 weeks. Cell damage was identified as increased membrane permeability using fluorescence microscopy by injection of propidium iodide prior to the killing of the rats. Capsule thickness and immunohistochemical quantification of macrophages were used as a further measure of the foreign-body reaction. There were no significant differences in capsular cell densities for macrophages, total cells (macrophages, fibroblasts, and other cells), or necrotic cells at the different time points for the four surfaces studied. However, significant differences in the kinetics of the response were found between plane surfaces compared with porous ones. Both types of plane surfaces developed a significant increase in capsule thickness over time in contrast to the porous implants. Porous silicon displayed a significant decrease in total cells in the reactive capsule over time. Furthermore, porous silicon and titanium surfaces displayed a significant decrease in total cell numbers at the implant interface between six and 12 weeks. The present study demonstrated that implanted silicon elicited soft-tissue reactions comparable to that of titanium.

Perforated silicon nerve chips with doped registration electrodes: in vitro performance and in vivo operation

An in vitro model was developed for the study of signal transduction between a Cu-wire, mimicking a neural signal source, and recording electrodes on perforated silicon chips. Phosphorous doped electrodes were used to achieve an all-silicon device. The model was used to study signal amplitude as a function of the spatial position, and distance to the signal source. Recordings of the signal crosstalk to neighboring electrodes on the chips were made. It was found that the amplitude decreased by a factor of two at a distance of 50 /spl mu/m between the electrode surface and the signal source. The chip electrode signal crosstalk was found to be 6 dB using an external reference electrode. Improvements were accomplished with an on chip reference electrode giving a crosstalk suppression of 20 dB. Impedance analysis showed that doped silicon electrodes displayed similar characteristics as Cu-electrodes at frequencies above 3 kHz. Sieve electrodes were implanted in the rat sciatic nerve and following a 10-week nerve regeneration period the dorsal and ventral (L5) roots in the spinal cord were stimulated. Compound action potentials were recorded via the chip. Stimulating the regenerated sciatic nerve via the sieve electrode also induced lower leg muscle contraction activity.

The Influence of Porous Silicon on Axonal Outgrowth in Vitro

Axonal outgrowth on smooth and porous silicon surfaces was studied in organ culture. The pore size of the silicon substrata varied between 100 and 1500 nm. We found that axons preferred to grow and elongate on porous silicon surfaces only when pores of (150-500 nm) are available.