Marta Duch

Electrodeposited Co-Ni alloys for MEMS

The electrodeposition process has been studied and optimized in order to obtain homogeneous Co-Ni deposits on Si/SiO2/Ti/Ni substrates. The electrochemical and magnetic characterization of the deposited layers shows the features of a soft magnetic material, i.e. high saturation magnetization (1.2 T) and low coercivity, which may be varied as a function of the electrodeposition parameters. Good adherence, brilliant aspect, smooth surfaces and a constant deposition rate have been obtained in all the processed samples. The compatibility of the Co-Ni plating process with the main microelectromechanical systems (MEMS) fabrication techniques, surface and bulk, has been evaluated and these results widely demonstrate the versatility of this magnetic layer in MEMS production. Due to the high selectivity and homogeneity of the deposition process, it has been possible to pattern the deposits with a definition down to 3 μm and an aspect ratio of 4.6 has been achieved. A novel method to liberate the patterned structures by sacrificial etching of the seed layer has been developed. Uniform and homogeneous deposits of Co-Ni alloy have also been obtained in devices fabricated by bulk technologies. For these devices an anisotropic wet etching procedure in tetramethyl ammonium hydroxide (TMAH) has been optimized to release the magnetic layer. Low stressed free-standing structures have been obtained by both surface and bulk methods and are presented in this paper.

Silicon chips detect intracellular pressure changes in living cells

The ability to measure pressure changes inside different components of a living cell is important, because it offers an alternative way to study fundamental processes that involve cell deformation. Most current techniques such as pipette aspiration, optical interferometry or external pressure probes use either indirect measurement methods or approaches that can damage the cell membrane. Here we show that a silicon chip small enough to be internalized into a living cell can be used to detect pressure changes inside the cell. The chip, which consists of two membranes separated by a vacuum gap to form a Fabry-Pérot resonator, detects pressure changes that can be quantified from the intensity of the reflected light. Using this chip, we show that extracellular hydrostatic pressure is transmitted into HeLa cells and that these cells can endure hypo-osmotic stress without significantly increasing their intracellular hydrostatic pressure.

Development and Characterization of Co-Ni Alloys for Microsystems Applications

A chloride plating bath, containing boric acid and saccharin has been optimized to obtain homogeneous Co-Ni deposits over Si/SiO 2 /Ti/Ni substrate. The goal is to explore the synergy of these layers in microsystems applications. Anomalous codeposition occurs and both electrochemical and structural results indicate that obtained Co-Ni layers correspond to solid solutions of face-centered cubic (fee) structure, whose composition may be varied as a function of the electrodeposition parameters. Results of the electrochemical and magnetic characterization carried out are presented, showing the trends of a soft magnetic material, i.e. high saturation magnetization (1.2 T) and low coercivity. Smooth surfaces and a steady deposition rate were observed in the Co-Ni deposits on silicon-based unpatterned substrates. In order to check the versatility and the compatibility of this Co-Ni electrodeposition with the standard microsystems fabrication technology, two processes, surface- and bulk-technology based, have been carried out. Due to the high selectivity and homogeneity of the Co-Ni layer, it has been possible to pattern the deposits with a definition down to 10 μm, as well as to fabricate 3D structures, Methods to liberate the Co-Ni films from the silicon-based substrate have been developed; as a result, low-stressed freestanding structures are presented.

Intracellular polysilicon barcodes for cell tracking.

During the past decade, diverse types of barcode have been designed in order to track living cells in vivo or in vitro, but none of them offer the possibility to follow an individual cell up to ten or more days. Using silicon microtechnologies a barcode sufficiently small to be introduced into a cell, yet visible and readily identifiable under an optical microscope, is designed. Cultured human macrophages are able to engulf the barcodes due to their phagocytic ability and their viability is not affected. The utility of the barcodes for cell tracking is demonstrated by following individual cells for up to ten days in culture and recording their locomotion. Interestingly, silicon microtechnology allows the mass production of reproducible codes at low cost with small features (bits) in the micrometer range that are additionally biocompatible.

Intracellular Silicon Chips in Living Cells

Spanish government; Grant Number: MINAHE 2 project MEC-TEC2005-07996-CO2-01, MINAHE 3 project MEC-TEC2008-06883-CO3-01, SAF2007-66175, INTRACELL project CSIC-200550F0241 8

A novel embryo identification system by direct tagging of mouse embryos using silicon-based barcodes

BACKGROUND Measures to prevent assisted reproductive technologies (ART) mix-ups, such as labeling of all labware and double-witnessing protocols, are currently in place in fertility clinics worldwide. Technological solutions for electronic witnessing are also being developed. However, none of these solutions eliminate the risk of identification errors, because gametes and embryos must be transferred between containers several times during an ART cycle. Thus, the objective of this study was to provide a proof of concept for a direct embryo labeling system using silicon-based barcodes. METHODS Three different types of silicon-based barcodes (A, B and C) were designed and manufactured, and microinjected into the perivitelline space of mouse pronuclear embryos (one to four barcodes per embryo). Embryos were cultured in vitro until the blastocyst stage, and rates of embryo development, retention of the barcodes in the perivitelline space and embryo identification were assessed every 24 h. Release of the barcodes after embryo hatching was also determined. Finally, embryos microinjected with barcodes were frozen and thawed at the 2-cell stage to test the validity of the system after cryopreservation. RESULTS Barcodes present in the perivitelline space, independently of their type and number, did not affect embryo development rates. The majority of embryos (>90%) retained at least one of the microinjected barcodes in their perivitelline space up to the blastocyst stage. Increasing the number of barcodes per embryo resulted in a significant increase in embryo identification rates, but a significant decrease in the barcode release rates after embryo hatching. The highest rates of successful embryo identification (97%) were achieved with the microinjection of four type C barcodes, and were not affected by cryopreservation. CONCLUSIONS Our results demonstrate the feasibility of a direct embryo labeling system and constitute the starting point in the development of such systems.

Internalization and cytotoxicity analysis of silicon-based microparticles in macrophages and embryos

Microchips can be fabricated, using semiconductor technologies, at microscopic level to be introduced into living cells for monitoring of intracellular parameters at a single cell level. As a first step towards intracellular chips development, silicon and polysilicon microparticles of controlled shape and dimensions were fabricated and introduced into human macrophages and mouse embryos by phagocytosis and microinjection, respectively. Microparticles showed to be non-cytotoxic for macrophages and were found to be localized mainly inside early endosomes, in tight association with endosomal membrane, and more rarely in acidic compartments. Embryos with microinjected microparticles developed normally to the blastocyst stage, confirming the non-cytotoxic effect of the particles. In view of these results silicon and polysilicon microparticles can serve as the frame for future intracellular chips development and this technology opens the possibility of real complex devices to be used as sensors or actuators inside living cells.

Optimized immobilization of lectins using self-assembled monolayers on polysilicon encoded materials for cell tagging.

Self-assembled monolayers (SAMs) have been used for the preparation of functional microtools consisting of encoded polysilicon barcodes biofunctionalized with proteins of the lectin family. These hybrid microtools exploit the lectins ability for recognizing specific carbohydrates of the cell membrane to give an efficient system for cell tagging. This work describes how the control of the methodology for SAM formation on polysilicon surfaces followed by lectin immobilization has a crucial influence on the microtool biofunction. Several parameters (silanization time, silane molar concentration, type of solvent or deposition methodology) have been studied to establish optimal function. Furthermore, silanes incorporating different terminal groups, such as aldehyde, activated ester or epoxide groups were tested in order to analyze their chemical coupling with the biomolecules, as well as their influence on the biofunctionality of the immobilized protein. Two different lectins - wheat germ agglutinin (WGA) and phytohemagglutinin (PHA-L) - were immobilized, because they have different and specific cell recognition behaviour and exhibit different cell toxicity. In this way we can assess the effect of intrinsic bulk toxicity with that of the cell compatibility once immobilized as well as the importance of cell affinity. A variety of nanometrical techniques were used to characterize the active surfaces, and lectin immobilization was quantified using ultraviolet-visible absorption spectroscopy (UV-vis) and optical waveguide light mode spectroscopy (OWLS). Once the best protocol was found, WGA and PHA were immobilized on polysilicon coded barcodes, and these microtools showed excellent cell tagging on living mouse embryos when WGA was used.

Efficient biofunctionalization of polysilicon barcodes for adhesion to the zona pellucida of mouse embryos.

Cell tracking is an emergent area in nanobiotechnology, promising the study of individual cells or the identification of populations of cultured cells. In our approach, microtools designed for extracellular tagging are prepared, because using biofunctionalized polysilicon barcodes to tag cell membranes externally avoids the inconveniences of cell internalization. The crucial covalent biofunctionalization process determining the ultimate functionality was studied in order to find the optimum conditions to link a biomolecule to a polysilicon barcode surface using a self-assembled monolayer (SAM) as the connector. Specifically, a lectin (wheat germ agglutinin, WGA) was used because of its capacity to recognize some specific carbohydrates present on the surface of most mammalian cells. Self-assembled monolayers were prepared on polysilicon surfaces including aldehyde groups as terminal functions to study the suitability of their covalent chemical bonding to WGA. Some parameters, such as the polysilicon surface roughness or the concentration of WGA, proved to be crucial for successful biofunctionalization and bioactivity. The SAMs were characterized by contact angle measurements, time-of-flight secondary ion mass spectrometry (TOF-SIMS), laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS), and atomic force microscopy (AFM). The biofunctionalization step was also characterized by fluorescence microscopy and, in the case of barcodes, by adhesion experiments to the zona pellucida of mouse embryos. These experiments showed high barcode retention rates after 96 h of culture as well as high embryo viability to the blastocyst stage, indicating the robustness of the biofunctionalization and, therefore, the potential of these new microtools to be used for cell tagging.

Comparative performance of static-mode ferrous MEMS gradiometers fabricated by a three-step DRIE process

Two MEMS structures—a cantilever beam and a quad-beam—have been designed and fabricated through a three-step deep reactive ion etching (DRIE) process. Devices feature target patterns to align with an external optical detection system and a micromachined cavity to embed an NdFeB hard mini-magnet, thus releasing the stress of structures. Structures are intended for magnetostatic gradient measurements. Induced magnetic fields generate an attracting force on the magnet that deflects the sensor. Deflection is optically detected through nanometer-resolution confocal microscopy. The static-mode sensitivity of up to 1.86 × 10−4 T m−1 demonstrates that MEMS gradiometers are able to perform in situ gradiometry with a single sensor and miniaturized size. Suitable techniques for integrated detection are discussed.