Massimo Grattarola

Micromechanical cantilever-based biosensors

Abstract The merging of silicon microfabrication techniques with surface functionalization biochemistry offers new exciting opportunities in developing microscopic biomedical analysis devices with unique characteristics. Micro-mechanical transducers for chemical and biosensing applications represent one possibility. Microcantilevers can transduce a chemical signal into a mechanical motion with high sensitivity. In this review we summarize how cantilever-based sensors can be operated, and their working principle is presented in few selected biosensing experiments which have been performed recently in our groups in the study of biotin–streptavidin and antigen–antibody interactions, and specific surface charge development of organic molecules. We also discuss the advantages of this novel technique as well as its potentials.

Development of ISFET array-based microsystems for bioelectrochemical measurements of cell populations

Monitoring the bioelectrochemical activity of living cells with sensor array-based microsystems represents an emerging technique in a large area of biomedical applications, ranging from basic research to various fields of pharmacological analyses. The main appeal is the ability of these miniaturised microsystems to perform, in real time, non-invasive in-vitro investigations of the physiological state of a cell population. In this paper, we present two different microsystems designed for multisite monitoring of the physiological state of a cell population. The first microsystem, intended for cellular metabolism monitoring, consists of an array of 12 spatially distributed ISFETs to detect small pH variations induced by the cell population. The second microsystem consists of an array of 40 ISFETs and 20 gold microelectrodes and it has been designed to monitor the electrical activity of neurons. This is achieved by direct coupling of the neuronal culture with the ISFET sensitive layer and by utilising gold microelectrodes for neuronal electrical stimulation.

Modeling H/sup +/-sensitive FETs with SPICE

A generalized physical model including two kinds of binding sites is presented on H/sup +/-sensitive ISFET devices. The model results in a set of equations which is introduced into a modified version of the electronic circuit simulation program SPICE. In this way, the effects induced on the device performances by varying several physico-chemical parameters are analyzed. The slope of V/sub out/ versus pH curves is predicted for SiO/sub 2/-, Al/sub 2/O/sub 3/-, and Si/sub 3/N/sub 4/-gate ISFETs. The model is then used to predict the behavior of a hypothetical, partially pH-insensitive (REFET) structure. Finally, the model is utilized to fit the slow response of the Al/sub 2/O/sub 3/-gate ISFET to a pH stop. >

Modeling the neuron-microtransducer junction: from extracellular to patch recording

A detailed characterization of the neuron-to-microtransducer junction, based on the equivalent electric-circuit approach, is provided. The recording of action potentials is then simulated with the general-purpose network-analysis program SPICE. Both noble-metal microelectrodes and insulated-gate FETs are considered. The responses of such devices are characterized as functions of several parameters, e.g. sealing impedance, density of ionic currents in the cell membrane, and spatial discontinuities of the adhesion process. It is shown that the various signal shapes reported in the literature can be reproduced and interpreted in terms of time derivatives of the action potential. In this way, the shape of any experimental signal can be interpreted on the basis of a specific sealing condition. Possible future improvements in microtransducer design, based on the proposed approach, are also suggested.<<ETX>>

Micromechanics senses biomolecules

Abstract How can microelectromechanical systems (MEMS) experts support molecular biologists in studying DNA hybridization? Cantilever-based devices are an example of how a ‘simple’ sensor can be tailored by microfabrication techniques and used to achieve an unprecedented performance. We review fascinating experiments, which use different mechanical transduction principles for detecting and analyzing small quantities of materials. The principles of these experiments allow biologists to study surface biochemistry on a nano-scale and offer new, exciting opportunities in developing microscopic biomedical analysis systems with unique characteristics. Cantilever sensors rely on relatively well known and simple transduction principles, and have attracted the interest of many researchers. This is, at least in part, because of the merging of silicon microfabrication techniques and surface functionalization biochemistry, together with the development of multi-cantilever sensing methods offering new opportunities in physical and (bio)chemical sensing.

Changes in surface stress at the liquid/solid interface measured with a microcantilever

Abstract The bending of microfabricated silicon nitride cantilevers was used to determine surface stress changes at solid–liquid interfaces. The radius of curvature of the bent cantilever is directly proportional to changes in the differential surface stress between its opposite sides. To demonstrate the possibilities and limitations of the technique, cantilevers coated on both sides with gold and densely packed monolayers of different thiols were put in a constant flow of aqueous electrolyte solution and the deflection was measured using a optical lever technique. Changes in the surface stress for the different thiol monolayers due to specific proton adsorption are presented. Possible applications and improvements of this technique are discussed.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Interfacing cultured neurons to planar substrate microelectrodes: characterization of the neuron-to-microelectrode junction☆

Abstract This paper focuses on the characterization of the bioelectrochemical interface that develops whenever neurons are cultured directly on top of planar arrays of noble metal microelectrodes. The characterization is based on the equivalent circuit analysis of a neuron membrane coupled to a metallic planar microelectrode. The membrane is described according to the Hodgkin-Huxley model, using a set of equations specialized for the description of action potentials generated by chick embryo dorsal root ganglia (DRG) neurons. An ad-hoc modified version of the circuit analysis program spice allows one to simulate the signal corresponding to an action potential as it should result from the microelectrode transduction. By varying the membrane-to-electrode coupling and other biophysical parameters, simulation signals of different durations, intensities and shapes are generated. The appropriateness of the model is verified by adapting simulation signals to experimental ones, obtained by preliminary experiments with DRG neurons cultured on a microelectrode array. The potentialities and limitations of the equivalent circuit approach as a tool for the characterization of a long-term (i.e. days) coupling of neurons to planar microelectrodes are discussed.

Mechanical and morphological properties of living 3T6 cells probed via scanning force microscopy

Scanning Force Microscopy (SFM) is utilized to study living confluent 3T6 cells. Images based on mechanical contrast are obtained and related morphological details, mostly regarding the cell cytoskeleton, are analyzed. Moreover, numerical estimates of the local mechanical properties of the living cells are given, by extensive use of the “force‐vs.‐distance” operation mode. On the basis of the results obtained, the potentialities of SFM as an optimal new technique available for probing the cell cytoskeleton of unstained living cells, and assessing related models, are shortly discussed. Microsc. Res. Tech. 36:165–171, 1997.

Comparison between a LAPS and an FET-based sensor for cell-metabolism detection

Several silicon-based biosensors have been developed for various applications, such as enzymatic and immunological activity determination and cell-metabolism detection. This work describes an electrochemical characterization of ion-sensitive field-effect transistor (ISFET) and light-addressable potentiometric sensor (LAPS) transducers and a test of such devices as detectors of 3T6 (Swiss albino mouse embryo, fibroblasts) metabolism. In particular, our investigation is devoted to some fundamental parameters of these transducers (e.g., pH sensitivity, drift, temperature dependence of the output signal, speed of response) for the purpose of comparing their performance related to cell-metabolism evaluation. The final goal is therefore to analyze the capabilities of these silicon-based transducers for use in a biosensing system for cell-metabolism detection.