Ion-Sensitive Field-Effect Transistor for Biological Sensing

In recent years there has been great progress in applying FET-type biosensors for highly sensitive biological detection. Among them, the ISFET (ion-sensitive field-effect transistor) is one of the most intriguing approaches in electrical biosensing technology. Here, we review some of the main advances in this field over the past few years, explore its application prospects, and discuss the main issues, approaches, and challenges, with the aim of stimulating a broader interest in developing ISFET-based biosensors and extending their applications for reliable and sensitive analysis of various biomolecules such as DNA, proteins, enzymes, and cells

Bioelectronic Nanosensor Devices for Environmental and Biomedical Analysis

A new type of Bioelectronic Nanosensor Device with potential applications in medicine,biotechnology and environmental analysis was designed. The nanosensor is based on RISFET (Regional Ion Sensitive Field Effect Transistor) technology. The design of the nanosensor involves use of a set of nano-sized electrodes on the surface of a silicon chip, giving the chip the ability to sense extremely low concentrations of specific analytes by means of an amplifying process. Analyte ions (or enzymatically generated product ions of these) are focused by a charged field to become concentrated in a narrow region – a conducting channel – located between two sensing electrodes. The focusing process leads to significant changes in conductivity, increasing the level of the current between the sensing electrodes. The current, registered by a pico-ammeter produced in-house, is a measure of analyte concentration. To achieve specificity, the RISFET nanosensor is provided with immobilized enzymes in the form of minicolumns within a flow system. Studies were conducted exploring possibilities of simplifying the system, improving its effectiveness and making it more compact. It appears that the enzymes should best be moved to an area of the chip in the vicinity of the sensing electrodes, and that branched nanowire structures (nanotrees) be placed on the chip-surface area located between the sensing electrodes to serve as carriers of the enzymes. The nanotrees would ensure an adequate load of enzymes without either the focusing of the ions or the sensing ability being disturbed. Features of this sort are seen as being particularly important for future high-density RISFET nanosensor arrays. The basic properties of the sensor were investigated using such analytes as glucose, gluconolactone, acetylcholine and carbofuran. Specificity was found to be achieved when the enzymes glucose oxidase and acetylcholine esterase were employed. The inhibition of acetylcholine esterase by carbofuran was detectable down to about 20 ng/L. An automatic online biosensor unit for the neurotoxic organocarbamate carbofuran was constructed and was found to work satisfactorily. A number of chip configurations, involving use of different silicon technologies and different electrode arrangements, were designed and characterized. In addition, a study of a Quartz Crystal Microbalance (QCM) biometric sensor was carried out; the sensor surface was functionalized by use of molecularly imprinted nanoparticles specific for (R)- or (S)-propranolol. Frequent use was made in the work as a whole of Atomic Force Microscopy (AFM), Scanning Kelvin Probe Microscopy (SKM) and Scanning Electron Microscopy (SEM). The usefulness of multifunctional nanobiosensor array systems for the surveillance of liquids regarding the presence of many different toxic and biomedically relevant analytes, and in medical, environmental and biotechnological analyses generally is discussed

Development of a tRNA-Synthetase Microarray for Protein Analysis

Proteins are composed of 20 different amino acids. In the translation process, each of these 20 amino acids is specifically recognized by their cognate aminoacyl-tRNA synthetase. The fidelity of this recognition system is essential if translation is to function properly. The development of an in vitro system based on this recognition scheme would make a powerful analytical tool with which to analyse translation, as well as providing an additional biomimetic scheme for protein analysis. Aminoacyl-tRNA synthetases microarrays could be applied to protein fingerprinting and sequence analysis. The fabrication of aminoacyl-tRNA synthetase arrays requires the use of advanced protein arraying technology that has only recently become available. In order to demonstrate the feasibility of this scheme, glutamyl-tRNA synthetase (GluRS) was immobilized on the streptavidinbased XNA on GoldTM biochip platform. The streptavidin layer provides a simple, efficient immobilization scheme that reduces nonspecific binding and improves the biocompatibility of the surface. Here, we demonstrate that biotinylated GluRS can be successfully immobilized on XNA on GoldTM. The immobilization efficiency was determined by double labelling GluRS with biotin and the fluorescent label Cy5. The CCD fluorescent microscopy images revealed that the GluRS was efficiently immobilized and evenly distributed over the surface. Control experiments indicate a very low degree of nonspecific binding which is essential if detection of these multicomponent, low-affinity interactions is to be realized. Furthermore, we show that immobilization does not significantly reduce the function of the enzyme. In addition to the specific aims of this study, this technology would provide valuable insights into the biomechanics of translation as well as being a tool for studying tRNA modifications and subclasses. Moreover, the implications for developing coupled transcription and translation systems should not be overlooked. Protein analysis schemes based on this approach would provide an urgently needed compliment to traditional methods. Finally, these arrays might also be useful tools in our efforts to understand the regulatory functions that small RNAs, i.e., iRNA, have been shown to play

The region ion sensitive field effect transistor, a novel bioelectronic nanosensor

A novel type of bioelectronic region ion sensitive field effect transistor (RISFET) nanosensor was constructed and demonstrated on two different sensor chips that could measure glucose with good linearity in the range of 0-0.6 mM and 0-0.3 mM with a limit of detection of 0.1 and 0.04 mM, respectively. The sensor is based on the principle of focusing charged reaction products with an electrical field in a region between the sensing electrodes. For glucose measurements, negatively charged gluconate ions were gathered between the sensing electrodes. The signal current response was measured using a low-noise pico ammeter (pA). Two different sizes of the RISFET sensor chips were constructed using conventional electron beam lithography. The measurements are done in partial volumes mainly restricted by the working distance between the sensing electrodes (790 and 2500 nm, respectively) and the influence of electrical fields that are concentrating the ions. The sensitivity was 28 pA/mM (2500 nm) and 830 pA/mM (790 nm), respectively. That is an increase in field strength by five times between the sensing electrodes increased the sensitivity by 30 times. The volumes expressed in this way are in low or sub femtoliter range. Preliminary studies revealed that with suitable modification and control of parameters such as the electric control signals and the chip electrode dimensions this sensor could also be used as a nanobiosensor by applying single enzyme molecule trapping. Hypotheses are given for impedance factors of the RISFET conducting channel. (c) 2007 Elsevier B.V. All rights reserved

Characterization of QCM sensor surfaces coated with molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolot in aqueous buffer

Signal frequency studies of an environmental application of a 65 nm region ion sensitive field effect transistor sensor

A rapid and sensitive novel type of bioelectronic Region Ion Sensitive Field Effect Transistor (RISFET) nanosensor was constructed on a chip with a 65 nm sensing electrode gap. The RISFET nanosensor was demonstrated for the environmental pesticide analysis of neurotoxic organocarbamate/carbofuran. The linear range for carbofuran analysis is ac signal frequency dependent, studied in the range (500 down-0.5 Hz, 50 mV(peak-peak) ac) and a bias voltage applied between the bottom capacitor plate and the electrodes. The signal current response is measured using a low-noise pico ammeter. The inhibition of acetylcholinesterase (AChE) by carbofuran was detectable in a logarithmic linear range (0.1-100nM) at 1.08 Hz, with a lower limit of detection of inhibition 0.1 nM with 10 min incubation time. The sensor is based on the principle of focusing charged reaction products with an electrical field in a region between the sensing electrodes. The current measurement by the sensor electrodes is correlated to the composition of the sample. The carbofuran detection is based on the ability to inhibit the enzyme AChE. The RISFET sensor chip is fabricated using conventional electron beam lithography. The encompassed sensor volume in the "nanocell" is in the attoliter range. (c) 2007 Elsevier B.V. All rights reserved

Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN(x)-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area and thereby higher enzyme binding capacity. A theoretical calculation is included to show how the enzyme kinetics and hence the sensitivity can be influenced and increased by the control of electrical fields in relation to the active sites of enzymes in an electronic biosensor. The possible effects of electrical fields employed in the RISFET on the function of acetylcholine esterase is investigated using quantum chemical methods, which show that the small electric field strengths used are unlikely to affect enzyme kinetics. Acetylcholine esterase activity is determined using choline oxidase and peroxidase by measuring the amount of choline formed using the chemiluminescent luminol reaction