Detection of DNA and proteins using amorphous silicon ion-sensitive thin-film field effect transistors

Amorphous silicon-based ion-sensitive field-effect transistors (a-Si:H ISFETs) are used for the label-free detection of biological molecules. The covalent immobilization of DNA, followed by DNA hybridization, and of the surface adsorption of oligonucleotides and proteins were detected electronically by the a-Si:H ISFET. The ISFET measurements are performed with an external Ag/AgCl microreference electrode immersed in 100mM phosphate buffer electrolyte with pH 7.0. Threshold voltage shifts in the transfer curve of the ISFETs are observed resulting from successive steps of surface chemical functionalization, covalent DNA attachment to the functionalized surface, surface blocking, and hybridization with a complementary target. The surface sensitivity achieved for DNA oligonucleotides is of the order of 1pmol/cm(2). Point-of-zero charge estimations were made for the functionalized surfaces and for the device surface after DNA immobilization and hybridization. The results show a correlation between the changes in the point-of-zero charge and the shift observed in the threshold voltage of the devices. Electronic detection of adsorbed proteins and DNA is also achieved by monitoring the shifts of the threshold voltage of the ISFETs, with a sensitivity of approximately 50nM.     

Modification of ISFETs with a monolayer of latex beads for specific detection of proteins

The so-called ion-step method is a novel potentiometric approach that can detect protein adsorbed onto the gate area of modified ion-sensitive field-effect transistors (ISFETs). In this report, a generic technology is described for immobilization of peptides and proteins to the ISFET gate in order to confer specific binding properties to the ISFET. For this, the surface of the ISFET was covered with a monolayer of Amino beads (diameter, 0.9 microm) followed by immobilization of protein ligands onto these beads. Amino beads are latex spheres that contain primary amino groups at the outer surface. Preactivation of the latex-bound amino groups with glutaraldehyde, and consecutive incubation with polylysine resulted in covalent immobilization of this polyamine, as revealed by ion stepping measurements. For ImmunoFET applications, human serum albumin (HSA) was immobilized onto the Amino bead-covered ISFETs, by passive adsorption but also by covalent coupling. Resulting devices were used for qualitative detection of alpha-HSA antibodies by means of the ion step method. The binding of antibody was very specific and fast (most of the binding was accomplished in 15 min) with signal yields up to 17 mV. Efforts to increase the antibody-binding capacity of the solid phase on the ISFET exploiting amino group activation (with glutaraldehyde or other homobifunctional cross linkers) before HSA coupling, did not improve signal yield. The bead technology described in this report is an easy, generic method for coating the ISFET with a solid phase that, using the ion-step method, can be applied to immunosensing.     

Molecular charge contact biosensing based on the interaction of biologically modified magnetic beads with an ion-sensitive field effect transistor

In this article, we report a novel method of biomolecular recognition based on the molecular charge contact (MCC). As one of the MCC biosensing method, the interaction between DNA-coated magnetic beads and a silicon-based semiconductor, an ion-sensitive field effect transistor (ISFET) could be detected for DNA molecular recognition events using the principle of the field effect, which enables detecting ionic or molecular charges. After DNA-coated magnetic beads had been introduced and brought in contact with the gate surface by a magnet, the threshold voltage of the ISFET was shifted in the positive direction by immobilization, hybridization and extension reaction of DNA molecules on magnetic beads. This positive shift was based on the increase in negative charges of the phosphate groups in them. Then, the ISFET device could be reused a couple of dozen times continuously and cost-effectively because the oligonucleotide probes were tethered to the magnetic beads, but this was not done directly on the gate surface of the ISFET. Moreover, the MCC biosensing method enabled discrimination of a single nucleotide polymorphism. By creating an interaction of magnetic beads with the semiconductor, we can expect enhancement of the reaction efficiency in a solution and reuse of the device by separating the reaction field from the sensing substrate.     

A novel ISFET-type biosensor based on P450 monooxygenases

We made a biosensor based on ion-sensitive field effect transistor (ISFET) using P450 monooxygenase. ISFETs are electrical devices and have been used as pH sensors. We used genetically engineered P450 monooxygenase for our research because of its high enzymatic activity. The fusion enzyme between rat CYP1A1P450 monooxygenase and yeast NADPH-cytochrome P450 oxidoreductase was expressed in yeast Saccharomyces cerevisiae strain AH22. Yeast microsomal membranes were immobilized in an agarose layer on the ISFET. o-Deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin was catalyzed by the enzyme in the presence of nicotinamide adenine dinucleotide phosphate reduced form (NADPH). Formation of 7-hydroxycoumarin from 7-ethoxycoumarin was also measured by fluorescence. The difference of the voltage between the ISFET device and control device without enzymes showed a voltage increase along with the enzymatic reaction of P450 monooxygenases, and this voltage increase in the device was inhibited by addition of MnCl(2), an inhibitor of P450 monooxygenase. There was a positive correlation between the voltage increase in the ISFET device and the fluorescence intensity. This is the first electrochemical biosensing using P450 monooxygenases immobilized on the ISFET, and is applicable to the sensing of chlorophenol compounds.     

Evaluation of the analytical performances of avidin-modified carbon sensors based on a mediated horseradish peroxidase enzyme label and their application to the amperometric detection of nucleic acids

In this study, neutravidin-coated screen-printed carbon sensors were fully characterized and further used for the amperometric detection of specific DNA sequences of human cytomegalovirus (HCMV DNA). For this purpose, we took advantage of an earlier established relationship between the amount of HRP affinity immobilized on the surface of the electrode and the steady-state current recorded in the presence of H₂O₂ as substrate and the single electron donor [Os^III(bpy)₂pyCl]²⁺ as cosubstrate. After incubating a saturating concentration of biotinylated horseradish peroxidase (Bio-HRP) onto the neutravidin-modified sensors, a surface concentration of active HRP of 3.6 pmol cm⁻² was calculated from the measurement of the electrocatalytic plateau current value. This result indicates that monolayers of neutravidin were adsorbed on the screen-printed carbon sensors. These neutravidin-covered platforms were then used to immobilize biotinylated nucleic acid targets. After hybridization with a complementary digoxigenin-labeled detection probe, the extent of hybrids formed was determined with an anti-digoxigenin HRP conjugate. The biosensor assay was applied to the detection of a synthetic oligonucleotide target, and then to the determination of an amplified viral DNA sequence. Monolayers of HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface whereas one third of the surface was covered with HCMV DNA hybrids. On the other hand, detection limits of 200 pM and 1 nM were obtained for the short oligonucleotide and the longer DNA targets, respectively. Finally, we demonstrated that the sensitivity of the electrochemical assay could be significantly improved by using high concentrations of the reduced form of the mediator [Os^II(bpy)₂pyCl]⁺, thus allowing one to detect as low as 30 pM of amplified HCMV DNA fragment.     

Lab-scale fermentation tests of microchip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration

This article shows the development and testing of a microchip with integrated electrochemical sensors for measurement of pH, temperature, dissolved oxygen and viable biomass concentration under yeast cultivation conditions. Measurements were done both under dynamic batch conditions as well as under prolonged continuous cultivation conditions. The response of the sensors compared well with conventional measurement techniques. The biomass sensor was based on impedance spectroscopy. The results of the biomass sensor matched very well with dry weight measurements and showed a limit of detection of approximately 1 g/L. The dissolved oxygen concentration was monitored amperometrically using an ultra-microelectrode array, which showed an accuracy of approximately 0.2 mg/L and negligible drift. pH was monitored using an ISFET with an accuracy well below 0.1 pH unit. The platinum thin-film temperature resistor followed temperature changes with approximately 0.1 degrees C accuracy. The dimensions of the multi sensor chip are chosen as such that it is compatible with the 96-well plate format.     

Characterization of proteins by means of their buffer capacity, measured with an ISFET-based coulometric sensor—actuator system

Proteins form the specific selector in many biochemical sensors. A change in one of the properties of such a protein has to be detected by an appropriate transducer, which completes the biochemical sensor. One of these properties is the buffer capacity of a protein. If the binding of a substance to a protein can significantly change the proton binding, which accounts for the buffer capacity of proteins, the detection of this changed buffer capacity enables the construction of a new type of biosensor.

It will be shown that the buffer capacity can be measured with an ISFET-based sensor—actuator device. The alternating generation of protons and hydroxyl ions by alternating current coulometry at a porous noble metal actuator electrode causes an associated small pH perturbation, which is detected by the underlying pH-sensitive ISFET. The amplitude of the measured signal is a function of the buffer capacity of the solute, in which proteins can be present (or these proteins can be adsorbed in the porous actuator electrode of the device). A model describing the transfer function from the electrical input signal of the actuator to the resulting chemical output, which is subsequently detected by the ISFET pH sensor, is presented. Preliminary results of the measured buffer capacity of ribonuclease and lysozyme are presented.     

Acetylcholinesterase-ISFET based system for the detection of acetylcholine and acetylcholinesterase inhibitors

A bioelectronic hybrid system for the detection of acetylcholine esterase (AChE) catalytic activity was assembled by way of immobilizing the enzyme to the gate surface of an ion-sensitive field-effect transistor (ISFET). Photometric methods used to characterize bonded enzyme and linker layers on silicon substrates confirm the existence of a stable amino-cyanurate containing AChE monolayer. The transduction of the enzyme-functionalized ISFET, in ionic solutions, is detected in response to application of acetylcholine (ACh). Recorded sensitivity of the modified ISFET to ACh has reached levels of up to 10(-5)M. The Michaelis-Menten constant of the immobilized AChE is only moderately altered. Nevertheless, the maximum reaction velocity is reduced by over an order of magnitude. The ISFET response time to bath or ionophoretic application of ACh from a micropipette was in the range of a second. The catalytic activity of the immobilized AChE is inhibited in a reversible manner by eserine, a competitive inhibitor of AChE. We conclude that the immobilized enzyme maintains its pharmacological properties, and thus the described bioelectronic hybrid can serve as a detector for reagents that inhibit AChE activity.     

An ISFET biosensor for the monitoring of maltose-induced conformational changes in MBP

Here we describe an ion sensitive field effect transistor (ISFET) biosensor, which was designed to monitor directly the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. This study is the first report of the application of a FET biosensor to the monitoring of conformationally changed proteins. Consequently, a significant drop in current on the basis of the charge-dependent capacitance measurement has been clearly observed in response to maltose, but not for the glucose control, thereby indicating that the substrate-specific conformational properties of the target protein could be successfully monitored using the ISFET. Collectively, our results clearly suggest that ISFET provide a high fidelity system for the detection of maltose-induced structural alterations in MBP.     

The study of genomic DNA adsorption and subsequent interactions using total internal reflection ellipsometry

The adsorption of genomic DNA and subsequent interactions between adsorbed and solvated DNA was studied using a novel sensitive optical method of total internal reflection ellipsometry (TIRE), which combines spectroscopic ellipsometry with surface plasmon resonance (SPR). Single strands of DNA of two species of fish (herring and salmon) were electrostatically adsorbed on top of polyethylenimine films deposited upon gold coated glass slides. The ellipsometric spectra were recorded and data fitting utilized to extract optical parameters (thickness and refractive index) of adsorbed DNA layers. The further adsorption of single stranded DNA from an identical source, i.e. herring ss-DNA on herring ss-DNA or salmon ss-DNA on salmon ss-DNA, on the surface was observed to give rise to substantial film thickness increases at the surface of about 20-21 nm. Conversely adsorption of DNA from alternate species, i.e. salmon ss-DNA on herring ss-DNA or herring ss-DNA on salmon ss-DNA, yielded much smaller changes in thickness of 3-5 nm. AFM studies of the surface roughness of adsorbed layers were in line with the TIRE data.     

ISFET based enzyme sensors

This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the dynamic range as well as the influence of the sample buffer capacity have not been solved. As a possible solution we introduce a coulometric system that compensates for the analyte buffer capacity. If the pH in the immobilized enzyme layer is thus controlled, the resulting pH-static enzyme sensor has an output that is independent of the sample pH and buffer capacity and has an expanded linear range.     

ISFET-neuron junction: circuit models and extracellular signal simulations

Purpose of this paper is to characterize the Ion-Sensitive Field-Effect Transistors (ISFET)-neuron junction, based on the equivalent electric-circuit approach. As a result, recording of action potentials can be simulated with a general-purpose circuit simulation program such as HSPICE. The neuronal electrical activity, extracellularly recorded by the ISFET, is analyzed as a function of the physical-chemical and geometric ISFET parameters, of the ionic currents in the neuron, and of the neuro-electronic junction parameters such as the sealing resistance, double-layer capacitance, and general adhesion conditions. The models of the neuron, of the coupling circuit, and of the ISFET implemented in HSPICE are first described. These models are then used to simulate the behavior of the junction between a patch of neuronal membrane (described by the compartmental model) and the ISFET.     

In-situ FTIR study on adsorption and oxidation of native and thermally denatured calf thymus DNA at glassy carbon electrodes

In-situ Fourier transform infra-red (FTIR) spectra of native and thermally denatured calf thymus DNA (CT DNA) adsorbed and/or oxidized at a glassy carbon (GC) electrode surface are reported. The adsorption of native DNA occurs throughout the potential range (- 0.2 approximately 1.3 V) studied, and the adsorbing state of DNA at electrode surface is changed from through the C=O band of bases and pyrimidine rings to through the C=O of cytosine and imidazole rings while the potential shifts negatively from 1.3 V to -0.2 V. An in-situ FTIR spectrum of native CT DNA adsorbed at GC electrode surface is similar to that of the dissolved DNA, indicating that the structure of CT DNA is not distorted while it is adsorbed at the GC electrode surface. In the potential range of -0.2 approximately1.30 V, the temperature-denatured CT DNA is adsorbed at the electrode surface first, then undergoes electrochemical oxidation reaction and following that, diffuses away from the electrode surface.     

Electrochemical sensor based on photopolymeric membranes for determining urea

A new electrochemical enzymatic sensor based on the ion selective field effect transistors (ISFETs) and photocurable membrane was developed for the determination of urea. For the immobilization of urease on the gate surface of the ISFET a simple method, involving the use of liquid photocurable compositions on the basis of vinylpirollidone, oligouretanemetacrylate and oligocarbonatemetacrylate, was applied. The linearange of the response of the developed electrochemical sensor lies in the range 0.05-20 mM. The latter corresponds to the claims of the medical practice. The overall time of the analysis is 5-10 min. The effects of the buffer concentration and its pH as well as temperature and presence of ammonia ions in the measuring medium on the amplitude of the sensor response were estimated. The duration of sensor work is as shortest 40 days. The proposed sensor on the basis of the ISFET is promising for the express analysis of the level urea in blood, while the developed method of membrane preparation with entrapped enzyme can be combined with the integral technology of producing of the biosensors semiconductor transducers.     

A new approach to immunoFET operation

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. The diffusion rate is determined by the immobile charge density in the amphoteric protein layer, which changes upon formation of antibody-antigen complexes. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin (HSA) and the immune reaction of HSA with the antibody anti-human serum albumin (alpha HSA). The influence of protein parameters on the amplitude of the transient can be described with an empirical equation. Assuming Langmuir behaviour, the protein concentration in the solution can well be correlated with the concentration in the membrane. This new detection method is unique concerning direct measurements of charge densities and isoelectric points of amphoteric macromolecules adsorbed in the membrane. The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied.     

Electrophoresis of absorbed RNA

The electrophoretic mobilities of adsorbed yeast ribonucleic acid have been measured as functions of pH, ionic strength, and biopolymer concentration and the results so obtained have been critically compared with those for adsorbed DNA. Like DNA, ribonucleic acid has also been found to reverse the positive charge of alumina owing to its adsorption on the solid-liquid interface. The mobilities of adsorbed RNA have been found to be less than those of adsorbed DNA under identical conditions. The observed mobilities of adsorbed heat- and alkali-denatured RNA are significantly less than those of adsorbed native RNA at a given pH and ionic strength of the medium. The electrophoretic mobilities as observed also show the evidence of RNA adsorption on the negatively charged surface of Dowex-50 resin, but practically no adsorption of RNA on the negatively charged glass surface has been predicted.     

Early detection of cell metabolism with a silicon microsensor

A silicon microsensor (ISFET — Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

Association of double-stranded DNA fragments into multistranded DNA structures.

We have previously observed that double-stranded DNA fragments containing a tract of the tandemly repeated sequence poly(CA). poly(TG) can associate in vitro to form stable complexes of low electrophoretic mobility, which are recognized with high specificity by proteins HMG1 and HMG2. The formation of such complexes has since been observed to depend on interactions of DNA with polypropylene surfaces, with the suggestion that the formation of low mobility complexes might be the result of strand dissociation followed by misaligned reassociation of the repetitive sequences. The data presented here show that at high ionic strength the interactions of DNA with polypropylene are sufficiently strong for DNA to remain bound to the polypropylene surface, which suggests that DNA might also be involved in interactions with hydrophobic molecules in vivo. Under such conditions, low-mobility complexes are found only in the material adsorbed to the polypropylene surface, and all DNA fragments are able to form low-mobility structures, whether or not they contain repetitive sequences. Preventing the separation of strands by ligating hairpin loop oligonucleotides at both ends of the fragments does not prevent the formation of low-mobility complexes. Our results suggest two different pathways for the formation of complexes. In the first, dissociation is followed by misaligned reassociation of repetitive sequences, yielding duplexes with single-stranded end regions that associate to form multimeric complexes. In the second, repetitive as well as nonrepetitive DNA molecules bound to polypropylene adopt a conformation with locally unwound regions, which allows interactions between neighboring duplexes adsorbed on the surface, resulting in the formation of low-mobility complexes.