Electrochemical fabrication and evaluation of highly sensitive nanorod-modified electrodes for a biotin/avidin system

Bioaffinity sensors need to be rapid, specific, and highly sensitive. To realize these features, electrodes that can elicit high electrochemical performance are necessary. In this study, we developed nanorod array electrode and performed cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments to study the interfacial properties of the nanorod array electrode with Fe(CN)(6)(3-/4-) as the redox molecules. Results showed that both the CV and EIS measurements captured very well the resistive and capacitive changes due to the adsorption of functionalizing molecules and the coupling between avidin and biotin. The EIS measurements were more sensitive in discriminating small changes caused by the surface adsorption of various molecules. The use of avidin-functionalized gold nanorod modified electrodes had led to much increased detection sensitivity along with a detection-limit as low as 1 ng/mL of biotin.     

电化学阻抗谱在生物传感器研究中的应用进展

电化学阻抗谱(EIS)是近年快速发展起来的一种电化学分析方法。它具有良好的界面表征作用,特别适合于分析电极表面生物敏感膜的制备、生物学反应的动力学机制,已逐渐成为生物传感器研究的有效辅助方法。简要概述了EIS技术的原理,及其在各种生物传感器中的应用进展。     

Assessment of the interaction between a synthetic epitope of troponin C and its specific antibody using a label-free faradaic impedimetric immunosensor and alpha-Keggin silicotungstic heteropolyacid as a redox probe

The development of an immunosensor for the direct probing of the interaction between a cysteine-modified synthetic peptide, which corresponds to the epitope cTnC-89-98 of troponin C, and its specific antibody is described. Following immobilization of the peptide onto gold electrodes through the formation of a self-assembled monolayer, the alteration of the interfacial properties of the electrodes upon peptide-antibody interaction was traced by faradaic electrochemical impedance spectroscopy (EIS) using a silicotungstic heteropolyacid, H(4)SiO(4).12WO(3), as a redox probe. The electrochemical behaviour of the redox probe was evaluated with cyclic voltammetry and EIS. The effect of milk protein or 4-mercaptophenol, which was used as post-blocking agents, on the performance of the immunosensor, was investigated. Treatment with 4-mercaptophenol resulted in immunoeffective electrodes that successfully tested in anti-serum samples. An optimum dilution ratio of the samples, where the effect of the matrix on the measuring signal is negligible, was also determined.     

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

Metallic oxide CdIn2O4 films for the label free electrochemical detection of DNA hybridization

DNA functionalised semiconductor metallic oxide electrodes have been developed for the direct electrochemical detection of DNA hybridization, without labelling or the introduction of a redox couple. Conductive CdIn(2)O(4) thin films with controlled properties were deposited on glass substrates using an aerosol pyrolysis technique. The films exhibit a polycrystalline microstructure with a surface roughness of 1.5 nm (r.m.s.) and an electrical resistivity ranging between 1 and 3 x 10(-3) Omega cm. These electrodes were functionalised using hydroxylation and silanisation steps, to allow the binding of DNA probe sequences (20 bases). The electrical detection of DNA hybridization with complementary sequences has been performed using electrochemical impedance spectrometry (EIS) measuring the variation of impedance before and after hybridization. Two set-ups were used, a standard set-up including three electrodes and a set-up including two symmetrical electrodes. In both configurations, a significant increase of the impedance modulus, more particularly of the real part of the impedance (160-225% according to the electrochemical cell used) has been obtained over a frequency range of 10-10(5)Hz. DNA hybridization has also been systematically confirmed using the fluorescence spectrometry. This study emphasizes the high sensitivity of the CdIn(2)O(4) as a working electrode for the detection of biological events occurring at the electrode surface.     

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.     

Investigation of interfacial capacitance of Pt,Ti and TiN coated electrodes by electrochemical impedance spectroscopy

Electrochemical processes at the electrode-electrolyte (body fluid) interface are of ultimate importance for stimulating/sensing electrode function. A high electrode surface area is desirable for safe stimulation through double-layer charging and discharging. Pt and Pt-Ir alloys have been the most common electrode materials. The use of TiN coating as the surface layer on the electrode has found increasing interest because of its metal-like conductivity, excellent mechanical and chemical properties, and the fact that it can be deposited with a high surface area. In this work, electrochemical impedance spectroscopy (EIS), which is a sensitive and non-destructive technique and widely used for characterization of electrical properties of electrode-electrolyte interfaces, was applied to investigate pure Pt and Ti, and TiN coated electrodes exposed to a phosphate-buffered-saline (PBS) solution. Platinized Pt and Ti were also studied for comparison. The capacitance value of the electrodes in PBS was obtained through quantitative analysis of the EIS spectra. The results reveal that the capacitance of the TiN coated electrodes with a rough surface is several hundreds times higher than that of a smooth Pt surface. Platinization of Ti can also increase the capacitance to the same extent as platina. EIS has been shown to be a powerful technique for characterization of stimulating/sensing electrodes.     

Interactions of amyloid beta-peptide (1-40) with ganglioside-containing membranes

Interactions between amyloid beta-peptides (Abeta) and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. To gain insight into the molecular details of this association, we investigated the interactions of Abeta (1-40) with ganglioside-containing membranes by circular dichroism (CD) and Fourier transform infrared-polarized attenuated total reflection (FTIR-PATR) spectroscopy. The CD study revealed that at physiological ionic strength Abeta (1-40) specifically binds to ganglioside-containing membranes inducing a two-state, unordered --> beta-sheet transition above a threshold intramembrane ganglioside concentration, which depends on the host lipid bilayers used. Furthermore, differences in the number and position of sialic acid residues of the carbohydrate backbone significantly affected the conformational transition of the peptide. FTIR-PATR spectroscopy experiments demonstrated that Abeta (1-40) forms an antiparallel beta-sheet, the plane of which lies parallel to the membrane surface, inducing dehydration of lipid interfacial groups and perturbation of acyl chain orientation. These results suggest that Abeta (1-40) imposes negative curvature strain on ganglioside-containing lipid bilayers, disturbing the structure and function of the membranes.     

Electrical frequency dependent characterization of DNA hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of electrochemical impedance spectroscopy (EIS) and quartz crystal microgravimetry (QCM). Synthetic 5'-amino terminated single stranded oligonucleotides (ssDNA) were attached to the exposed glass surface between the digits of microlithographically fabricated interdigitated microsensor electrodes using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA immobilization was achieved to the surface of the gold driving electrodes of AT-cut quartz QCM crystals using 3-mercaptopropyl-trimethoxysilane. Significant changes in electrochemical impedance values (both real and imaginary components) (11% increase in impedance modulus at 120 Hz) and resonant frequency values (0.004% decrease) were detected as a consequence of hybridization of the bound ssDNA upon exposure to its complement under hybridization conditions. Non-complementary (random) sequence sowed a modest decrease in impedance and a non-detectable change in resonant frequency. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA, has been demonstrated. The potential for development of label-free, low density DNA microarrays is demonstrated and is being pursued.     

Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

Exploiting the interactions of PNA-DNA films with Ni2+ ions: detection of nucleobase mismatches and electrochemical genotyping of the single-nucleotide mismatch in apoE 4 related to Alzheimer's disease

The presence of Ni(2+) enables us to distinguish the presence of single-nucleotide mismatches in PNA (peptide nucleic acids)-DNA films on gold electrodes by electrochemical impedance spectroscopy (EIS). With the help of a modified Randles' equivalent circuit, differences in the charge transfer resistance (ΔR(CT)) before and after the addition of Ni(2+) are a diagnostic measure for the presence of single-nucleotide mismatch. The approach works under real-life conditions with concentrations of the DNA target strand down to 10 fM, and a PNA capture probe is used to genotype the single-nucleotide mismatch in apoE 4 related to Alzheimer's disease (AD).     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1).     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.     

A label-free electrochemical immunoassay for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and nonconductive polymer film

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) has been developed. CEA antibody (CEAAb) was covalently attached on glutathione (GSH) monolayer-modified gold nanoparticle (AuNP) and the resulting CEAAb-AuNP bioconjugates were immobilized on Au electrode by electro-copolymerization with o-aminophenol (OAP). Electrochemical impedance spectroscopy and cyclic voltammetry studies demonstrate that the formation of CEA antibody-antigen complexes increases the electron transfer resistance of [Fe(CN)(6)](3-/4-) redox pair at the poly-OAP/CEAAb-AuNP/Au electrode. The use of CEA antibody-AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode. The preliminary application of poly-OAP/CEAAb-AuNP/Au electrode for detection of CEA was also evaluated.     

A cytokine immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy

A biosensor for the serum cytokine, interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy monitoring is described. Overexpression of IL-12 has been correlated to the diagnosis of multiple sclerosis (MS). The prototype biosensor was fabricated on a disposable gold-coated silver ribbon electrode by immobilizing anti-IL-12 monoclonal antibodies (mAbs) onto the surface of the electrode. This technique was advantageous as the silver electrodes provided a more rigid and conductive substrate than thin gold foil electrodes and helped in obtaining more reproducible data when used with the electrode holder. Results indicate that IL-12 can be detected at physiological levels, <100 fM with p<0.05 in a label-free and real-time manner. The cost-effective approach described here can be used for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.     

Direct evidence that all three histidine residues coordinate to Cu(II) in amyloid-beta1-16

We provide direct evidence that all three histidine residues in amyloid-beta 1-16 (Abeta 1-16) coordinate to Cu(II). In our approach, we generate Abeta 1-16 analogues, in each of which a selected histidine residue is isotopically enriched with (15)N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopy clearly show that all three histidine imidazole rings at positions 6, 13 and 14 in Abeta 1-16 bind to Cu(II). The method employed here does not require either chemical side chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We find that the histidine coordination in the Abeta 1-16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Abeta 1-40 peptide. The ESR results also suggest tight binding between the histidine residues and the Cu(II) ion, which is likely the reason for the high binding affinity of the Abeta peptide for Cu(II).     

Two types of Alzheimer's beta-amyloid (1-40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation

The 39-42 amino acid long, amphipathic amyloid-beta peptide (Abeta) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Abeta presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Abeta(1-40) membrane assemblies for two different scenarios with potential implication in AD: Abeta peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Abeta(1-40) by stabilizing its hydrophobic transmembrane C-terminal part (residue 29-40) in an alpha-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Abeta(1-40) is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Abeta(1-40) peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide-membrane assemblies are involved in Abeta's pathophysiology with the finely balanced type of Abeta-lipid interactions against release of Abeta from neuronal membranes being overcompensated by an Abeta-membrane assembly which causes toxic beta-structured aggregates in AD. Therefore, pathological interactions of Abeta peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Abeta-membrane assemblies inherited from Abeta's origin.