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.     

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.     

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).     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

Enzymatically biocatalytic precipitates amplified antibody-antigen interaction for super low level immunoassay: an investigation combined surface plasmon resonance with electrochemistry

We demonstrated a simple and efficient strategy, which based on the enzymatically biocatalytic precipitates amplified antibody-antigen interaction, for improving the response signals of surface plasmon resonance (SPR) immunosensing. The antibody-antigen-alkaline phosphatase (AP) labeled secondary antibody sandwich were successfully prepared and characterized by SPR, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The SPR signal amplification was accomplished through probing resonance angle shift and Faradaic electron impedance of [Fe(CN)(6)](3-/4-) redox pair after the enzymatically biocatalytic products precipitating on the immunosensing electrode surface. As a result, the accumulation of the enzymatically biocatalytic precipitates leads to significantly resonance angle shift and increase of electron transfer impedance of [Fe(CN)(6)](3-/4-) probe. The precipitates-enhanced sandwich SPR immunoassay for mouse immunoglobulin G (m-IgG) can easily detect solution protein concentrations in the linear range of 0.02-40 ng mL(-1) and with a detection limit of 200 fg mL(-1), which is more than four-orders and 10 times better compared with the values using streptavidin-biotinylated protein complex and biotinylated HRP biocatalyzation amplification methods. Moreover, this method is generally applicable to other sandwich immunoassays and also can be expanded to monitor other antibody-antigen interaction for immunosensing detection at low concentrations.     

Determination of nanomolar uric and ascorbic acids using enlarged gold nanoparticles modified electrode

Individual and simultaneous determination of 50 nM uric acid (UA) and ascorbic acid (AA) using enlarged, citrate-stabilized gold nanoparticles (AuNPs) self-assembled to 2,5-dimercapto-1,3,4-thiadiazole (DMT) monolayer modified Au (Au/DMT) electrode by an amperometric method is described for the first time. Self-assembly of AuNPs on the electrode surface was confirmed by atomic force microscopy (AFM), attenuated total reflectance FT-IR and diffuse reflectance spectral measurements. The electron transfer reaction (ETR) of [Fe(CN)₆]^3−/4− was blocked at Au/DMT electrode, whereas it was restored with a peak separation of 200 mV after the attachment of AuNPs on the Au/DMT (Au/DMT/AuNPs) electrode, which was confirmed from the ETR of the [Fe(CN)₆]^3−/4− redox couple. When the self-assembled AuNPs were enlarged by hydroxylamine seeding, the ETR of [Fe(CN)₆]^3−/4− was improved significantly with a peak separation of 100 mV. Tapping mode AFM showed that the average size of the enlarged-AuNPs (E-AuNPs) was 50-70 nm. The E-AuNPs modified electrode catalyzes the oxidation of AA and UA, separates their voltammetric signals by 200 mV, and has excellent sensitivity towards AA and UA with a detection limit of 50 nM. The practical application of the modified electrode was demonstrated by measuring the concentration of UA in blood serum and urine.     

Nanoporous gold film based immunosensor for label-free detection of cancer biomarker

Nanoporous gold (NPG) film modified electrode for the construction of novel label-free electrochemical immunosensor for ultrasensitive detection of cancer biomarker prostate specific antigen (PSA) is described. Due to its high conductivity, large surface area, and good biocompatibility, NPG film modified electrode was used for the adsorption of anti-PSA antibody (Ab). The sensing signal is based on the monitoring of the electrode's current response towards K(3)Fe(CN)(6), which is extremely sensitive to the formation of immunocomplex within the nanoporous film. Under optimum conditions, the amperometric signal decreases linearly with PSA concentration (0.05-26 ng/mL), resulting in a low limit of detection (3 pg/mL). We demonstrated the application of the novel immunosensor for the detection of PSA in real sample with satisfactory results.     

Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes

The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigitated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the presence of K(3)[Fe(CN)(6)]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K(3)[Fe(CN)(6)], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN)(6)(3-) ions through defects in the ODN monolayer determines the impedance of Au-ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K(3)[Fe(CN)(6)] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms.     

Electrical recognition of label-free oligonucleotides upon streptavidin-modified electrode surfaces

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in Fe(CN)₆ ³⁻/Fe(CN)₆ ⁴⁻ solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.     

AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7

The microscopic surface molecular structures and macroscopic electrochemical impedance properties of the epoxysilane monolayer and anti-Escherichia coli antibody layer on an indium-tin oxide (ITO) electrode surface were studied in this paper. Characterization of stepwise changes in microscopic features of the surfaces and electrochemical properties upon the formation of each layer were carried out using both atomic force microscopy (AFM) and electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-) as a redox couple. AFM images of the self-assembled monolayer (SAM) evidenced the dense, complete, and homogeneous morphology of the epoxysilane monolayer on the ITO surface. The uniformity of the epoxysilane SAM allowed antibodies to attach to the epoxy surface groups of the silanes in a similarly uniform fashion. The effects of epoxysilane monolayer and the antibody layer on the electrochemical properties of the electrode were quantitatively analyzed in terms of double layer capacitance, electron transfer resistance, Warburg impedance and solution resistance using Randles model as the equivalent circuit. It was demonstrated that the epoxysilane monolayer and the antibody layer act as barriers for the electron transfer between the electrode surface and the redox species in the solution, resulting in most significant increases in the electron transfer resistance compared to all the electric elements. Immunoreaction with E. coli O157:H7 cells demonstrated specific recognition of the immobilized anti-E. coli antibodies as evidenced by AFM imaging and impedance spectroscopy. It was found that the binding of E. coli cells mainly affected the electron transfer resistance and Warburg impedance.     

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin (BSA) by piezoelectric quartz crystal impedance analysis

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin by the piezoelectric quartz crystal impedance (PQCI) analysis was carried out for the first time. Three kinds of electrodes were investigated. Compared with bare gold (Au) electrode, the gold electrode self-assembled of nanogold colloids exhibits maintained biocompatibility, increased capacity and more bioactivity. Additionally, on the basis of the multi-dimensional information provided by the PQCI analysis, the real-time interaction information and the kinetics of the binding process was investigated and a response model was deduced. At 37 degrees C, the binding rate (k1), dissociation rate (k(-1)) and equilibrium constants (Ka) were 4.19x10(2) (mol l(-1))(-1) s(-1), 1.01x10(-3) s(-1) and 4.15x10(5) (mol l(-1))(-1) for the electrode modified by nanogold particles; 3.83x10(2) (mol l(-1))(-1) s(-1), 9.70x10(-4) s(-1) and 3.95x10(5) (mol l(-1))(-1) for the bare gold electrode, respectively.     

Strategy of red blood cells immobilisation onto a gold electrode: Characterization by electrochemical impedance spectroscopy and quartz crystal microbalance

This study describes the grafting of red blood cells (RBC) onto a gold electrode. The erythrocytes were immobilised using antigen/antibody crosslinking based on the bonding of anti-D with the corresponding antigen of the RBC membrane that is shared by all erythrocytes from the positive rhesus group (O⁺). To optimise the reproducibility of the modified electrode and to avoid nonspecific interactions, the anti-D layer was deposited onto a protein G layer. The bridge between the protein G and the gold transducer was formed using mixed thiol-based self-assembled monolayer (SAM) (a mixture of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol in a 1/10 ratio). Each layer deposited was characterized, firstly, with a quartz crystal microbalance to obtain the deposited mass and the corresponding number of moles per square centimetres and secondly, by electrochemical impedance spectroscopy (EIS) using a redox couple Fe(CN)₆^3−/4− (1:1) as an EIS probe. Subsequent modelling with appropriate circuitry enabled the values for each component representing the interface (electrode/film/solution) to be calculated at each step of the grafting process. From these results the surface coverage has been calculated to range from 95 to 98%.     

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.     

Electrochemical recognition for sugars on the chitosan-poly(diallyldimethylammonium chloride)-nano-Prussian blue/nano-Au/4-mercaptophenylboronic acid modified glassy carbon electrode

A new amperometric biosensor for the detection of sugars was prepared. A glassy carbon electrode was modified with Prussian blue (PB) nanoparticles protected by chitosan (CS) and poly(diallyldimethylammonium chloride) (PDDA), and then gold nanoparticles were assembled onto the electrode followed by the assembly of 4-mercaptophenylboronic acid (MPBA) onto the surface of gold nanoparticles through a sulfur–Au bond to fabricate a self-assembled biosensor. The PB nanoparticles protected by CS and PDDA were characterized using transmission electron microscopy and UV–vis absorption spectroscopy. The characterization of the self-assembled electrode was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The pK _a values of the MPBA monolayer before and after combining with sugars were determined. The fabricated electrode exhibited excellent performances for determining d(+)-glucose, d(+)-mannose, and d(−)-fructose on the basis of the change in i _p of the Fe(CN)₆^3−/4− ion in the presence of sugars.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.     

Electrochemical immunosensor for casein based on gold nanoparticles and poly(L-Arginine)/multi-walled carbon nanotubes composite film functionalized interface

In this paper, a novel electrochemical immunosensor for the determination of casein based on gold nanoparticles and poly(L-Arginine)/multi-walled carbon nanotubes (P-L-Arg/MWCNTs) composite film was proposed. The P-L-Arg/MWCNTs composite film was used to modify glassy carbon electrode (GCE) to fabricate P-L-Arg/MWCNTs/GCE through electropolymerization of L-Arginine on MWCNTs/GCE. Gold nanoparticles were adsorbed on the modified electrode to immobilize the casein antibody and to construct the immunosensor. The stepwise assembly process of the immunosensor was characterized by cyclic voltammetry and differential pulse voltammetry. Results demonstrated that the peak currents of [Fe(CN)(6)](3-/4-) redox pair decreased due to the formation of antibody-antigen complex on the modified electrode. The optimization of the adsorption time of gold nanoparticles, the pH of supporting electrolyte and the incubation time were investigated in details. Under optimal conditions, the peak currents obtained by DPV decreased linearly with the increasing casein concentrations in the range from 1 × 10(-7) to 1 × 10(-5) g mL(-1) with a linear coefficiency of 0.993. This electrochemical immunoassay has a low detection limit of 5 × 10(-8) g mL(-1) and was successfully applied to the determination of casein in cheese samples.     

Novel potentiometry immunoassay with amplified sensitivity for diphtheria antigen based on Nafion, colloidal Ag and polyvinyl butyral as matrixes

A novel potentiometry immunoassay with amplified sensitivity has been developed for the detection of diphtheria antigen (Diph) via immobilizing diphtheria antibody (anti-Diph) on a platinum electrode based on Nafion, colloidal Ag (Ag), and polyvinyl butyral (PVB) as matrixes in this study. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log Diph concentrations, high sensitivity (51.4 mV/decade), wide linear range from 8 to 800 ng ml(-1) with a detection limit of 1.5 ng ml(-1), rapid potentiometric response (<3 min) and long-term stability (>6 months). 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 diphtheria antigen in the clinical diagnosis.     

Conducting polyamic acid membranes for sensing and site-directed immobilization of proteins

A biosensor platform based on polyamic acid (PAA) is reported for oriented immobilization of biomolecules. PAA, a functionalized conducting polymer substrate that provides electrochemical detection and control of biospecific binding, was used to covalently attach biomolecules, resulting in a significant improvement in the detection sensitivity. The biosensor sensing elements comprise a layer of PAA antibody (or antigen) composite self-assembled onto gold (Au) electrode via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linking. The modified PAA was characterized by Fourier transform infrared (FTIR), (1)H nuclear magnetic resonance (NMR), and electrochemical techniques. Cyclic voltammetry and impedance spectroscopy experiments conducted on electrodeposited PAA on Au electrode using ferricyanide produced a measurable decrease in the diffusion coefficient compared with the bare electrode, indicating some retardation of electron transfer within the bulk material of the PAA. Thereafter, the modified PAA surface was used to immobilize antibodies and then to detect inducible nitric oxide synthase and mouse immunoglobulin G (IgG) using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and amperometric techniques. ELISA results indicated a significant amplified signal by the modified PAA, whereas the SPR and amperometric biosensors produced significant responses as the concentration of the antigen was increased. Detection limits of 3.1×10(-3)ng/ml and 2.7×10(-1)ng/ml were obtained for SPR and amperometric biosensors, respectively.     

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe(3)O(4), which was synthesized with easy handling feature of Fe(3)O(4) by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe(3)O(4) magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 μg mL(-1), with the detection limit of 0.025 μg mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c.     

A simple assay to amplify the electrochemical signal by the aptamer based biosensor modified with CdS hollow nanospheres

A simple method to amplify the electrochemical signal by an aptamer with 22 bases modified with CdS hollow nanospheres (CdSHNs) was described. Using the thrombin as a model, the interaction between the aptamer and CdSHNs was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and circular dichroism spectroscopy. CdSHNs promoted the electron transfer between the gold electrode and K(3)[Fe(CN)(6)] and facilitated the conformation conversion of the aptamer from hairpin to G-quadruplex after the aptamer interacted with thrombin. Under optimal conditions, the modified electrode could be used for the determination of thrombin from 0 to 33 μg mL(-1) and the sensitivity was 1.34 μA mL μg(-1)cm(-2), while the linear range of the modified electrode without the immobilization of CdSHNs was from 2.75 to 27.5 μg mL(-1) and the sensitivity was 0.062 μA mL μg(-1)cm(-2). This constructed biosensor also had a good stability, specificity, reproducibility and accuracy which could provide a promising platform for fabrication of aptamer based biosensors.