Electrochemical immunoassay for subgroup J of avian leukosis viruses using a glassy carbon electrode modified with a film of poly (3-thiophene boronic acid), gold nanoparticles, graphene and immobilized antibody

We have modified a glassy carbon electrode (GCE) with a film of poly(3-thiophene boronic acid), gold nanoparticles and graphene, and an antibody (Ab) was immobilized on its surface through the covalent bond formed between the boronic acid group and the glycosyl groups of the Ab. Subgroup J of avian leukosis viruses (ALV-J) were electrochemically determined with the help of this electrode. There is a linear relationship between the electron transfer resistance (R _et) and the concentration of ALV-J in the range from 527 to 3,162 TCID₅₀?mL^?1 (where TCID₅₀ is the 50?% tissue culture infective dose). The detection limit is 210 TCID₅₀?mL^?1 (at an S/N of 3), and the correlation coefficient (R) is 0.9964. The electrochemical immunoassay showed good selectivity, stability and reproducibility.

Glassy carbon electrode modified with poly-Neutral Red for photoelectrocatalytic oxidation of NADH

A new approach is described for the photoelectrocatalytic oxidation of Reduced ß-Nicotinamide Adenine Dinucleotide (NADH). It is based on a glassy carbon electrode (GCE) modified with a film of poly-Neutral Red (poly-NR) that is obtained by electropolymerization. Electrochemical measurements revealed that the modified electrode displays electrocatalytic and photo-electrocatalytic activity towards oxidation of NADH. If irradiated with a 250-W halogen lamp, the electrode yields a strongly increased electrocatalytic current compared to the current without irradiation. Amperometric and photo-amperometric detection of NADH was performed at +150 mV vs. Ag/AgCl/KCl_sat and the currents obtained are linearly related to the concentration of NADH. Linear calibration plots are obtained in the concentration range from 1.0 μM to 1.0 mM for both methods. However, the slope of the current-NADH concentration curve of the photo-electrocatalytic procedure was 2-times better than that obtained without irradiation.

An efficient electrochemical disinfection of E. coli and S. aureus in drinking water using ferrocene–PAMAM–multiwalled carbon nanotubes–chitosan nanocomposite modified pyrolytic graphite electrode

This work reported an efficient electrochemical treatment for drinking water disinfection using a pyrolytic graphite electrode modified with ferrocenyl tethered poly(amidoamine) dendrimers–multiwalled carbon nanotubes–chitosan nanocomposite. The influence parameters of electrochemical disinfection of Escherichia coli and Staphylococcus aureus, such as applied potential and sterilization time, were investigated. Further investigation indicated that almost all (99.99 %) of the initial bacteria were killed after applying a low potential of 0.4 V for 10 min. During the electrochemical disinfection process, the oxidized form of ferrocene was formed on electrode, which played a key role in the disinfection towards E. coli and S. aureus. Hence, the proposed method may provide potential application for the disinfection of drinking water.

Electrochemiluminescence immunoassay at a nanoporous gold leaf electrode and using CdTe quantun dots as labels

An electrochemiluminescence-based immunoassay using quantum dots (QDs) as labels for the carcinoembryonic antigen (CEA) was developed using an electrode modified with leafs of nanoporous gold. CEA was initially immobilized on the electrode via a sandwich immunoreaction, and then CdTe quantum dots capped with thioglycolic acid were used to label the second antibody. The intensity of the ECL of the QDs reflects the quantity of CEA immobilized on the electrode. Thus, in the presence of dithiopersulfate as the coreactant, the ECL serves as the signal for the determination of CEA. The intensity of the electroluminescence (ECL) of the electrode was about 5.5-fold higher than that obtained with a bare gold electrode. The relation between ECL intensity and CEA concentration is linear in the range from 0.05 to 200?ng.mL^-1, and the detection limit is 0.01?ng.mL^-1. The method has the advantages of high sensitivity, good reproducibility and long-term stability, and paves a new avenue for applying quantum dots in ECL-based bioassays.

Voltammetric determination of bisphenol A in food package by a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes

A highly sensitive and mercury-free method for determination of bisphenol A (BPA) was established using a glassy carbon electrode that was modified with carboxylated multi-walled carbon nanotubes. A sensitive oxidation peak is found at 550?mV in linear sweep voltammograms at pH?7. Based on this finding, trace levels of bisphenol A can be determined over a concentration range that is linear from 10?nM to 10⁴?nM, the correlation coefficient being 0.9983, and the detection limit (S/N?=?3) being 5.0?nM. The method was successfully applied to the determination of BPA in food package.

Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate

We report on the modification of a graphene paste electrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve as a platform for the construction of a novel electrochemical immunosensor for the detection of hepatitis B surface antigen (HBsAg). To obtain the immunosensor, an antibody against HBsAg was immobilized on the surface of the electrode, and this process was followed by cyclic voltammetry and electrochemical impedance spectroscopy. The peak currents of a hexacyanoferrate redox system decreased on formation of the antibody-antigen complex on the surface of the electrode. Then increased electrochemical response is thought to result from a combination of beneficial effects including the biocompatibility and large surface area of the AuNPs, the high conductivity of the graphene paste electrode, the synergistic effects of composite film, and the increased quantity of HBsAb adsorbed on the electrode surface. The differential pulse voltammetric responses of the hexacyanoferrate redox pair are proportional to the concentration of HBsAg in the range from 0.5–800?ng?mL^?1, and the detection limit is 0.1?ng?mL^?1 (at an S/N of 3). The immunosensor is sensitive and stable.

Ultrasensitive electrochemiluminescent immunoassay for morphine using a gold electrode modified with CdS quantum dots, polyamidoamine, and gold nanoparticles

We report on a novel electrochemiluminescent (ECL) immunoassay for the ultrasensitive determination of morphine by making use of a gold electrode which was modified with a nanocomposite film containing self-assembled polyamidoamine (PAMAM) CdS quantum dots and electrodeposited gold nanoparticles (Au-NPs). The highly uniform and well-dispersed quantum dots were capped with PAMAM dendrimers. Due to the synergistic effect of the modified quantum dots and the electrodeposited Au-NPs, the ECL response is dramatically enhanced. Under optimal experimental conditions, the immunoreaction between morphine and anti-morphine antibody resulted in a decrease of the ECL signal because of steric hindrance. The calibration plot is linear in the morphine concentration range from 0.2 to 180 ng?mL^?1, with a detection limit as low as 67 pg?mL^?1. The sensor was successfully applied to the determination of morphine in blood plasma. This kind of assay is expected to pave new avenues in label-free drug assays.

Immunoassay for SKOV-3 human ovarian carcinoma cells using a graphene oxide-modified electrode

We describe a highly sensitive electrochemical immunoassay for the tumor maker HER2 on the surface of SKOV-3 human ovarian cancer cells. Following the binding of the cancer cells, ssDNA-labeled anti-HER2 antibody (ssDNA-Ab; the detection antibody) was added to conjugate unbound antigen on the target cells. Following hybridization of ssDNA with its complementary DNA, daunorubicin was injected in order to intercalate into the duplex. This enables electron transfer between daunorubicin and electrode to take place. The GO film strongly amplifies the redox signal of daunorubicin. This new assay has a detection limit of 5.2 cells per mL and in our opinion holds great promise for clinical screening of cancer biomarkers and point-of-care diagnostics.

Selective recognition and electrochemical detection of p-nitrophenol based on a macroporous imprinted polymer containing gold nanoparticles

We have combined the molecular imprinting and the layer-by-layer assembly techniques to obtain molecularly imprint polymers (MIPs) for the electrochemical determination of p-nitrophenol (p-NPh). Silica microspheres functionalized with thiol groups and gold nanoparticles (Au-NPs) were assembled on a gold electrode surface layer by layer. The electrode was then immersed into a solution of pyrrole and p-NPh (the template), and electropolymerization led to the creation of a polymer-modified surface. After the removal of the silica spheres and the template, electrochemical impedance spectroscopy and differential pulse voltammetry (DPV) were employed to characterize the surface. The results demonstrated the successful fabrication of macroporous MIPs embedded with Au-NPs on the gold electrode. The effects of monomer concentration and scan rate on the performance of the electrode were optimized. Excellent recognition capacity is found for p-NPh over chemically similar species. The DPV peak current is linearly related to concentration of p-NPh in the 0.1 μM to 1.4 mM range, with a 0.1 μM limit of detection (at S/N?=?3).

The impact of antibody/epitope affinity strength on the sensitivity of electrochemical immunosensors for detecting small molecules

A displacement immunoassay involves having a labelled analogue of the analyte (the epitope) already bound to the antibody. The presence of the analyte causes a competition for antibodies, and some of the antibodies dissociates from the epitope so that it can bind with the analyte. Herein, the influence of the affinity of the surface-bound epitope for the antibody on the sensitivity and selectivity of a displacement immunosensor is explored both theoretically and experimentally. An electrochemical immunosensor described previously [1], where the dissociation of antibodies from an electrode surface causes an increase in current from surface-bound ferrocene species, is used for this purpose. As expected, the ease and effectiveness of the bound antibody being displaced is inversely related to the affinity of the antibody to the surface-bound epitope relative to the analyte in solution as expected. However, if the affinity constant is too low, selectivity and/or sensitivity are compromised. Experimental results are qualitatively compared with a simple mass-action model.

Disposable electrochemical immunosensor for myeloperoxidase based on the indium tin oxide electrode modified with an ionic liquid composite film containing gold nanoparticles, poly(o-phenylenediamine) and carbon nanotubes

A disposable electrochemical myeloperoxidase (MPO) immunosensor was fabricated based on the indium tin oxide electrode modified with a film composed of gold nanoparticles (AuNPs), poly(o-phenylenediamine), multi-walled carbon nanotubes and an ionic liquid. The composite film on the surface of the electrode was prepared by in situ electropolymerization using the ionic liquid as a supporting electrolyte. Negatively charged AuNPs were then adsorbed on the modified electrode via amine-gold affinity and to immobilize MPO antibody. Finally, bovine serum albumin was employed to block possible remaining active sites on the AuNPs. The modification of the electrode was studied by cyclic voltammetry and scanning electron microscopy. The factors affecting the performance of the immunosensor were investigated in detail using the hexacyanoferrate redox system. The sensor exhibited good response to MPO over two linear ranges (from 0.2 to 23.4 and from 23.4 to 300 ng.mL^?1), with a detection limit of 0.05 ng.mL^?1 (at an S/N of 3).

A novel pH sensing membrane based on an ionic liquid-polymer composite

A novel pH sensing membrane was developed that consists of the ionic liquid n-cetylpyridinium hexafluorophosphate (CPFP), poly(vinyl chloride), and quinhydrone. The membrane is stable and flexible and can be easily deposited on the electrode. Electrochemical impedance spectroscopy was used to study the interfacial charge transfer of this membrane. Compared to a traditional plasticizer-based membrane electrode, the new electrode possesses excellent potentiometric characteristics for monitoring pH, such as a response time of less than 10 s, high sensitivity, stability, and reproducibility. The response is almost Nernstian, with a slope of ?57.5?±?0.2 mV pH^?1 in the pH range from 2 to 9.5. The new electrode was used for direct monitoring of pH in real food samples.

Electrochemical immunoassays for the detection the activity of DNA methyltransferase by using the rolling circle amplification technique

We report on an electrochemical method for the determination of the activity of the enzyme methyltransferase (MTase). The methyl-binding domain-1 protein was applied to recognize symmetrically methylated cytosine in CpG (-C-phosphate-G-) islands of ds-DNA which then specifically bind to anti-His tag antibody. Hyperbranched rolling circle amplification (RCA) was used to improve sensitivity. When the dsDNA was treated with M.Sss I methyltransferase, the sequence 5′-CCGG-3′ was methylated and recognized by the methyl binding protein. In turn, the anti-His tag, biotinylated IgG, streptavidin and biotinylated oligonucleotide were captured successively on the surface of an electrode. Subsequently, the RCA reaction was initiated and streptavidin-labeled alkaline phosphatase immobilized on the surface of the electrode. ALP was able to catalyze the hydrolysis of 1-naphthyl phosphate to form 1-naphthol at pH 9.8. The oxidation peak current of 1-naphthol was used to monitor the methylation process. The response obtained by differential pulse voltammetry was linearly related to the concentration of M.Sss I MTase in the range from 0.1 to 40 unit mL^?1, and the detection limit was 0.03 unit mL^?1 (at an SNR of 3). The inhibitory action of paclitaxel on the activity of M.Sss I MTase also was investigated.

Direct electrochemistry of hemoglobin on an ionic liquid carbon electrode modified with zinc tungstate nanorods

We have constructed a new electrochemical biosensor by immobilization of hemoglobin (Hb) and ZnWO₄ nanorods in a thin film of chitosan (CTS) on the surface of carbon ionic liquid electrode. UV–vis and FT-IR spectra reveal that Hb remains in its native conformation in the film. The modified electrode was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. A pair of well-defined redox peaks appears which indicates direct electron transfer from the electrode. The presence of CTS also warrants biocompatibility. The electron transfer coefficient and the apparent heterogeneous electron transfer rate constant were calculated to be 0.35 and 0.757 s^?1, respectively. The modified electrode displays good electrocatalytic activity for the reduction of trichloroacetic acid with the detection limit of 0.613 mmol L^?1 (3σ). The results extend the protein electrochemistry based on the use of ZnWO₄ nanorods.

A carbon nitride electrode for highly selective and sensitive determination of lead(II)

We have constructed a carbon nitride electrode modified with a bismuth film and show that it can be used for the electroanalysis of lead(II) by differential pulse anodic stripping voltammetry. The combination of such materials is shown to greatly improve the sensing capability of the electrode. Under the optimal conditions, the electrochemical response of the modified electrode is linearly related to the concentration of Pb(II) in the 6 – 1,000 nM concentration range (R = 0.9983). The detection limit is 2.0 pM (at an SNR of 3), and the sensitivity is 8 times better of that of respective graphite electrodes. The sensor enables rapid, highly sensitive, continuous, and environmentally friendly determination of trace levels of Pb(II) at affordable costs.

Electrochemical sensor for bisphenol A based on a nanoporous polymerized ionic liquid interface

The ionic liquid 1-butyl -3-[3-(N-pyrrole)-propyl]imidazolium tetrafluoroborate was employed to fabricate a glassy carbon electrode (GCE) modified with a porous film of a polymerized ionic liquid. The resulting film electrode was treated with sodium dodecyl sulfonate solution to exchange the terafluoroborate anions by dodecyl sulfonate groups. This was confirmed by X-ray photoelectron spectroscopy. The morphology of the modified GCE was characterized by scanning electron microscopy and revealed a nanoporous surface. The electrochemical properties of this film electrode were studied by electrochemical impedance spectroscopy using the hexacyanoferrate(II/III) system as an electroactive probe. The response to bisphenol A was investigated by voltammetry. Compared to the unmodified GCE, the oxidation potential is positively shifted, and the oxidation peak current is strongly increased. Experimental conditions were optimized and resulted in an oxidation peak current that is linearly related to concentration of bisphenol A in the 10 nM to ~ 10 μM range. The detection limit is 8.0 nM (at S/N?=?3). The electrode was successfully applied to the determination of bisphenol A in leachates of plastic drinking bottles, and its accuracy was verified by independent assays via HPLC.

Kappa carrageenan-g-poly (acrylic acid)/SPION nanocomposite as a novel stimuli-sensitive drug delivery system

In this article, a novel triple-stimuli hydrogel was prepared by simultaneous formation of super paramagnetic iron oxide nanoparticles (SPION) and crosslinking of poly (acrylic acid) grafted onto kappa carrageenan (κC-g-PAA). The structure, thermal stability, surface morphology, and magnetic property of the κC-g-PAA/SPION hydrogel were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy with energy dispersive X-ray analysis (SEM–EDAX), transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), and vibrating sample magnetometer (VSM). Moreover, swelling capacity of the obtained hydrogel was measured at different temperature, pH, and magnetic-field to assess the sensitivity of κC-g-PAA/SPION hydrogel. This synthetic hydrogel was also examined as a controlled drug delivery system and defrasirox release was investigated at different temperature, pH, and magnetic-field. The in vitro antibacterial activity of κC-g-PAA/SPION hydrogel was studied against Escherichia coli and Staphylococcus aureus bacteria where the results showed no antibacterial activity of this new hydrogel. In vitro biocompatibility experiments were undertaken using human bladder epithelial cell line HTB 5637. These results indicated the synthesized κC-g-PAA/SPION hydrogel are nontoxic that will be useful for biomedical applications.

Bilayer lipid membranes from falling droplets

We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid–liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid–alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.

Bioelectrochemical probing of intracellular redox processes in living yeast cells—application of redox polymer wiring in a microfluidic environment

Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. “Wiring” of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione–PVI-Os double mediator system provides an effective means to conduct automated microbial assays.

Direct electron transfer and biocatalytic activity of iron storage protein molecules immobilized on electrodeposited cobalt oxide nanoparticles

Ferritin was immobilized on a glassy carbon electrode with electrodeposited cobalt oxide nanoparticles, and its direct electron transfer behavior was studied. It exhibits a pair of redox peaks due to direct electron transfer between ferritin and the nanoparticles. Electrochemical parameters including the formal potential (E^0??), the charge transfer coefficient (??), and the apparent heterogeneous electron transfer rate constant (k_s) were determined. The sensor displays excellent biocatalytic activity in terms of reduction of hydrogen peroxide, and this was applied to electrochemical sensing of hydrogen peroxide.