Disposable immunosensor for the determination of domoic acid in shellfish

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.     

Development of an immunochromatographic strip test for the rapid detection of okadaic acid in shellfish sample

A rapid detection technology for okadaic acid (OA) in shellfish with one-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe was developed. OA is one of the diarrhetic shellfish toxins. Firstly, OA was conjugated to bovine serum albumin, and the conjugations as immunogen were injected into mice to raise the polyclonal antibody against OA. Hybridoma cells fused between spleen cells from immunized mouse and myeloma cells (Sp2/0) were prepared and injected into mice intraperitoneally at 1?×?10⁶?cells to produce monoclonal antibody in the ascitic fluid. With the monoclonal antibody against OA, the idc-ELISA assay was established to detect OA. The calibration curve for OA was linear over the concentration range of 0.31–50 ng mL^?1, and the detection limit for OA was 0.45 ng mL^?1. On that basis, paper test strips for detecting OA were prepared, and a fast detection method for okadaic acid using gold-labeled immunological assay was established. With the paper test strips, the detection limit was 6.25 ng mL^?1, and whole detection process for OA in shellfish samples needed only about 40 min.     

A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid in shellfish products

A lateral flow immunochromatographic (LFIC) test strip based on a colloidal gold-monoclonal antibody (McAb) conjugate was developed for on-site rapid detection of okadaic acid (OA) in shellfish. It applies a competitive format using an immobilized toxin conjugate and free toxin present in samples. The McAb against OA was conjugated with 20-nm colloidal gold as detector reagent. The toxin in the sample competed with the immobilized toxin to bind to the gold conjugated with McAb. The colloidal gold/McAb/toxin mobile complex was not captured by OA-bovine serum albumin (BSA) on the test line, but it was captured by goat anti-mouse immunoglobulin G (IgG) on the control line. The color density of the test line correlated with the concentration of toxin in the range of 10-50 ng ml(-1). The qualitative detection limit of 150 μg kg(-1) sample was close to the European Union (EU) regulatory limit (160 μg kg(-1)). Therefore, these strips were able to directly and qualitatively estimate the consuming safety of shellfish. They require no equipment because of available visual results, and they screened numerous samples within 10 min. The results were further confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). As a food safety screening tool, the test strips are convenient and useful to rapidly on-site test the presence of OA in shellfish products.     

A mediatorless and label-free amperometric immunosensor for detection of h-IgG

A novel experimental methodology for studying a mediatorless and label-free immunosensor is proposed by immobilizing antibody on gold nanoparticle/L-cysteine coated electrode (nano-Au/L-cysteine electrode). Differential pulse voltammograms (DPV) resulting from the assembled immunosensor indicate that the immunosensor shows excellent electrochemical response to dopamine so that the electrochemical response is utilized for the signal generation step of the immunosensor. Therefore, by means of unenzymatic-labeling procedure combined with the amperometric detection using dopamine as substrate, the immunological reaction can be detected. After the immunosensor is incubated with h-IgG solution, the access of electrocatalytic behavior center of the immunosensor to dopamine is partly inhibited, which leads to a linear decrease in amperometric response of the immunosensor with h-IgG concentration over a range 0.82-90 ng mL(-1) by DPV.     

Highly sensitive amperometric immunosensor for the detection of Escherichia coli

With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH₂-anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole (Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl β-d-galactopyranoside (PAPG), the bacterial enzyme, β-d-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC) electrode at 0.22 V. The operational procedure consists in first adding the test sample containing the bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte (being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates an improved detection limit of more than 10 times less bacteria detected than other immunosensing techniques without the need for multi step pretreatments of the test sample and/or incubation as found in some of the existing methods.     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.     

Development of an amperometric immunosensor for detection of staphylococcal enterotoxin type A in cheese

This paper reports a novel electrochemical immunosensor for the sensitive detection of staphylococcal enterotoxin A (SEA) based on self-assembly monolayer (SAM) and protein A immobilization on gold electrode. Three different methods of protein A immobilization were tested: physical adsorption, cross-linking using glutaraldehyde and covalent binding after activation with N-hydroxysuccinimide (NHS)/N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) on cysteamine-modified gold electrode. The EDC/NHS method for protein A immobilization was selected to lead development of the biosensor. The coating steps of the surface modification were characterized by cyclic voltammetry and the biosensor response by chronoamperometry. The advantages of the immunosensor were exposed in its high sensitivity and specificity. The proposed amperometric immunosensor was successfully used for determination of SEA in contaminated and non-contaminated cheese samples with excellent responses.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain tissue

The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1μg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors.     

Carbon nanotube-based ultrasensitive multiplexing electrochemical immunosensor for cancer biomarkers

A multiplexing electrochemical immunosensor was developed for ultrasensitive detection of cancer related protein biomarkers. We employed disposable screen-printed carbon electrode (SPCE) array as the detection platform. A universal multi-labeled nanoprobe was developed by loading HRP and goat-anti-rabbit IgG (secondary antibody, Ab₂) onto multiwalled carbon nanotube (MWNT). This universal nanoprobe was available for virtually any sandwich-based antigen detection and showed superiority in several areas. By using the SPCE array and the universal nanoprobe, we could detect as low as 5 pg mL⁻¹ of prostate specific antigen (PSA) and 8 pg mL⁻¹ of Interleukin 8 (IL-8) with the electrochemical immunosensor. We also demonstrated simultaneous detection of two protein biomarkers with this platform. With these attracted features, our immunoassay system shows promising applications for in-field and point-of-care test in clinical diagnostics.     

Disposable amperometric immunosensor for the detection of polycyclic aromatic hydrocarbons (PAHs) using screen-printed electrodes

An amperometric immunosensor for polycyclic aromatic hydrocarbons (PAHs) was developed. The immunosensor was based on disposable screen-printed carbon electrodes. The coating antigen used was phenanthrene-9-carboxaldehyde coupled to bovine serum albumin (BSA) via adipic acid dihydrazide. Antibodies were monoclonal mouse anti-phenanthrene. The enzyme alkaline phosphatase (AP) was used in combination with the substrate p-aminophenyl phosphate (pAPP) for detection at +300 mV (vs. Ag/AgCl). Various assay types were compared. Good results were achieved with an indirect co-exposure competition assay with a LOD of 0.8 ng/ml (800 ppt) and an IC(50) of 7.1 ng/ml (7.1 ppb) for phenanthrene. An indirect competition assay could detect phenanthrene with a LOD of 2 ng/ml (IC(50): 15 ng/ml) and an indirect displacement assay with a LOD of 2 ng/ml (IC(50): 11 ng/ml) at a 5 microl surface coating of 8.8 microg/ml phenanthrene-BSA conjugate. A coating concentration of 2.2 microg/ml allowed detection with a LOD of 0.25 ng/ml (250 ppt) with the indirect competition assay. The influence of the coating concentration on the sensor performance was investigated. Cross-reactivities were tested for 16 important PAHs. Anthracene and chrysene showed strong cross-reactivity, whereas benzo[g,h,i]perylene and dibenzo[a,h]anthracene showed no cross-reactivity.     

Study on an amperometric immunosensor based on Nafion-cysteine composite membrane for detection of carcinoembryonic antigen

In this work, protonated l-cysteine was entrapped in Nafion (Nf) membrane by cation exchange function, forming Nf-Cys (cysteine) composite membrane, which was more stable, compact, biocompatible, and favorable for mass and electron transfer compared with Nf film solely. Then gold (Au) nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/ml with a detection limit of 3.3 pg/ml (signal/noise = 3) was observed. Moreover, the morphology of the modified Au substrates was investigated with atomic force microscopy, and the electrochemical properties and performance of modified electrodes were investigated by cyclic voltammograms and electrochemical impendence spectroscopy. The results exhibited that the immunosensor has advantages of simple preparation, high sensitivity, good stability, and long life expectancy. Thus, the method can be used for CEA analysis.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

A label-free amperometric immunosensor for fast and sensitive assay of human serum chorionic gonadotropin (hCG) is presented. hCG was immobilized on nanoporous gold (NPG) foils and using hydroquinone (HQ) redox species as indicator. The variation of amperometric response to the concentration of hCG, the target antigen, was evaluated by cyclic voltammetry in phosphate-buffered solution. Taking advantage of dual-amplification effects of the NPG foils and graphene sheets (GSs), the immunosensor exhibited a specific response to hCG in the range of 0.5–40.00 ng ml⁻¹ with a detection limit of 0.034 ng ml⁻¹ under optimal conditions. It was demonstrated that our proposed method possesses good accuracy, acceptable precision, and reproducibility. The NPG showed a better sensitizing effect and stability as immobilization matrices.     

A novel amperometric immunosensor based on acetone-extracted propolis for the detection of the HIV-1 p24 antigen

A novel amperometric immunosensor for the detection of the p24 antigen (p24Ag) from HIV-1 was constructed using gold nanoparticles (GNP), multi-walled carbon nanotubes (MWCNTs), and an acetone-extracted propolis film (AEP). First, amino-functionalized MWCNTs (MWCNTNH?) were prepared and dispersed in an HAuCl? solution to synthesize GNPs in situ. Next, the GNP/CNT/AEP nanocomposite was prepared by mixing an AEP solution and the GNP/CNT powder. The nanocomposite was dripped onto a gold electrode (GE), and then p24 antibody (anti-p24 Ab) was immobilized on the resulting modified gold electrode to construct the immunosensor. The assembly process was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The factors that were likely to influence the performance of the proposed immunosensor were studied in detail. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to the presence of p24 in a concentration range of 0.01 to 60.00 ng/mL, with a relatively low detection limit of 0.0064 ng/mL (S/N = 3). Moreover, the proposed immunosensor showed a rapid (≤ 18 s) and highly sensitive amperometric response (0.018 and 1.940 μA/ng/mL) to p24 with acceptable stability and reproducibility.     

Graphene-assisted dual amplification strategy for the fabrication of sensitive amperometric immunosensor

A sensitive electrochemical immunosensor with graphene-assisted signal amplification has been developed. In order to construct the base of the immunosensor, a novel hybrid architecture was initially fabricated by combining poly (diallyldimethylammonium chloride) functionalized graphene nanosheets (PDDA-G) and gold nanoparticles (AuNPs) via a simple sonication-induced assembly. The formed hybrid architecture provided an effective matrix for antibody immobilization with good stability and bioactivity. Subsequently, a smart, multilabel, and graphene-based nanoprobe that contains gold nanoparticles functionalized exfoliated graphene oxide and horseradish peroxidase-secondary antibodies was designed for constructing a novel sandwiched electrochemical immunosensor. Enhanced sensitivity was obtained by combining the advantages of high-binding capability and excellent electrical conductivity of hybrid architecture with the multilabel signal amplification. On the basis of the dual signal amplification strategy of graphene-based architecture and the multilabel, the immunosensor displayed excellent analytical performance for the detection of human IgG (HIgG) range from 0.1 to 200 ng/mL with a detection limit of 0.05 ng/mL at 3σ. Moreover, the proposed method showed good precision, acceptable stability and reproducibility, and could be used for the detection of HIgG in real samples. Therefore, the present strategy definitely paves a way for the wide application of graphene in clinical research.     

Application of an amperometric immunosensor for the enumeration of Nitrobacter in activated sludge

A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments. Received: 4 October 1999 / Received revision: 20 February 2000 / Accepted: 25 February 2000     

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods

Summary A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 30⁴ cfu/ml, and at ca. 10⁵ cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperomatric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.Offprint requests to: R. G. Kroll