Amperometric immunosensor for diagnosis of BLV infection

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody-antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N',N'-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.     

Continuous-flow/stopped-flow system using an immunobiosensor for quantification of human serum IgG antibodies to Helicobacter pylori

Conventional methods, such as gastric biopsy, enzyme-linked immunosorbent assay (ELISA), culture, require a long time for the determination of Helicobacter pylori infections. This study reports an amperometric immunoreactor for rapid and sensitive quantification of human serum immunoglobulin G (IgG) antibodies to H. pylori. Antibodies in the serum sample are allowed to react immunologically with the purified H. pylori antigens that are immobilized on a rotating disk. The bound antibodies are quantified by horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to human IgG. HRP in the presence of hydrogen peroxide catalyzes the oxidation of hydroquinone to p-benzoquinone. The electrochemical reduction back to hydroquinone is detected on a glassy carbon electrode surface at -0.15 V. The electrochemical detection can be done within 1 min, and the analysis time does not exceed 30 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.6 and 1.9 U ml-1, respectively. The amperometric immunoreactors showed higher sensitivity and lower time consumed than did the standard spectrophotometric detection ELISA method. It can also be used for rapid analysis in conventional and field conditions in biological, physiological, and analytical practices.     

A carbon nanotube-based high-sensitivity electrochemical immunosensor for rapid and portable detection of clenbuterol

Carbon nanotubes have shown their unique advantages of mechanical, chemical and electronic properties in bioanalysis. We herein report a new method to efficiently and reproducibly prepare multi-walled carbon nanotubes (MWNTs)-protein sensing layers for electrochemical immunosensors. This method employs centrifugation to prepare a conjugate of MWNTs and goat anti mouse-immunoglobulin G (IgG) (secondary antibody). The conjugates were then deposited on screen-printed electrodes to form a nanostructured layer (MWNT-I layer). CLB monoclonal antibody was assembled through its binding to the secondary antibody. The MWNT-I layer-based electrodes were used for rapid and sensitive amperometric immunosensing detection of clenbuterol (CLB) in swine urine samples. Horseradish peroxidase-coupled CLB (CLB-HRP) competed with free CLB in the samples to bind the monoclonal antibody. It has shown significantly higher sensitivity and better reproducibility than the chemical conjugation method. This MWNT-based immunosensor is highly sensitive, leading to a limit of detection of 0.1 ng/mL within a rapid assay time of 16 min. Its sensitivity is at least 1 order of magnitude higher than that of a normal immunosensor (without MWNTs). The sensing device is portable with disposable screen-printed electrode, satisfactorily meeting the requirements for field detection of food security-related species.     

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.     

Amperometric immunosensor based on multiwalled carbon nanotubes/Prussian blue/nanogold-modified electrode for determination of α-fetoprotein

In this article, a conspicuously simple and highly sensitive amperometric immunosensor based on the sequential electrodeposition of Prussian blue (PB) and gold nanoparticles (GNPs) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrode (GCE) surface is proposed for the detection of α-fetoprotein (AFP). By comparison with PB, the MWCNT/PB composite film had been proven to show much better electrochemical stability and a larger response current. The electrodeposited GNP film can be used not only to immobilize biomolecules but also to avoid the leakage of PB and to prevent shedding of MWCNT/PB composite film from the electrode surface. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal experimental conditions, the proposed immunosensor for AFP was observed with an ultralow limit of detection (LOD) equal to 3 pg/ml (at 3δ), and the linear working range spanned the concentrations of AFP from 0.01 to 300 ng/ml. Moreover, the immunosensor, as well as a commercially available kit, was examined for use in the determination of AFP in real human serum specimens. More significant, the assay mentioned here is simpler than the traditional enzyme-linked immunosorbent assay (ELISA), and an excellent correlation of levels of AFP measured was obtained, indicating that the developed immunoassay could be a promising alternative approach for detection of AFP and other tumor markers in the clinical diagnosis.     

Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies by up‐converting phosphor technology‐based lateral‐flow assay

In this study, an up‐converting phosphor technology‐based lateral‐flow (UPT‐LF) assay was developed to detect severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies rapidly and specifically. SFTSV recombinant N protein (SFTSV‐rNP) was coated on analytical membrane for sample capture, up‐converting phosphor (UCP) particles were used as the reporter, the luminescence emitted by UCP particles was converted to a measurable signal by a biosensor. The performance of UPT‐LF assay was evaluated by testing 302 field serum samples by ELISA (enzyme‐linked immunosorbent assay), Western blotting and UPT‐LF assay. UPT‐LF assay exhibited a lower detection limit than ELISA, and a satisfied level of agreement was exhibited by Kappa statistics (Kappa coefficient = 0.938). Considering Western blotting as the reference for comparison, the sensitivity and specificity of UPT‐LF assay could reach 98.31% and 100%. UPT‐LF assay showed no specific reaction with hantavirus total serum antibodies, which avoids the misdiagnosis of SFTSV from hantavirus that could cause similar clinical symptoms. UPT‐LF assay was able to achieve acceptable results within 15 min and needed only 10 μL sample for each test. As a whole, UPT‐LF assay is a candidate method for on‐site surveillance of SFTSV total antibodies owing to its excellent sensitivity, specificity, stability, easy operation and for being less time consuming.     

Novel potentiometric immunosensor for hepatitis B surface antigen using a gold nanoparticle-based biomolecular immobilization method

A novel potentiometric immunosensor for the detection of hepatitis B surface antigen has been developed by means of self-assembly to immobilize hepatitis B surface antibody on a platinum disk electrode based on gold nanoparticles, Nafion, and gelatin as matrices in this study. The modification procedure of the immunosensor was further characterized by using cyclic voltammetry and the enzyme-linked immunosorbent assay (ELISA) method. The detection is based on the change in the electric potential before and after the antigen-antibody reaction. In contrast to the commonly applied methods (e.g., the glutaraldehyde crosslinking procedure), this strategy could allow for antibodies immobilized with a higher loading amount and better retained immunoactivity, as demonstrated by the potentiometric measurements. A dynamic concentration range of 4-800 ng ml(-1) and a detection limit of 1.3 ng ml(-1) were observed. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by ELISA. In addition, the technique presents some distinct advantages over the traditional sandwich format in that the analyzing performances are direct, rapid, and simple without multiple separation and labeling steps.     

Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

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.     

Amperometric immunosensor for the determination of IgA deficiency in human serum samples

An electrochemical immunosensor for the detection of human IgA deficiency in real human blood serum has been developed. The performance of the immunosensor presents a large but sensitive dynamic range that allows the determination of non-deficient IgA levels (>70 μg/mL) as well as of severe IgA deficiencies (0.5-5.0 μg/mL). The assay architecture involves the immobilisation of a coating antibody on an electrode surface using carboxylic-ended bipodal alkane-thiol self-assembled monolayers (SAMs). The long chain bipodal SAM presents intercalated poly(ethylenglycol) groups that confer the immunosensor the ability to retain its optimum performance in very complex matrices and serum with negligible non-specific adsorption phenomena. Amperometric optimisation of the assay resulted in limits of detection of 142 ng/mL in just 30 min total assay time. Real patients' serum samples were analysed using the developed electrochemical immunosensor demonstrating an excellent correlation in terms of sensitivity and reproducibility compared with standard enzyme linked immunosorbent assays (ELISA).     

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.     

重金属离子的免疫检测研究进展

农畜产品中残留的重金属离子已对人类安全构成严重威胁,急需快速、高效的重金属残留检测方法。重金属离子免疫检测是一种新型的检测方法,与传统检测方法相比,具有省时、省力、费用低廉、便于携带、易于操作等优点。除了化学螯合剂之外,植物螯合肽和金属硫蛋白也可用来制备重金属免疫原。重金属离子的免疫检测可分为多克隆抗体免疫检测和单克隆抗体免疫检测,前者包括荧光偏振免疫检测,后者包括间接竞争性ELISA、一步法竞争性免疫检测和KinExA免疫检测。作为一种辅助方法,胶体金快速免疫层析法可初步检测样品中的重金属离子浓度。     

委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立

目的:表达委内瑞拉马脑炎病毒E2重组蛋白,并结合胶体金免疫层析技术建立一种简便检测委内瑞拉马脑炎病毒特异性抗体的方法。方法:利用已经构建的表达E2抗原的工程菌, 用IPTG诱导, 表达蛋白主要以包涵体的形式存在。通过一系列条件的变性、复性、透析,所制抗原用以包被硝酸纤维素膜,利用胶体金标记和免疫层析技术,建立委内瑞拉马脑炎病毒快速检测方法。对该方法的敏感性、特异性和稳定性作出评价。结果:重组工程菌可表达分子质量为 40 kDa的目的蛋白, 纯化后的蛋白质经SDS-PAGE显示纯度达95%以上。建立的检测方法可在20 min内完成检测。对症状相似及近缘的其他病毒进行检测,均无非特异反应。试纸条在37℃下保存2周,检测结果不变。该方法与R&;D公司商品化的ELISA试剂盒灵敏度检测无明显差异;对92份阴性血清进行检测,两种检测方法的符合率为96.7%。结论:重组委内瑞拉马脑炎病毒蛋白产生的包涵体变性复性后生具有良好的重复性和稳定性, 可作为委内瑞拉马脑炎病毒多种检测方法的抗原原料。胶体金免疫层析法具有快速、灵敏、特异、稳定的特点,适用于现场检测。     

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.     

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.     

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.     

CRP determination based on a novel magnetic biosensor

The c-reactive protein (CRP) is a very significant human blood marker for inflammatory processes and is routinely determined for many clinical purposes. The widespread and well established detection method for this approximately 115 kDa hepatic protein is the high-sensitivity ELISA assay (hsCRP-ELISA) in blood serum. New approaches in medical CRP diagnosis (e.g. for CVD, inflammatory bowel disease) require rapid quantification in native matrices. A novel CRP determination method based on magnetic detection is described and tested for human blood serum, saliva and urine. The detection principle is based on two different anti-CRP antibodies (monoclonal, IgG) for CRP trapment and labelling. The linear detection range of this immunosensor ranged from 25 ng/ml to 2.5 microg/ml and is therefore much more sensitive than typical hsCRP-ELISA-assays.     

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.     

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.