Cadmium ion-doped magnetic poly(styrene-acrylic acid) nanospheres for sensitive electrochemical immunoassay

A novel class of molecular tags, cadmium ion-doped magnetic poly(styrene-acrylic acid) nanospheres (Cd-MPSA), was first synthesized and functionalized with polyclonal rabbit anti-human luteinizing hormone antibodies (PAb(2)) for highly efficient electrochemical immunoassay of luteinizing hormone (LH). Transmission electron microscope (TEM) and Fourier transform infrared spectroscope (FTIR) were employed to characterize the prepared Cd-MPSA. By using Cd-MPSA-labeled PAb(2) as molecular tags, a novel sandwich-type immunoassay protocol was built for determination of LH on monoclonal mouse anti-human luteinizing hormone antibody (MAb(1))-functionalized gold electrode. The assay was carried out in pH 5.3 HAc-NaAc buffer solution by square wave voltammetry (SWV). The signal was obtained by the reduction of the doped cadmium ions in the Cd-MPSA. Under optimal conditions, the currents increased with the increasing LH level in the sample, and exhibited a linear range from 0.25 to 240 mIU mL(-1) with a detection limit of 0.08 mIU mL(-1) LH at 3s(B). The precision, reproducibility, and specificity were acceptable. No obvious difference was encountered in the analysis of spiking LH samples into newborn calf serum with the referenced values.     

Production of ultrasensitive antibodies against aflatoxin B1

AIMS: To produce specific antibodies against the haptenic fungal toxin aflatoxin B1 (AFB1) and apply these antibodies in immunochemical assays for aflatoxins. METHODS AND RESULTS: Rabbits were immunized using an AFB1-bovine serum albumin conjugate and serum titres determined by double-antibody enzyme immunoassay. High titres of antibodies with very high affinity for AFB1 were obtained 15 and 4 weeks after the initial immunization and the first booster immunization respectively. The antibodies were employed in enzyme immunoassay (EIA) and immunoaffinity chromatography (IAC) methods for aflatoxins. With a detection limit of 15.8 pg ml(-1) for AFB1, the EIA employing these antibodies is the most sensitive test for AFB1 described so far. In IAC columns, these antibodies provided high binding capacity for all major aflatoxins, including AFB1, AFB2, AFG1 and AFG2. CONCLUSION: The antibodies described here are useful for the analysis of trace levels of aflatoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyclonal antibody-based EIA and IAC methods for aflatoxin analysis offer a suitable alternative to the more expensive monoclonal antibody-based methods.     

The chemiluminescence immunoassay for aflatoxin B1 based on functionalized magnetic nanoparticles with two strategies of antigen probe immobilization

A rapid and sensitive chemiluminescence immunoassay (CLIA) based on magnetic nanoparticles (MNPs) was developed to detect aflatoxin B1 (AFB1), which is a potent carcinogen in nature. We prepared monodisperse MNPs (300 nm in diameter) according to the solvothermal synthesis reaction and the MNPs were coated with silica by the Stöber method. Triethox was used as a one‐step carboxylation reagent, and 3‐aminopropyltriethoxysilane (APTES) an amination reagent, to modify the MNPs. We prepared two types of solid phase antigens using the above synthesized functionalized MNPs coupled with the later prepared AFB1‐oxime active ester and the purchased BSA–AFB1 respectively. 2′,6′‐dimethylcarbonylphenyl‐10‐sulfopropylacridinium‐9‐carboxylate 4′‐N‐hydroxysuccinimide (4′‐NHS) ester (NSP–DMAE–NHS), as a novel luminescent reagent, was used to label anti‐AFB1 antibodies. The two CLIA calibration curves based on the two types of solid phase antigens were obtained and compared. The acquired limit of detection (LOD) was about 0.001 ng/mL for the two functionalized MNPs‐based immunoassays, and the half maximal inhibitory concentration (IC₅₀) was 0.51 ng/mL for the MNPs–AFB1‐based method and 0.72 ng/mL for the MNPs–BSA–AFB1‐based method.     

Degradation of aflatoxin B1 during anaerobic digestion and its effect on process stability

The effect of aflatoxin B1 (AFB1) on an anaerobic digestion process (AD) was studied. Batch anaerobic digestion trials were performed with both non-contaminated AFB1 corn grain (Control A) and contaminated-AFB1 corn grain at different doses (AFB1 contents of 0.54, 66.2, and 110 μg kg⁻¹ wet weight). Both cumulative biogas production and the degradation rate of AFB1 were studied. Results indicated that no adverse effects on AD were detected during the processes which could be attributed to the presence of AFB1. AFB1 degradation ranged from 69% to 87% of the total initial AFB1 content.Anaerobic digestion trials using Completely Stirred Tank Reactors (CSTR) were also carried out, comparing the biogas production of a mix of contaminated corn grain plus pig slurry (AFB1 content of 7.2 μg kg⁻¹ wet weight) with a mix of non-contaminated corn grain plus pig slurry (Control B). No adverse effect of AFB1 on biogas production was detected. The CSTR trial resulted in an average degradation of AFB1 of 42%. The further storage of the digestate for 30 days resulted in an overall degradation (CSTR plus storage) of AFB1 of 61% of the starting content.     

Reactivity of aflatoxin B2a antibody with aflatoxin B1-modified DNA and related metabolites.

Aflatoxin B2a (AFB2a) antiserum has been previously used in an enzyme-linked immunosorbent assay (ELISA) for the quantitation of AFB1 and AFB2a. The present investigation examined the reactivity of the antiserum toward those adducts and metabolites of AFB1 believed to play a major role in aflatoxicosis and carcinogenesis. 2,3-Dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua), the putative 2,3-(N5-formyl-2-2', 5',6'-triamino-4-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-FAPyr), 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol), AFB1-N7-Gua-modified DNA, and AFB1-FAPyr-modified DNA were prepared by in vitro incubation or chemical methods and subjected to competitive AFB2a ELISA. The antiserum showed significant reactivity with all five compounds, indicating that it had a high degree of specificity for both the cyclopentenone and the methoxy group of the parent aflatoxin molecule. Sensitivity for AFB-N7-Gua-modified DNA, AFB1-FAPyr-modified DNA, and AFB1-diol by the ELISA method was 0.1 pmol per assay. To test the applicability of immunological detection of covalent binding of AFB1 to DNA, the ELISA was compared with a conventional radioisotopic assay in two in vitro studies. The results showed that estimates of the kinetics and substrate dependence of covalent binding to calf thymus DNA in rat microsomal incubation mixtures by both methods were comparable. The broad specificity AFB2a antibody might be of considerable value in the detection of AFB1 macromolecular adducts and related metabolites in epidemiological investigations or in the diagnosis of aflatoxicosis.     

Impedimetric nanoimmunosensor platform for aflatoxin B1 detection in peanuts

This article developed a novel electrochemical immunosensor for the specific detection of aflatoxin B1 (AFB1). Amino-functionalized iron oxide nanoparticles (Fe₃O₄-NH₂) were synthesized. Fe₃O₄-NH₂ were chemically bound on self-assembly monolayers (SAMs) of mercaptobenzoic acid (MBA). Finally, polyclonal antibodies (pAb) were immobilized on Fe₃O₄-NH₂-MBA. The sensor system was evaluated through atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). A reduction in the anodic and cathodic peak currents was observed after the assembly of the sensor platform. The charge transfer resistance (R_ct) was increased due to the electrically insulating bioconjugates. Then, the specific interaction between the sensor platform and AFB1 blocks the electron transfer of the [Fe(CN)₆]^3−/4− redox pair. The nanoimmunosensor showed a linear response range estimated from 0.5 to 30 μg/mL with a limit of detection (LOD) of 9.47 μg/mL and a limit of quantification (LOQ) of 28.72 μg/mL for AFB1 identification in a purified sample. In addition, a LOD of 3.79 μg/mL, a LOQ of 11.48 μg/mL, and a regression coefficient of 0.9891 were estimated for biodetection tests on peanut samples. The proposed immunosensor represents a simple alternative, successfully applied in detecting AFB1 in peanuts, and therefore, represents a valuable tool for ensuring food safety.     

Effect of glutathione and uridine 5'-diphosphoglucuronic acid on mutagenesis by benzo[a]pyrene and aflatoxin B1 in the Salmonella/microsome assay

In the Salmonella/microsome plate or liquid assay, the addition of glutathione (GSH) and uridine 5'-diphosphoglucuronic acid (UDPGA), both cofactors for GSH-S-transferases or UDPGA-transferases, altered the rat-liver microsome-mediated mutagenesis of benzo[a]pyrene (BP) and aflatoxin B1 (AFB). With either BP or AFB, an increased, unchanged or decreased number of revertant colonies of S. typhimurium was observed, depending on the substrate concentration, the source of rat-liver 9000 X g supernatant (S9), the time of incubation and the type of mutagenicity test (liquid or plate assay). Several factors responsible for quantitative changes in the pattern of BP and AFB metabolites under various assay conditions in vitro, which alter the overall mutagenic activity of the parent compound, are discussed.     

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.     

抗黄曲霉毒素B1单克隆抗体的制备及特性

用杂交瘤技术制备了5株产生抗黄曲霉毒紊B₁单克隆抗体的杂交瘤细胞株。对其中之一AFB1－2H8进行了较系统的研究。AFB1一2H8属IgC3。纯化腹水抗体效价约5×10⁶。ELISA检测标准毒素的线性范围为0．5～50ng／ml。最低检出量为0．01ng／ml。该单抗与参试的其它黄曲霉代谢物的交叉反应系数为0～0．21,该抗体有较大的应用价值。     

Fiber-optic immunosensor for mycotoxins

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.     

Effect of phenolic antioxidants on the mutagenicity of aflatoxin B1.

The Ames assay employing Salmonella typhimurium TA100 and TA98 was used to investigate potential interactions between aflatoxin B1 (AFB1) and the phenolic antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate. AFB1 doses were within the linear response range, and the antioxidants were used at levels of 0 to 50 micrograms per plate. All three antioxidants were nonmutagenic in either bacterial tester strain, with or without the hepatic S-9 enzyme preparation; toxic effects were observed at doses higher than 20 micrograms per plate. Butylated hydroxytoluene and butylated hydroxyanisole substantially increased AFB1-induced mutagenesis in the two tester strains with microsomal activation. The addition of 5 to 20 micrograms of butylated hydroxytoluene or hydroxyanisole to 5 to 20 ng of AFB1 per plate caused more than a twofold increase in the number of His+ revertants. Addition of propyl gallate resulted in only a moderate increase in the number of revertants. Whereas several anticarcinogenic and antimutagenic effects by phenolic antioxidants have been reported, particularly in studies with polycyclic aromatic hydrocarbons, the enhancement of mutagenic potency of AFB1 by these compounds suggests a specificity with respect to the chemical nature of AFB1.     

Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.     

Tetramethyl-6-carboxyrhodamine quenching-based aptasensing platform for aflatoxin B1: Analytical performance comparison of two aptamers

In this study, a simple TAMRA (tetramethyl-6-carboxyrhodamine) quenching-based aptasensing platform was designed for the detection of aflatoxin B1 (AFB1). Here, we compared the analytical performance of two aptamer sequences: seqA and seqB. The AFB1 detection was based on the interactions of FAM (carboxyfluorescein)-labeled aptamer with TAMRA-labeled DNA complementary strand in the presence and absence of target analyte. Under optimized experimental conditions, TAMRA-labeled strand quenched the fluorescence response of FAM-labeled aptamer due to the noncovalent interaction between the two DNA strands. The binding of AFB1 induced the complex formation and weakened the interaction between FAM-labeled aptamer and TAMRA-labeled complementary strand, resulting in the fluorescence recovery. By using this principle concept, an assay was constructed for the detection of AFB1. The method exhibited good sensitivity, good selectivity with a limit of detection of 0.2 ng ml⁻¹, and a wide linear range from 0.25 to 32 ng ml⁻¹. For real sample application, the aptasensors were tested in beer and wine samples, with good recovery rates obtained for AFB1 detection.     

Electrochemical immunosensor based on Pd–Au nanoparticles supported on functionalized PDDA-MWCNT nanocomposites for aflatoxin B1 detection

This paper reports a label-free electrochemical immunosensor for the determination of aflatoxin B1 (AFB1), which is based on a gold electrode modified by a biocompatible film of carbon nanotubes/poly(diallyldimethylammoniumchloride)/Pd–Au nanoparticles (CNTs/PDDA/Pd–Au). The nanocomposite was characterized by transmission electron microscopy and the electrochemical behavior of modified electrodes was investigated by cyclic voltammetry. The CNTs/PDDA/Pd–Au nanocomposites film showed good electron transfer ability, which ensured high sensitivity to detect AFB1 in a range from 0.05 to 25 ng mL⁻¹ with a detection limit of 0.03 ng mL⁻¹ obtained at 3σ (where σ is the standard deviation of the blank solution, n = 10). The proposed immunosensor provides a simple tool for AFB1 detection. This strategy can be extended to any other antigen detection by using the corresponding antibodies.     

Development of immunochromatography strip-test using nanocolloidal gold-antibody probe for the rapid detection of aflatoxin B1 in grain and feed samples

An immunochromatography (ICG) strip test using a nanocolloidal gold-antibody probe was developed and optimized for the rapid detection of aflatoxin B1 (AFB1). A monoclonal antibody specific to AFB1 was produced from the cloned hybridoma cell (AF78), coupled with nanocolloidal gold, and distributed on the conjugate pad of the ICG strip test. The visual detection limit of the ICG strip test was 0.5 ng/ml, and this method showed a cross-reaction to aflatoxin B2, G1, and G2. In total, 172 grain and feed samples were collected and analyzed by both the ICG strip test and HPLC. The results of the ICG strip test showed a good agreement with those obtained by HPLC. These results indicated that the ICG strip test has a potential use as a rapid and cost-effective screening tool for the determination of AFB1 in real samples and could be applied to the preliminary screening of mycotoxin in food and agricultural products, generating results within 15 min without complicated steps.     

Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts

An ultrasensitive immunochromatographic (IC) assay for simultaneous detection of total aflatoxins (AFB1, AFB2, AFG1, and AFG2) was developed to meet the requirement for rapidly monitoring aflatoxins in agro-products. The assay was based on a competitive format and its sensitivity was improved by using a novel criterion to screen the optimal amount of monoclonal antibody (MAb) labeled to nanogold particles. The visual detection limits (VDLs) for aflatoxins B1, B2, G1, and G2 in peanut matrix were 0.03, 0.06, 0.12, and 0.25 ng mL(-1), respectively, which were lower than those of published literatures. The results of IC assay were in good agreement with those of high performance liquid chromatography (HPLC) in the analysis of aflatoxins in peanuts, demonstrating the practical applicability of the developed assay in real samples. This qualitative test based on the visual evaluation of results did not require any equipment. Overall, to our knowledge, this is the first report of qualitative detection for total aflatoxins by immunochromatographic assay.     

假单胞菌胞外酶降解黄曲霉毒素B1的酶学性质

[背景]黄曲霉毒素B1(AflatoxinB1,AFB1)毒性强、污染普遍,目前尚无有效的防治办法.[目的]为了发掘高效的AFB1降解菌并探索其降解特性,对红树林污泥样品中一株AFB1降解菌株(HAI2)的酶学性质进行分析.[方法]以AFB1结构类似物为唯一碳源,筛选出一株高效的AFB1降解菌,利用16SrRNA基因测...     

Development of aflatoxin B(1)-lysine adduct monoclonal antibody for human exposure studies

Mouse monoclonal antibodies were developed against a synthetic aflatoxin B(1) (AFB)-lysine-cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(lambda). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB > aflatoxin M(1) > aflatoxin Q(1). IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when (3)H-AFB-lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and (3)H-AFB-lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer.     

一种新的检测黄曲霉毒素B1的酶生物传感器的制作

本文报道了一种新的检测黄曲霉毒素B1的生物传感器,该传感器以开管的多壁纳米碳管固定化黄曲霉毒素氧化还原酶制作传感电极检测黄曲霉毒素B1,其线性范围达到0.16μM-3.2μM,当把特异性的黄曲霉毒素B1抗体与黄曲霉毒素氧化还原酶通过多壁纳米碳管共固定化制作修饰电极,传感器的检测限提高到16nM,灵敏度提高了10倍。用这种方法制作黄曲霉毒素酶生物传感器,使黄曲霉毒素酶生物传感器向实用化迈进了一步。