Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2 pg mL(-1) with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples.     

Blood plasma biomarkers of citrinin and ochratoxin A exposure in young adults in Bangladesh

Citrinin (CIT) and Ochratoxin A (OTA) are nephrotoxic mycotoxins which can co-occur in food commodities, resulting in internal exposure. Studies in many countries reported on the presence of OTA in human blood; however, such biomonitoring data for CIT is still scarce. This study was conducted to characterize both CIT and OTA biomarker levels in plasma of volunteers since food analysis data are insufficient to assess human exposure in Bangladesh. In total 104 blood samples were collected from university students in 2013 (sampling 1: n?=?64, midsummer) and 2014 (sampling 2: n?=?40, end winter) for analysis of CIT and OTA and their metabolites HO-CIT and OTα by LC-MS/MS and HPLC-FD techniques, respectively. CIT and HO-CIT were detected in 90% (max 2.70 ng/mL) and 85% (max 1.44 ng/mL) of all samples. Mean levels in sampling 2 (CIT 0.47 ng/mL; HO-CIT 0.40 ng/mL) were higher than in sampling 1 (0.25 ng/mL; 0.37 ng/mL) indicative of variable CIT exposure. OTA was present in all (max 6.63 ng/mL) and OTα in 98% (max 0.99 ng/mL) of the samples. In sampling 1, mean OTA (0.85 ng/mL) was higher than in sampling 2 (0.51 ng/mL); the reverse situation was found for OTα mean levels. The calculated dietary OTA intake among the students (mean 9.9; max 91.7 ng/kg bw/week) was lower than the tolerable weekly intake for this mycotoxin (120 ng/kg bw/week) set by EFSA. But frequent co-exposure to CIT should be considered, and the results of this study indicate the necessity to identify major sources of CIT and OTA intake in the Bangladeshi population.     

Ochratoxin A blood concentration in healthy subjects and bladder cancer cases from Pakistan

Astract  The mycotoxin ochratoxin A (OTA) is a public health issue in many countries. Data on OTA concentrations in foods and in blood are available for several European countries including the Balkan area, as well as for Canada and Japan. Yet, for developing countries such data are scarce. In this study we determined OTA blood levels as biomarker of exposure in bladder cancer patients and in healthy controls from Pakistan. OTA in blood was analyzed after extraction by HPLC with fluorescence detection (limit of detection: <0.03 ng/mL) in 96 patients and in 31 controls. Over 92% of all blood samples (87 patients, 30 controls) contained quantifiable amounts of OTA: The mean OTA concentrations were 0.33 ng/mL (SD 0.42; range: 0.03 to 3.41 ng/mL) in bladder cancer patients, and 0.31 ng/mL (SD 0.29; range: 0.04 to 1.25 ng/mL) in healthy controls. These OTA concentrations are comparable to those reported for the general population in the European Union. Presented at the 27^th Mykotoxin-Workshop, Dortmund Germany, done 13–15, 2005. The IfADo is accredited as WHO Cellaporating Center for Occupational Health.     

A Simple and Sensitive Approach for Ochratoxin A Detection Using a Label-Free Fluorescent Aptasensor

Ochratoxin A(OTA) is found to be one of the predominant contaminating mycotoxins in a wide variety of food commodities. To avoid the risk of OTA consumption, the detection and quantitation of OTA level are of great significance. Based on the fact that ssDNA aptamer has the ability to form a double-strand structure with its complementary sequence, a simple and rapid aptamer-based label-free approach for highly sensitive and selective fluorescence detection of OTA was developed by using ultra-sensitive double-strand DNA specific dyes PicoGreen. The results showed that as low as 1 ng/mL of OTA could be detected with a dynamic range of more than 5 orders of magnitude which satisfies the requirements for OTA maximum residue limit in various food regulated by European Commission. With the specificity of aptamer, the assay exhibited high selectivity for OTA against two other analogues (N-acetyl-l-phenylalanine and zearalenone). We also tested the aptasensor practicability using real sample of 1% beer spiked with a series of concentration of OTA and the results show good tolerance to matrix effect. All detections could be achieved in less than 30 min, which provides a simple, quick and sensitive detection method for OTA screening in food safety and could be easily extend to other small molecular chemical compounds detection which aptamer has been selected.     

Aptamer-based depletion of small molecular contaminants: A case study using ochratoxin A

Based on the increasing demand for detection and depletion of small molecules like mycotoxins or pesticides in food, water, or pharmaceuticals, aptamers are gaining more importance as sensitive, specific-depletion molecules. Here, we present an aptamer-based method for depletion of ochratoxin A (OTA) as a model system and show the advantages and the limitations of aptamers in the depletion of small molecular contaminants. OTA is a mycotoxin produced by various Penicillium and Aspergillus strains and is often found in grain and grain derivatives. We immobilized a well-described DNA aptamer against OTA on an agarose gel and used the column as a clean-up system. The aptamer shows a high specificity and sensitivity for OTA: Ochratoxin B, a molecule similar to OTA, was not bound by the aptamer; and a control oligonucleotide was not able to bind OTA. After optimizing the process for better economic feasibility, the column could be used for several times without loss of aptamer activity. We investigated the location of immobilized aptamer within the gel using fluorescent-labeled aptamers. Furthermore, beer samples spiked with OTA were used to investigate aptamer activity in complex samples. Using these complex samples we have observed a significant loss of aptamer activity. We have further investigated this limitation by performing microscale thermophoresis experiments to determine the KD values of the aptamer in different complex samples like beer, coffee, juice and wine. Our results indicate that the applicability of aptamers to real processes is currently restricted by the selection buffer used during its selection process (SELEX). We therefore suggest using conditions closer to those of the later application of the aptamer during future SELEX experiments.     

Simultaneous analysis of ochratoxin A and its major metabolite ochratoxin alpha in plasma and urine for an advanced biomonitoring of the mycotoxin

Ochratoxin A (OTA) is a frequent mycotoxin contaminant found worldwide in foods and feedstuffs. Biomonitoring has been used to assess internal OTA exposure resulting from dietary intake and from other sources. Mycotoxin levels in blood and/or urine provide good estimates of past and recent exposure since OTA binds to serum proteins and is also partly excreted via the kidney. But, measuring OTA alone does not reflect its biotransformation. In light of scarce data on its metabolites in humans, it was the aim of this study to develop a method that allows analysis of OTA and its detoxication product ochratoxin alpha (OTα) in urine and in blood plasma. The method involves enzymatic hydrolysis of conjugates, liquid–liquid extraction, and analysis of sample extracts by liquid chromatography with fluorescence detection. Application of the validated method in a pilot study with 13 volunteers revealed the presence of OTA and OTα in all samples (limit of quantification: 0.05 ng/mL in urine, and 0.1 ng/mL in plasma). In line with negative findings of others, an OTA glucuronide was not detected, neither in urine nor in plasma. By contrast, conjugates of OTα (glucuronide and/or sulfate) are major products in these samples. This was confirmed by mass spectrometry detection. As OTα represents a large fraction of ingested mycotoxin, we propose to include analyses of this metabolite in future biomonitoring studies, also in light of the observed variations for urine OTα-levels that suggest different interindividual abilities for OTA-detoxification in humans.     

Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A

In this work, a new signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamer complementary (DNA2) were immobilized onto a magnetic bead (MB). In the presence of OTA, DNA2 was dissociated and released from the MB. The released DNA2 then hybridized with DNA3, which was linked at the 5' terminus of the amplification template and can extend along the template in the presence of Phi 29 DNA polymerase. The formed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI and produced a short single-stranded DNA. The cleaved DNA strand generated a new site by Phi 29 DNA polymerase and the process of extension and cleavage was cyclical. Thus, a amount of the short single-stranded DNA were produced. Using DNA and ABEI labeled carboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-stranded DNA could be sensitively detected. The CL intensity (ΔI) versus the concentration of OTA was linear in the range from 1.0×10(-12) to 5.0×10(-8)gmL(-1). The detection limit was 3.0×10(-13)gmL(-1), and the RSD was 3.4% at 1.0×10(-10)gmL(-1) (n=7). The developed method has been applied to detect OTA in naturally contaminated wheat samples. Due to its simplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CL bioassay offers a promising approach for the detection of OTA and other biomolecules.     

Development of an immuno-affinity column for ochratoxin analysis using an organic solvent-tolerant monoclonal antibody

Two kinds of monoclonal antibodies (MoAbs), OCA-10A and OCA-1B, were prepared based on their specificity to ochratoxin A (OTA) and ochratoxin B (OTB) and on their tolerance to 40% methanol. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) value of OCA-10A was 27ng/mL for OTA and 17ng/mL for OTB, and that of OCA-1B was 28ng/mL for OTA and 13ng/mL for OTB. Immuno-affinity columns (IACs) using these MoAbs were prepared with agarose gel beads. The IAC with OCA-1B showed a NaCl-dependent binding ability to OTA and OTB, while interestingly, the IAC with OCA-10A bound to them without NaCl. The IAC with OCA-10A showed a high methanol tolerance when compared with existing IACs, as expected from the high methanol tolerance of OCA-10A itself. Such tolerance was maintained for the application of the cocoa extract with 70% methanol and the wheat extract with 60% acetonitrile, while the tolerance was slightly altered by interference from the cocoa extract. Examinations with organic solvents at higher concentrations than the allowable level in existing IACs showed that OTA and OTB spiked with wheat, cocoa and red wine could be purified with high recovery. The newly developed IAC is expected to show sufficient clean-up ability for food analyses.     

A new method for the detection of the H5 influenza virus by magnetic beads capturing quantum dot fluorescent signals

A new method to detect H5 influenza virus using quantum dots (QDs) and magnetic beads (MBs) is described. QDs conjugated with oligonucleotide probes were used to produce fluorescent signals and MBs, that were also conjugated with probes, were used to isolate and concentrate the signals. Target viral RNAs led to a sandwich hybridization between the functionalized QDs and MBs. One-step hybridization facilitated the subtype determination. As little as 0.1 ng viral RNA could be detected.     

Low levels of ochratocin A in wines from Piedmont

Ochratoxin A (OTA) is a mycotoxin mainly produced by a number of species of Aspergillus, commonly found in warm and tropical climates. OTA poses risks for the human health because of its nephrotoxic, teratogenic, immunotoxic and neurotoxic activity. The mycotoxin, classified as possible human carcinogen (Group 2B) by the IARC, naturally occurs in a wide range of foods, including wine, where the main producer is A. carbonarius. The aim of this work was the validation of a procedure for the analysis of OTA in Piedmontese red and white wines produced after vintage 2003 and 2004, in relationship with the limit of 2.0 microg l(-1) introduced by European Union for wine, must or grape juice (Regulation CE N. 123/2005). An analytical method based on immunoaffinity column (IAC) for clean-up and liquid chromatography with fluorescence detection (LC-FLD) was used to determine the occurrence of OTA in wines. Detection limit (LOD) and quantification Limit (LOQ) were 7.18 pg/ml and 9.31 pg/ml based on statistical method (IUPAC). Average recoveries of OTA from wine samples spiked at levels from 0.1 to 10 ng/ml ranged from 90.8% to 92.4%, with relative standard deviations (RSDs) between 2.64 and 2.71%. Repeatability limit was 8.73 pg/ml for samples spiked with 0.1 ng/ml of OTA. Ninety-one Denomination of Controlled Origin (DOC) wines were analysed, including 41 Barbera (red), 38 Dolcetto (red), and 16 white wines, such as Erbaluce, Cortese and Roero Arneis. The study focused on wines commercialized in Italian supermarkets and wine shops. The white wines resulted, as expected, less contaminated than the red ones. Wines produced after vintage 2003, a season particularly conducive to the growth of A. carbonorius, contained higher levels of OTA than the wines produced in 2004. The samples, resulting positive, contained a concentration of OTA highly inferior to the threshold limits introduced by the European Union. The sample of the highest level of OTA was a Dolcetto produced in 2004, with 1.10 ng/ml of mycotoxin.     

基于纳米颗粒的赭曲霉毒素A高灵敏酶联免疫检测方法的建立及应用

赭曲霉毒素(ochratoxins)主要是由青霉菌Penicillium和曲霉菌Aspergillus产生的有毒次级代谢产物,常见于发霉或发酵的农产品中,其中赭曲霉毒素A(ochratoxin A,OTA)毒性最强且最为普遍。OTA是粮食作物和饲料的重要污染物,在加工、储存或运输过程中均可产生,具有肾毒性和免疫毒性,可通过蓄积作用发挥毒性效应,对人类和动物健康造成严重威胁。本研究通过将OTA单克隆抗体包被于纳米磁珠(magnetic nanoparticles,MNPs)表面,获得具有免疫活性的磁珠抗体复合物(MNPs-Anti OTA),并制备生物素标记的偶联抗原OTA-BSA-Bio,后续采用链酶亲和素标记的纳米金颗粒(Strep-HRP-AuNPs)催化底物进行信号检测,最终建立了OTA高灵敏检测方法(MNPs-bs-AuNPs-ELISA)。在最优条件下,经计算该方法检测下限(IC10)为0.01ng/mL,检测区间(IC20-IC80)为0.02-0.73ng/mL,半数抑制率(IC50)为0.13ng/mL。与OTA类似物OTB、OTC交叉反应性为4.3%和8.1%,对其他常见真菌毒素AFB1、ZEN、FB1、DON、CIT和PAT均无交叉反应。玉米、面粉和大豆样本中的加标回收率可达85.6%-115.7%,对天然样本中OTA含量的检测结果表明,该方法与LC-MS/MS相关性良好。本研究建立的MNPs-bs-AuNPs-ELISA可满足谷物及饲料样本中OTA的快速、高灵敏度定量检测,成本较低,具有很好的应用前景。     

Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels

A novel and sensitive immunoassay for the simultaneous detection of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in food samples was developed by using artificial antigen-modified magnetic nanoparticles (MNPs) as immunosensing probes and antibody functionalized upconversion nanoparticles (UCNPs) as signal probes. NaY_0.78F₄:Yb_0.2, Tm_0.02 and NaY_0.28F₄:Yb_0.7,Er_0.02 UCNPs were prepared and functionalized, respectively, with immobilized monoclonal anti-AFB₁ antibodies and anti-OTA antibodies as signal probes. Based on a competitive immunoassay format, the detection limit for both AFB₁ and OTA under optimal conditions was as low as 0.01 ng mL⁻¹, and the effective detection range was from 0.01 to 10 ng mL⁻¹. The proposed method was successfully applied to measure AFB₁ and OTA in naturally contaminated maize samples and compared to a commercially available ELISA method. The high sensitivity and selectivity of this method is due to the magnetic separation and concentration effect of the MNPs, the high sensitivity of the UCNPs, and the different emission lines of Yb/Tm and Yb/Er doped NaYF₄ UCNPs excited by 980 nm laser. Multicolor UCNPs have the potential to be used in other applications for detecting toxins in the field of food safety and other fields.     

Specific detection of DNA using quantum dots and magnetic beads for large volume samples

Here we present a sensitive DNA detection protocol using quantum dots (QDs) and magnetic beads (MBs) for large volume samples. In this study, QDs, conjugated with streptavidin, were used to produce fluorescent signals while magnetic beads (MBs) were used to isolate and concentrate the signals. The presence of target DNAs leads to the sandwich hybridization between the functionalized QDs, the target DNAs and the MBs. In fact, the QDs-MBs complex, which is bound using the target DNA, can be isolated and then concentrated. The binding of the QDs to the surface of the MBs was confirmed by confocal microscopy and Cd elemental analysis. It was found that the fluorescent intensity was proportional to concentration of the target DNA, while the presence of non-complementary DNA produced no significant fluorescent signal. In addition, the presence of low copies of target DNAs such as 0.5 pM in large volume samples up to 40 mL was successfully detected by using a magnet-assisted concentration protocol which consequently results in the enhancement of the sensitivity more than 100-fold.     

Simply amplified electrochemical aptasensor of ochratoxin A based on exonuclease-catalyzed target recycling

A new "signal-on" aptasensor for ultrasensitive detection of Ochratoxin A (OTA) in wheat starch was developed based on exonuclease-catalyzed target recycling. To construct the aptasensor, a ferrocene (Fc) labeled probe DNA (S1) was immobilized on a gold electrode (GE) via Au-S bonding for the following hybridization with the complementary OTA aptamer, with the labeled Fc on S1 far from the GE surface. In the presence of analyte OTA, the formation of aptamer-OTA complex would result in not only the dissociation of aptamer from the double-strand DNA but also the transformation of the probe DNA into a hairpin structure. Subsequently, the OTA could be liberated from the aptamer-OTA complex for analyte recycling due to the employment of exonuclease, which is a single-stranded DNA specific exonuclease to selectively digest the appointed DNA (aptamer). Owing to the labeled Fc in close proximity to the electrode surface caused by the formation of the hairpin DNA and to the analyte recycling, differential pulse voltammetry (DPV) signal could be produced with enhanced signal amplification. Based on this strategy, an ultrasensitive aptasensor for the detection of OTA could be exhibited with a wide linear range of 0.005-10.0ngmL(-1) with a low detection limit (LOD) of 1.0pgmL(-1) OTA (at 3σ). The fabricated biosensor was then applied for the measurement of OTA in real wheat starch sample and validated by ELISA method.     

Development of DNA Aptamers to a Foot-and-Mouth Disease Peptide for Competitive FRET-Based Detection

We sought to develop a novel competitive fluorescence resonance energy transfer (FRET)-aptamer-based strategy for detection of foot-and-mouth (FMD) disease within minutes. A 14-amino-acid peptide from the VP1 structural protein, which is conserved among 16 strains of O-serotype FMD virus, was synthesized and labeled with Black Hole Quencher-2 (BHQ-2) dye. Polyclonal FMD DNA aptamers were labeled with Alexa Fluor 546-14-dUTP by polymerase chain reaction and allowed to bind the BHQ-2-peptide conjugate. Following purification of the FRET–aptamer–peptide complex, a “lights off” response was observed within 10 minutes and was sensitive to a level of 25–250 ng/mL of FMD peptide. Ten candidate aptamers were sequenced from the polyclonal family. The aptamer candidates were screened in an enzyme-based plate assay. A high- and low-affinity aptamer candidate were each labeled with Alexa Fluor 546-14-dUTP by asymmetric polymerase chain reaction and used in the competitive FRET assay, but neither matched the sensitivity of the polyclonal FRET response, indicating the need for further screening of the aptamer library.     

基于纳米磁珠和双标记抗体的玉米赤霉烯酮快速、高灵敏检测方法的建立及应用

玉米赤霉烯酮(zeralenone,ZEN)具有雌激素活性,主要污染谷物和饲料,大量聚积可导致流产和死胎,给动物和人类健康带来严重威胁。本研究通过将ZEN偶联抗原ZEN-BSA包被于纳米磁珠(magnetic nanoparticles,MNPs),制备纳米磁珠-偶联抗原复合物(MNPs-BSA-ZEN),同时使用金颗粒(Au nanoparticles,AuNPs)和辣根过氧化物酶(horseradish peroxidase,HRP)双标记的ZEN单克隆抗体,建立新型酶联免疫检测方法(MNPs-HRP-AuNPsIC-ELISA)。检测下限(IC10)达到0.03ng/mL,检测区间(IC20–IC80)为0.05–0.89ng/mL,半数抑制率(IC50)为0.22ng/mL,与ZEN类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol和β-zearalanol)的交叉反应性依次为19.2%、11.7%、8.3%、1.2%和4.3%,与黄曲霉毒素B1、赭曲霉毒素A、伏马毒素B1、桔青霉素和展青霉毒素几乎不存在交叉反应。在玉米、面粉和大豆样本中的加标回收率可达81.6%–113.5%,与LC-MS/MS同时对天然样本中ZEN含量的检测结果表明,两种方法相关性良好。本研究建立的MNPs-HRP-AuNPs IC-ELISA具备快速和高灵敏的双重优势,也可为其他霉菌毒素精准检测技术的开发提供参考。     

Airborne mycotoxins in dust from grain elevators

Workers in grain elevators are exposed to grain dust and may therefore have an increased risk of inhalatory contact with mycotoxins. To study the mycotoxin burden of such environments, settled grain dust samples (n=35) were collected from several locations of a total of 13 grain elevators in Germany, and analysed for ochratoxin A (OTA, detection limit 0.01 ng/g), deoxynivalenol (DON, detection limit 15 ng/g), and zearalenone (ZEA, detection limit 6 ng/g), respectively. Cytotoxicity of these samples was assessed by a MTT bioassay with a swine kidney target cell line. Additionally, the airborne dust concentration of these locations was determined. Nearly all settled dust samples contained OTA (96%), DON (100%), and ZEA (100%) with median concentrations of 0.4 ng/g, 416 ng/g, and 126 ng/g, respectively. Cytotoxic effects in varying degrees from weakly to highly toxic were caused by crude extracts of 86% of the dust samples. However, cytotoxicity did not correlate with mycotoxin levels in these samples and thus indicated the presence of cytotoxic compounds of unknown origin. Based on the mycotoxin findings in settled dust samples and the airborne dust concentrations, the average airborne mycotoxin concentrations were estimated to be 0.002 ng/m³ (OTA), 2 ng/m³ (DON), and 1 ng/m³ (ZEA), respectively. The relevance of these findings for occupational health was assessed by comparison with WHO recommendations for the maximum tolerable daily (oral) intake (TDI). Even in a worst case scenario, the calculated inhalatory intake was far below the TDI values. However, considering the uncertainties resulting from different exposure pathways, namely oral ingestion versus inhalation, further research should primarily address the problem of how adequate assessment criteria for airborne exposure to mycotoxins could be established. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Exposure of neonates to ochratoxin A: first biomonitoring results in human milk (colostrum) from Chile

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OTα) were determined in milk and blood from nine lactating women who provided samples soon after delivery at a hospital in southern Chile. The analytical method applied liquid–liquid extraction with chloroform, and in the case of blood, an extra purification with solid phase extraction prior to HPLC analysis with fluorescence detection. OTA was detected in all human milk samples, with an average concentration of 106?±?45 ng/L (range 44–184 ng/L). Levels of OTα were 40?±?30 ng/L (LOQ 40 ng/L), but increased considerably upon enzymatic hydrolysis with ß-glucuronidase/sulfatase (up to 840?±?256 ng/L) in human milk. By contrast, there was no evidence for conjugates of OTA. The data on OTA in breast milk and levels reported in blood from women in Chile are indicative of an efficient lactational transfer of the mycotoxin. Infant exposure to OTA was estimated by considering their daily OTA intake with human milk at early stages of nursing. For the majority of milk samples, the calculated OTA intake of infants exceeded the tolerable daily intake (TDI) of 5 ng/kg body weight (bw)/day proposed by the Nordic Expert Group, and infant exposure approached the provisional tolerable doses of 14–16 ng/kg bw/day suggested by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) and by EFSA for adults. The present study documents and confirms the presence of OTA in human milk at levels where the TDI can be exceeded. These results point out the need to continue food and biological monitoring and to develop strategies, e.g. dietary recommendations to pregnant and lactating women, aimed to reduce OTA exposure in early periods of life.     

Chitosan/polyaniline hybrid conducting biopolymer base impedimetric immunosensor to detect Ochratoxin-A

Chitosan (CS)-polyaniline (PANI) hybrid conducting biopolymer film was obtained on indium-tin-oxide (ITO) electrode using electrochemical polymerization process. Fourier transform infrared (FT-IR) spectra of PANI-CS had showed covalent and hydrogen binding between PANI and CS molecules. Electrochemical impedance spectroscopy (EIS) measurements had showed low charge transfer resistance (R(CT)) of PANI-CS and PANI. Successive rabbit antibody (IgGs) immobilization on PANI-CS, CS and PANI matrixes surface were confirmed with FT-IR and EIS measurements. Ochratoxin-A (OTA) interaction with IgGs had increased R(CT) values and showed linear response up to 10 ng/mL OTA concentration in electrolyte. Relative change in R(CT) was higher in PANI-CS due to higher proportion of carboxylic and hydroxyl functionalities at PANI-CS matrix surfaces. The absolute sensitivity of PANI, CS, and PANI-CS were 16+/-6, 22+/-9 and 53+/-8 Omega mL/ng, respectively derived from slope of linear response up to 10 ng/mL with 1 ng/mL minimum detection limit.     

Ochratoxin A Analysen im Blut von Arbeitnehmern in der Abfallwirtschaft

Tasks such as manual sorting of domestic wastes for recyclable goods and the deposition of various materials may result in inhalation of mycotoxin-containing aerosols. Ochratoxin A (OTA) was analyzed in blood samples from workers employed at waste handling facilities in Southern Germany to assess the potential impact of this mycotoxin, and explore its use as a biomarker of exposure to bioaerosols. Results from this analysis are reported: OTA serum levels (median values) in subgroups of workers involved in waste deposition (n=76 ‘Deponierer’) or in waste sorting (n=60 ‘Wertstoffsortierer’) were 0.36 and 0.53 ng/ml, respectively. Both groups are natives of countries within the European Community (EU). In waste sorters who were born in other European (non-EU) countries (n=72) or elsewhere (n=12 from Asia, Africa), the OTA serum levels were 0.50 and 0.37 ng/ml, respectively. In controls (n=84 office clerks at the facilities; EU citizens) the median OTA value was 0.39 ng/ml. Comparing the different groups, and previously published data on median OTA levels in the general population (0.21 ng/ml) which result from dietary (background) exposure to OTA in Germany, our data point to an additional uptake of this mycotoxin by inhalation in workers with exposure to bioaerosols. The results support the view that apart from the pathogenic and allergological relevance of microbial emissions from garbage, secondary fungal metabolites, and thus toxicological aspects, deserve further attention.

Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003