Biosensors for Detection of Ochratoxin A

Conference paper
Part of the NATO Science for Peace and Security Series A: Chemistry and Biology book series (NAPSA)

Abstract

Mycotoxins such as ochratoxin A, aflatoxin B and others are dangerous food contaminants that usually occur in trace amounts from nanograms to micrograms per gram of food. Therefore high sensitive methods are necessary for their detection. The conventional methods such are high-performance liquid chromatography (HPLC), mass spectroscopy are rather expensive and time consuming, therefore biosensor technology is rather promising for rapid detection of toxicants in field conditions, far from specialized laboratories. Among biosensors based on affinity of monoclonal antibodies or DNA aptamers to mycotoxins are of special interest, because provide sensitivity of detection that is better than allowable quantities of toxicants in food. While antibodies are traditional receptors in biosensors, aptamers are novel biopolymers with the affinity comparable to that of antibodies. However in contrast with antibodies, aptamers are more stable and the biosensors based on DNA aptamers can be regenerated which allowing their multiple use. This contribution reviews recent achievements in development affinity biosensors for detection ochratoxin A.

Keywords

Mycotoxin Ochratoxin A Antibodies DNA aptamers Biosensors 

10.1 Introduction

The natural toxins, such are mycotoxins, abrin, ricin, saxitoxin, palytoxin, batrachotoxin, botulinum neurotoxin type A, mycrocystin–RC represent considerable ­hazard for health and could be considered as potential warfare agents [1]. Among ­mycotoxins the ochratoxin A (OTA) is of special interest. OTA belongs to toxical fungal metabolites that can occur in primary food products. OTA is produced by Aspergillus ochraceus and Penicillium verrucosum and generally appears during improperly storage of cereals, coffee, cocoa, dried fruit, pork etc. and occasionally in the field of grapes. It may also be present in blood and kidneys of animals that have been fed on contaminated feeds. In a blood OTA is bound to the serum proteins and is redistributed to various tissues. The most susceptible to OTA are kidneys. It has been shown, that accumulation of OTA in this organ causes acute and chronic lesions by affecting the anion transport [2]. This molecule causes also other toxicological effects including hepatotoxic, neurotoxic, teratogenic, immunotoxic. The toxicity of OTA is in particularly connected with inhibition of protein synthesis because it competes with phenylalanine in the cells that utilizing this amino acid. Animal studies indicated that OTA is carcinogenic [3]. This is connected with the genotoxicity of OTA because it induces oxidative stress in the cells [2, 4], oxidative base damage [5] and the cleavage of single stranded DNA [6, 7]. OTA interacts also with double stranded DNA (dsDNA), but the damage of double helix has not been observed [8]. The mechanism of OTA-induced cancer is not clear yet. Moreover the contradiction results were reported in OTA behavior in vivo and in vitro conditions. While in vivo OTA is poorly metabolized [9], in vitro investigations suggest that a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine adducts are formed by electrochemical oxidation and photoreaction of OTA which may be the reason of OTA carcinogenicity [10].

Due to high toxicity of OTA, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) already in 1991 evaluated a provisional tolerable weekly intake (PTWI) of 112 ng/kg body weight (b.w.) for this mycotoxin. This evaluation was based on the porcine nephropathies data [3]. Most recently, the European Commission has fixed maximum concentration of OTA in foodstuffs: 3 μg/kg (7.4 nM) for cereal products, 5 μg/kg (12.4 nM) for roasted coffee and up to 10 μg/kg (25 nM) for instant coffee. Similar contamination limit was fixed for dry grapes (10 μg/kg) (EC No. 466/2001, 1881/2006), but 2 μg/L (5 nM) contamination limit is valid for wine (EC No. 123/2005). Even stronger limit of contamination by OTA was fixed for all food preparation for babies (0.5 μg/kg, EC No. 466/2001). The cereals, especially in countries with hot climate are contaminated by OTA ­produced by Aspergillus ochraceus. But in countries with colder climate the contamination is due to Penicillium verrucosum. The studies performed by Pittet [11] suggest that 25–40% of cereals are contamined by mycotoxins worldwide. Many works reports also on contamination of grape juices with approx 7 μg/L OTA (see [3] and references herein).

OTA is weak organic acid of a molar mass 403.8 g/mol. The chemical structure is presented on Fig. 10.1. It is a pentaketide derived from the dihydrocoumarins family coupled to β-phenylalanine.
Fig. 10.1

Chemical structure of ochratoxin A

So far 15 naturally occurred ochratoxins were identified. Most frequently appeared are ochratoxins A, B, C, from which OTA is more prevalent. The differences between ochratoxins are in some cases minor. For example replacement of Cl on H yields in ochratoxin B [3]. At neutral pH (pH  =  7) in a water OTA is negatively charged. This is due to ionization of carboxyl (pKa  =  4.2–4.4) and phenolic moieties (pKa  =  7.0–7.3) [12, 13]. Therefore both mono (OTA) and dianions of OTA (OTA2−) are present at physiological pH.

OTA contamination is typically in trace amount from ng to μg per gram of foodstuff. Therefore sensitive analytical methods of detection should be applied. At present the OTA detection is performed mostly by high-performance liquid chromatography (HPLC) with fluorescence detection (OTA posses natural fluorescence) [14], gas chromatography connected with mass spectrometry (GC-MS) [15], capillary electrophoresis [16], radioimmunoassay [17] or enzyme-linked immunosorbent assay (ELISA) [18, 19]. Association of Official Analytical Chemists (AOAC) official methods for determination of OTA in food are based on HPLC. However, traditional methods are rather expensive, time consuming and could be performed by qualified staffs only in specialized laboratories. In addition these methods usually require organic solvents for extraction toxines from food, which represent additional pollution for environment. ELISA belongs to the rapid detection techniques, however the disadvantage is in necessity of using enzyme-labeling reagents, which are expensive. Therefore, there is urgent requirement for direct, rapid, and low costs methods for OTA detection.

The biosensor technology fulfills the above-mentioned requirements. The ­biosensor is portable device consisting of sensing element, which has usually biological nature, for example antibody, enzymes, DNA, DNA aptamers, natural receptors. However, even the systems that mimic the biological structures, for example calixarenes incorporated into the lipid films can also be considered as belonging to these devices [20, 21]. The next part of the biosensor is transducer that transform usually chemical signal to the electrical, optical or mass. This signal is analyzed by separate instruments, for example potentiostats, network analyzers, spectrometers, surface plasmon resonance (SPR) etc. The tendency however exists in the integration of sensing element and transducer into one chip, such as field effect transistor. The principles of biosensor construction have been explained in many books and reviews (see for example [22, 23, 24]).

The label free detection of OTA can be based on its redox properties which allowing OTA detection at certain type of surfaces using electrical methods, such as cyclic, differential pulse or square wave voltammetry. However, more sensitive are the methods utilizing specific receptors, for example the enzymes, antibodies or DNA aptamers, immobilized at surfaces. This contribution reviews recent ­achievements in development biosensors for detection OTA and contains also results obtained in author’s laboratory.

10.2 The Biosensors for Detection OTA

10.2.1 OTA Oxidation and Its Detection by Amperometry

We already mentioned above that OTA carcinogenic effect may be connected with its oxidation, which causes appearance of reactive oxidation species in the cells. Oxidation of OTA is connected with its phenolic moiety [8, 25, 26]. It has been shown, that OTA can be oxidized at glassy carbon electrode (GCE) at specific conditions in organic solvents or aqueous media with pH between 6 and 8. Oxidation of OTA was studied also in presence of transition metal ions [10] and of a Fe-porphyrin system. In later case a hydroquinone species were detected by HPLC.

Detailed study of OTA oxidation at GCE at wide pH range (2–12) was performed by Oliveira et al. [8]. Using cyclic, differential pulse and square wave voltammetry they observed well resolved redox peaks. The peaks appeared at acidic conditions (pH  =  4.0) at potentials +1.05 and 0.45 V vs. Ag/AgCl reference electrode were used for analytical purposes. It has been shown that using square wave voltammetry it is possible to detect OTA with the limit of detection (LOD) 0.26 μM at optimal conditions. They also analyzed possible interferences with catechol, phenol and reveatrol, which significantly affected the OTA detection. Despite the fact that the LOD obtained by voltammetry at bare GCE is insufficient for practical applications, the method is useful for analysis of interaction of OTA with dsDNA. As mentioned above, authors observed interaction of OTA with dsDNA without causing its damage.

The electrochemical studies performed by Calcutt et al. [25] predicted that peroxidases could participate in OTA oxidation. This has been approved in work by Alonso-Lomillo et al. [27, 28] and used for detection OTA. Oxidation of horseradish peroxidase (HRP) at presence of hydrogen peroxide resulted in oxidation of OTA in aqueous solution. At the surface of screen printed carbon electrode (SPCE) at certain potential (around −0.3 V vs Ag/AgCl reference electrode) further ­oxidation of OTA took place. It is observed as an increase of anodic current in chronoamperometry experiment. It has been shown that immobilization of HRP has substantial effect on the sensor sensitivity. In Ref. [27] the HRP was immobilized at the surface of polypyrrol layer electropolymerized at SPCE. The OTA was in this case detected with LOD 0.25 nM (0.1 ng/mL). In most recent work the SPCE was prepared using carbon ink containing HRP [28]. However, this resulted worse detection properties of OTA (LOD 26.8  ±  3.6 nM). On the other hand in both cases the matrix effect of beer or roasted coffee was rather small and sensor revealed recovery 103% and 99%, respectively [27]. Rather high sensitivity in OTA detection was reported by Perrotta et al. [29]. They used square wave voltammetry for detection of OTA at the surface of gold electrode modified by cysteamine self-assembled monolayers. The detection and quantification limit was 4 ng/L and 12 ng/L, respectively. This is rather high sensitivity, but selectivity of this assay was not investigated,. They also performed test of the sensor in a red wine with a recovery ranging from 94% to 145%. However, without specific receptors the amperometric detection of OTA can not be considered as a selective method due to various potential interferences from electroactive species that are present in large amount in a real samples, for example ascorbic acid, phenolic compounds etc. Large (145%) value of recovery in ref. [29] suggests that such interferences are very likely.

10.2.2 Amperometry OTA Biosensor Based on ELISA

Immunochemical methods of detection are of high sensitivity and allowing detection of OTA at concentrations below the EC regulatory values. The most popular immunochemical method is ELISA. The conventional method is based on ELISA optical microplate reader using spectrophotometric detection. The detection is performed in a small volume (around 100 μL) in a well of microplate. Microplate containing usually large number of wells (typically 96), which allowing fast and simultaneous detection of several analytes. The product of enzyme reaction that posses absorbance or fluorescence signal is detected by optical reader. The detection can be indirect or direct and is based on competitive assay [30].

In indirect detection format usually 100 μL solution of OTA conjugated with bovine serum albumin (BSA) is added into the well and kept at 4°C overnight. Then 100 μL of 1% polyvinilalcohol (PVA) is added to block the microwells (1 h at 37°C). The anti-OTA IgG conjugated with enzyme alkaline phosphatase (AP) is added into the well in a sample containing unknown concentration of OTA. With increased amount of OTA in a sample less number of anti-OTA IgG is bounded to the OTA-BSA. AP transforms substrate 1-naphtyl phosphate into the product 1-naphtol (NP). The product absorbs light at wavelength 405 nm and thus can be detected by colorimetry. Therefore after addition of the substrate NP is detected. The amount of NP is indirectly proportional to the OTA.

In direct ELISA approx. 100 μL solution of anti OTA IgG is added into the well and incubated overnight at 4°C. The well is then blocked by PVA like in indirect assay. The OTA-AP conjugate is then added in a sample containing unknown concentration of OTA. OTA-AP will compete with free OTA in a sample. Thus, with increased concentration of OTA, less number of OTA-AP complexes will bind to anti OTA IgG. The concentration of OTA-AP is detected by the same method like in an indirect assay.

In a biosensor format the ELISA approach can be easily adapted for amperometric detection of the enzyme reaction. NP is electroactive and at certain potential (approx. 0.3 V vs. Ag/AgCl) is oxidized at SCPE into 1-iminoquionone. The scheme of indirect and direct electrochemical detection in ELISA format is presented on Fig. 10.2.
Fig. 10.2

The scheme of (a) indirect and (b) direct electrochemical ELISA assay (for explanation see the text)

The conventional colorimetry based ELISA has been used so far in a large number of works focused on detection various toxins, such are pesticides, marine toxins [31, 32], fungal toxins (aflatoxin, tricothecene) [33], OTA [34, 35, 36, 37, 38], ricin [39, 40] bacterial toxins [41, 42, 43, 44]. See also [1, 45] for review. Simultaneous enzyme assay for screening the aflatoxin and OTA was also reported [46]. In this assay monoclonal antibodies were immobilized onto the nitrocellulose membranes. The method allowed detection of aflatoxin and OTA with LOD 2 and 10 μg/kg for aflatoxin and OTA respectively and has been successfully used for determination these mycotoxin in chili samples. High sensitive indirect ELISA in a flow format with chemiluminiscence detection was reported in Ref. [47]. In this assay the OTA was immobilized at the glass plate using peptide linker. The LOD 0.01 μg/L (approx. 74 pM) belongs to the most sensitive reported so far. The method was useful for practical applications and has been approved for detection OTA in a roasted coffee.

The amperometric biosensors for detection OTA appeared only in 1987 when Aizawa [18] reported biosensor for detection OTA in ELISA like format. The sensor was composed of amperometric oxygen electrode and OTA covalently bound to a membrane that covered this electrode. The anti-OTA antibody labeled with enzyme catalase has been added in a fixed quantity to the sample solution and allowed to react competitively with the OTA immobilized at the membrane and with free OTA The catalase activity (production of oxygen) was inversely proportional to the ­concentration of OTA in a sample. The sensor allowed OTA detection with LOD 0.1 μg/L (0.25 nM, or 0.1 ppb).

In contrast with traditional methods such is HPLC in which the analyte to be detected is extracted from real sample using various solvents, the ELISA is different. The analyte is detected in a real sample. Therefore the matrix effect could affect the detection and should be therefore specially analyzed and detection assay optimized. The most sensitive methods detect OTA in real samples with sensitivity in μg/kg range in a batch [48, 49] or in a flow format [50]. Even 0.05 μg/kg LOD was reported for indirect immunoassay in microfluidic format with antibodies immobilised on magnetic nanoparticles. This assay was approved for detection OTA in apples [51]. High sensitivity in an indirect enzyme assay was reported in Ref. [52]. In this work the OTA-ovalbumin conjugates were immobilized on a gold colloid layer. The competitive amperometric detection was performed by addition of free OTA and specific antibodies conjugated by alkaline phosphatase with detection limit 8.2 ng/L and validated in a corn samples. The appearance of screen-printed electrodes that integrate the working electrode (carbon, gold, etc.) with reference Ag/AgCl and counter electrode resulted in further advantage of immobilization of the specific receptors [53, 54]. The recognition elements can be even entrapped into the ink during preparation of the sensing layer [28]. Optimization of immobilization and detection condition allowed detecting OTA with substantially improved sensitivity in a direct ELISA format with LOD up to 0.05 μg/L [30].

The enzyme based biosensors for detection OTA are rather useful, but still require enzyme conjugation and laborious preparation. Currently, only conventional ELISA kits are available at the market. But several hours are typically required for obtaining the result. Faster kits appeared recently (Veratox, Neogen, Lasing, USA) allowing detection of OTA within 30 min, but sensitivity is lower. Therefore there is attempt to develop another specific immunoassay that does not require application of antibody/enzyme conjugates. In this respect the focus is mostly on application of the surface plasmon resonance (SPR), quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS) for detection OTA at surfaces modified by specific antibodies. The overview of achievements in development of SPR, QCM and EIS based immunosensors is presented in next section.

10.2.3 Immunosensors Sensitive to OTA

In this part we will analyze the works focused on development SPR, QCM and EIS based immunosensors for detection OTA. The main advantage of these devices is lack of enzymes and in some cases possibility of direct detection of OTA.

SPR method belongs currently to the standard one for detection affinity interactions at surfaces. This is due to high sensitivity, selectivity, possibility of surface regeneration and due to existence of powerful commercial instruments; such are Biacore (Sweden) or Spreeta (Texas Instruments, USA). The principles of SPR are described in many textbooks and reviews (see for example [23]). SPR is based on generation of plasmons in a thin layer adjacent to the glass prism by irradiation of laser beam at certain angle of incidence. The angle of reflected beam shifts with increasing the thickness of sensing layer. Thus, measurement of this angle allowing determination of the surface concentration of adsorbed molecules as well as the thickness of the layer [55]. This method has been used for multiple detection of mycotoxins, including OTA within 25 min with sensitivity approx. 0.5 ppb (0.5 μg/L) [56]. SPR device combined with molecularly imprinted polymer method (MIP) allowed detection of OTA within a linear range from 0.05 to 0.5 mg/kg [57]. Yuan et al. [58] proposed interesting approach. They used IgG-gold nanoparticle conjugates for amplification the SPR signal following competitive interaction of OTA and monoclonal antibodies with the surface of specifically immobilized OTA. The indirect competitive assay was based on immobilization of OTA conjugated to BSA or OTA connected to polyethylene-based linker (PEG) to a Biacore chip. Simultaneous addition of OTA and monoclonal antibody specific to OTA (mAb) resulted in SPR signal due to binding of mAb to a surface. With increased concentration of OTA, more mAb bind to free OTA in comparison with that at the surface. Thus SPR signal was indirectly proportional to the OTA concentration. Approx. ten fold better detection limit was obtained for OTA immobilized to a surface through PEG linker (1.5 μg/L) in comparison with OTA-BSA conjugate. This has been attributed to higher density of OTA at surface. The PEG linker was also important in providing less nonspecific interactions of species with the sensor surface and allowed substantially improving surface regeneration (up to 600 binding cycles) using 5 M guanidine in 50 mM glycine (pH  =  2). The assay as well as surface regeneration was performed in a flow format (25 μL/min). Using gold nanoparticles of a diameter 40 nm conjugated to anti mouse IgG it was possible to amplify the SPR detection reaching LOD 0.042  ±  0.004 μg/L. The sensitivity is higher in comparison with requirements of allowable contamination of foodstufs indicated by food and environmental agencies. The method was successfully applied for detection OTA in oat, corn, white and red wine, grape and apple juice spiked samples. Despite rather high detection limit there is still disadvantage consisting in indirect assay and in necessity to use OTA-mAb conjugates.

Most recently the direct SPR biosensor for OTA detection was reported by Zamfir et al. [59]. The monoclonal antibodies specific to OTA were immobilized at SPR chip surface using magnetic beads. The addition of OTA resulted in increase of SPR angle. The detection limit obtained (0.94 μg/L) is higher in comparison with previous indirect approach, but still sufficient for practical applications.

QCM method belongs to powerful tools for analyzing the affinity interactions at surfaces. The method is based on using specially cutting quartz crystal (AT cut) and modification of its one side by sensing layer. At certain frequency, typically between 5 and 10 MHz the shear oscillation of the crystal takes place. The resonant frequency of the oscillations is indirectly proportional to the mass of the sensing layer [60]. It should be, however, note that in a liquid the crystal oscillations are affected by viscosity forces, which resulted in damping of the acoustic wave amplitude [61, 62]. However, in certain cases the measurement of only oscillation frequency can be useful for analytical purposes. The advantage of QCM is in much lower price of instrument in comparison with SPR and in easy operation. QCM immunosensor for detection OTA by indirect assay was reported in paper by Tsai and Hsieh [63]. They immobilized anti OTA Ab at the surface of 16-mercaptohexadecanoic acid (16-MHDA). Then the OTA conjugated with BSA in a solution containing free OTA in different concentrations was added to a QCM surface. This competitive assay exhibited a working range of 50–1,000 μg/L and a detection limit of 16.1 μg/L. The ­sensor was applied to several real samples with recovery in a range of 76–142%. The detection limit is lower in comparison with SPR method. However, ­amplification by nanoparticles could improve this.

EIS method has been so far extensively used for detection affinity interactions in biosensors [64]. It is based on high sensitivity of impedance to the surface modification. The sensitivity is substantially enhanced at presence of redox probe, for example [Fe(CN)6]−3/−4. At certain redox potential the probe provides enhanced electron exchange between the probe and the electrode surface. EIS allowing determination of the charge transfer resistance Rct that is measure of the intensity of charge ­transfer. Higher electron transfer corresponds to lower Rct and vice versa. The modification of the sensor surface resulted in changes of Rct. Addition of analyte also yields in Rct changes and may be affected by analyte charge. For example, because OTA is negatively charged at neutral pH, its adsorption to a sensor surface will result in repealing the redox probe. Thus, increased concentration of OTA will cause increase in Rct. This approach has been used in recently reported OTA immunosensors, which differ mostly in the method of immobilization of OTA specific antibodies. Chronologically, the EIS sensor based on OTA specific Ab immobilized on a surface of chitosan-polyaniline conducting polymer prepared by electropolymerization on a indium–tin-oxide (ITO) electrode was reported by Khan and Dhayal [65, 66]. The highest sensitivity of 1 μg/L was obtained, which is comparable with direct SPR assay. Shortly after these works Radi et al. [67] published paper in which Ab was immobilized on a gold surface using carbodiimide chemistry (LOD 0.5 μg/L). Rather high sensitivity (LOD 6 ng/L) was obtained with EIS immunosensor in which the OTA specific antibodies were immobilized on nanostructured zinc oxide deposited onto ITO covered glass plate [68] or using carbon nanotubes as immobilization matrix (LOD 2.5 nM/L) [69]. Comparable LOD (0.01 μg/L) was obtained in Ref. [58] in which Ab were immobilized by means of magnetic beads. Rather high sensitivity of EIS biosensor based on platinum electrode with electropolymerized sulfonated polyaniline with incorporated polyclonal anti OTA antibody was reported most recently [70]. The detection limit indicated 10 pg/kg, however seems to be overestimated considering the sensor response at the OTA concentrations studied 2–10 μg/L. The problem arises also with calibration curve presented in this paper as a plot of charge transfer resistance, Rct, vs OTA concentrations. While Nyquist plot indicates the decrease of Rct value, the calibration plot revealed opposite direction. From Nyquist plot it is also evident saturation of Rct at higher OTA concentrations, but in a corresponding concentration range the Rct value is presented as a linear plot vs, OTA content. In most works the analysis was performed also in real samples with minor matrix effect. However in certain cases the discrepancy between the concentrations of OTA in samples provided by vendor was substantially different in comparison with that determined by EIS biosensor and ELISA test. For example see [70] for detection OTA in roasted coffee and wheat. Thus, EIS immunosensors are rather useful and sensitive tools for detection OTA. Unfortunately in most cases the sensors were disposable without possibility of surface regeneration. Another disadvantage of immunosensors is necessity of rather laborious and expensive preparation of monoclonal antibodies and hapten conjugates. Antibodies are unstable in multiple uses. As an alternative the DNA aptamers are of high promising receptors in biosensing ­applications that could replace antibodies. In a next section the biosensors based on these novel biopolymers sensitive to OTA are presented.

10.2.4 Aptamer Based Biosensors for OTA Detection

Biosensors based on DNA aptamers (aptasensors) are of growing interest due to their high sensitivity and selectivity [71, 72, 73, 74, 75]. This is particularly due to unique properties of DNA or RNA aptamers – the single stranded nucleic acids with high affinity to proteins or to other low and macromolecular compounds, which is ­comparable with that of antibodies. In contrast with antibodies, aptamers are synthesized in vitro by the SELEX procedure [76, 77]. Aptamers are thermally stable, reusable and once selected they can be produced by chemical synthesis in necessary quantity by means of conventional oligonucleotide chemistry. Aptamers can be chemically modified by biotin, thiol or amino groups, allowing them to be immobilized on various solid supports. In contrast with antibodies aptamers are more stable and the aptasensors can also be regenerated. This opens new routes for construction of biosensors for practical applications, for example for diagnosis purpose in medicine or for detection toxicants in food or in the environment. Recently the DNA aptamer sensitive to OTA has been developed [78]. This aptamer has the following oligonucleotide sequence: 5′ GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA 3′. The analysis of this sequence using mfold program [79] shows two structures containing loops that slightly differ by Gibbs energy. Energetically more favorable structure (ΔG  =  −1.2 kcal/mol) contains loop between 16 and 28 nucleotides. Larger loop was found for second structure (ΔG  =  −0.88 kcal/mol). The analysis of the aptamer structure using the QGRS Mapper program predicting the existence of guanine quadruplexes [80] we showed that the OTA sensitive aptamer contains one guanine quadruplex connected by loops [81]. The existence of quadruplex is supported also by our recent data on the study of OTA aptamers thermodynamic properties [82]. The phase transition temperature for most stable aptamers at presence of 20 mM Ca2+ has been 48.3  ±  0.5°C, which is close to the melting temperature of DNA aptamers sensitive to fibrinogen binding site of thrombin that also contain one guanine quadruplex. For this aptamers the quadruplex structure has been well established using various methods including circular dichroism (CD) [83]. Most recently the existence of quadruplex in OTA aptamer has been approved by CD method [84]. The changes in Gibbs energy for OTA aptamers determined from melting data was 3.8 kcal/mol, which is higher in comparison with that obtained from mfold program. However, this program does not taking into account the quadruplexes, which are rather stable also thanks to stabilizing role of K+ ions.

The binding site for OTA in the aptamers is not known yet. However, as it has been shown in original work by Cruz-Aguado and Penner [78] this aptamer has rather high affinity and selectivity to OTA. The affinity substantially increases at presence of 20 mM Ca2+ (constant of dissociation KD  =  49 nM). At the same time, no binding of OTA was observed without calcium or magnesium ions. However, we have recently shown that OTA sensitive aptamers modified by thiol groups and immobilized at gold surface by chemisorption can bind OTA even when no Ca2+ is present. Moreover, the constant of dissociation is lower in comparison with that in solution, which is evidence of improved affinity properties of the aptamers at the surface [81].

According to WOS database the first aptasensor for OTA was reported most recently and utilized electrochemiluminiscence method of detection (LOD 17 pM) [85]. The sensitivity of chemiluminiscence method of detection OTA by aptasensor was recently substantially improved by using Fe3O4 based magnetic nanoparticles (MNPs) and upconversion nanoparticles (UCNPs) as sensitive labels [86]. The assay was based on immobilization of aptamer DNA 1 sequence onto the surface of MNPs, which allowed capturing and concentrating OTA from bulk samples. The aptamer DNA 1 sequence then hybridized with UCNPs modified with DNA 2 sequence, which could dissociate from DNA 1 and result in a decreased luminescent signal when aptamer DNA 1 recognized and bound to OTA. Under the optimal conditions, the decreased luminescent intensity was proportional to the concentration of OTA in the range of 0.1 ng/L to 1 μg/L with a detection limit of 0.1 ng/L (0.25 pM). This method allowed measurements of OTA in naturally contaminated maize samples.

Aptasensor utilizing amperometric detection based on methylene blue (MB) as a redox probe has also been reported [87]. In later case the effective detection range of OTA was 0.25–49.5 nM (sensitivity of detection: 74.3 pM). Such a high sensitivity has been achieved by signal amplification using gold nanoparticles. Three DNA oligonucleotides were used: 12-mer DNA 1 modified by amino group at 3′ end was immobilized to the activated surface of glassy carbon electrode (GCE). The aptamer, 36 – mer DNA 2, containing complementary part to DNA 1 was then added and allowed to hybridize with DNA 1. Finally, 12-mer DNA 3 thiolated at 5′ end containing complementary part at 3′ end and modified by gold nanoparticles at 5′ end was added and allowed to hybridize with DNA 2. Addition of MB, that selectively binds to guanine residues posses well resolved CV signal with two redox peaks at −0.23 and −0.18 V vs. saturated calomel electrode (SCE). Addition of OTA resulted in folding of the aptamers into 3D configuration and caused removing of the DNA 2 and DNA 3 from the sensor surface. Because MB was bounded mostly with DNA 2 this removal also caused decrease of the amplitude of redox peaks that served as analytical signal. The sensor selectively detected OTA in comparison with aflatoxin B. However slight interaction was observed with OTA analogue – ochratoxin B. Sensor was validated in a red wine with a good recovery in a range of 95–110%.

Further the electrochemical aptasensor based on indirect and direct competitive assay (LOD 0.27 nM) has been developed [88]. In an indirect assay the biotin-OTA conjugates were immobiližed on a surface of magnetic beads coated by streptavidin. The magnetic beads were attached to the SPCE by magnet. The competitive assay was performed by addition of various concentration of OTA in a buffer containing fixed concentration of OTA-sensitive aptamers conjugated with HRP. The presence of HRP was detected chronoamperometrically at presence of the H2O2, substrate of HRP at potential −0.2 V vs. Ag/AgCl electrode. The amplitude of current was ­proportional to a surface density of HRP and indirectly proportional to the concentration of OTA in a solution. The LOD in an indirect assay (1.1 μg/L) was similar to that obtained in competitive indirect immunoassay [89]. In a direct assay the aminated aptamers were immobilized in a magnetic beads coated by carboxylic acid using carbodiimide chemistry. The beads were attached to a surface of SPCE by magnet. Free OTA was added in a solution containing fixed concentration of AP-OTA conjugates. The surface density of AP was measured by differential pulse voltammetry (DVP) at presence of the non-electroactive substrate 1-naphtyl phosphate (1-NP). 1-NP has been dephosphorylated by AP into electroactive 1-naphtol, which was oxidized at electrode to 1-iminoquinone. Oxidation current was ­measured at the range 0–0.4 V vs. Ag/AgCl. Similarly like for indirect assay the amplitude of current was indirectly proportional to the concentration of OTA. The indirect assay was ten fold more sensitive in comparison with indirect one (LOD 0.11 μg/L or 0.27 nM). The sensitivity of this sensor was validated in spiked wine with a good recovery in a range 94–97%.

Slightly improved sensitivity (LOD 0.17 nM) has been obtained with aptasensor based on the similar magnetic beads technology, but instead of aminated the biotinylated aptamers were used for modification of streptavidin coated magnetic beads and HRP-OTA conjugates were used in a direct DVP assay [90]. In this work indirect and direct competitive assay has been used. The best sensitivity was obtained using direct competitive assay using streptavidin coated magnetic beads with immobilized biotinylated aptamers. (biotin has high affinity to streptavidin, KD  ∼ 10−15 M). The competitive binding of OTA and OTA conjugated with HRP (OTA-HRP) to the aptamers adsorbed at the magnetic beads surface took place. By means of magnet the beads were separated from unbounded compounds. Then the beads were immobilized at the surface of SPCE with help of magnet placed at the bottom part of the electrode. The detection of OTA was performed by DVP at the potential −0.125 mV vs. Ag/AgCl. At this potential the maximal current was observed due to the electron transfer between the electrode surface and the p-benzoquinon (p-BQ) – the product of enzymatic degradation of hydroquinon (HQ) (at presence of H2O2) (Fig. 10.3).
Fig. 10.3

The scheme of direct competitive assay based on biotinylated aptamers immobilized at magnetic beads covered by streptavidin. Competition between OTA and OTA-HRP was detected amperometrically by DVP method (for explanation see the text) [90]

The current was inversely proportional to the concentration of OTA. Authors also confirmed that at presence of 20 mM Ca2+ the signal increased by approx. 12% due to improved binding of OTA to the aptamers. At presence of Ca ions negatively charged OTA probably easier binds to the negatively charged aptamers. The binding of OTA was selective. Approx. 100 fold less binding took place for ochratoxin B and structural components of OTA – L-phenylalanine and warfarin. The sensor was validated in OTA containing wheat standard with recovery ranged from 102% to 104%. The sensor revealed higher sensitivity in comparison with immunosensor utilizing similar detection method [91]. The disadvantage of the sensor consisted in necessity of using enzyme conjugates as well as in possible non-specific interactions of conjugates with SPCE.

The direct, one step detection of OTA would be, however, more advantageous for practical applications. Most recently the simple colorimetric method of OTA detection has been reported [84]. In this work the gold nanoparticles were modified by OTA sensitive aptamers. Addition of OTA resulted in removal of the aptamers from the surface of nanoparticles and after addition of salts the changes in color has been observed due to nanoparticle aggregation. This method allowed detection of OTA with LOD of 20 nM.

In our recent work we reported acoustic aptasensor for detection OTA using biotinylated aptamers that have been adsorbed on a surface of a thin gold layer of quartz crystal transducer covered by neutravidin (neutravidin, similarly to streptavidin has very high affinity to biotin) (Fig. 10.4a). This sensor allowed direct detection of OTA with LOD 30 nM [82]. In this method we used network analyzer for measurement changes of series resonant frequency, ΔfS, and the so-called motional resistance, ΔRm. The later value is sensitive to the viscosity contribution due to the friction between the sensing layer and the buffer. This approach is important especially for detection molecules with relatively low molecular mass like OTA. For such molecules changes of thickness in a sensing layer are negligible. However due to negative charge, OTA can alter the surface properties. This has been certainly confirmed. Addition of OTA to the sensor surface resulted in increase of the Rm and decrease in resonant ­frequency (Fig. 10.4b). The shape of the frequency and resistance changes is typical for Langmuir adsorption isotherm. This means that the OTA binds to the aptamers independently. The binding of OTA to the aptamers can be quantitatively characterized by Langmuir equation [24]: For example for changes of Rm one can write:
$$ -\Delta {\rm{R}}_{\rm{m}}=-{\left(\Delta {\rm{R}}_{\rm{m}}\right)}_{\mathrm{max}}\left[\rm{c}/\left({\rm{K}}_{\rm{D}}+\rm{c}\right)\right]$$
(10.1)
where (ΔRm)max are the maximal changes of the motional resistance. The KD value is a measure of the affinity of OTA to the aptamers at the sensor surface. Using Eq. 10.1 and the least square method the KD value has been determined as KD  =  43.9  ±  30 nM. This value is in good agreement with that reported in Ref. [77] for free aptamers in a volume at presence 20 mM Ca2+ (fluorescence detection of OTA, KD  =  49  ±  3 nM). The limit of detection (LOD) for acoustic sensor was 30 nM. It has been determined using common criteria of significant analyte determination at the level corresponding to signal to noise ratio, S/N  =  3. The obtained LOD was comparable with QCM immunosensor based on indirect competitive detection method using OTA specific antibodies [63].
Fig. 10.4

(a) The scheme of immobilization biotinylated aptamers on a thin gold layer of the quartz crystal transducer covered by neutravidin. (b) The representative plot of the dependence of the changes of resonant frequency, Δfs, and motional resistance, ΔRm as a function of OTA for TSM sensor. Dashed line is the fit according to Langmuir isotherm (Eq. 10.1) [82]

Another approach for direct detection of OTA is based on EIS electrochemical aptasensors. The results in development of such aptasensor for detection OTA utilizing thiolated OTA specific aptamers chemisorbed on a gold surface have been presented by us in AISEM conference in February 2011 [92]. The approach is similar to those for EIS immunosensor at presence of redox probe [Fe(CN)6]−3/−4. As we mentioned above, the EIS method can sensitively monitor the changes of charge transfer resistance, Rct, due to the alterations at the sensor surface. The binding of negatively charged OTA to the aptamers resulted in increase of negative surface charge and in repealing of the redox probe from the surface. This causes increase in Rct value. At the same time other parameters of the circuit such are capacitance and Warburg impedance changed only slightly. The changes in Rct can serve as an analytical signal. This value sharply increased at relatively low OTA concentration range 0.1–3 nM with saturation at concentrations approx. 100 nM. This dependence had the shape of Langmuir isotherm and can be described by the equation analogical to that presented above (Eq. 10.1). The analysis of this dependence allowed to obtain KD  =  8.3  ±  0.8 nM [81]. This value is lower in comparison with that determined by fluorescence method for free aptamers in a volume [78] as well as that determined by acoustic method [82]. But in EIS sensor the aptamers have been immobilized by different method, using chemisorption, which may affect the aptamers affinity properties. This biosensor exhibited comparable sensitivity with above-mentioned indirect assay (LOD 0.4 nM) and selectively detected OTA. The sensor was regenerable in 1 mM HCl and successfully validated in coffee and flour with recovery of 88% and 104%, respectively for spiked samples containing 10 nM OTA [81, 92]. Most recently the EIS biosensor based on DNA aptamer specific to OTA covalently immobilized onto mixed Langmuir–Blodgett monolayer composed of polyanilyne-stearic acid and deposited on ITO coated glass plates has been reported [93]. This sensor revealed similar detection limit (0.24 nM), however the fabrication procedure has been more complicated in comparison with simple chemisorption used in our work. The sensor discriminated between OTA and aflatoxin, however it has not been validated in real food samples. In addition the Rct changes were of opposite direction, i.e. with increased OTA concentration the Rct value decreased at presence of redox probe. This is in contradiction with already published papers.

Thus, the aptasensors are of high perspective even for detection small molecules such are OTA. The sensitivity of detection in most cases is similar to those of antibodies. Substantial advantage of aptasensors is possibility of surface regeneration which allowing their multiple use. Moreover, recent papers on application SPR, thickness shear mode acoustic method and EIS are evidence of possibility of direct detection OTA without using OTA-protein conjugates or other labels.

10.3 Conclusion

The biosensor technology is certainly powerful tool for detection food mycotoxins such as ochratoxin A. The achievements reported in this review revealed that most of the approaches allowing detect OTA with high sensitivity, which is better than allowable contamination of food by this toxin. Rather perspective direction in biosensor development consisting in application of DNA aptamers. Using these novel biopolymers even most sensitive biosensor assay was demonstrated, allowing detection of OTA with LOD 0.25 pM. Direct detection methods such are acoustic and electrochemical impedance spectroscopy are rather challenging, due to fast response and high sensitivity which is substantial advantage over traditional methods such are HPLC or mass spectroscopy. We believe that further effort should result in appearance of low cost, portable and easy to use biosensor for detection OTA and other toxins applicable in food factories and agricultural farms.

Notes

Acknowledgments

The work was supported by the Slovak Research and Development Agency (contracts No. APVV-0410-10, LPP-0341-09).

References

  1. 1.
    Paddle BM (1996) Biosensors for chemical and biological agents of defence interest. Biosens Bioelectron 11:1079CrossRefGoogle Scholar
  2. 2.
    Ringot D, Chango A, Schneider Y-J, Larondelle Y (2006) Toxicokinetics and toxicodynamics of ochratoxin A, an update. Chem Biol Interact 159:18CrossRefGoogle Scholar
  3. 3.
    El Khoury A, Atoui A (2010) Ochratoxin A: General overview and actual molecular status. Toxins 2:461CrossRefGoogle Scholar
  4. 4.
    Gautier J-C, Holzhaeuser D, Markovic J, Gremaud E, Schilter B, Turesky RJ (2001) Oxidative damage and stress response from ochratoxin A exposure in rats. Free Rad Biol Med 30:1089CrossRefGoogle Scholar
  5. 5.
    Schaaf GJ, Nijmeijer SM, Maas RFM, Roestenberg P, de Groene EM, Fink-Gremmels J (2002) The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells. Biochim Biophys Acta 1588:149Google Scholar
  6. 6.
    Creppy EE, Kane A, Dirheimer G, Lafarge-Frayssinet C, Mousset S, Frayssinet C (1985) Genotoxicity of ochratoxin A in mice: DNA single strand break evaluation in spleen, liver and kidney. Toxicol Lett 28:29CrossRefGoogle Scholar
  7. 7.
    Lebrun S, Follmann W (2002) Detection of ochratoxin A–induced DNA damage in MDCK cells by alkaline single cell electrophoresis (comet assay). Arch Toxicol 75:734CrossRefGoogle Scholar
  8. 8.
    Oliveira SCB, Diculescu VC, Palleschi G, Compagnone D, Oliveira-Brett AM (2007) Electrochemical oxidation of ochratoxin A at a glassy carbon electrode and in situ evaluation of the interaction with deoxyribonucleic acid using an electrochemical deoxyribonucleic acid-biosensor. Anal Chim Acta 588:283CrossRefGoogle Scholar
  9. 9.
    Mally A, Zepnik H, Waken P, Eder E, Dingley K, Ihmels H, Volkel W, Dekat W (2004) Ochratoxin A: lack of formation of covalent DNA adducts. Chem Res Toxicol 17:234CrossRefGoogle Scholar
  10. 10.
    Gillman IG, Clark TN, Manderville RA (1999) Oxidation of ochratoxin A by an Fe-porphyrin system: model for enzymatic activation and DNA cleavage. Chem Res Toxicol 12:1066CrossRefGoogle Scholar
  11. 11.
    Pittet A (1998) Natural occurrence of mycotoxins in foods and feeds: an updated review. Rev Med Vet 149:479Google Scholar
  12. 12.
    Uchiyama S, Saito Y (1987) Protein-binding potential of ochratoxin A in vitro and Its fluorescence enhancement. J Food Hyg Soc Jpn 28:453CrossRefGoogle Scholar
  13. 13.
    Whitley RD, Wachter R, Liu F, Wang NH (1989) Ion exchange equilibria of lysozyme, myoglobin and bovine serum albumin. J Chromatogr 465:137CrossRefGoogle Scholar
  14. 14.
    Zimmerli B, Dick R (1995) Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup methodology and Swiss data. J Chromatogr B 666:85CrossRefGoogle Scholar
  15. 15.
    Olsson J, Borjesson T, Lundstedt T, Schnurer J (2002) Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC–MS and electronic nose. Int J Food Microbiol 72:203CrossRefGoogle Scholar
  16. 16.
    Penas EG, Leache C, de Cerain AL, Lizarraga E (2006) Comparison between capillary electrophoresis and HPLC-FL forochratoxin A quantification in wine. Food Chem 97:349CrossRefGoogle Scholar
  17. 17.
    Obrecht-Pflumio S, Dirheimer G (2000) In vitro DNA and dGMP addcuts formation caused by ochratoxin A. Chem Biol Interact 127:29CrossRefGoogle Scholar
  18. 18.
    Aizawa M (1987) Immunosensors. Philos Trans R Soc Lond B316:121Google Scholar
  19. 19.
    Scott PM (2002) Methods of analysis for ochratoxin A. Mycotoxins Food Safe 504:117CrossRefGoogle Scholar
  20. 20.
    Nikolelis DP, Petropoulou S-SE, Mitrokotsa MV (2002) A minisensor for the rapid screening of atenolol in pharmaceutical preparations based on surface-stabilized bilayer lipid membranes with incorporated DNA. Bioelectrochemistry 58:107CrossRefGoogle Scholar
  21. 21.
    Mohsin MA, Banica F-G, Oshima T, Hianik T (2011) Electrochemical impedance spectroscopy for assessing the recognition of cytochrome c by immobilized calixarenes. Electroanalysis 23:1229CrossRefGoogle Scholar
  22. 22.
    Hall EAH (1991) Biosensors. Prentice-Hall, Englewood Press, Englewood CliffsGoogle Scholar
  23. 23.
    Eggins BR (2002) Chemical sensors and biosensors. Wiley, ChichesterGoogle Scholar
  24. 24.
    Wang J (2006) Analytical electrochemistry. Wiley, HobokenCrossRefGoogle Scholar
  25. 25.
    Calcutt MW, Gillman IG, Noftle RE, Manderville RA (2001) Electrochemical oxidation of ochratoxin A. Correlation with 4-chlorophenol. Chem Res Toxicol 14:1266CrossRefGoogle Scholar
  26. 26.
    Manderville RA, Calcutt MW, Dai J, Park G, Gillman IG, Noftle RE, Mohammed AK, Dizdaroglu M, Rodriguez H, Akman SA (2003) Stoichiometric preference in copper-promoted oxidative DNA damage by ochratoxin A. J Inorg Biochem 95:87CrossRefGoogle Scholar
  27. 27.
    Alonso-Lomillo MA, Domínguez-Renedo O, Ferreira-Goncalves L, Arcos-Martínez MJ (2010) Sensitive enzyme-biosensor based on screen-printed electrodes for Ochratoxin A. Biosens Bioelectron 25:1333CrossRefGoogle Scholar
  28. 28.
    Alonso-Lomillo MA, Domínguez-Renedo O, del Torno-de Roman L, Arcos-Martínez MJ (2011) Horseradish peroxidase-screen printed biosensors for determination of Ochratoxin A. Anal Chim Acta 688:49CrossRefGoogle Scholar
  29. 29.
    Perrotta PR, Vettorazzi NR, Arevalo FJ, Granero AM, Chulze SN, Zon MA, Fernandez H (2011) Electrochemical studies of ochratoxin A mycotoxin at gold electrodes modified with cysteamine self-assembled monolayers. Its ultrasensitive quantification in red wine samples. Electroanalysis 23:1585CrossRefGoogle Scholar
  30. 30.
    Alarcon SH, Palleschi G, Compagnone D, Pascale M, Visconti A, Barna-Vetro I (2006) Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat. Talanta 69:1031CrossRefGoogle Scholar
  31. 31.
    Hokama Y (1991) Immunological analysis of low molecular weight marine toxins. J Toxicol Toxin Rev 10:1Google Scholar
  32. 32.
    Bignami GS, Raybould TJG, Sachinvala ND, Grothaus SB, Simpson SB, Lazo CB, Byrnes JB, Moore E, Vann DC (1992) Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples. Toxicon 30:687CrossRefGoogle Scholar
  33. 33.
    Van Emon JM, Lopez-Avila V (1992) Immunochemical for environmental analysis. Anal Chem 64:79ACrossRefGoogle Scholar
  34. 34.
    Candlish AAG, Stimsom WH, Smith JE (1988) Determination of ochratoxin A by monoclonal antibody based enzyme immunoassay. J Assoc Off Anal Chem 71:961Google Scholar
  35. 35.
    Ramakrishna N, Lacey J, Candlish AAG, Smith JE, Goodbrand IA (1990) Monoclonal antibody based enzyme linked immunosorbent assay of aflatoxin B, T-2 toxin and ochratoxin A in barley. J Assoc Off Anal Chem 73:71Google Scholar
  36. 36.
    Clarke JR, Marquardt RR, Oosterveld A, Frohlich AA, Madrid FJ, Dawood M (1993) Development of quantitative and sensitive enzyme-linked immunosorbent assay for ochratoxin A using antibodies from the yolk of laying hen. J Agric Food Chem 41:1784CrossRefGoogle Scholar
  37. 37.
    Barna Vetro L, Solti J, T´eren A, Gyongyosi E, Szabo A, Wolfling J (1996) Sensitive ELISA test for determination of ochratoxin A. Agric Food Chem 44:4071CrossRefGoogle Scholar
  38. 38.
    Kwak B, Shon D (2000) Detection of ochratoxin A in agricultural commodities using enzyme-linked immunosorbent assay. Food Sci Biotechnol 9:168Google Scholar
  39. 39.
    Ogert RA, Burans J, O’ Brien T, Ligler FS (1994) Comparative analysis of toxin detection in biological and environmental samples. Proc SPIE Int Soc Opt Eng 2068:151Google Scholar
  40. 40.
    Poli MA, Rivera VR, Hewetson JF, Merril GA (1994) Detection of ricin by colorimetric and chemiluminescence ELISA. Toxicon 32:1371CrossRefGoogle Scholar
  41. 41.
    Wieneke AA, Gilbert RJ (1985) The use of sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning. J Hygiene 95:131CrossRefGoogle Scholar
  42. 42.
    Nielsen P, Koch C, Friis H, Heron I, Prag J, Schmidt J (1987) Double-antibody sandwich enzyme-linked immunosorbent assay for rapid detection of toxin-producing. J Clin Microbiol 25:1280Google Scholar
  43. 43.
    Bhatti AR, Siddiqui YM, Micusan VV (1994) Highly sensitive fluorogenic enzyme-linked immunosorbent assay: detection of staphylococcal enlerotoxin B. J Microbiol Meth 19:179CrossRefGoogle Scholar
  44. 44.
    Doellgast GJ, Beard GA, Bottoms JD, Cheng T, Roh BH, Roman MG, Hall PA, Triscott MX (1994) Enzyme-linked immunosorbent assay-enzyme-linked coagulation assay for detection of antibodies to Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes. J Clin Microbiol 32:105Google Scholar
  45. 45.
    Monaci L, Palmisano F (2004) Determination of ochratoxin A in foods: State- of-the-art and analytical challenges. Anal Bioanal Chem 378:96CrossRefGoogle Scholar
  46. 46.
    Saha D, Acharya D, Roy D, Shrestha D, Dhar TK (2007) Simultaneous enzyme immunoassay for the screening of aflatoxin B-1 and ochratoxin A in chili samples. Anal Chim Acta 584:343CrossRefGoogle Scholar
  47. 47.
    Sauceda-Friebe JC, Karsunke XYZ, Vazac S, Biselli S, Niessner R, Knopp D (2011) Regenerable immuno-biochip for screening ochratoxin A in green coffee extract using an automated microarray chip reader with chemiluminescence detection. Anal Chim Acta 689:234CrossRefGoogle Scholar
  48. 48.
    Clarke JR, Marquardt RR, Frohlich AA, Pitura RJ (1994) Quantification of ochratoxin A in swine kidneys by enzyme-linked immunosorbent assay using a simplify sample preparation procedure. J Food Protect 57:991Google Scholar
  49. 49.
    Sibanda L, De saeger S, Barna-Vetro I, Van Petheghem CJ (2002) Development of a solid-phase clean-up and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin A in roasted coffee. J Agric Food Chem 50:6964CrossRefGoogle Scholar
  50. 50.
    De Saeger S, Van Peteghem C (1999) Flowthrough membrane-based enzyme immunoassay for rapid detection of ochratoxin A in wheat. J Food Protect 62:65Google Scholar
  51. 51.
    Fernandez-Baldo MA, Bertolino FA, Fernandez G, Messina GA, Sanz MI, Raba J (2011) Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles. Analyst 136:2756CrossRefGoogle Scholar
  52. 52.
    Liu X-P, Deng Y-J, Jin X-Y, Chen L-G, Jiang J-H, Shen G-L, Yu R-Q (2009) Ultrasensitive electrochemical immunosensor for ochratoxin A using gold colloid-mediated hapten immobilization. Anal Biochem 389:63CrossRefGoogle Scholar
  53. 53.
    Micheli L, Radoi A, Guarrina R, Massoud R, Bala C, Moscone D, Palleschi G (2004) Disposable immunosensor for the determination of domoic acid in shellfish. Biosens Bioelectron 20:190CrossRefGoogle Scholar
  54. 54.
    Ammida NHS, Micheli L, Palleschi G (2004) Electrochemical immunosensor for determination of aflatoxin B1 in barley. Anal Chim Acta 520:159CrossRefGoogle Scholar
  55. 55.
    Stenberg E, Persson B, Roos H, Urbaniczky C (1991) Quantitative determination of surface concentration of protein with surface plasmon resonance by using radiolabeled proteins. J Colloid Interface Sci 143:513CrossRefGoogle Scholar
  56. 56.
    van der Gaag B, Spath S, Dietrich H, Stigter E, Boonzaaijer G, van Osenbruggen T, Koopal K (2003) Biosensors and multiple mycotoxin analysis. Food Control 14:251CrossRefGoogle Scholar
  57. 57.
    Yu JCC, Lai EPC (2005) Interaction of ochratoxin A with molecularly imprinted polypyrrole film on surface plasmon resonance sensor. React Funct Polym 63:171CrossRefGoogle Scholar
  58. 58.
    Yuan J, Deng D, Lauren DR, Aguilar M-I, Wu Y (2009) Surface plasmon resonance biosensor for the detection of ochratoxin A in cereals and beverages. Anal Chim Acta 656:63CrossRefGoogle Scholar
  59. 59.
    Zamfir L-G, Geana I, Bourigua S, Rotariu L, Bala C, Errachid A, Jaffrezic-Renault N (2011) Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection. Sens Actuat B. 159:178Google Scholar
  60. 60.
    Sauerbrey G (1959) The use of oscillator for weighing thin layers and for microweighing. Z Phys 155:206CrossRefGoogle Scholar
  61. 61.
    Auge J, Hauptmann P, Hartmann J, Rosler S, Lucklum R (1995) New design for QCM sensors in liquid. Sens Actuat B 24–25:43–48CrossRefGoogle Scholar
  62. 62.
    Ellis JS, Thompson M (2010) Viscoelastic modelling with interfacial slip of a protein monolayer electrode-adsorbed on an acoustic wave biosensor. Langmuir 26:11558CrossRefGoogle Scholar
  63. 63.
    Tsai WC, Hsieh CK (2007) QCM-based immunosensor for the determination of ochratoxin A. Anal Lett 40:1979CrossRefGoogle Scholar
  64. 64.
    Lisdat F, Schäfer D (2008) The use of electrochemical impedance spectroscopy for biosensing. Anal Bioanal Chem 391:1555CrossRefGoogle Scholar
  65. 65.
    Khan R, Dhayal M (2008) Nanocrystalline bioactive TiO2-chitosan impedimetric immunosensor for ochratoxin-A. Electrochem Commun 10:492CrossRefGoogle Scholar
  66. 66.
    Khan R, Dhayal M (2009) Chitosan/polyaniline hybrid conducting biopolymer base impedimetric immunosensor to detect Ochratoxin-A. Biosens Bioelectr 24:1700CrossRefGoogle Scholar
  67. 67.
    Radi A-E, Munoz-Berbel X, Lates V, Marty J-L (2009) Label-free impedimetric immunosensor for sensitive detection of ochratoxin A. Biosens Bioelectr 24:1888CrossRefGoogle Scholar
  68. 68.
    Ansari AA, Kaushik A, Solanki PR, Malhotra BD (2010) Nanostructured zinc oxide platform for mycotoxin detection. Bioelectrochemistry 77:75CrossRefGoogle Scholar
  69. 69.
    Kaushik A, Solanki PR, Pandey MK, Kaneto K, Ahmad S, Malhotra BD (2010) Carbon nanotubes chitosan anobiocomposite for immunosensor. Thin Solid Films 519:1160CrossRefGoogle Scholar
  70. 70.
    Muchindu M, Iwuoha E, Pool E, West N, Jahed N, Baker P, Waryo T, Williams A (2011) Electrochemical ochratoxin A immunosensor system developed on sulfonated polyaniline. Electroanalysis 23:122CrossRefGoogle Scholar
  71. 71.
    Xiao Y, Piorek BD, Plaxco KW, Heeger AJ (2005) A reagentless, signal-on design for electronic aptamer-based sensors via target-induced strand displacement. J Am Chem Soc 127:17990CrossRefGoogle Scholar
  72. 72.
    Hianik T, Wang J (2009) Electrochemical aptasensors – recent achievements and perspectives. Electroanalysis 21:1223CrossRefGoogle Scholar
  73. 73.
    Cheng AKH, Sen D, Yu H-Z (2009) Design and testing of aptamer-based electrochemical biosensors for small molecules and proteins. Bioelectrochemistry 77:1CrossRefGoogle Scholar
  74. 74.
    Ferapontova EE, Gothelf KV (2011) Recent advances in electrochemical aptamer-based sensors. Curr Org Chem 15:498CrossRefGoogle Scholar
  75. 75.
    Wei F, Ho C-M (2009) Aptamer-based electrochemical biosensor for Botulinum neurotoxin. Anal Bioanal Chem 393:1943CrossRefGoogle Scholar
  76. 76.
    Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505CrossRefGoogle Scholar
  77. 77.
    Elington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature (Lond) 346:818CrossRefGoogle Scholar
  78. 78.
    Cruz-Aguado JA, Penner G (2008) Determination of ochratoxin A with a DNA aptamer. J Agr Food Chem 56:10456CrossRefGoogle Scholar
  79. 79.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406CrossRefGoogle Scholar
  80. 80.
    Kikin O, D’Antonio L, Bagga PS (2006) QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic Acids Res 34:W676CrossRefGoogle Scholar
  81. 81.
    Castillo G, Lamberti I, Mosiello L, Hianik, T (2011) Impedimetric DNA aptasensor for sensitive detection of ochratoxin A in a food. Electroanalysis doi:10.1002/elan.201100485.Google Scholar
  82. 82.
    Lamberti I, Mosiello L, Hianik T (2011) Development of thickness shear mode biosensor based on DNA aptamers for detection of ochratoxin A. Chem Sensors 1:11Google Scholar
  83. 83.
    Poniková S, Tlučková K, Antalík M, Víglaský V, Hianik T (2011) The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers. Biophys Chem 155:29CrossRefGoogle Scholar
  84. 84.
    Yang C, Wang Y, Marty J-L, Yang X (2011) Aptamer-based colorimetric biosensing of ochratoxin A using unmodified gold nanoparticles indicator. Biosens Bioelectron 26:2724CrossRefGoogle Scholar
  85. 85.
    Wang ZP, Duan N, Hun X, Wu SJ (2010) Electrochemiluminescent aptamer biosensor for the determination of ochratoxin A at a gold-nanoparticles-modified gold electrode using N-(aminobutyl)-N-ethylisoluminol as a luminescent label. Anal Bioanal Chem 398:2125CrossRefGoogle Scholar
  86. 86.
    Wu S, Duan N, Wang Z, Wang H (2011) Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin A using upconversion nanoparticles as labels. Analyst 136:2306CrossRefGoogle Scholar
  87. 87.
    Kuang H, Chen W, Xu D, Xu L, Zhu Y, Liu L, Chu H, Peng C, Xu C, Zhu S (2010) Fabricated aptamer-based electrochemical “signal-off” sensor of ochratoxin A. Biosens Bioelectron 26:710CrossRefGoogle Scholar
  88. 88.
    Barthelmebs L, Hayat A, Limiadi AW, Marty J-L, Noguer T (2011) Electrochemical DNA aptamer-based biosensor for OTA detection, using superparamagnetic nanoparticles. Sens Actuat B 156:932CrossRefGoogle Scholar
  89. 89.
    Prieto-Simon B, Campas M, Marty J-L, Noguer T (2008) Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples. Biosens Bioelectron 23:995CrossRefGoogle Scholar
  90. 90.
    Bonel L, Vidal JC, Duato P, Castillo JR (2011) An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer. Biosens Bioelectron 26:3254CrossRefGoogle Scholar
  91. 91.
    Bonel L, Vidal JC, Duato P, Castillo JR (2010) An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer. Anal Methods 2:335CrossRefGoogle Scholar
  92. 92.
    Castillo G, Lamberti I, Mosiello L, Hianik T (2012) High-sensitive impedimetric aptasensor for detection ochratoxin A in food. In: Di Natale C (ed) Sensors and microsystems. Lecture Notes in Electrical Engineering. Springer, New York/Heilderberg doi:10.1007/978-1-4614-0935-9_6.
  93. 93.
    Prabhakar P, Matharu Z, Malhotra BD (2011) Polyaniline Langmuir-Blodgett film based aptasensor for ochratoxin A detection. Biosens Bioelectron 26:4006CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Faculty of Mathematics, Physics and InformaticsComenius UniversityBratislavaSlovakia

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