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Aptamer based detection and separation platforms for ochratoxin A: A systematic review


1 Laboratory of Bioanalysis, Institute of Biochemistry & Biophysics, University of Tehran, Tehran, Iran
2 Baqiyatallah Research Center for Gastroenterology and Liver Diseases (BRCGL), Baqiyatallah University of Medical Sciences, Tehran, Iran
3 Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
4 Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

* Corresponding Authors: REZA RANJBAR. Email: email; MOHAMMAD HEIAT. Email: email

BIOCELL 2022, 46(12), 2537-2557. https://doi.org/10.32604/biocell.2022.021373


Ochratoxin A (OTA), one of the most dangerous mycotoxins for human health, has been subjected to numerous studies for separation and detection in minimal amounts. Aptamers as novel recognition elements have been employed to fabricate ultrasensitive biosensors for the detection of OTA and designing delicate analytical tools. This review attempted to comprehensively examine all reported aptamer-based detection and separation platforms for ochratoxin. The most relevant databases were considered to discover all specific aptamers for dealing with OTA. Aptamer-based detection and separation devices specified for OTA were searched for, analyzed, discussed, and classified based on their specifications. The optical aptasensors have gathered a higher interest than electrochemical aptasensors, which can achieve a lower limit of detections. Moreover, some extraction platforms based on these aptamers were also found. However, aptamer-based devices seem to have some challenges in their application.



Ochratoxins belong to a family of toxic secondary metabolites produced by several species of fungi such as Aspergillus and Penicillium spp. Ochratoxins A, B, and C are members of this family with abundance in specimens. Ochratoxins are ubiquitous fungal toxins in a wide variety of poorly stored agricultural supplies, ranging from cereal grains to dried fruits to wine and coffee (Bui-Klimke and Wu, 2015). They threaten human and animal health by impacting food and nutrition safety and can affect food and agricultural economics (Heussner and Bingle, 2015).

The structure of ochratoxin A (OTA), a chlorophenolic mycotoxin, is chemically stable (Fig. 1) and comprises a phenylalanine moiety and dihydro-isocoumarin ring.


Figure 1: Chemical structure of ochratoxin A (OTA).

OTA exerts several toxic effects such as immunotoxicity, nephrotoxicity, hepatotoxicity, neurotoxicity, teratogenicity, and carcinogenicity (el Khoury and Atoui, 2010). It enters the human food chain through livestock products as a result of animal feeding on contaminated nutrition.

Children who consume large amounts of milk daily may have higher daily intakes of OTA (Muñoz et al., 2014).

Avoiding the risk of OTA exposure and the detection and quantitation of OTA levels are issues of great significance.

Currently, there are various analytical methods to detect OTA in as low as microgram levels. The most typical methods to detect and analyze OTA include immunological, such as enzyme-linked immunosorbent assay (ELISA) (Sun et al., 2019), Radio-immunoassay (RIA) (Rousseau et al., 1985), and chromatographic methods such as thin-layer chromatography (Pittet and Royer, 2002), liquid chromatography (LC), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) (Chung and Kwong, 2019), gas chromatography (Olsson et al., 2002), and high-performance liquid chromatography (HPLC) (Sibanda et al., 2001).

In addition to their high cost, these methods take a long time and require a trained person and specific instruments to perform.

With the increasing need to develop a simple method to detect and separate OTA, various biosensors have been developed to detect this toxin in cereal products, food materials, and beverages. The development of novel biosensors has created an opportunity in the agriculture and food industries to improve food quality and safety assurance.

Hitherto numerous antibody-based biosensors have been reported for the detection of OTA (Huang et al., 2017; Ren et al., 2018; Alhamoud et al., 2019). In most biosensors, antibodies are used to identify targets. However, several new recognition elements have been introduced as alternatives for antibodies.

Aptamers, oligonucleotides, or peptide molecules that bind to a specific target can be isolated against amino acids, drugs, proteins, and other molecules. These are potent in their use in a variety of tools, including biosensors and analytical techniques (Zhao et al., 2008). Aptamers, known as chemical antibodies (Zhou et al., 2016a), are worthwhile alternatives for antibodies with greater benefits. They can bind specifically with a high affinity to their targets, ranging from ions to a complex targets such as whole cells. The specificity of aptamers is such that they can even distinguish between chiral molecules. These characteristics have enabled aptamers into a promising tool to construct diagnostic and analytical platforms with low limits of detection ranging from picomoles to nanomoles of targets (Song et al., 2008).

An interesting issue to address OTA contaminations is the development of aptasensors and biosensors. Some studies applied OTA aptamer for separation approaches such as aptamer-assisted real-time polymerase chain reaction (PCR), HPLC, suspension arrays, microfluidic devices, and affinity columns.

In © present review, we have tried to systematically study and classify OTA-specific isolated aptamers based on their applications and specifications.

Materials and Methods

Search for available OTA-specific aptamers and their separation and detection platforms

Data mining and searches were carried out in PubMed and Scopus for articles from 2008 to 2020. The main search keywords included OTA-specific aptamers, aptamer-based biosensors, aptamer-based detection, OTA aptasensors, ochratoxin A separation, ochratoxin A analysis, and other similar compositions. Keywords were selected using the medical subject headings (MeSH) terms and composed using proper Boolean operators (AND/OR/NOT).

The data mining was performed independently by two researchers, and the results were checked by the other researchers.

Study eligibility

The following inclusion criteria were considered: only articles published in the English language, articles with methods for separation or detection of OTA, and use of aptamer-based sensors.

For data collection and extraction, selected publications that met inclusion criteria were reviewed. The methods were categorized and reviewed based on the separation approach, sensor platform, label, detection limit, detection range, and sequences of aptamers. The analytical methods used to measure OTA were also evaluated.

Study selection

A total of 269 records (up to October 2020) were recovered; among these, 15 studies were removed after the first screening. The remaining 254 studies were reviewed in detail. Studies addressing other aspects of the title and deviation from inclusion criteria were excluded. The remaining studies that met the inclusion criteria and were classified for inclusion in the systematic review (Fig. 2).


Figure 2: PRISMA flow-diagram.

Docking studies

OTA structure was prepared from (PDB: 6J2W), and the tertiary structures of OTA-specific aptamers were designed according to the findings in our recent article. In summary, Secondary structures of four OTA-specific aptamers were predicted using the Mfold web server (Zuker, 2003) (version 3.0, http://www.unafold.org/mfold/applications/dna-folding-form.php). Then, based on the resulting secondary structure of the Vienna file format, their PDB structure was predicted using RNA composer (Antczak et al., 2016) and refined by UCSF Chimera (Pettersen et al., 2004) and discovery studio visualizer. In summary, the uracil was replaced by thymine (T), and the ribose sugars were substituted with deoxyribose sugars in the primary chain. The OTA was docked on each aptamer by HDOCK webserver (Yan et al., 2020) to predict complex models. Then, discovery studio visualizer software (2016) was employed to find the most appropriate and final structure of OTA and each aptamer complex and to monitor possible intermolecular interactions.


Specific Aptamers against OTA

Some aptamers have been reported for specific detection of OTA (Table 1).


Cruz-Aguado and Penner isolated 13 DNA aptamers against OTA by SELEX after twelve reiterative rounds, between which an aptamer named OTA binding aptamer (OBA; 1.12) showed the highest affinity (Kd = 0.36 μM). They also developed buffer conditions by adding divalent cations to reduce the Kd value of the aptamer to 50 nM. Their results found that the Kd value could be reduced by substituting calcium for magnesium in the buffer.

Presumably, OTA forms a coordination complex with magnesium or calcium with the aid of its carboxyl and 8-hydroxyl groups, and this complex increases the binding to the aptamer. This buffer was subsequently used for the separation of the OTA from wheat samples using an aptamer affinity column. The column removed more than 97% of OTA from 1 mL of a 100 nM OTA solution (Cruz-Aguado and Penner, 2008). Other scientists applied a structure-guided post-SELEX approach to improve the affinity of this reported aptamer. They achieved this goal by forming a novel hairpin structure containing an intramolecular triple helix in the aptamer structure by mutating T and adenine (A) in critical points in aptamer sequence to cytosine (C) and guanine (G), respectively, and adding a C in the 5’–terminus of the aptamer. Using such an approach increased the affinity aptamers’ by up to 50-fold (Xu et al., 2019).

In 2011, another research group (Barthelmebs et al., 2011) isolated five aptamers against OTA by employing in vitro SELEX method. They selected an aptamer with the lowest IC50 (0.051 µg/mL), called H12, with Kd of 96 nM as the best one (Barthelmebs et al., 2011). They found two important conserved sequences (GGGTGTGGG) and (AGGGAGT) in the stem region and the single-strand terminal loop, respectively, in these aptamers for binding to OTA in the stem and loop regions similar to aptamer OBA (Cruz-Aguado and Penner, 2008).

A08 and B08, were other novel aptamers reported for the detection of OTA with relatively high affinity (Kd = 290 ± 150 and 110 ± 50 nM respectively), after fifteen reiterative rounds of SELEX (McKeague et al., 2014) without any similarity with previously reported aptamers (Cruz-Aguado and Penner, 2008; Barthelmebs et al., 2011).

Recently, a study used the in vitro evolution technique and introduced a threose nucleic acid aptamer for OTA with high stability and low Kd (71 nM) (Rangel et al., 2018).

Docking results

In silico investigation on binding structures confers valuable data about intermolecular interactions. Knowledge of the interaction mechanism can help us design purposeful diagnostic and analytical systems. Each binding—partial or complete binding or pocket formation—of the aptamer has features that help researchers to design their devices intelligently and accurately.

To know more about OTA-aptamer interactions, the OTA molecule was docked on reported aptamers (aptamers with the highest affinity in each study). Docking of OTA on its specific aptamers by the HDOCK web server (http://hdock.phys.hust.edu.cn/) (Yan et al., 2020) resulted in over 100 probable models. From these structures, the top models ranked by HDOCK were summarized in Table 2. We also calculated the strength of interaction of each top model of docking of OTA with the highest affinity OTA aptamers ranked by HDOCK using the PreDDICTA tool (https://www.scfbio-iitd.res.in/software/drugdesign/preddictanew.jsp) http://www.scfbio-iitd.res.in/software/drugdesign/preddictanew.jsp and compared them (Shaikh and Jayaram, 2007). The OBA (1.12) (Cruz-Aguado and Penner, 2008) showed the lowest predicted binding free energy (−3.0 kcal/mol).


After minimizing the energy of these models for each aptamer, final models were achieved and considered for further analysis (Fig. 3). Hydrogen bonds in the final complex structures was monitored by Discovery studio visualizer software (2016) (Wallace et al., 1995). The illustration revealed the possible hydrogen bonds between OTA and its aptamers (Fig. 3 and Table 3).


Figure 3: Illustration of docking Ochratoxin A (OTA) (purple) on its specific aptamers (white tubes): a) OTA binding aptamer (OBA; Cruz-Aguado and Penner, 2008), b) H12 (Barthelmebs et al., 2011), c) B08 (McKeague et al., 2014) and d) OBA3 (Xu et al., 2019). The OTA bonded nucleotides of each aptamer are displayed in the magnified form on the right side of each figure. Green dashed lines: Conventional hydrogen, Blue dashed lines: Carbon-hydrogen bonds.


Docking results revealed that these aptamers could surround and bind OTA through some hydrogen bonds by creating binding pockets. The carbonyl oxygen and hydrogens from the amide and chlorine of OTA showed high potential for forming intermolecular hydrogen bonds between the OTA and the aptamers.

Different binding interactions could explain their distinct predicted affinity for binding OTA (Table 2). Some studies showed comparable binding strength of carbon hydrogen bonds to that of the conventional hydrogen bonds. The attractive dispersion interaction could explain the unusual stabilities of these hydrogen bonds (Ghosh et al., 2020). However, aptamers are more complex to be interpreted with preliminary molecular studies, and such a basic analysis cannot totally disclose the mechanism of aptamer-target binding.

Aptamer-based methods for OTA

Various aptamer-based sensors have been developed for the quantitative measurement of OTA. In this regard, electrochemical, optical, and mass-sensitive transducers have been used to fabricate aptasensors. In addition to the biosensors, some other approaches such as aptamer assisted real-time PCR, HPLC, suspension array, microfluidic devices, and extraction columns have been developed.

All studies can be divided into two main categories of detection (aptasensor or non-biosensor detection methods) and separation.

Most of the fabricated aptasensors for OTA have been developed based on the first reported aptamer (OBA, Kd = 0.36 µM) (Cruz-Aguado and Penner, 2008). Of reported aptasensors based on OBA aptamer, 38 were electrochemical (Table 4), and 70 were optical aptasensor (Table 5). An optical aptasenor was also fabricated based on B08 (McKeague et al., 2014).


Other studies (17 original studies) have used either the first reported aptamer (OBA aptamer, 15 studies) (Cruz-Aguado and Penner, 2008) or H12 aptamer (two bioassays including direct and indirect Enzyme-Linked Aptamer Assay) (Barthelmebs et al., 2011) in non-biosensor detection approaches, isolation, and extraction.

In the following, the most prominent studies in two categories of aptasensors and non-biosensor aptamer-based detection and separation methods were discussed. We have also mentioned a few reported lateral flow and Lab-on-Chip-based assays. However, all the relevant studies are listed in the Tables.

a) Aptasensors

i) Electrochemical Aptasensors

In electrochemical biosensors, the biologically active materials are combined with an electrochemical sensing element transducer. The transducer transduces a chemical signal into an electrical signal by amperometric, voltammetric, potentiometric, or conductometric methods. There are thirty-eight reports on electrochemical aptrasensors for the detection of OTA (Table 4).

In these studies, gold electrodes were employed as the working electrode in twenty electrochemical aptasensors (n = 20) that exhibited favorable outcomes using this type of electrode. The advantages of this electrode include high surface area, ease of synthesis, preparation, and modification, tunable pore size, conductivity, and a bicontinuous open pore network (Angnes et al., 2000).

The other popular electrode for this type of OTA specific electrochemical aptasensor (n = 10) is a screen-printed carbon electrode (SPCE) with a wide potential window, low background current, and low-cost properties. SPCE consists of a reference electrode, a counter electrode, and a carbon working electrode. The area, thickness, and composition of these electrodes can be readily controlled. Catalysts can be simply incorporated by pasting them to the screen printing ink (Cumba et al., 2020).

Glassy carbon electrodes (GCEs), which were employed in six of the OTA electrochemical biosensors are less sensitive than GE because of having a wider window of electrochemical activity. Since these electrodes are highly resistant to heat and corrosion and can be easily cleaned and polished due to their tightly knit atomic structure and glassy exterior, they are widely applied in electrochemical biosensors.

The indium tin oxide electrode exhibited a wide range of current density implemented in two of the reported devices (n = 2) (Bouden et al., 2016).

The electrochemical properties of electrodes could be improved by various techniques. Nanomaterials were used to develop electrochemical sensors by signal amplification to improve both sensitivity and selectivity. These materials produce a synergistic effect among catalytic activity, conductivity, and biocompatibility to accelerate the signal transduction. The surface of nanomaterials could be efficiently activated and easily functionalized, making them an ideal surface for immobilization of biomolecules such as enzymes and bioreceptors, including aptamers, antibodies, and also electroactive labels.

For example, Chrouda et al. (2015) used an electrochemically oxidated carboxyl end of the long spacer chain of polyethylene glycol (NH2-PEG-COOH) on a boron-doped diamond (BDD) microcell through which sensitivity of the system was increased to the range of pg/mL. For immobilization of biomolecules on the BDD surface of microcell, that is inactive, the surface should be activated by reactive groups. This procedure helps to control the surface easily. In their aptasensor, immobilized amino-aptamer on PEG was utilized. In the presence of OTA, the conformation of the aptamer changed to G-quadruplex structures, and the electron transfer rate of the redox probe was decreased. Their results showed a wide linear range (0.00001–0.0132 ng/mL) with a LOD of 0.00001 ng/mL. This sensor needed a shorter time (1 h) to detect OTA in real samples of rice in comparison with the other designed methods using the same aptamer (6–16 h).

Kaur et al. (2019) fabricated a simple and efficient functionalized graphene (f-graphene) doped chitosan (CS) nanocomposites based electrochemical aptasensor for the detection of OTA. The use of f-graphene increased the electroactive surface area of the electrode, and CS prevented the leaching of the aptamer molecules. CS is a suitable matrix due to its biocompatibility, hydrophilicity, non-toxicity, excellent mechanical stability, cost-effectiveness, and availability of reactive functional groups for chemical modifications. Besides, f-graphene increased the electroactive surface area of the electrode. The dual use of CS and f-graphene overall improved the sensor performance. This aptasensor exhibited LOD of 1 fg/mL for standard and 0.01 ng/mL for real samples within a response time of 8 min.

Yang et al. (2019) developed a label-free ultrasensitive electrochemical aptasensor based on NH2/Janus particles for the detection of OTA. Janus particles are special types of nanoparticles (NPs) with two or more surfaces having distinct physical or chemical properties. This allows two different types of chemicals to occur on the same particle. They prepared Janus particles by coating a layer of Au onto the hemisphere of amino polystyrene microspheres and immobilized the thiolated OTA aptamer on the Au layer. This aptasensor showed a very low LOD (0.0000001 ng/mL: 3.3 × 10−3 pM) and a wide dynamic linear range of OTA concentration (0.000004–4.0381 ng/mL: 1 × 10−5 to 10 nM) with high selectivity due to highly specific molecular recognition between OTA and aptamer and also good stability and reproducibility. They also applied this electrochemical sensor for the detection of OTA in red wine.

The common issue in these devices is employing nanomaterials. Nanoparticles provide a more accessible surface for immobilization of biorecognition elements which increase the sensitivity of biosensor to as low as pico/femtomolar levels.

Interestingly, there were some devices with an extremely low limit of detection (LOD) down to pico/femtogram/mL of OTA. The key point of some of these devices is the use of amplification techniques that enhance the signal to fabricating ultrasensitive biosensors such as PCR, real-time PCR, and recently, isothermal amplification-detection strategies such as loop-mediated isothermal amplification (LAMP).

For the first time, Yuan et al. (2014) integrated the LAMP technique with an electro-chemiluminescent (ECL) system to fabricate an ultrasensitive aptasensor. They immobilized a dsDNA composed of an OTA aptamer and its capture DNA on the electrode. With the presence of OTA, some of the aptamers were separated, and the remaining aptamers on the electrode served as an inner primer to initiate the LAMP reaction. The amplification procedure was tracked by monitoring the intercalation of DNA-binding Ru(phen)32+ ECL indicators into newly formed amplicons. Therefore, the presence of more OTA was equal to the release of more aptamer, less remaining aptamer on the electrode for producing LAMP amplicons, less Ru(phen)32+ intercalating into the formed amplicons, and thus increasing the ECL signal. They achieved a detection limit as low as 10 fM of OTA with good reproducibility and stability.

The integration of nanomaterials with these nucleic acid amplification strategies also could have a positive impact on the sensitivity of aptasensors.

Wang et al. (2017b) applied NH2@Cobalt-Metal–organic frameworks (MOFs) with their μ3-O linked trigonal prism structures to construct a sensitive biosensor. These frameworks efficiently possess intrinsic electrocatalytic activities for redox molecules, like thionine, which could intercalate into grooves of dsDNA via electrostatic adsorption and generate ultrasensitive square wave voltammetry signal. For amplification of the signal, they also employed a versatile exonuclease I (Exo I)-assisted target recycling for the production of sufficient numbers of cDNA sequences. Exo I catalyzes the hydrolysis of ssDNA instead of dsDNA from its 3′ end and digests the aptamer in the OTA-aptamer complex to release OTA again for further reaction. In brief, the gold nanoparticles (AuNPs), which effectively promote the electron transfer, were linked to the surface of NH2-Co-MOFs on the Au electrode. Biotin-modified OTA aptamer and cDNA were immobilized onto streptavidin magnetic beads. A series of OTA concentrations were incubated, and then Exo I-assisted target recycling reaction was performed. Eventually, the aptamer was dissociated from the matched double-stranded DNA in the presence of OTA and digested by Exo I, which led to the release of OTA. Then, the released OTA participated in another specific recognition reaction with the remaining aptamers, achieving the target recycling and signal amplification. The dissociated cDNA in the supernatant solution was collected. The mixture of the collected complementary DNA, and SH-Capture probe was incubated on the Au electrode to form the ternary DNA Y-junction structure on the NH2-Co-MOFs sensing surface. At the signal generation step, large amounts of thionine could intercalate into the three complementary DNA sequences. Further hybridization of the SH-Capture probe and cDNA to form a “Y” junction structure on the electrode surface resulted in significant signal recovery. They successfully applied this sensor to determine OTA in red wine with a linear range of 0.000001 to 1 ng/mL and LOD of 0.00000033 ng/mL (0.33 fg/mL). It also showed 90.0–105.0% recoveries for OTA in the red wine.

ii) Optical Aptasensors

Based on our search, 70 papers have reported optical aptrasensors for the detection of OTA (Table 5). Here we have attempted to discuss the most prominent construction.


A sensitive luminescent aptasensor was designed by Wu et al. (2011a). They utilized aptamer-conjugated magnetic nanoparticles (MNPs) and sodium yttrium fluoride (NaYF4): Yb (Ytterbium), Erbium (Er) labeled upconversion nanoparticles (UCNPs) as the recognition element and highly sensitive label, respectively. They immobilized OBA aptamer (Cruz-Aguado and Penner, 2008) on the surface of Fe3O4 MNPs hybridized with UCNPs coated with complementary DNA. Once OTA was bound to the aptamer, the complementary ssDNA was released, and subsequently, the luminescent signal was reduced. By fabricating this simple method, they could achieve very low LOD (0.0001 ng/mL) and a wide linearity range (0.0001–0.1 ng/mL) (Fig. 4a).


Figure 4: Schematic illustration of the most sensitive reported optical aptasensors. a) The immobilized aptamer on the surface of magnetic nanoparticles (MNPs) hybridized with cDNA immobilized on sodium yttrium fluoride (NaYF4): Yb (Ytterbium), Erbium (Er) labeled upconversion nanoparticles. Once OTA was bound to the aptamer, the cDNA was released, and subsequently, the luminescent signal was reduced (Wu et al., 2011a). b) Simultaneous detection of ochratoxin A (OTA) and fumonisin B1 (FB1) based on aptamer−SPCMs suspension arrays (Yue et al., 2014).

Song et al. (2018) introduced a Surface-Enhanced Raman Spectroscopy (SERS)-based aptasensor for the detection of OTA by hybridizing cDNA coated Fe3O4@Au magnetic nanoparticles (MGNPs), and the detection of OBA aptamer (Cruz-Aguado and Penner, 2008) modified Au@Ag nanoprobes labeled with the Raman reporter 5,5′-Dithiobis(2-nitrobenzoic acid) (DNTB) (Au-DTNB@Ag NPs). In the absence of OTA, the peak of SERS was high, but with the presence of OTA, due to the binding of OTA to the aptamer and the release of cDNA, the signal decreased proportionally to the concentration of OTA. This platform showed picogram levels (0.48 pg/mL) of LOD with good recovery and accuracy in evaluating real samples of wine and coffee.

A label-free fluorescent aptasensor which was developed by Lv et al. (2014), gives one of the widest reported dynamic ranges of detection. In this highly-sensitive (0.025 ng/mL) and selective fluorescent sensor, they applied PicoGreen (PG), an asymmetric cyanine dye, which exhibits its fluorescence property only after ultra-selective binding to the minor groove of dsDNA. In this platform, if there is no OTA, the ssDNA aptamers (Cruz-Aguado and Penner, 2008) hybridize to cDNAs. The PG dyes can bind the formed dsDNA and exhibit fluorescence. In the presence of OTA, the aptamer binds to the target and generates a G-quadruplex structure, and the signal intensity decreases. In this approach, a wide dynamic range (1 to 100000 ng/mL) was achieved to determine OTA concentrations in beer samples.

Yue et al. (2014) developed a simple and novel aptamer-based photonic crystal encoded suspension which could simultaneously recognize and quantify OTA and fumonisin B1 (FB1) in cereal samples by immobilizing the OBA aptamers (Cruz-Aguado and Penner, 2008) on the surfaces of different kinds of silica photonic crystal microsphere (SPCM) by a covalent bond. The different OTA and FB1 aptamer probes hybridized with their fluorescence-labeled complementary DNA and those immobilized on the surfaces of SPCMs could bind to their specific targets (OTA and FB1). Binding each aptamer to its target induced a structural switch of the aptamer, causing the release of the fluorescence-labeled aptamer complementary DNA and a marked decrease in fluorescent intensity of each kind of SPCM (Fig. 4b).

One interesting method in the line of optical aptasensors was developed by Song et al. (2018). They utilized a dual-color fluorescence resonance energy transfer (FRET) based aptasensor for simultaneous detection of the mycotoxins aflatoxin M1 (AFM1) and OTA. They labeled AFM1 and OTA aptamers with two different fluorophores as the signaling probes. In the blank samples, the aptamers were hybridized to their specific quencher labeled cDNA, resulting in weak fluorescent signals. Simultaneously, the fluorescent labels of the aptamers bound to their targets in milk samples could produce strong signals under optimized conditions, as this aptasensor selectively detected AFM1 and OTA with a LOD value of 0.021 ng/mL over a wide linear range (0.001–1000 ng/mL).

In another study, an optical aptasensor was constructed based on a B08 aptamer by McKeague et al. (2014). In this diagnostic system, the SYBR Green I dye, which can intercalate to the minor groove of dsDNA, was used to enhance fluorescent emission. In their designed sensor, OTA competed with SYBR Green I for binding to aptamer, and consequently led to a decline in SYBR Green I fluorescence emission. SYBR Green intercalates with the free DNA aptamer and fluoresces at 520 nM. However, increasing concentrations of the OTA and binding to aptamers displaces SYBR Green I and a concentration-dependent loss of emitted fluorescence. This label-free SYBR Green I-based aptasensor showed Kd (9 nM) and linear range in the nanomolar scales (9–100 nM).

There are some reports that introduced lateral flow assay (LFAs)-based optical biosensors (Anfossi et al., 2012; Anfossi et al., 2013; Moon et al., 2013; Zhou et al., 2016b; Jiang et al., 2017; Ren et al., 2018; Velu and DeRosa, 2018; Oh et al., 2019; Hao et al., 2021; Zhao et al., 2021).

LFAs are popular among commercial paper-based assay products for quality control, food safety assessment, and in medical and clinical centers and laboratories (Majdinasab et al., 2022). However, this method has limited sensitivity in the detection of low concentrations of analytes. The samples also require pretreatment, including sample extraction, filtration, and dilution in some analytes, such as complex matrix or solid samples. Thus, LFA strips are not ideal for these samples. Positive results of LFA tests also need confirmatory analysis (Majdinasab et al., 2022).

b) Non-biosensor Aptamer based separation and detection methods

Due to the specificity and selectivity of the OTA aptamer isolated by Cruz-Aguado and Penner (OBA aptamer) (Cruz-Aguado and Penner, 2008), several studies have applied it as a detection element in a column to isolate, extract and clean up this toxin from samples, including wine, beer, or food (Table 6).


In some studies, various oligosorbents on solid-phase columns were constructed, such as magnetic nanospheres (MNS) (Wu et al. 2011b), coupling gel for the preparation of the aptamer-SPE (solid-phase extraction) columns (de Girolamo et al., 2011), cyanogen bromide-activated sepharose (Hadj Ali and Pichon, 2014), chitosan magnetic nanoparticles (Wang et al., 2020), or monolithic columns with high coverage density of aptamer (Chen et al., 2018; Yu et al., 2018; Lyu et al., 2020).

i) Liquid chromatography

In one of the considerable methods, an ultra-fast liquid chromatography with tandem mass spectrometry method was reported based on aptamer-affinity column and vortex-assisted solid-liquid microextraction, which can promote the diffusive motion of analyte from the sample to the extraction solvent (Yang et al., 2014). In another method, polyhedral oligomeric silsesquioxane (POSS) was applied to construct aptamer monoliths. POSS is a special organic-inorganic material with highly stereoscopic nano-cage and massive function sites that provide high coverage density of aptamer. Polyethyleneimine (PEI) was also used for the immobilization of AuNPs aptamer due to abundant amino groups. This fabrication resulted in a highly efficient, sensitive, and selective recognition system (LOD = 0.06 ng/mL) (Yu et al., 2018).

In a similar study, a POSS-PEI monolith was prepared with 2,4,6-trichloro-1,3,5-triazine (TCT) as a linker for binding high levels of the aptamer that enable them to achieve a LOD as low as 0.01 ng/mL in beer samples (Yu et al., 2019).

ii) Polymerase chain reaction-based assays

Real-time PCR is an amplification method and a high throughput screening method that could shorten operation time, decrease detection limit for low concentration analyte samples and have a favorable reproducibility.

The most sensitive device was reported with a LOD of 0.000001 ng/mL (1 fg/mL) for the detection of OTA by employing real-time PCR (Ma et al., 2013). It has shown a satisfactory recovery rate (99–112%) in red wine samples. The recruited strategy was based on the conformational change of the OBA aptamer (Cruz-Aguado and Penner, 2008). At first, the biotinylated aptamers were immobilized on the surface of the streptavidin-coated PCR tubes. Then, in the absence of the OTA, ssDNA aptamer was hybridized with complementary DNA strands and subjected to the same treatment as the PCR template. The forward and reverse primers and other PCR components were added and the PCR procedure was performed. Then, the emitted fluorescence was measured after each annealing step. In the presence of OTA, a structural switch of the aptamer was induced by the target binding, leading to the formation of an antiparallel G-quadruplex, which resulted in complementary ssDNA release. This could reduce the amount of the template for amplification and increase the cycle threshold (Ct). Thus, the concentration of the OTA was measured by the change in the PCR amplification (Ma et al., 2013).

iii) Enzyme-Linked Aptamer Assay (ELAA)

ELAA is employed for biorecognition of analytes by replacing antibody with aptamer, with its benefits over antibody through (Vargas-Montes et al., 2019).

Among other reported aptamers, H12 aptamer (Barthelmebs et al., 2011) was also applied for the detection of OTA. The H12 aptamer was used for both direct and indirect ELAA for detection of OTA in spiked red wine samples by Barthelmebs et al. (2011). In this approach, the fluorescein-labeled aptamers are specifically bound to biotinylated OTA. This complex could attach to the OTA-HRP conjugate, and the emitted fluorescence could be simply detected (Barthelmebs et al., 2011).

Chip-based aptasensors

Miniaturization is one of the important issues in the fabrication of biosensors to produce portable and user-friendly devices. Lab-on-a-chip (LoC) is also recently considered in this field. Some Efforts have also been made to design LoC-aptasensors for the detection of OTA.

Recently, a novel ACSB was described for the detection of OTA via FRET with an LOD of 0.005 ng/m in a linearity range of 0.01–10 ng/mL. They immobilized a Cy3-labeled OTA-specific biotinylated aptamer on an epoxy-coated chip via streptavidin-biotin binding. A black hole quencher 2 (BHQ2) labeled complementary DNA strand to OTA aptamer. In the presence of OTA, the Cy3-labeled OTA aptamer bound specifically to OTA and led to the physical separation of Cy3 and BHQ2, which resulted in an increase in fluorescence signal. This aptasensor was tested in rice samples spiked with OTA with a mean recovery rate of 91% (Li et al., 2021).

A fluorescent label-free LoC aptamer portable assay, integrated into the microfluidic network, was constructed on a single glass substrate, comprising an array of amorphous silicon photosensors and a long pass interferential filter. The employed fluorescent molecule was a “light switch” complex [Ru(phen)2(dppz)](2+) which intercalated between the base pairs of the aptamer. The aptamer was directly anchored into a layer of poly(2-hydroxyethyl methacrylate) polymer brushes grown inside the channels. The presence of OTA changed the aptamer conformation and released the fluorophore, causing a decline in fluorescence. This device performed detection in 5 min with an LOD of 1.3 ng/mL and 5–200 ng/mL linear detection in real samples (beer and wheat samples) (Costantini et al., 2019).

Nekrasov et al. (2022) reported an advanced aptasensor based on an array of graphene field-effect transistors integrated on a single silicon chip. Graphene with electrochemical, thermal, optical, electronic, and mechanical properties holds enormous potential for LOC devices. The G-rich aptamer was covalently attached to the graphene surface via pyrenebutanoic acid, succinimidyl ester (PBASE) chemistry. PBASE created efficient π-π stacking to graphene via an electric field stimulation. In the absence of OTA, the aptamer strands created π-π stacking on graphene, and after the addition of OTA molecules, aptamers reconfigured in G-quadruplex to bind OTA molecule. This fast assay (10 s) graphene-aptasensor showed an LOD of 1.4 pM for OTA with a demonstrated performance of wine in real-time.

An ultrasensitive label-free liquid crystal (LCs) OTA aptasensor was also designed with the lowest reported LOD (0.63 Am) based on the conformational switch of the immobilized π-shaped aptamer on the glass substrate in the presence of the OTA. A shift in the orientation of LCs from random to a homeotropic state altered the optical appearance of the aptasensor platform, which could be examined by polarized light microscopy for the detection of OTA in grape juice, coffee, and human serum samples (Khoshbin et al., 2021).

Expert Opinion and Conclusion

OTA-specific recognition aptamers show good affinity, making them applicable recognition elements in different devices. Docking results reveal that these aptamers could surround the OTA through some hydrogen bonds by creating binding pockets. However, a logical and straight relationship between docking scores and monitored hydrogen bonds has not yet been found, possibly because aptamer-target interaction has more intricacy and requires more in silico analyses to discover critical features in this interaction.

Aptamers as alternative recognition elements to antibodies exhibit several advantages, including the ease of in vitro isolation, high specificity and selectivity, longer duration of stability, ability to isolate a variety of small, toxic, and non-immunogenic molecules, amenable to modification, rare immunogenicity, and limited batch-to-batch variation. Aptamer-based systems also have several advantages over conventional methods due to their high selectivity and sensitivity, low cost, and stability.

Overview of this database could show that, by employing signal amplification strategies such as nanomaterials for increasing surface accessible area or catalytic effects on reaction, could be ultrasensitive devices with LODs as low as pico/femto level. Most of the aptasensors show reproduced responses with a good confidence. They are also applied frequently for the detection of real samples such as beverages, particularly wine, beer, and also cereals such as wheat. This could confirm the efficiency of these devices for consumers.

Nucleic acid amplification strategies are the other techniques for enhancing signals and, consequently, increasing sensitivity. PCR, real-time PCR and recently, LAMP as isothermal amplification-detection strategies were employed in OTA-specific aptasensors. Utilizing other nucleic acid amplification techniques such as helicase-dependent amplification, strand displacement amplification, rolling circle amplification, and recombinase polymerase amplification could be considered for further aptasensor fabrication.

Gold electrodes act as favorable working electrodes among electrochemical OTA aptasensors due to their unique properties such as good conductivity and chemical inertness.

Aptamers as alternative recognition elements to antibodies exhibit several advantages, including the ease of in vitro isolation, high specificity, and selectivity, long duration of stability, ability to isolate a variety of small, toxic, and non-immunogenic molecules, the capability of modification, rare immunogenicity, and limited batch-to-batch variation. Aptamer-based systems also have several advantages over conventional methods due to their high selectivity and sensitivity, low cost, and stability. However, the application of aptamer as a recognition element has some challenges. The most important issue is maintaining the desired affinity of the aptamer. Changing conditions in different detection methods could impact the affinity of the aptamer. For instance, immobilization techniques could result in microstructure changes which may alter the binding affinity. Other circumstances such as ionic strength and pH could influence aptamer configuration which may reduce its affinity properties. On the other hand, complex samples such as foods include various components such as nucleases which may have an adverse effect on the structure of aptamers and affect their activity.

Despite hundreds of reported separation methods and aptasensors with very low LODs for OTA, none have received approval for commercial applications, posing major challenges for improving research prototypes to reliable instruments.

Achieving the simple, rapid, sensitive, specific, and cost-effective methods for measuring OTA are the primary requisites for industrial, analytical, and medical assessments. Numerous aptamers are just capable of detecting samples in the aqueous solutions but not in solid analytes. It is necessary to improve some aspects, including ease of application, sample preparation, and cost of the production to reduce the gap between research and large-scale industrial applications (Schmitz et al., 2020).

Almost all of the sensors and separation approaches have used ssDNA aptamer reported by Cruz-Aguado and Penner (2008). They demonstrated a well-designed isolated aptamer which can meet merits for application.

Electrochemical and optical biosensors are two types of aptasensors developed for detection of OTA. Although the electrochemical methods have shown the highest sensitivity and selectivity, the optical aptasensor exhibited the widest linear ranges. However, several ultra-sensitive aptasensors have used nanomaterials to increase accessible surface area to enhance the OTA-aptamer interaction for signal amplification.

The current systematic review demonstrated the possibility, simplicity, and high selectivity of using aptamers to detect and analyze OTA in different real samples that are highly needed in agriculture, food industry, and water management.

Acknowledgement: The authors would like to thank the Clinical Research Development Unit of Baqiyatallah Hospital, Tehran, Iran, for guidance and advice.

Author Contribution: The authors confirm contribution to the paper as follows: study conception and design: M. Heiat and R. Ranjbar; data collection, analysis and interpretation of results, draft manuscript preparation: R. Torabi, A. A. Rezvanipour, H. Esmaeili Gouvarchinghaleh. All authors reviewed the results and approved the final version of the manuscript.

Ethics Approval: Not applicable.

Funding Statement: The authors received no specific funding for this study.

Conflicts of Interest:The authors declare that they have no conflicts of interest to report regarding the present study.


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APA Style
TORABI, R., REZVANIPOUR, A.A., GOUVARCHINGHALEH, H.E., RANJBAR, R., HEIAT, M. (2022). Aptamer based detection and separation platforms for ochratoxin A: A systematic review. BIOCELL, 46(12), 2537-2557. https://doi.org/10.32604/biocell.2022.021373
Vancouver Style
TORABI R, REZVANIPOUR AA, GOUVARCHINGHALEH HE, RANJBAR R, HEIAT M. Aptamer based detection and separation platforms for ochratoxin A: A systematic review. BIOCELL . 2022;46(12):2537-2557 https://doi.org/10.32604/biocell.2022.021373
IEEE Style
R. TORABI, A.A. REZVANIPOUR, H.E. GOUVARCHINGHALEH, R. RANJBAR, and M. HEIAT "Aptamer based detection and separation platforms for ochratoxin A: A systematic review," BIOCELL , vol. 46, no. 12, pp. 2537-2557. 2022. https://doi.org/10.32604/biocell.2022.021373

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