Review Examines Immunosensing and Aptasensing Progress for Ochratoxin A Detection

Agricultural production and human nutritional safety face a persistent challenge from ochratoxin A, a highly toxic secondary metabolite generated by fungal species within the Aspergillus and Penicillium genera. Chemically structured as a weak organic acid comprising phenylalanine and dihydro-isocumarin linked through a stable amide bond, ochratoxin A exhibits high thermal stability, environmental persistence, and resistance to standard industrial food processing. The molecule possesses a molecular weight of 403.8 grams per mole and exists in non-ionic, monoanionic, and dianionic forms, remaining capable of surviving secondary manufacturing and prolonged storage across essential commodities including wheat, barley, corn, coffee, wine, dried fruits, and animal feed. In a comprehensive review published in Frontiers in Nutrition, researchers Chao Lin, Renxiong Yao, Yue Li, Xiaoming Sun, and Dongxu Wang systematically analyzed biosensing innovations reported from 2020 through 2026. Their assessment underscores that while conventional instrumental platforms provide rigorous analytical benchmarks, the operational demands of agricultural supply chains require sensitive, rapid, and field-deployable detection systems capable of quantifying trace concentrations of this resilient fungal contaminant.

The urgency surrounding ochratoxin A surveillance stems directly from its profound multi-organ toxicity, characterized by documented carcinogenic, nephrotoxic, hepatotoxic, neurotoxic, and immunotoxic activities. Once ingested, the toxin is metabolized within liver microsomes by mixed-function oxidases into 4-hydroxy and 8-hydroxy derivatives, which subsequently disrupt normal cellular function by inhibiting the biological synthesis of proteins, ribonucleic acid, and deoxyribonucleic acid. Toxicokinetic evaluations indicate that ochratoxin A binds extensively to plasma and serum albumin proteins, conferring an exceptionally prolonged vascular circulation half-life reaching up to 35 days following single exposures, alongside high oral bioavailability demonstrated across mammalian species, including estimated values of 60 percent in pigs and 93 percent in humans. Toxicological risk characterization conducted by the European Food Safety Authority established a benchmark dose lower confidence limit of 4.73 micrograms per kilogram body weight per day for non-neoplastic microscopic renal tubular lesions in female pigs, alongside a benchmark threshold of 14.5 micrograms per kilogram body weight per day for rat renal carcinogenicity. Consequently, regulatory authorities enforce strict maximum residue boundaries, spanning 0.5 to 10 micrograms per kilogram under European Commission rules and 5.0 micrograms per kilogram for grains under Chinese national standards.

Managing the persistent risk of mycotoxin contamination remains constrained by the inherent limitations of established mitigation and analytical techniques. Existing decontamination strategies, categorized broadly into physical, chemical, and biological interventions, frequently present unavoidable operational trade-offs during commercial food processing. Physical treatments such as heat, irradiation, and adsorption separation often yield incomplete detoxification while degrading nutritional content and matrix flavor profiles; chemical degradation strategies using acidic, alkaline, or redox reagents risk leaving harmful chemical residues; and microbial or enzymatic biological degradations typically suffer from prolonged reaction cycles and inadequate operational stability. In parallel, standard analytical methodologies including high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry achieve high precision and reproducible measurements, yet their heavy reliance on complex multistep sample preparation, bulky instrumentation, and specialized technical expertise precludes rapid on-site inspection. Even traditional enzyme-linked immunosorbent assays remain hindered by high reagent costs, lengthy operational procedures, and susceptibility to matrix interference, accelerating the research community toward miniaturized biosensors integrating biological recognition elements with sensitive physical transducers.

Electrochemical immunosensors have emerged as a prominent analytical direction by pairing immobilized antibodies with conductive transducers that convert targeted binding interactions into measurable electrical currents, interfacial impedances, or potentials. Investigators have explored diverse functional nanomaterials and amplification cascades to enhance electrocatalytic performance and biological loading capacities. For example, resistive-pulse sensing systems grounded in the Coulter counting principle have demonstrated robust quantitative detection by converting target-induced microparticle aggregation into distinct voltage pulse variations. In one design, horseradish peroxidase-mediated polydopamine deposition on magnetic nanoparticles captured polystyrene microspheres within microchannels, translating ochratoxin A concentrations into discrete electrical signals with a 77 picogram per milliliter detection limit across a 90-minute assay. Other researchers successfully replaced labile enzymatic catalysts with cuprous oxide and cupric oxide heterostructures or copper-catalyzed azide-alkyne click reactions to aggregate microspheres for direct electronic counting. Furthermore, self-supported flexible carbon paper electrodes modified with cuprous oxide, cupric oxide, and gold nanoparticle heterostructures yielded a detection limit down to 0.2 picograms per milliliter, while label-free electrochemical impedance spectroscopy on gold-coated glass substrates functionalized with cysteamine self-assembled monolayers provided direct, reagentless target quantification.

Electrochemiluminescence immunosensors further advance trace analytical capabilities by coupling electrochemical stimulation with photon emission, achieving low optical backgrounds and exceptionally wide dynamic ranges. In these analytical configurations, specific electrode potentials drive electron transfer between luminophores and coreactants to generate excited-state species that emit measurable light upon radiative relaxation to the ground state. Recent investigations have developed both quenching-type and signal-enhanced electrochemiluminescence architectures. A signal-on quenching sensor engineered with gold-doped carbon nitride nanosheet luminophores combined with polydopamine-modified zeolite imidazole frameworks achieved an ultra-trace detection limit of 4.8 femtograms per milliliter in flour and rice matrices. In parallel, enhanced-type sensing platforms have utilized manganese-coordinated porphyrin zirconium metal-organic frameworks supported by gold-platinum nanoflowers on glassy carbon electrodes, realizing a limit of detection of 0.067 picograms per milliliter with robust signal stability across grain samples. Although electrochemiluminescence devices attain extraordinary analytical sensitivity, the synthesis of customized nanomaterial probes remains intricate, and the luminescence emission remains susceptible to operational variables including environmental temperature, ambient pH fluctuations, and electroactive interferents present in untreated agricultural extracts.

Optical immunosensors represent another major branch of ochratoxin A monitoring, translating biological recognition into ultraviolet, visible, or infrared signals across colorimetric, fluorescent, and luminescent detection modes. A critical advancement in this domain involves replacing conventional monoclonal antibodies with genetically engineered camelid nanobodies, which consist exclusively of heavy chains that offer superior thermal stability, elevated chemical resistance, and cost-effective microbial production. Leveraging these single-domain antibodies, researchers engineered ratiometric bioluminescence sensors and split-luciferase NanoBiT platforms that reassemble functional enzymes upon target competition, achieving quantitative measurements within 5 to 10 minutes in cereal grains. Optical configurations frequently incorporate fluorescence resonance energy transfer, pairing nanobodies with quantum dots or superfolder green fluorescent protein fusions to monitor donor-to-acceptor energy migration with 5 picogram per milliliter sensitivity. Additional optical strategies utilize terbium-chelated mesoporous silica nanoparticles that leverage the distinct photophysical antenna effect of rare-earth ions to bypass endogenous sample autofluorescence, while microfluidic fluoroimmunoassays deploying superparamagnetic microbeads and laser excitation achieve rapid separation and high-throughput screening in small liquid volumes.

To overcome the false-positive tendencies and vulnerability to matrix interference that frequently compromise single-signal platforms, investigators have designed multi-modal immunosensors that combine distinct transduction pathways within single assay architectures. Integrating complementary detection channels allows simultaneous mutual validation, bolstering diagnostic reliability across complex agricultural substrates. For instance, researchers synthesized silver-doped amino-silica core-shell nanohybrids to formulate bifunctional probes capable of operating through ultraviolet-visible spectrophotometric absorption alongside fluorescence quenching modes in corn, soybean, and wheat matrices. Similarly, combining genetic engineering with material science yielded colorimetric and fluorescent lateral flow strips based on heptameric nanobodies and gold nanoflower fusions, providing clear naked-eye visual screening alongside instrument-verified fluorescence quantification. Bridging distinct transduction categories, portable paper-based dual-mode devices utilizing bovine serum albumin-stabilized gold nanoclusters have demonstrated simultaneous electrochemical signal measurement and smartphone-assisted fluorescence imaging, enabling point-of-care screening in field environments without requiring complex laboratory infrastructure.

Aptamer-based biosensors, or aptasensors, have gained equal prominence by substituting proteinaceous antibodies with artificial single-stranded DNA or RNA oligonucleotides generated through systematic evolution of ligands by exponential enrichment. Because aptamers are synthetic oligonucleotides, they offer unmatched molecular programmability, superior batch-to-batch reproducibility, and seamless compatibility with enzyme-free isothermal nucleic acid amplification strategies. In electrochemical aptasensing systems, target recognition frequently initiates programmable DNA walking machines or rolling circle amplification cascades, driving significant signal amplification directly on conductive interfaces. In one illustrative design, a non-targeted triggering rolling circle amplification system utilizing padlock probes and gold electrodes generated distinct current outputs upon ochratoxin A binding. Other electrochemical strategies enhance signals through target-induced probe desorption from reusable gold-based screen-printed electrodes, in-situ silver metallization coupled with exonuclease I background cleavage, or integration with titanium carbide MXene and polyvinylidene fluoride composite nanofibers. Furthermore, field-effect transistor arrays fabricated from graphene monolayers modified with pyrenebutanoic acid succinimidyl esters have demonstrated rapid, label-free electronic quantification, highlighting strong prospects for monolithic microchip integration.

Optical aptasensors exploit the localized surface plasmon resonance of noble metal nanoparticles alongside advanced molecular switches to deliver accessible, visual, and spectroscopic readouts. Hybrid platforms assembling gold nanostars and dye-labeled complementary DNA nanospheres provide dual surface-enhanced Raman scattering and fluorescence tracking capable of differentiating ochratoxin A from structurally similar mycotoxins. Simpler colorimetric assays utilize alkaline phosphatase-induced aggregation of gold nanoparticles to yield visible color shifts discernible without specialized instrumentation, although these naked-eye tests generally exhibit higher detection limits suited primarily for initial qualitative screening. To achieve higher analytical sensitivity, researchers have implemented enzyme-free catalytic circuits, DNA-templated silver nanoclusters, and exponential rolling circle amplification coupled with G-quadruplex molecular switches that resist sample pigment interference. More recently, the field has integrated clustered regularly interspaced short palindromic repeats and Cas12a endonuclease systems into aptasensing frameworks. Utilizing the non-specific single-stranded collateral trans-cleavage activity of Cas12a triggered by aptamer-target recognition, researchers have paired CRISPR systems with hybridization chain reactions and ratiometric fluorescence probes to achieve ultra-sensitive mycotoxin quantification.

Despite the notable analytical milestones documented across immunosensors and aptasensors between 2020 and 2026, significant technical and practical hurdles continue to impede their transition from laboratory proof-of-concept demonstrations to commercialized agricultural monitoring. The authors of the review emphasize that most validation experiments rely exclusively on clean matrices artificially fortified with ochratoxin A standards, leaving biosensing performance largely uncharacterized in naturally contaminated grains containing complex matrix backgrounds, degraded fungal metabolites, and chemical analogs. Non-specific adsorption of matrix proteins, pigments, and polysaccharides frequently causes electrode fouling or microfluidic channel clogging, demanding rigorous sample pre-extraction steps that undermine the portability of point-of-care devices. Additionally, the field faces unresolved challenges concerning batch-to-batch variability in nanomaterial synthesis, unverified long-term storage stability of functionalized bio-interfaces, and an absence of standardized cross-laboratory validation protocols. Bridging these industrial translation barriers will require coordinated progress in synthetic nanobody engineering, robust dual-mode multi-signal fusion platforms, and microfluidic integration with ubiquitous consumer smartphones, ultimately establishing resilient analytical tools capable of safeguarding the global food supply chain against mycotoxin exposure.

Subject of Research: Biosensing technologies for trace detection of ochratoxin A

Article Title: Research progress on immunosensing and aptasensing of ochratoxin A

Article References: Lin, C., Yao, R., Li, Y., Sun, X., & Wang, D. (2026). Research progress on immunosensing and aptasensing of ochratoxin A. Frontiers in Nutrition, 13, Article 1950981. https://doi.org/10.3389/fnut.2026.1950981

Image Credits: AI Generated

DOI: 10.3389/fnut.2026.1950981

Keywords: ochratoxin A, mycotoxins, biosensors, immunosensors, aptasensors, food safety, electrochemical biosensors, optical biosensors, nanobodies, electrochemiluminescence, fluorescence resonance energy transfer, rolling circle amplification, CRISPR-Cas12a, limit of detection, matrix interference, dual-mode sensing, surface plasmon resonance, point-of-care testing