Dual-Modal Immunosensor Made with the Multifunction Nanobody for Fluorescent/Colorimetric Sensitive Detection of Aflatoxin B₁ in Maize

Abstract

In recent years, dual-modal immunosensors based on synthetic nanomaterials have provided accurate and sensitive detection. However, preparation of nanomaterial probes can be time-consuming, laborious, and not limited to producing inactive and low-affinity antibodies. These challenges can be addressed through the multifunction nanobody without conjugation. In this study, a nanobody-enhanced green fluorescent (Nb26-EGFP) was novel produced with a satisfactory affinity and fluorescent properties. Then, a dual-modal fluorescent/colorimetric immunosensor was constructed using the Nb26-EGFP-gold nanoflowers (AuNFs) composite as a probe, to detect the aflatoxin B1 (AFB1). In the maize matrix, the proposed immunosensor showed high sensitivity with a limit of detection (LOD) of 0.0024 ng/mL and a visual LOD of 1 ng/mL, which is 20-fold and 325-fold compared with the Nb26-EGFP-based single-modal immunosensor and original nanobody Nb26-based immunoassay. The performance of the dual-modal assay was validated by a high-performance liquid chromatography method. The recoveries were between 83.19 and 108.85%, with the coefficients of variation below 9.43%, indicating satisfied accuracy and repeatability. Overall, the novel Nb26-EGFP could be used as the detection probe, and the dual-modal immunosensor could be used as a practical detection method for AFB1 in real samples.

A Novel and Highly Efficient Microextraction Method for the Determination of Aflatoxin Precursor Averantin in Fatty Grain Samples

“…Mycotoxins are toxic secondary metabolites produced by fungal species that may contaminate food commodities. The Food and Agriculture Organization estimated global mycotoxin contamination of food crops at 25%, specifically due to aflatoxins (AFs). , AFs are the most toxic and carcinogenic compounds and produced through multiple enzymatic reactions. − In AF biosynthesis pathways, approximately 15 precursors, including norsoloric acid, versicolorin A (Ver A), and sterigmatocystin (ST), have been identified. − These precursors also contaminate food and agricultural products, posing a significant risk to human and animal health, which are necessary to be determined accurately. The development of detection methods for these AF precursors is mainly focused on Ver A and ST. − Wu et al established an in-tube solid-phase microextraction with high-performance liquid chromatography (HPLC) and successfully determined ST in rice samples .…”

Self-Assembly Multivalent Fluorescence-Nanobody Coupled Multifunctional Nanomaterial with Colorimetric Fluorescence and Photothermal to Enhance Immunochromatographic Assay

“…Lateral flow immunoassay (LFIA) is a widely utilized tool for point-of-care (POC) detection. Multimodal LFIA technology has developed rapidly and has been successfully applied to food testing, , in vitro diagnostics, , and environmental monitoring recent years. , Multisignal LFIA sensors are constructed by integrating antibodies (monoclonal, polyclonal, and nanobody) and nanomaterials (noble metal nanomaterials, carbon nanomaterials, quantum dots and nanoenzymes, etc. ), which can provide multiple signal outputs, mainly including colorimetric signals, fluorescent signals, Raman signals, electrochemical signals and thermal signals .…”

“…In antibody-based lateral flow immunoassays (LFIA), external fluorescent material labeling is required to obtain fluorescent signals, such as the use of fluorescent gold nanoparticles and lanthanide nanoparticles, but this is significantly limiting in constructing fluorescent multipeak signal outputs for LFIA. Therefore, the introduction of fluorescent protein, , luciferase, and alkaline phosphatase into nanobodies by genetic engineering not only eliminates the synthesis step of immunofluorescent labeling complexes but also provides a method for constructing multimodal LFIA for precise quantitative detection.…”

Multiple Isothermal Amplification Coupled with CRISPR–Cas14a for the Naked-eye and Colorimetric Detection of Aflatoxin B1

“…Researchers are therefore focused on developing new technologies that simplify sample preparation and yield more portable detection devices. These include colorimetric, fluorescent, and electrochemical methods combined with various biosensors for AFB1 detection. − Among these, colorimetry has gained wide acceptance due to its portability, rapid analysis, and high sensitivity. − Magnetic nanoparticles (MNPs) offer advantages such as easy preparation, a large specific surface area, and superparamagnetic properties, making them useful for efficient target enrichment and rapid sample matrix separation . Additionally, gold nanoparticles (AuNPs) exhibit local surface plasmon resonance characteristics; their color change can be used as a biosensor response signal .…”