The natural occurrence of mycotoxins in food and feedstuff is a major issue across the world which can cause severe health problems in humans, animals and other livestock (Richard, 2007). Mycotoxins are toxic fungal secondary metabolites produced by various fungi and moulds, particularly Aspergillus, Fusarium, and Penicillium (Goyal, Ramawat, & Merillon, 2016). It has been estimated that about 25% of the world’s food crops every year get contaminated by mycotoxins, resulting in huge industrial and agricultural loss per year (Mateo et al., 2018). Among the various mycotoxins, AFB₁, which is a low molecular mass compound produced by Aspergillus flavus and Aspergillus parasiticus, is considered to be a potent, strong teratogen, mutagen, and carcinogen. AFB₁ is commonly found in various food and feedstuffs including cereals, oilseeds, spices, coffee beans, and nuts (Omrani et al., 2016, Negash, 2018). AFB₁ is classified by the International Agency for Research on Cancer (IARC) as a Group-1 Carcinogen that shows a high risk for Hepatocellular Carcinoma (Kensler & Groopman, 2018). The European Commission has set a maximum permissible level of AFB₁ in food content to 5–10 µg/kg (Heshmati, Zohrevand, Khaneghah, Nejad, & Sant’Ana, 2017). Conventional, well-equipped laboratory techniques, such as high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are commonly used for aflatoxins detection but usually lack sensitivity and sensibility (high limit of detection) (Wang et al., 2019). Advanced and more complex techniques also exist such as enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR) based assays which usually show higher sensitivity and sensibility but suffer cross-reactivity issues and difficulties of implementation for routine analysis (Koedrith et al., 2015).

In the past two decades, optical biosensors based on SPR appeared to be one of the most powerful technique for interaction kinetic analysis and label-free quantification of small biomolecules (<1000 Da) (Mullett et al., 2000, Nikolovska-Coleska, 2015). Thus, SPR based biosensors have gained great attention in recent years due to their application in various areas such as biotechnology and life science research, agro-food analysis, environmental monitoring and medical diagnostics (Jia et al., 2018, Zhang et al., 2014).

In real-time monitoring, signal amplification for label-free detection of small molecules such as AFB₁ is a real challenge. However, only seldom efforts have been devoted to improving biosensing characteristics such as sensitivity and limit of detection for SPR based AFB₁ detection. Upon these few examples, Sun et al. proposed an SPR aptamer-based assay for AFB₁ detection ranging from 0.4 to 200 nM, with a limit of detection of 0.4 nM (Sun, Wu, & Zhao, 2017). Recently, Park et al. developed bifunctional protein crosslinker-based SPR biosensor to detect AFB₁ where the genetically engineered fusion of gold binding protein and protein-G has been used to fabricate gold sensor chip. The limit of detection was 1 µg/mL in the detection range from 0.5 to 100 µg/mL (Park, Kim, Kim, & Ko, 2014).

Noble metal NPs have also been used to enhance SPR signal response, leading to the enhancement of specificity and sensitivity for small biomolecules detection. Among the various described metallic NPs, spherical AuNPs are the most favorable for biosensing amplification thanks to their plasmonic properties, large dielectric constant, high surface-to-volume ratio and surface energy. Moreover, their isotropic structure allows the strong optical coupling to occur in every direction with incident light rather than to take place only in one direction, thereby enhancing the sensitivity of the SPR biosensor (Devi et al., 2015, Martinsson et al., 2014, Szunerits et al., 2014). Interestingly, AuNPs surface modification using suitable capping agents or ligands can provide functionality for bio-recognition elements immobilization. In this work, surface functionalization of AuNPs was modified by solvent extraction-based ligand exchange method, slightly modified from a literature reported method (Dewi, Laufersky, & Nann, 2014). Briefly, the two-step process was utilized to complete the replacement of Cetyltrimethylammonium bromide (CTAB) surfactant to lipoic acid by maintaining their colloidal stability in an aqueous solution. In the first step, CTAB surfactant was partially removed from the aqueous phase of AuNPs to immiscible organic solvent of dichloromethane. Second, complete replacement of CTAB to lipoic acid was achieved through direct chemisorption, resulting in a higher degree of exchange. This approach is a versatile method for AuNPs surface modification also preserving colloidal stability, shape, and size of the particles.

Another important parameter for SPR biosensing performance is the quality of the bio-recognition element immobilization onto the sensor surface. Indeed, direct and random immobilization of antibodies on a solid sensing surface can generate antibodies conformational changes, leading to instability or loss of antigen-binding (Tsekenis, Chatzipetrou, Massaouti, & Zergioti, 2019). The immobilization of antibody binding protein such as protein-A which specifically bind with Fc region of an antibody without any chemical modification is one of the smart approaches leading to the immobilization of orientated bio-recognition elements (Liu & Yu, 2016).

We are presenting here the development of an AuNPs based SPRi biosensor for AFB₁ detection. To do so, chemically modified AuNPs were grafted over a SAM Au chip surface through an amine linkage. Protein-A was used as an orientating immobilization tool of AFB₁ specific antibodies. For comparative study, bare Au sensor chips using the same immobilization protocol were used.