Natural milk possesses high nutritional value as it is an excellent source of high-quality proteins, fats, carbohydrates, vitamins, and minerals [1]. However, milk is susceptible to contamination by mycotoxins and antibiotic residues. Aflatoxin M1 (AFM1) is a hydroxylated mammalian metabolite of aflatoxin B1 [2,3]. Meanwhile, tetracycline antibiotics (TC), commonly administered as veterinary drugs for treating bovine mastitis, readily form persistent residues in dairy products [4]. On one hand, AFM1 is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) due to its potent carcinogenicity, prompting the European Commission (EC) to establish a stringent maximum residue limit (MRL) of 0.05 μg/kg in milk [[5], [6], [7]]. On the other hand, TC may trigger allergic reactions in sensitive individuals and promote antimicrobial resistance (AMR), leading the European Union (EU) to set its MRL in milk at 100 μg/L [5]. Compared to EU standards, China implements more stringent MRLs for TC and AFM1 in milk, set at 20 μg/kg and 0.35 μg/kg (NY/T 3314−2018 and GB 31 658.6 − 2021) [5,8,9]. Therefore, in order to further ensure the physical health of residents, conducting tests on these two compounds has certain practical significance.

Currently, most detection methods for TC and AFM1 in milk employ independent analytical approaches. Purification of TC primarily relies on adsorbents such as hydrophilic-lipophilic balanced (HLB) materials, weak cation-exchange (WCX) materials, C18 columns, or silica columns. Their sole reliance on a single mechanism (hydrophobic or ion-exchange interactions) often results in insufficient selectivity [[10], [11], [12]]. Although AFM1 can be purified using highly specific immunoaffinity columns (IACs), their high cost limits widespread application [13]. Recent studies have reported various efficient enrichment materials, such as porous polythiophene materials, three-dimensional sponge-architecture covalent organic frameworks-aerogel, aptamer-functionalized magnetic nanoparticles, and PEGylated multi-walled carbon nanotube magnetic nanoparticles, which can be used for the enrichment of TC and AFM1 [[14], [15], [16], [17]]. However, these materials are only capable of individually detecting one of the target analytes. To address this, Janus-MIP-Apt was synthesized in this study to achieve the simultaneous enrichment and detection of both TC and AFM1.

To date, methods for the synchronous detection of multiple pollutants (such as antibiotics and toxins) remain inadequate. Molecularly imprinted polymers (MIPs), capable of specifically recognizing the spatial configuration and size of target analytes (e.g., antibiotics) and their analogues, have been widely applied [18]. Aptamers (Apt), owing to their ease of preparation, stability, and high selectivity, serve as efficient and low-cost solutions for detecting small-molecule toxins [19]. Although some researchers have improved the specificity and adsorption capacity for target substances by combining MIPs with Apt, they are still limited to the detection of a single substance [20,21]. For the simultaneous detection of TC and AFM1, the dual-MIPs strategy has drawbacks because during the elution process of TC and AFM1, it tends to result in incomplete elution of one template or excessive loss of the other, which impairs the accuracy of detection. Additionally, in the same polymerization system, the two template molecules may interact with each other, reducing the specificity of imprinted sites [22,23]. The dual-Apt strategy, on the other hand, faces an issue because the immobilization of Apt on the surface of the same nanoparticle leads to steric hindrance, which affects the adsorption capacity. This study innovatively utilizes Janus particles as the substrate material. Their unique physical heterogeneity and surface compartmentalization characteristics break the limitations of traditional symmetric materials, enabling a single particle system to exhibit different functionalities [24]. By leveraging the abundant aromatic rings and cis-diol groups in TC, phenylboronic acid modification was performed on the Janus particle surface, synergistically establishing multiple recognition mechanisms including hydrophobic interactions, π-π stacking, and boronate affinity. Following template molecule elution, a three-dimensional imprinted cavity with exceptional selectivity for TC was generated. Simultaneously, Apt, as a short-chain nucleic acid, achieves highly specific binding with AFM1 through its unique secondary structures, such as bulges, G-quadruplexes, and hairpins. By integrating the efficient enrichment capability of solid-phase extraction (SPE) with the advantage of multiple interactions offered by the adsorbent, this study successfully constructed a highly selective SPE material capable of synchronous enrichment and detection of TC and AFM1.

Common detection methods for AFM1 in milk include thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), and reversed-phase high-performance liquid chromatography (RP-HPLC). For TC residues, gas chromatography (GC) or high-performance liquid chromatography-mass spectrometry (HPLC-MS) are typically employed. However, these methods exhibit significant limitations: TLC suffers from inadequate separation and quantification accuracy; ELISA is prone to false positives due to antibody cross-reactivity; RP-HPLC, while operationally simple and sensitive, requires time-consuming sample pretreatment, specialized operation, and incurs high costs [[25], [26], [27]]. More critically, these methods are generally applicable only to single-target detection. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS/MS), leveraging its exceptional sensitivity and specificity, enables synchronous and accurate quantification of trace TC and AFM1 residues in milk [28,29].

Therefore, in this experiment, anisotropic Janus particles were successfully synthesized by using poly(GMA) (poly(glycidyl methacrylate)) as seed, 4-vinylbenzyl chloride (VBC) as monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent, p-xylene and n-decyl alcohol as porogen through photoinitiation. According to the anisotropic characteristics, boronic acid-affinity Janus-MIP was successfully synthesized with TC as the template molecule and 4-vinylphenylboronic acid as the functional monomer. AFM1 aptamer was subsequently modified through epoxy-amine reaction, ultimately constructing Janus-MIP-Apt functional material capable of synchronously detecting TC and AFM1. A stepwise elution approach optimized the elution process, significantly reducing time costs and establishing a simple, sensitive, and rapid dual-contaminant detection method. This method demonstrated favorable applicability in milk sample detection and provides a novel approach for analyzing trace contaminants in food.