“Lollipop” particle counting immunoassay based on antigen-powered CRISPR-Cas12a dual signal amplification for the sensitive detection of deoxynivalenol in the environment and food samples

Deoxynivalenol (DON) is one of the subgroup B mycotoxins, mainly produced by Fusarium species, and reported to be the predominant contaminant in the environment and food [17], [26]. The toxic effects of DON include immune modulation, disruption of intestinal barrier function, and cytotoxicity leading to cell death [13], [8]. Due to its water solubility, DON can spread from contaminated grains to water and soil, causing serious damage to agricultural resources and the surrounding environment [2], [37]. Recent studies have shown that low doses of DON intake can also be harmful to the health of the body [10], [9]. At present, the classical DON detection methods include enzyme-linked immunosorbent assay (ELISA) [14], thin-layer chromatography (TLC) [22], [7], high performance liquid chromatography (HPLC) [31] and HPLC tandem mass spectrometry [6]. However, these methods still have some deficiencies: For example, ELISA methods have the limitation of insufficient sensitivity; other methods require expensive large-scale equipment [36]. Therefore, it is important to develop high-sensitivity, specific, and simple detection methods to meet the detection needs.

Electrical biosensors are known for their accessibility, high sensitivity and low cost. The resistance-based particle counter (ERPC) is a scientific measurement instrument that uses the Coulter counter principle to convert each particle into a pulsed signal proportional to its volume [1], [23], [25]. Particle counters are widely used to analyze the size and quantity of particles because of their sensitivity, accuracy, and low cost [12], [28]. Among the microparticles, polystyrene (PS) microspheres are a polymer with controllable particle size, low cost, and good stability; moreover, they can be easily modified and can be used as a stable signal readout for particle counters [16], [33], [5]. In our previous work, we have developed a series of PS particle counter-based assays. Wang et al. Wang et al., ($year$) [30], meanwhile, developed a multifunctional bioassay based on PS microsphere signal probes and electronic particle counters, which opens a new pathway to detect multi-targets based on immunomagnetic separation-mediated changes in the number of PS microsphere signaling probes. The introduction of magnetic separation techniques achieves good phase separation, however, the large number of magnetic separation steps in the detection process may lead to distortion of the signal transmission. Furthermore, Ren et al. Ren et al., ($year$) [21] established a one-step homogeneous micro-orifice resistance immunoassay to detect chlorpyrifos. They greatly simplify the detection steps by the homogeneous assay, but the complex composition of food matrices is a challenge for this detection method. Also, their work does not introduce an effective signal amplification strategy which may cause deficiencies in the detection of different detection sensitivity requirements for different targets. In brief, improving sensitivity, achieving efficient phase separation, and managing interference from complex matrices are the key challenges in developing particle counter-based detection methods.

Introducing an efficient signal amplification strategy is considered to be an effective means to improve the detection sensitivity. Recently, the CRISPR-Cas system has been widely applied in the field of detection due to its high specificity and signal amplification ability [19], [20], [29], [35]. Cas12a is an RNA-guided endonuclease with trans-cleavage activity for single-stranded DNAs (ssDNAs), which has received particular attention for its ease of use, low cost, and high stability [15], [27], [3]. However, how to combine it with other strategies (immunoassay) efficiently and even improve the performance is the main challenge to expand its application scope.

To achieve efficient phase separation and to overcome the interference from complex matrices, we first proposed a “Lollipop” structure as the carrier for immunoassay. The head of the lollipop is made of polystyrene, which means it can bind the antibody by simply inserting it into the sample with the efficient physical adsorption. Unlike the time-consuming and laborious phase separation conducted in previous studies [30], here it is only necessary to put the lollipop into the washing solution and stir it for 3–5 seconds, which greatly simplifies the washing steps (Fig. 1A). Moreover, based on the facile phase separation process, the “Lollipop” array structure coated with the antibody can achieve an efficient multiple sample pre-treatment simultaneously to overcome the complex matrix interference in the actual sample.

In this work, we developed a highly sensitive “Lollipop” particle counting immunoassay (LPCI) for DON detection based on an antigen-powered CRISPR-Cas12a dual signal amplification strategy. First, signal probes were constructed by linking PS microspheres with magnetic nanoparticles (MNPs) via single-stranded reporter DNA (PS-Reporter-MNP), and capture probes were constructed by co-incubating the “Lollipop” carrier with DON-Ab. The lollipop-style capture probes were incubated with DON for specific capture, enabling a sample pre-treatment process (Fig. 1A). The DON-BSA-Activator related to DON content was obtained in the lollipop-style capture probes and further activated the cleavage activity of Cas 12a and cut off PS-Reporter-MNP. After magnetic separation of the reaction liquid, the supernatant was taken to measure the number of PS microspheres by particle counters for quantitative analysis of DON (Fig. 1B). The antigen-powered CRISPR system and the lollipop-structured carrier bestow the immunoassay method with the significant advantages of high sensitivity, high selectivity, and simple operation.

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