*Article* **Development of an Ultrasensitive and Rapid Fluorescence Polarization Immunoassay for Ochratoxin A in Rice**

**Xiaorong Huang 1,2,3,4,5,**†**, Xiaoqian Tang 1,2,3,4,5,**†**, Abdoulie Jallow 1,2,3, Xin Qi 5, Wen Zhang 5, Jun Jiang 5, Hui Li 2,5, Qi Zhang 1,2,3,4,5,\* and Peiwu Li 1,2,3,4,5,\***


Received: 28 August 2020; Accepted: 18 October 2020; Published: 29 October 2020

**Abstract:** Ochratoxin A (OTA) is a known food contaminant that affects a wide range of food and agricultural products. The presence of this fungal metabolite in foods poses a threat to human health. Therefore, various detection and quantification methods have been developed to determine its presence in foods. Herein, we describe a rapid and ultrasensitive tracer-based fluorescence polarization immunoassay (FPIA) for the detection of OTA in rice samples. Four fluorescent tracers OTA-fluorescein thiocarbamoyl ethylenediamine (EDF), OTA-fluorescein thiocarbamoyl butane diamine (BDF), OTA-amino-methyl fluorescein (AMF), and OTA-fluorescein thiocarbamoyl hexame (HDF) with fluorescence polarization values (δFP = FPbind-FPfree) of 5, 100, 207, and 80 mP, respectively, were synthesized. The tracer with the highest δFP value (OTA-AMF) was selected and further optimized for the development of an ultrasensitive FPIA with a detection range of 0.03–0.78 ng/mL. A mean recovery of 70.0% to 110.0% was obtained from spiked rice samples with a relative standard deviation of equal to or less than 20%. Good correlations (*r*<sup>2</sup> = 0.9966) were observed between OTA levels in contaminated rice samples obtained by the FPIA method and high-performance liquid chromatography (HPLC) as a reference method. The rapidity of the method was confirmed by analyzing ten rice samples that were analyzed within 25 min, on average. The sensitivity, accuracy, and rapidity of the method show that it is suitable for screening and quantification of OTA in food samples without the cumbersome pre-analytical steps required in other mycotoxin detection methods.

**Keywords:** FPIA; mycotoxin; OTA; detection methods; food safety; monoclonal antibody (mAb); tracer; HPLC

**Key Contribution:** Herein, we synthesized an OTA-AMF fluorescent tracer with 207 δFP value, and established an ultrasensitive fluorescence polarization immunoassay with a detection range of 0.03 to 0.78 ng/mL, providing an ultrasensitive, simple, and rapid detection method for on-site monitoring of OTA.

#### **1. Introduction**

Ochratoxin A (OTA) is the poisonous secondary metabolite excreted by *Penicillium* and *Aspergillus* species, which is often found in a wide range of foods, such as rice, beans, wine, beer, coffee, cocoa, dried fruit, and animal products. OTA is categorized by the International Agency for Research on Cancer (IARC) as a group 2B possible human carcinogen. It is hepatotoxic, teratogenic, immunosuppressive, nephrotoxic, and nephrocarcinogenic [1,2]. A number of countries have moved to establish regulatory limits on OTA in food products destined for human consumptions [3]. For instance, the European Commission has imposed regulatory limits on OTA in corn and corn products. A maximum of 5 μg/kg for natural corn grain, 3 μg/kg for all other corn products destined for direct human consumption, and 0.5 μg/kg for baby food and corn-based products intended for young children is allowed [4].

To safeguard human health against the food safety risks associated with OTA, advanced, sensitive, and accurate analytical methods are required for its detection and quantification [4]. Instrument-based methods like HPLC connected to a fluorescence detector (HPLC/FLD) and liquid chromatography/mass spectrometry (LC/MS) are some of the most widely-used mycotoxin detection techniques. While instrument-based methods offer precision and reliability, compared to newer analytic techniques, they have some weaknesses: they are costly, require a certain level of expertise to operate them, and are not suitable for on-site use [5,6]. To overcome these drawbacks, immunoassays have recently gained popularity as an alternative to the above-described methods. Based on the binding of antigen to antibody, immunoassay-based techniques are cheap, simple, and sensitive [7,8].

Certain immunoassay techniques, such as ELISA, require tedious and time-consuming assay development [8]. Alternatively, FPIA is a simple and user-friendly immunoassay as it does not require tedious and time-consuming pre-analytical steps [9,10]. FPIA is a widely used homogeneous-based immunoassay with simple and rapid operational procedures. Currently, the method is widely applied in the monitoring of small molecules in variety of matrices [11–13]. Fluorescence polarization (FP) is commonly used to excite fluorescent molecules with polarized light in a vertical direction, and then measure the fluorescence intensity Iv and Ip of polarized light emitted in the vertical and horizontal directions, respectively. FP = (IV-IP)/(Iv + Ip), where FP is a dimensionless quantity, and the unit is usually expressed in mP. The principle of fluorescence polarization detection is based on the different sizes of the fluorescence molecules and the different intensities of the fluorescence polarization signal [14].

The use of FPIA to detect mycotoxins such as aflatoxins (AFTs), fumonisins (FBs), deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEN), and HT-2 and T-2 toxins in various matrices as reviewed by Maragos [4]. Additionally, Li et al. reported the development of a multiplexed FPIA for the simultaneous determination of deoxynivalenol, T-2 toxin, and fumonisin in maize samples. With regards to OTA, an OTA-ethylenediamine fluorescence (EDF) conjugate-based FPIA with a limit of detection (LOD) of 0.3 ng/mL of OTA in unpolished rice was reported [15]. In this study, we synthesized four tracers, among which the OTA-AMF tracer was chosen for further optimization to improve the detection sensitivity.

In this work, four new tracers with different fluorophores were synthesized. Based on fluorescence intensity, we selected two apparently better tracers for the subsequent experiments. Two FPIAs were then researched for the determination of OTA in buffer by optimizing the reaction conditions. Based on the optimum tracers and sensitive antibody against OTA, we successfully built and applied a simple, fast, and sensitive FPIA for the detection of OTA in rice. Based on optimal conditions, we further validated the results that were obtained by FPIA using HPLC as a reference method. The developed FPIA, as a result, is a promising method for the rapid analysis of OTA-contaminated rice samples.
