**1. Introduction**

Pesticides are widely used to protect plants from insects and diseases caused by bacteria and fungi in agricultural production all over the world. Among them, organophosphate insecticides, including malathion and phoxim, can effectively kill insects by targeting their nervous system [1]. Organosulfur fungicides such as thiram are also used for the preservation of fruits and vegetables [2]. Therefore, the combination of these pesticides is commonly used in agriculture [3]. However, the overuse of pesticides has already caused serious problems in terms of environmental pollution [4]. Moreover, their residues in agricultural products pose a direct threat to human health [5,6]. For example, there are reports of several health issues caused by thiram, with symptoms such as lethargy and motor disturbances [7]. Exposure to a high enough level of malathion could lead to various symptoms, including nausea, vomiting, muscle cramps, abdominal pain, and diarrhea [8]. Phoxim may have an acute effect on the nervous systems of humans and livestock through acetylcholinesterase inhibition [9]. Typical symptoms caused by phoxim are similar to malathion, and include nausea, vomiting, diarrhea, and so forth. Because of the potential

**Citation:** Zhai, W.; Cao, M.; Xiao, Z.; Li, D.; Wang, M. Rapid Detection of Malathion, Phoxim and Thiram on Orange Surfaces Using Ag Nanoparticle Modified PDMS as Surface-Enhanced Raman Spectroscopy Substrate. *Foods* **2022**, *11*, 3597. https://doi.org/10.3390/ foods11223597

Academic Editors: Evaristo Ballesteros and Thierry Noguer

Received: 4 October 2022 Accepted: 9 November 2022 Published: 11 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to cause those severe health issues, strict regulations have been introduced to minimize exposure to the aforementioned pesticides in many countries [10].

To better protect humans and livestock from the harmful effects of pesticides, it is urgently needed to develop effective analytical methods, especially those that can target mixed pesticides [11]. So far, malathion, phoxim, and thiram in agricultural products can be accurately measured using high-performance liquid chromatography (HPLC) [12], gas chromatography tandem mass spectrometry (GC-MS) [13], and liquid chromatography tandem mass spectrometry (LC-MS) [14]. However, these techniques require sophisticated and expensive instruments, as well as fully-trained technicians, to operate. The pretreatment processes for food samples are also time-consuming [15]. In order to achieve rapid and on-site screening for these pesticides, many analytical methods have been developed in recent years, including electrochemical sensors [16,17], enzymatic inhibitionbased sensors [18], enzyme-linked immunosorbent assay (ELISA) [19], lateral flow strip [20], surface-enhanced Raman spectroscopy (SERS) [21], microfluidics [22], and so forth. Among them, in recent decades SERS has been developed as a promising analytical platform for rapid and on-site detection of various target molecules [23,24]. Highly sensitive SERS detection depends on the enhancement of the Raman scattering signal generated from the localized surface plasmon resonance (LSPR) effect of Au or Ag nanomaterials. It presents unique advantages, including high sensitivity, instant data acquisition, highly specific molecular fingerprint from the SERS spectrum, low operating cost, and portability of devices [25,26]. Therefore, many SERS-based strategies have been reported for applications in the fields of food safety [27–29], environment monitoring [30], clinical diagnosis [31], security checking [32], and so forth.

In order to accomplish rapid and on-site detection of pesticides on agricultural products, a great deal of effort has been devoted to designing and fabricating Ag or Au nanomaterials as SERS substrates [33]. Recently, a new trend has been developed by combining a flexible substrate with SERS-active nanoparticles [34–36]. Simple sampling processes can be carried out by swabbing the target surface with the flexible substrate, while qualitative and even quantitative detection can be realized by SERS [37]. For example, a flexible SERS substrate was introduced by applying Au and Ag nanoparticles to an aluminum foil-based ZnO nanosheet. Extraction and identification of thiram on fruits and vegetables were accomplished using this substrate [38]. Apart from metal foils, polymer films have also been studied to fabricate flexible substrates for SERS [39]. Bai et al. spin-coated a monolayer of Ag island on a waterborne polyurethane emulsion film and applied the substrate for in-situ detection of thiram on apples [40]. In another study, a flexible Au nanostars/polydimethylsiloxane (PDMS) substrate was recently developed for in-situ determination of methyl parathion on fruit [41]. Wang et al. also reported the rapid detection of mixed chlorpyrifos and 2,4-dichlorophenoxyacetic acid residues on apple surfaces by using Ag colloid as a SERS substrate [42]. All these studies show the outstanding performance and great potential of applying a flexible substrate for fast and on-site SERS detection of pesticides on fruit surfaces. However, many studies still focus on a single type of pesticide, especially thiram, as the target [43,44]. New methods capable of rapidly determining mixed pesticides on one substrate are more desirable in practical applications [45].

Herein, we report the preparation of a flexible SERS substrate for the fast and simultaneous determination of three pesticides on orange surfaces. The proposed SERS substrate was fabricated by anchoring SERS-active Ag nanoparticles (AgNPs) on chemically modified PDMS thin film. The functionalization of PDMS and the surface density of AgNPs were optimized to minimize interference and achieve high SERS activity. Rhodamine 6G (R6G) was employed to evaluate the sensitivity, time stability, and signal uniformity of the prepared substrate. For pesticide detection, two organophosphate insecticides, malathion and phoxim, and the fungicide thiram were chosen as the targets. Qualitative and semiquantitative measurement of each pesticide was established using a standard solution. Benefitting from the molecular fingerprint property of SERS spectra, the presence of each target compound can be identified according to the characteristic SERS bands, allowing

simultaneous identification of the mixture of three pesticides on orange surfaces. In general, this method provides a facile and affordable platform for rapid and on-site determination of different hazardous chemicals on the surfaces of food samples.
