**1. Introduction**

Fungal contamination causes significant damages to the crops for human consumption every year, resulting in poor yield, deficient food quality, and huge economic loss. To circumvent these problems, the use of fungicides has been intensified over the last decades [1].

 Karachaliou, C.-E.; Raptis, I.; Petrou, P.; Livaniou, E.; Kakabakos, S. Fast and Sensitive Determination of the Fungicide Carbendazim in Fruit Juices with an Immunosensor Based on White Light Reflectance Spectroscopy. *Biosensors* **2021**, *11*, 153.https://doi.org/10.3390/bios 11050153

Koukouvinos,

G.;

Received: 22 March 2021 Accepted: 10 May 2021 Published: 13 May 2021

**Citation:**

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Carbendazim (methyl 1H-benzimidazol-2-ylcarbamate) is a synthetic, systemic, broadspectrum, benzimidazole-type fungicide used worldwide as pre- and post-harvest treatment to control fungi that compromise the quality of various food commodities such as vegetables, fruits, cereals, and seeds [2,3]. Despite the unquestionable benefits regarding crop yield, carbendazim is a major pollutant, which induces acute and chronic effects on humans and livestock. In this context, carbendazim has been documented to induce infertility, embryotoxicity, teratogenicity, hepatocellular dysfunction, endocrine-disrupting effects, disruption of hematological functions, and mutagenicity [2]. Additionally, the World Health Organization has classified carbendazim as a possible human carcinogen [4]. Due to its aforementioned severe toxicities and its persistence in food and the environment, carbendazim has been officially banned in most of the European Union countries, USA, and Australia. However, its production and use in various formulations are still permitted in some countries, such as UK, Portugal, India, China, and Brazil [5], raising a growing concern for its adverse effects on the health of both humans and animals. To protect consumers from food products contaminated with carbendazim, regulatory authorities have established maximum residue limits (MRLs) for this pesticide in several matrices (fresh fruits and vegetables, oil seeds, cereals, spices, etc.). For example, the carbendazim MRL in fruit juices is 200 ppb and the MRL sum of benzimidazole pesticides has been set at 500 ppb by European Union [6,7]. The presence of carbendazim in fruit juice products has raised many concerns due to their worldwide popularity and the fact that children are their primary consumers [8].

The protection of the public health from pesticide residues in food requires, in addition to relevant legislation, accurate analytical methodologies. Determination of carbendazim is routinely performed by instrumental analytical techniques, mainly high-performance liquid chromatography coupled to mass spectroscopy or ultraviolet spectroscopy [9,10]. Alternatively, immunochemical techniques have been developed and used, offering lowcost and short-time analyses, simple assay protocols, and minimum sample pretreatment, and high-throughput sample analysis capacity. In this context, classic enzyme-linked immunosorbent assays (ELISA) [11] and immunochromatographic strips [12] have been reported in the literature, while immunosensors have lately attracted much attention due to their simplicity, rapidity, portability, and potential for the point-of-need application [13].

In this work, a real-time immunosensor based on white light reflectance spectroscopy (WLRS) is employed for the accurate, fast, and sensitive determination of carbendazim in fruit juice samples with the potential for use at the point-of-need. The transducer is a Si chip with a 1-μm thick SiO2 layer on top and it is transformed to a versatile biosensing element through immobilization of a suitable recognition molecule. The optical set-up includes a white light source, a reflection probe consisting of a bundle of seven optical fibers; six at the periphery of the probe and one at the center, and a spectrometer. The six fibers at the periphery of the reflection probe guide the light from the source to the chip surface, while the seventh central fiber collects the light reflected by the chip and guides it to the spectrometer. As the light strikes the chip surface vertically, it is reflected by the silicon surface and by the transparent materials adlayers (silicon dioxide and biomolecular layer) of different refractive index. This way interference takes place at each wavelength resulting in an interference spectrum that is collected by the central fiber of the reflection probe. The increase of the biomolecular adlayer thickness due to binding reactions taking place onto the chip surface causes a shift of the interference spectrum. The software receives the interference spectrum from the embedded spectrometer and the effective biomolecular adlayer thickness (that is the signal of the WLRS sensor) is determined implementing the Levenberg–Marquart algorithm [14]. As this conversion is done by the software in real-time, the evolution of the effective biomolecular adlayer thickness in the course of the binding reactions occurring on a biochip surface could be monitored in real-time (Scheme 1). Thus, the WLRS biosensing platform allows for the label-free, real-time monitoring of biomolecular interactions carried out onto the Si/SiO2 chip with a detectable effective adlayer thickness <0.1 nm. A presentation of WLRS set-up

and operation principle is presented in Scheme 1. The WLRS sensing principle has been successfully applied to the quantitative determination of both high and low molecular weight analytes into a plethora of matrices, after proper biofunctionalization of the sensing surface [14–16].

**Scheme 1.** WLRS set-up and operation principle: (**a**) a schematic of the incident light beam (blue dotted line) reflection at the layers of different refractive index of a WLRS chip (red lines); (**b**) typical reflectance spectrum (black line) and its fitting by the sensor software (red line); (**c**) a depiction of the real-time signal monitoring resulting by the recorded spectrum processing by dedicated software.

For carbendazim determination, a competitive immunoassay involving the delivery of a mixture of standards or fruit juice samples with a carbendazim-specific antibody to a benzimidazole conjugate-modified chip was conducted, as it is schematically depicted in Figure 1a. The primary immunoreaction, i.e., the competitive reaction between the immobilized onto the chip benzimidazole moieties and carbendazim in the standards or samples for the limited binding sites of the carbendazim-specific antibody [17], was followed by two signal enhancement steps. The first step included reaction with a biotinylated secondary antibody and the second one with streptavidin so as to further increase the thickness of the adlayer formed. The implementation of these two reactions aimed at the increase of the effective biomolecule adlayer thickness, i.e., the sensor signal, thus increasing the detection sensitivity. The benzimidazole conjugate [17] used for chip coating is also schematically presented in Figure 1b. Initially, a lysine-core peptidyl moiety was prepared using a fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis strategy previously described by us [18] with slight modifications. This moiety was then functionalized with 3- maleimidopropionic acid [19]. A benzimidazole derivative, 2-mercaptobenzimidazole, was then coupled to the 3-maleimidopropionic acid-functionalized peptidyl moiety through its thiol functional group. Notably, 2-mercaptobenzimidazole was used here because it is structurally similar to, but less toxic than, carbendazim. All assay parameters were optimized to achieve the highest possible detection sensitivity and the shortest assay duration. A simple sample preparation procedure was also developed to demonstrate the analysis of several commercially available fruit juice samples without any detectable "matrix-effect". The accuracy of measurements with the proposed methodology was evaluated through recovery experiments using carbendazim-spiked samples prepared in commercially available fruit juices. Finally, the potential of regeneration and re-use of the biofunctionalized sensor chips was investigated, as a means to reduce the total cost of analysis. The novelty of the present work is mainly based on the combined use of an in-house prepared antibody for carbendazim recognition [17] and a benzimidazole derivative as a coating conjugate [17–19] in the WLRS immunoassay for carbendazim in different fruit juices.

**Figure 1.** Schematic representation of (**a**) assay procedure for the detection of carbendazim with the WLRS sensor and (**b**) benzimidazole conjugate structure.

#### **2. Materials and Methods**

#### *2.1. Reagents and Materials*

The rabbit polyclonal antibody for carbendazim (primary antibody) and the benzimidazole conjugate used for surface functionalization were in-house developed, as previously described [17]. Biotinylated goa<sup>t</sup> anti-rabbit IgG (secondary antibody), streptavidin, streptavidinhorseradish peroxidase conjugate (streptavidin-HRP), 3,3,5,5-tetramethylbenzidine (TMB), carbendazim pestanal®, highly pure ethanol, acetone, isopropanol, and (3-aminopropyl) triethoxysilane (APTES) were from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from Acros Organics (Geel, Belgium). Polytetrafluoroethylene (PTFE) syringe filters of 0.45 μm pore size were the product of Membrane Solutions (Auburn, WA, USA). All other chemicals were purchased from Merck (Darmstadt, Germany). Highbinding 96-well polystyrene microtiter plates (Code No. 3590) were purchased from Corning-Costar (Corning, NY, USA). Four-inch Si wafers (< 100 >) were purchased from Si-Mat Germany (Kaufering, Germany). These wafers were sequentially sonicated in acetone and isopropanol before a 1000-nm thick SiO2 layer was thermally oxidized on them at 1100 ◦C using the clean room facility at the Institute of Nanoscience and Nanotechnology of NCSR "Demokritos". Then, the wafers were diced to chips with dimensions of 5 mm × 15 mm.

#### *2.2. Preparation of Carbendazim Standard Solutions and Fruit Juice Samples*

A 5 mg/mL carbendazim stock solution in absolute ethanol was prepared and stored at −20 ◦C. Standard solutions ranging from 20 ng/mL to 20 μg/mL prepared in 10 mM phosphate buffer, pH 7.4, containing 0.9% (*w/v*) NaCl, and 0.4% (*w/v*) BSA (assay buffer), were kept at −20 ◦C for up to 2 months. Fruit juices used in this study were purchased from local markets. In particular the following juices have been purchased: Amita orange and orange–lemon–carrot juice, and nine fruits Amita Motion juice from Coca-Cola 3E SA (Maroussi, Greece); orange, orange–apple–carrot, and nine fruits juice from Olympos Greek Dairies SA (Larissa, Greece); Eviva orange (short and long self-life) and lemon juice from Lidl Hellas (Sindos, Greece); Marata orange juice from Sklavenitis SA (Peristeri, Greece). All juices were separately filtered through a 0.45 μm-PTFE filter and the pH of the filtrate was adjusted to 7.4 ± 0.2 with 1 M NaOH solution.
