*2.3. Carbendazim-ELISA Assay*

An indirect competitive ELISA was set up to conduct carbendazim detection. In brief, ELISA wells were incubated overnight in 100 μL of a 1 μg/mL benzimidazole conjugate in 0.05 M carbonate buffer, pH 9.2 (coating buffer). The plates were then washed three times with 10 mM phosphate buffered saline (PBS), pH 7.4, containing 0.05% (*v/v*) Tween-20 (PBS-T) using an ELISA plate washer (DIA Source), and incubated with 10 mM PBS, pH 7.4, containing 2% (*w/v*) BSA (200 μL/well), at room temperature for 1 h to block any remaining binding sites. After three washes with PBS-T, to each well were added 100 μL of 1:1 (*v/v*) mixtures of carbendazim standards (0.02–20 μg/mL in assay buffer) or samples and the anti-carbendazim antibody solution (2 μg/mL in assay buffer), which have been preincubated at room temperature for 1 h. After incubation at 37 ◦C for 90 min, the wells were washed three times with PBS-T, and incubated at 37 ◦C for 1 h with the biotinylated secondary antibody diluted 1:2000 in assay buffer (100 μL/well). The wells were then washed again with PBS-T and incubated at 37 ◦C for 45 min with a 750 ng/mL streptavidin-HRP solution in assay buffer (100 μL/well). The wells were washed again with PBS-T and incubated at room temperature for 20 min in 100 μL of chromogenic HRP substrate (TMB/H2O2). Finally, 50 μL of a 2 M aqueous sulfuric acid solution were added per well to terminate color development, and the absorbance of the wells at 450 nm (A450) was measured using a microtiter plate reader (Sirio S, Seak). An absorbance percentage was then calculated by dividing the absorbance of each standard (Ax) to that of zero standard (A0). An absorbance percentage versus carbendazim concentration calibration plot was then constructed.

#### *2.4. Evaluation of Primary Antibody—Specificity with the Carbendazim-ELISA Assay*

The specificity of the primary antibody was evaluated through cross-reactivity studies with the pesticides carbaryl, imazalil, atrazine, and paraquat as follows. ELISA microwells were coated, blocked and washed as described in Section 2.3. Then, the wells were incubated at 37 ◦C for 90 min in 100 μL of a 1:1 (*v/v*) mixture (preincubated at room temperature for 1 h) of carbendazim standard solutions or standard solutions containing 0.02–5.0 μg/mL of each cross-reactant in assay buffer, and a 2 μg/mL anticarbendazim antibody solution in assay buffer. Microwells were washed, incubated with biotinylated secondary antibody, streptavidin-HRP, and TMB/H2O2 solution as described in Section 2.3. Finally, the absorbance was read at 450 nm, the calibration plots were constructed and the percent cross-reactivity (%CR) with each pesticide tested was determined according to the equation:

$$\text{\textquotedblleft\textquotedblright} \text{CR} = \text{(IC}\_{50} \text{ carbeddzim}/\text{IC}\_{50} \text{ cross-reactant positive)} \times 100$$

where IC50 carbedazim and IC50 cross-reactant represent the concentrations of carbendazim and cross-reactant in question, respectively, which provided 50% signal drop with respect to zero standard.

### *2.5. WLRS Instrumentation*

WLRS instrumentation involves an FR-Pro tool operating in the 450–720 nm spectral range (ThetaMetrisis SA; Egaleo, Greece). The tool is equipped with a stabilized visible/near infrared light source and a high-performance miniaturized spectrometer tuned to provide very high optical resolution in the dedicated spectral range. The reflection probe delivers the incident light emitted by the source to the biofunctionalized chip surface through six fibers (diameter 400 μm) positioned to its periphery and collects the reflected light directing it to the spectrometer through a seventh fiber (diameter 400 μm) positioned

at the center of the probe. The sensing surface consists of a transparent SiO2 film over a Si reflecting substrate covered by a custom-designed microfluidic cell (Jobst Technologies GmbH; Freiburg, Germany) providing the fluidic connections to the solutions. The FR-Pro tool was accompanied by a dedicated software that evaluates the initial thickness of the SiO2/protein adlayer and transforms in real-time the spectral shift due to the binding reactions in effective thickness of biomolecular adlayer expressed in nm. In more detail, a reference [ *R*(*λ*)) and a dark spectrum ( *D*(*λ*)] were acquired prior to the continuous recording of the reflectance spectrum [*S*(*λ*)], and the absolute reflectance spectrum is calculated by Equation (1):

$$R(\lambda) = \frac{S(\lambda) - D(\lambda)}{R(\lambda) - D(\lambda)} \tag{1}$$

The normalized spectrum is then processed applying Levenberg–Marquart algorithm to calculate the thickness of the biomolecular adlayer, *d*1, from the shift in the interference spectrum wavelength, *δλ*, according to Equation (2):

$$
\delta\lambda = r\_1 \frac{1 - r\_2^2}{(r\_1 + r\_2)(1 + r\_1 r\_2)} \frac{n\_1 d\_1}{n\_2 d\_2} \lambda\_{0m} \tag{2}
$$

where, *r*1 and *r*2, and *n*1 and *n*2 are the Fresnel coefficients and refractive indices of the biomolecular and the silicon dioxide layer, respectively, *d*1 and *d*2 are the thickness of the two layers, and *λ* the wavelength.

#### *2.6. Biochip Preparation and Biosensor Assay Performance*

Chips were first cleaned and hydrophilized by O2 plasma treatment (10 mTorr) for 30 s in a reactive ion etcher. Then, they were immersed in a 2% (*v/v*) aqueous APTES solution for 20 min, gently washed with distilled water, dried under a nitrogen (N2) stream and cured by heating at 120 ◦C for 20 min. Chips were kept in a desiccator at room temperature for at least 48 h prior to use. For chip biofunctionalization, the benzimidazole conjugate (500 μg/mL, in coating buffer) was deposited on the chips and incubated at room temperature overnight. The following day, chips were rinsed with 10 mM PBS, pH 7.4 (washing buffer), blocked through immersion in 10 mM PBS, pH 7.4, containing 2% (*w/v*) BSA, for 3 h, rinsed with washing buffer and distilled water, dried with N2, and used for the assay (Figure 1). Prior to assay, each biofunctionalized chip was assembled with the fluidic module, placed on the docking station and equilibrated with assay buffer to acquire a stable baseline. For the assay, 1:1 (*v/v*) mixtures of standards (0.02–20 μg/mL in assay buffer) or samples with the rabbit anticarbendazim antibody (2 μg/mL in assay buffer), preincubated for 60 min, were passed over the chip for 18 min, followed by a 1:200 dilution of biotinylated anti-rabbit IgG antibody solution for 7 min, and a 10 μg/mL streptavidin solution in assay buffer for 3 min. The flow rate throughout the assay was 50 μL/min. Finally, the biochip was regenerated by running a 0.1 M glycine-HCl buffer, pH 2.5, for 3 min, followed by re-equilibration with assay buffer. The calibration plot was constructed by plotting the effective thickness of the built-up biomolecular adlayer (signal) corresponding to different standards, Sx, expressed as percentage of the zero standard signal, S0 (maximum signal), against the carbendazim concentration in the standard solutions.

#### **3. Results and Discussion**

#### *3.1. ELISA Assay for Carbendazim*

Prior to the development of the carbendazim WLRS immunosensor assay, an ELISA in microtiter plates was set up to evaluate the basic immunoreagents used on the sensor platform, e.g., specificity of the anti-carbendazim antibody, as well as to determine optimal conditions for each assay step, e.g., optimum assay buffer. Moreover, the results obtained from the analysis of fruit juice samples with the ELISA assay were compared with those of the WLRS immunosensor.
