*2.1. Materials*

Poly(ε-caprolactone) (PCL) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). AChE (200 U/g, from head of fly) and IA were purchased from ShanghaiYuanye Bio-Technology Co., Ltd. (Shanghai, China). Trichlorfon and carbaryl were purchased from Aladdin (Shanghai, China). Malathion and carbofuran were purchased from the National Information Center for Certified Reference Materials (Beijing, China). The commercial rapid detection card was obtained from Dayuan Oasis Food Safety Technology Co., Ltd. (Guangzhou, China). Analytical pesticide standards (100 μg/mL) were stored at 4 ◦C and spiked to the desired concentrations. Samples of cabbage and apples were purchased from a local market (Guangzhou, China).

#### *2.2. Preparation of the Detection Card Matrix*

PCL solutions in the concentration range of 100–150 g/L were prepared by dissolving certain amounts of PCL into a mixed solvent of methanol and chloroform at different volume ratios (0, 1:9, 3:7, 5:5). Then, the sealed solution was placed on a magnetic stirrer and stirred at room temperature in the dark for more than one hour to obtain the stable solution. The conductivity and viscosity of different spinning solutions were measured by Brookfield digital viscometer (Model DV-II t Pro, Brookfield Engineering, Inc., Middleboro, MA, USA) and conductivity meter (DDS-11A, Shanghai Leici Chuangyi Instrument Co., Ltd., Jiading, Shanghai, China), respectively. Then, electrospinning was conducted by putting the solution into a plastic syringe (5 mL) with a 20-gauge steel needle which was

connected to a high voltage power supply. The voltage and the distance between the needle tip and collector were set in the range of 11–17 kV and 11–15 cm, respectively. The injection rate was controlled by a syringe pump (NE-300, New Era Pump Systems Inc., Farmingdale, NY, USA) in the range of 1.5 to 3.5 mL/h. The fiber mat was fabricated at 24 ± 2 ◦C and under 55 ± 5% relative humidity for a period of time.

#### *2.3. Hydrophilic Modification of the Matrix*

Here, the PCL fiber mat was modified by one step or two steps of treatment. For the one-step modification, the PCL fiber mat was immersed in the NaOH solution with different concentrations (1 M, 2 M, 4 M, and 6 M) for a certain period of time, while the two-step modification was performed by initially infiltrating in the 70% ethanol for 15 min before the same NaOH immersion process. Then, the fiber mat was washed with deionized water and dried in a vacuum drying oven.

#### *2.4. Characterization of the Matrix*

The morphologies of the original and modified PCL fiber were observed by scanning electron microscopy (SEM, S-3700N, Hitachi High-Tech Ltd., Chiyoda-ku, Japan). Before the observation, the electrospun fiber mat was sputter-coated under vacuum conditions and observed at 15 kV. Then, the obtained SEM image was processed by the Nano Measure 1.2 software, and the distribution of the fibers was further calculated by analyzing around 50 fibers. The thickness of the fiber mat was measured by a digital thickness gauge (Syntek, Deqing Shengtai Electronic Technology Co., Ltd., Deqing, China).

The changes that occurred in the polymer molecules were examined by employing attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) (VERTEX 70, Bruker Co., Ettlingen, Germany). All the spectra were recorded in the frequency region of 500–4000 cm<sup>−</sup><sup>1</sup> at a spectral resolution of 4 cm<sup>−</sup>1.

To characterize the surface wettability of the fiber mat, the surface contact angle tester (OCA40, DataPhysics Instruments GmbH, Filderstadt, Germany) was used to determine the contact angles of different samples. Briefly, the measurement was conducted by dropping 2 μL of deionized water onto the surface of the fiber mat with a drop rate of 2 μL/s. After 10 s, the contact angle of the fiber mat was measured by taking a screenshot. Each sample was tested 3 times, and the average value was calculated.

#### *2.5. Optimization of the Immobilization and Determination Conditions*

In this study, two fiber mats, namely the enzyme (AChE) fiber mat (AFM) and substrate (IA) fiber mat (IFM), were used for the preparation of the nano/micro-structured detection card. For optimizing the suitable AChE concentration for the preparation of AFM, the IFM was prepared by immersing the fiber mat in 5 mg/mL IA solution, and the cards were then dried in a vacuum oven. Similarly, the fiber mat was treated with 8 mg/mL of AChE solution when the optimal IA solution was investigated. Here, PBS (pH 7.4) and 1 mg/L of malathion were selected as control and positive sample, respectively. On the other hand, the absorption efficiencies of the fiber mat and other commonly used absorption materials (qualitative and quantitative filter paper) were examined by determining the amount of released enzyme from the carrier materials according to the Bradford method. Before doing this, the carrier materials were immersed into 5 mg/mL of AChE solution for 24 h and dried in a vacuum drying oven.

The principle of this detection method is that AChE can catalyze the hydrolysis of colorless IA to produce indoxyl, as depicted in Figure 1, which then becomes blue due to the rapid oxidation by air. The blue color of indigo can be well distinguished with the naked eye. Hence, the existence of pesticides can be analyzed according to the color change as a consequence of the inhibition of the pesticide on AChE activity. On the basis of understanding the principle of this determination method, inhibition and color development time were two critical factors that significantly influenced the result. Herein, the inhibition time was optimized in the range of 5–15 min, where the color development

time was set at 15 min. Similarly, the inhibition time was set at 10 min for exploring the appropriate color development time (5–25 min). PBS (pH 7.4) and 0.5 mg/L malathion were applied as control and positive samples, respectively.

**Figure 1.** The reaction of indoxyl acetate hydrolysis, catalyzed by AChE.

#### *2.6. Performance of the Detection Card and Its Real-Life Application*

To evaluate the efficacy of this detection card, a series of concentrations of these two classical pesticides, including organophosphorus (OP) (omethoate and malathion) and carbamate (CM) (carbaryl and carbofuran), were determined and compared with their corresponding low limit of detection values reported in SAC. In brief, the pesticides were diluted to a series of concentrations by PBS. An aliquot (50 μL) of sample solution was dropped on the AFM for analysis, and PBS served as the control group. All the detection procedures were performed according to the conditions optimized in the above section.

The storage stabilities of the detection card under 4 ◦C and room temperature (RT) were evaluated for 60 days. Malathion and PBS were served as the positive and control group to examine its detection efficacy periodically.

To further verify the determination performance of this card, organic cabbage and carrots were tested as samples with different concentrations of malathion (0, 5, 10, 20 μg/mL). Different volumes of malathion were sprayed on sample surfaces based on the sample weight (1 mL/g) and stored at room temperature for 24 h. Subsequently, 5 g of the samples were immersed in 10 mL PBS and then shaken by hand. After the stabilization for 2 min, the supernatant was analyzed using self-made and commercial rapid detection cards.
