*2.1. Reagents and Instruments*

DEX, hydrocortisone, prednisone, triamcinolone, betamethasone, 1-ethyl-3-(3-dimethy laminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(Nmorpholino) ethanesulfonic acid (MES), ovalbumin (OVA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRFM, europium chelates (365/610), with 1% solid content (*w/v*) and 0.2 μm particle size, was purchased from Bangs Laboratories, Inc (Fishers, IN, USA). Anti-DEX monoclonal A and DEX-OVA coating antigen (Ag) were prepared in our laboratory. The nitrocellulose filter (NC) membrane (Sartorius, UniSart CN95) was purchased from Sartorius Stedim Biotech GmbH (Goettingen, Germany). The microtiter plates were supplied by the JET BIOFIL Co. (Guangzhou, China). The polyvinylchloride (PVC) backing plate (SMA31-40), sample pad (GF-2), and absorbent pad (CH37) were purchased from Shanghai Kinbio Tech. Co., Ltd. (Shanghai, China).

Sucrose, sodium chloride, and other chemicals reagents were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

The strip cutter ZQ-2000 and the slitting machine SPT300 were purchased from Shanghai kinbio Tech. Co., Ltd. (Shanghai, China). The XYZ-3060 Dispensing Platform was bought from BioDot, Inc. (Irvine, CA, USA). The UV spectrometer was provided by Thermo Fisher Scientific Co. (Waltham, Massachusetts, USA). The Lynx-4000 centrifuge was obtained from Thermo Fisher Scientific GmbH (Berlin, Germany). The time-resolved fluorescence quantitative analysis reader (FQ-S2, 254 nm, 365 nm) was purchased from WDWK Biotechnology Co., Ltd. (Beijing, China).

## *2.2. Preparation of TRFM Immunoprobe*

The preparation of TRFM immunoprobe mainly includes two steps: activation of carboxyl groups on the surface of TRFM and covalent coupling with DEX Ab [28] (Figure 1A). Briefly, with constant stirring (300 rpm/min), 0.1 mg of TRFM was added in 1 mL of MES buffer (50 mM, pH 5.5). Then, 15 μL of freshly prepared 0.5 mg/mL EDC and NHS solution were sequentially added. The mixture was centrifuged at 14,000× *g* for 15 min at 4 ◦C after reaction for 15 min. The supernatant was discarded, and the bottom sediment was redissolved with 1 mL of borate buffer (BB, 50 mM, pH 8.0). Anti-DEX Ab (1 μL, 1.0 mg/mL), which was dissolved in 60 μL of BB (2 mM, pH 8.0), was added. The reaction solution was well mixed and incubated at room temperature for 45 min, and then 20 μL of 20% BSA (*w/v*) were added for blocking. The above solution was centrifuged at 14,000× *g* for 15 min at 4 ◦C after another 60 min of blocking reaction. The bottom sediment was dissolved in 200 μL of resuspension, which was stored at 4 ◦C for later use. The key technical parameters are shown in Table S1.

**Figure 1.** Schematic diagram of TRFM-ICG for DEX detection in milk and pork: ( **A**) the preparation principle of TRFM-DEX Ab immunoprobe and structure of test strip; (**B**) the detection principle of test strip; and ( **C**) the qualitative and quantitative test results.

#### *2.3. Preparation of the Test Strips*

The goa<sup>t</sup> anti-mouse secondary Ab and coating Ag (DEX-OVA) were diluted to the optimal concentration, and then sprayed on the NC film to form the control (C) and test (T) lines, respectively. The technical parameters of the spray film were as follows: spray length, 30 cm; distance between T-C line, 8 mm; and spray volume, 0.8 μL/cm. The processed NC films were placed in a 37 ◦C oven to dry overnight. The sample pads were immersed in the designed sample pad treatment solution for 30 s, and then dried for 2 h at 60 ◦C. Finally, the dried NC film, sample pad, and absorbent pad were pasted on the PVC backing pad with 2 mm overlap each other (Figure 1A). The PVC sheet was cut into 3.5 mm strips for use. The working principle is that the analyte competes with coating Ag for the limited Abs. The binding of the analyte to the Abs inhibited that of the coating Ag to the Abs, which was judged by the fluorescence intensity of T-line. The greater is the amount analyte, the weaker is the T-line signal, or there is even no color (Figure 1B). The working parameters of the goa<sup>t</sup> anti-mouse secondary Ab and coating Ag are shown in Table S1.
