**3. Results and Discussion**

## *3.1. Optimization of the TRFM-ICG*

Several important technical parameters were optimized to achieve the best detection performance of the established TRFM-ICG, including the particle size and activated pH value of TRFM, the optimal pH value and ion concentration of Ab coupling with TRFM, Ab amount, sample pad treatment solution formula, etc.

#### 3.1.1. Particle Size of TRFM

The particle size of the microsphere determines the specific surface area, which affects not only the binding efficiency of the carboxyl groups on the surface of the microsphere with Ab but also the chromatographic release of the immunoprobe [8,28]. The detection results of 200 and 300 nm microspheres were compared. The results show that the release effect of the 300 nm microsphere immunoprobe was not as good as that of the 200 nm microsphere immunoprobe. There were many residues in the reaction zone, which led to the background being very red and fuzzy (Figure S1). However, high background values will have a negative impact on the subsequent visual judgment and quantitative detection. Therefore, 200 nm microspheres were selected as DEX-Ab labeling tracers.

#### 3.1.2. Activation pH Value of TRFM

TRFM are modified with carboxyl groups and need to be activated before coupling with Ab. The appropriate pH value can improve the activation efficiency of carboxyl group, and thus improve the binding efficiency with Ab [24]. The carbodiimide method was applied to activate the carboxyl groups under four different pH conditions, including pH 5.0, 5.5, 6.0, and 6.5. The results show that the fluorescence signal intensity of T-line increased with the increase of pH value, but the inhibitory effect was difficult to distinguish with the naked eyes. With the help of quantitative analysis results, the inhibition rates were the highest in the detection of milk and pork samples under pH 5.5 (Figure S2). Therefore, the optimal activation pH was selected as 5.5.

#### 3.1.3. The Ab Dilution Buffer

The Ab dilution buffer can not only maintain the biological activity of Ab but also prevent non-specific reactions and improve the sensitivity of the method [7,32]. Five different Ab dilution buffers, namely ultrapure water, 0.01 M PB (pH 7.4), 0.01 M PB (pH 7.4, 0.5% BSA), 0.5% BSA, and 0.002 M BB (pH 8.0), were used to dilute DEX Ab, adding a control group with no Ab diluent. The photos of the test strips and quantitative detection results show that 0.002 M BB (pH 8.0) as an Ab diluent had outstanding advantages in inhibition

effect, and the fluorescence intensity was also satisfactory (Figure 2). This result shows that pH was a key factor affecting the binding of Ab-FMs, and BSA would hinder the coupling of Ab-FMs. Therefore, BSA should not be contained in the Ab diluent. Interestingly, this result is exactly the opposite of our previous research [27]. Therefore, there was no doubt that 0.002 M BB (pH 8.0) was our target Ab dilution.

**Figure 2.** The fluorescence intensity, inhibition effect, and inhibition rate results of the Ab dilution buffer: (**A**) ultraviolet lamp results for milk detection; (**B**) ultraviolet lamp results for pork detection; (**C**) fluorescence quantitative results for milk detection; and (**D**) fluorescence quantitative results for pork detection.
