*3.4. Detection of ALP*

The absorption peak of Cr (VI) at about 380 nm disappears after the addition of AA, which indicates that AA can reduce Cr (VI) to a low-valent Cr species, thereby eliminating the IFE between Cr (VI) and SQDs and restoring the fluorescence of SQDs. ALP can hydrolyze AAP to generate AA, which can also recover the fluorescence of SQDs-Cr (VI) system. Hence, the principle can be applied for detecting ALP. The selectivity of the system to AA was investigated. Other potential reductants including glutathione (GSH), cysteine (Cys), glucose and some common ions had little effect on the fluorescence of SQDs-Cr (VI) system, indicating that SQDs-Cr (VI) system had good selectivity for AA (Figure S2). As described in Figure 6a, the fluorescence intensity of SQDs increases with the increase of ALP activity. When the ALP activity is more than 10 U/mL, the fluorescence intensity of the system tends to be stable, and the fluorescence strength of SQDs was almost fully

recovered. The fluorescence intensity of SQDs had a good linear relationship with ALP activity in the range of 1.5–5.0 U/mL, and the linear correlation coefficient R<sup>2</sup> was 0.992. Based on a signal–noise ratio of 3, the calculated LOD was 0.13 U/mL. Table 1 summarizes previously reported strategies for detecting ALP using different probes, indicating that the methods are comparable to those reported in other studies.

**Figure 6.** (**a**) Fluorescence response of the assay to ALP of different activities. Inset: linear response of SQDs to ALP in the activity range of 1.5–5.0 U/mL; (**b**) Selectivity of the assay to ALP.

Then, the selectivity of the assay for ALP was analyzed. Responses of the assay to 2 U/mL of alkaline phosphatase (ALP) and other enzymes with an activity of 5 U/mL were tested. As described in Figure 6b, when ALP is added, the fluorescence change is obvious, while the responses of the assay to other enzymes are negligible, similar to blanks. These data show that the assay has good selectivity to ALP sensing. In addition, the assay is used for the recovery testing of ALP in serum. With the serum signal as the background, ALP with the activity of 1, 2, 2.5 and 3.5 U/mL were added into the serum sample, and then the level of ALP was measured. The results are shown in Table 2. The recoveries for ALP in the serum were in the range of 102.01~119.61%, with the relative standard deviations (RSD) less than 5%. Therefore, the assay has potential application for clinical detection of ALP.



**Table 2.** Recovery analysis of ALP in human serum.

