**2. Experimental Section**

## *2.1. Materials and Apparatus*

Potassium dichromate, sodium hydroxide, polyethylene glycol and sulfur powder were purchased from Aladdin (Shanghai, China). ALP and AA were obtained from Sigma-Aldrich (Shanghai, China). All reagents are of analytical grade and do not require further purification. The solutions used in the experiment were prepared by ultrapure water.

Transmission electron microscopy (TEM) images of the SQDs were obtained under a FEI Titan G2 60-300 microscope. Fluorescence measurements were performed on an F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). UV-visible absorption spectra were recorded on a UV-2450 spectrophotometer (Hitachi, Tokyo, Japan).

## *2.2. Synthesis of SQDs*

The synthesis of SQDs was based on previously reported methods with minor modifications [43–45]. Briefly, 1 g NaOH, 0.175 g sulfur powder, and 1.5 mL PEG-400 were mixed under stirring at 50 ◦C until the solution was clear. Then, the clear solution was placed in a microwave oven and reacted at 200 W for 1.5 h. Next, the obtained solution was centrifuged at 6000 rpm for 15 min, and the supernatant was centrifuged for two more times. Finally, the obtained supernatant was the SQDs solution, which was placed at 4 ◦C before use.

## *2.3. Determination of ALP*

Initially, 20 μL of SQDs solution was mixed with various concentrations of aqueous K2Cr2O7, then a PBS buffer (pH 7.4, 0.1 M) was added until the volume of the mixture reached 200 μL. The fluorescence spectrum of the solution was then measured. The excitation wavelength was maintained at 380 nm throughout the detection.

For the detection of ALP, 40 μL of solution containing different concentrations of ALP were mixed with 40 μL of 30 mM AAP. After mixing, the solution was incubated for 30 min in a water bath at 37 ◦C, then 10 μL of 10 mM K2Cr2O7 was added. After incubating for another 10 min, 20 μL of SQDs was added into the mixture. Fluorescence spectra of the solution were then collected.

## **3. Results and Discussion**

#### *3.1. Preparation and Characterization of the SQDs*

As shown in Scheme 1, using sulfur powder as a precursor, and PEG-400 as stabilizer, SQDs were synthesized by microwave-assisted heating. The whole synthesis process is easy to operate, the raw materials used being basically non-toxic to the environment, and the synthesis time was shortened to 90 min. In order to understand the size and morphology of SQDs, the morphology of SQDs was imaged by transmission electron microscope (TEM). As shown in Figure 1a, SQDs can be well dispersed into water. The particles display

spherical shape, and the particle size is 2.27 ± 0.76 nm, which is similar to the fluorescent SQDs previously reported.

**Figure 1.** Morphological characterizations of the SQDs: (**a**) TEM images; (**b**) size distribution histogram.

The excitation and emission positions and fluorescence intensity of SQDs can be obtained by fluorescence tests. Fluorescence properties of the synthesized SQDs were studied (Figure S1). When excited under different wavelength, with the increase of wavelength from 320 nm to 380 nm, the emission intensity of SQDs increases with the increase of excitation wavelength. By further increasing the wavelength from 380 nm to 420 nm, the emission intensity decreases with the increase of excitation wavelength. In addition, the fluorescence emission peak is redshifted with the increase of excitation wavelength. Therefore, 380 nm was used as the optimal excitation wavelength in subsequent experiments.

#### *3.2. Feasibility of the Assay for ALP Analysis*

The feasibility of the assay for ALP analysis was tested. As described in Figure 2, SQDs has a strong fluorescence emission peak at about 470 nm. However, the fluorescence of the SQDs is quenched after adding a certain concentration of K2Cr2O7, and can then recovered with further addition of AA. The recovery of fluorescence intensity is in accordance with the amount of AA added; this is because AA can reduce Cr (VI) to Cr (III). The fluorescence intensity of SQDs, mixed with a certain amount of chromium chloride solution, has no obvious difference with that of the single SQDs solution, which indicates that Cr (III) generated due to the reduction of K2Cr2O7 by AA has no quenching effect on the fluorescence signal of the SQDs. Meanwhile, after the K2Cr2O7 is added into the SQDs, the fluorescence signal is not changed by following addition of AAP, indicating AAP can not react with K2Cr2O7 and AAP alone will not affect the fluorescence intensity of the system. However, in the presence of ALP, the fluorescence signal was restored, which verified that ALP can catalyze the hydrolysis of AAP to generate AA, thus achieving the same effect as adding AA. Based on the above experimental results, it can be seen that fluorescence sensing of ALP activity can be achieved based on SQDs.

UV-vis absorption spectra further verified the above experimental results (Figure 3). Cr (VI) has a strong absorption at 380 nm. In contrast, the absorption of SQDs is weak. The competion of Cr (VI) ion with SQDs for the absorption of 380 nm light resulted in the quenching of the fluorescence of SQDs. Adding SQDs to Cr (VI), the absorption at 380 nm is slightly enhanced compared to Cr (VI) alone which is due to the absorption of SQDs at 380 nm. After the addition of AA, the absorption at this point almost disappeared because AA reduced Cr (VI) to Cr (III). These results are consistent with the fluorescence data.

**Figure 2.** Feasibility analysis of the assay for ALP detection.

**Figure 3.** UV-vis absorption spectra of different solutions.

#### *3.3. Detection of Cr (VI)*

The quenching of the fluorescence of SQDs by Cr (VI) is due to fact that the excitation spectrum of SQDs overlaps well with the absorption band of Cr (VI). Therefore, a strong IFE occurred between SQDs and Cr (VI), because Cr (VI) can shield the excitation light of SQDs. As shown in Figure 4a, the fluorescence intensity of SQDs decreased gradually with the increase of the amount of Cr (VI) added. When the Cr (VI) concentration reaches 5 mM, the fluorescence of SQDs is almost completely quenched. The fluorescence intensity of SQDs was linearly correlated with Cr (VI) concentration in the range of 10–100 μM and has a good linear correlation coefficient (*R*<sup>2</sup> = 0.997) (Figure 4b).

**Figure 4.** (**a**) Emission spectra of SQDs after adding different concentrations of Cr (VI) solution from 0 to 5 mM; (**b**) Fluorescence value of the SQDs in response to different concentrations of Cr (VI) in the range of 0 to 2 mM. Inset: the calibration curve to Cr (VI) in the linear range of 10–100 μM.

The selectivity of the synthesized SQDs for Cr (VI) was evaluated. Under the same experimental conditions, 2 mM Cr (VI) and 6 mM, other interfering ions, were added into SQDs and the fluorescence intensity was measured under 380 nm excitation. As described in Figure 5, when 2 mM Cr (VI) was added to the SQDs, the fluorescence intensity of the SQDs was reduced by more than 90%. However, even when 6 mM of other common metal ions were added, the fluorescence intensity of the SQDs was not affected significantly. These data show that the SQDs have good selectivity for Cr (VI).

**Figure 5.** Response of SQDs to different ions.
