*3.2. Verification of the Feasibility of the Sensing Strategy*

*3.2. Verification of the Feasibility of the Sensing Strategy*  In order to demonstrate the feasibility of the proposed strategy, a series of experiments were carried out with or without ALP. As illustrated in Figure 1A, the CuNPs couldn't form in the presence of ALP and Exo I (curve a). However, in the absence of ALP or Exo I, high fluorescence signals were observed (curve b, curve c). In addition, 20% Urea-PAGE stained by silver was employed to verify the feasibility of the proposed approach as well. Figure 1B shows that T30 disappeared in the presence of ALP and Exo I (lane 3). In order to demonstrate the feasibility of the proposed strategy, a series of experiments were carried out with or without ALP. As illustrated in Figure 1A, the CuNPs couldn't form in the presence of ALP and Exo I (curve a). However, in the absence of ALP or Exo I, high fluorescence signals were observed (curve b, curve c). In addition, 20% Urea-PAGE stained by silver was employed to verify the feasibility of the proposed approach as well. Figure 1B shows that T30 disappeared in the presence of ALP and Exo I (lane 3). Nevertheless, when there was no ALP and Exo I (lane 2) or no ALP present (lane1), T30 could be stained by silver. These results verified the feasibility of the proposed strategy to detect ALP.

detect ALP.

detect ALP.

*Biosensors* **2021**, *11*, x FOR PEER REVIEW 4 of 10

**Figure 1.** Investigation of the feasibility of the sensing strategy. (**A**) The fluorescence emission spectra of CuNPs under different conditions. Curve a: T30 + ALP + Exo I + Cu2+ + sodium ascorbate (T30, 2 µM; ALP, 500 U/L; Exo I, 40 U/mL; Cu2+, 200 µM; sodium ascorbate, 5 mM); curve b: without Exo I; curve c: without ALP. (**B**) Urea-PAGE with silver staining analysis under different conditions. Lane 1: T30+Exo I (T30, 2 µM; Exo I, 40 U/mL); lane 2: T30 (T30, 2 µM); lane 3: T30+ALP+Exo I (T30, 2 µM; ALP, 500 U/L; Exo I, 40 U/mL). **Figure 1.** Investigation of the feasibility of the sensing strategy. (**A**) The fluorescence emission spectra of CuNPs under different conditions. Curve a: T30 + ALP + Exo I + Cu2+ + sodium ascorbate (T30, 2 µM; ALP, 500 U/L; Exo I, 40 U/mL; Cu2+, 200 µM; sodium ascorbate, 5 mM); curve b: without Exo I; curve c: without ALP. (**B**) Urea-PAGE with silver staining analysis under different conditions. Lane 1: T30+Exo I (T30, 2 µM; Exo I, 40 U/mL); lane 2: T30 (T30, 2 µM); lane 3: T30+ALP+Exo I (T30, 2 µM; ALP, 500 U/L; Exo I, 40 U/mL). **Figure 1.** Investigation of the feasibility of the sensing strategy. (**A**) The fluorescence emission spectra of CuNPs under different conditions. Curve a: T30 + ALP + Exo I + Cu2+ + sodium ascorbate (T30, 2 µM; ALP, 500 U/L; Exo I, 40 U/mL; Cu2+, 200 µM; sodium ascorbate, 5 mM); curve b: without Exo I; curve c: without ALP. (**B**) Urea-PAGE with silver staining analysis under different conditions. Lane 1: T30+Exo I (T30, 2 µM; Exo I, 40 U/mL); lane 2: T30 (T30, 2 µM); lane 3: T30+ALP+Exo I (T30, 2 µM; ALP, 500 U/L; Exo I, 40 U/mL).

#### *3.3. Optimization of Experimental Conditions 3.3. Optimization of Experimental Conditions 3.3. Optimization of Experimental Conditions*

We investigated the effects of different assay conditions, including the concentration of T30, the concentration of Exo I, the concentration of Cu2+, the reaction time of ALP, and the reaction time of Exo I. The fluorescence intensity ratios of the controlled group to the experimental group (F0/F) changed with varying assay conditions. As illustrated in Figure 2A–E, we found the optimal reaction condition as follows: 2 µM T30, 40 U/mL Exo I, 10 µM Cu2+ solution, a reaction time of 10 min between ALP and T30, and Exo I reaction time of 10 min. We investigated the effects of different assay conditions, including the concentration of T30, the concentration of Exo I, the concentration of Cu2+, the reaction time of ALP, and the reaction time of Exo I. The fluorescence intensity ratios of the controlled group to the experimental group (F0/F) changed with varying assay conditions. As illustrated in Figure 2A–E, we found the optimal reaction condition as follows: 2 µM T30, 40 U/mL Exo I, 10 µM Cu2+ solution, a reaction time of 10 min between ALP and T30, and Exo I reaction time of 10 min. We investigated the effects of different assay conditions, including the concentration of T30, the concentration of Exo I, the concentration of Cu2+, the reaction time of ALP, and the reaction time of Exo I. The fluorescence intensity ratios of the controlled group to the experimental group (F0/F) changed with varying assay conditions. As illustrated in Figure 2A–E, we found the optimal reaction condition as follows: 2 µM T30, 40 U/mL Exo I, 10 µM Cu2+ solution, a reaction time of 10 min between ALP and T30, and Exo I reaction time of 10 min.

Nevertheless, when there was no ALP and Exo I (lane 2) or no ALP present (lane1), T30 could be stained by silver. These results verified the feasibility of the proposed strategy to

Nevertheless, when there was no ALP and Exo I (lane 2) or no ALP present (lane1), T30 could be stained by silver. These results verified the feasibility of the proposed strategy to

**Figure 2.** *Cont*.

**Figure 2.** Optimization of reaction conditions. (**A**) The concentration of T30; (**B**) Exo I concentration; (**C**) Cu2+ concentration; (**D**) the reaction time of ALP; (**E**) the reaction time of Exo I. **Figure 2.** Optimization of reaction conditions. (**A**) The concentration of T30; (**B**) Exo I concentration; (**C**) Cu2+ concentration; (**D**) the reaction time of ALP; (**E**) the reaction time of Exo I.

#### *3.4. Quantitative Fluorescence Measurement of ALP Activity 3.4. Quantitative Fluorescence Measurement of ALP Activity*

In the present study, we have investigated the fluorescence responses of the proposed analytical method in varying concentrations of ALP under optimized conditions. As displayed in Figure 3A, the peak of fluorescence intensities decreased gradually when the concentrations of ALP increased from 0 to 20 U/L. The relationship between the F0-F [the fluorescence intensities (at 615 nm) of the controlled group minus the fluorescence intensities (at 615 nm) of the experimental group] and the activity of ALP is plotted in Figure 3B, where we can observe that F0-F values linearly increased with the concentration of ALP ranging from 0.01 to 5 U/L (regression coefficient R2 = 0.9979). The evaluated detection limit of the proposed strategy is 0.0098 U/L according to the 3σ rule, which is comparable or better than the existing methods (Table 1). Therefore, these results demonstrated the satisfactory sensitivity of the proposed method towards ALP. In the present study, we have investigated the fluorescence responses of the proposed analytical method in varying concentrations of ALP under optimized conditions. As displayed in Figure 3A, the peak of fluorescence intensities decreased gradually when the concentrations of ALP increased from 0 to 20 U/L. The relationship between the F0-F [the fluorescence intensities (at 615 nm) of the controlled group minus the fluorescence intensities (at 615 nm) of the experimental group] and the activity of ALP is plotted in Figure 3B, where we can observe that F0-F values linearly increased with the concentration of ALP ranging from 0.01 to 5 U/L (regression coefficient R<sup>2</sup> = 0.9979). The evaluated detection limit of the proposed strategy is 0.0098 U/L according to the 3σ rule, which is comparable or better than the existing methods (Table 1). Therefore, these results demonstrated the satisfactory sensitivity of the proposed method towards ALP.

**Figure 3.** The quantitative measurement of ALP. (**A**) Fluorescence spectra of the assay system response to different activity units of ALP (0, 0.01, 0.1, 1, 2, 3, 4, 5, 10, and 20 U/L); (**B**) the plot between F0-F and the concentration of ALP. Inset: calibration linear curve for ALP detection. **Figure 3.** The quantitative measurement of ALP. (**A**) Fluorescence spectra of the assay system response to different activity units of ALP (0, 0.01, 0.1, 1, 2, 3, 4, 5, 10, and 20 U/L); (**B**) the plot between F<sup>0</sup> -F and the concentration of ALP. Inset: calibration linear curve for ALP detection.



#### *3.5. Selectivity Assay 3.5. Selectivity Assay*

To demonstrate the selectivity of the proposed strategy, the interfering proteins such as UDG, T4 DNA Ligase and Nb. BtsI, in the same concentration as ALP [13,14,36], were evaluated using the present method. As illustrated in Figure 4, the interfering proteins all arouse strong fluorescence responses except ALP, indicating the prominent specificity of the proposed approach. To demonstrate the selectivity of the proposed strategy, the interfering proteins such as UDG, T4 DNA Ligase and Nb. BtsI, in the same concentration as ALP [13,14,36], were evaluated using the present method. As illustrated in Figure 4, the interfering proteins all arouse strong fluorescence responses except ALP, indicating the prominent specificity of the proposed approach. *Biosensors* **2021**, *11*, x FOR PEER REVIEW 7 of 10

*3.6. ALP Inhibition Investigation* 

inhibitors potentially.

**Figure 4.** Selectivity assay. The concentrations of ALP, UDG, T4 DNA Ligase, and Nb. BtsI were 5 U/L. **Figure 4.** Selectivity assay. The concentrations of ALP, UDG, T4 DNA Ligase, and Nb. BtsI were 5 U/L.

applied. As shown in Figure 5, the value of relative activity of ALP decreased upon increasing the concentration of Na3VO4 from 0 to 0.75 mM. The inset graph indicated a linear relationship (R2 = 0.9126) between the relative activity and low Na3VO4 concentrations. The half-maximal inhibitory concentration (IC50) of Na3VO4 was calculated to be 0.433 mM. The results demonstrate that the proposed method can be applied to screen ALP

#### *3.6. ALP Inhibition Investigation*

Na3VO4, reported to be one of the inhibitors of ALP, was chosen as the inhibitor to be applied. As shown in Figure 5, the value of relative activity of ALP decreased upon increasing the concentration of Na3VO<sup>4</sup> from 0 to 0.75 mM. The inset graph indicated a linear relationship (R<sup>2</sup> = 0.9126) between the relative activity and low Na3VO<sup>4</sup> concentrations. The half-maximal inhibitory concentration (IC50) of Na3VO<sup>4</sup> was calculated to be 0.433 mM. The results demonstrate that the proposed method can be applied to screen ALP inhibitors potentially. *Biosensors* **2021**, *11*, x FOR PEER REVIEW 8 of 10

**Figure 5.** The inhibitory effects of Na3VO4 (0, 0.15, 0.25, 0.5, and 0.75 mM) on ALP. Concentration of ALP was 5 U/L. Inset: calibration linear curve for the inhibitory effects of Na3VO4 on ALP. **Figure 5.** The inhibitory effects of Na3VO<sup>4</sup> (0, 0.15, 0.25, 0.5, and 0.75 mM) on ALP. Concentration of ALP was 5 U/L. Inset: calibration linear curve for the inhibitory effects of Na3VO<sup>4</sup> on ALP.

#### *3.7. ALP Assay in Diluted Human Serum Samples 3.7. ALP Assay in Diluted Human Serum Samples*

To investigate the practical feasibility of the proposed strategy, a variety of concentrations of ALP, including 0.5 U/L, 3 U/L, and 5 U/L, were tested by the proposed method while adding a human serum into the reaction buffer to simulate the complex biological environment during the experimental procedures. As illustrated in Table 2, the recovery rates of various concentrations of ALP in 1% human serum diluted were 97.15% for 0.5 U/L, 102.11% for 3 U/L and 99.89% for 5 U/L with R.S.D of 7.95%, 8.73%, and 1.09%, respectively. Therefore, the results displayed that the proposed strategy has great potential in practical applications. To investigate the practical feasibility of the proposed strategy, a variety of concentrations of ALP, including 0.5 U/L, 3 U/L, and 5 U/L, were tested by the proposed method while adding a human serum into the reaction buffer to simulate the complex biological environment during the experimental procedures. As illustrated in Table 2, the recovery rates of various concentrations of ALP in 1% human serum diluted were 97.15% for 0.5 U/L, 102.11% for 3 U/L and 99.89% for 5 U/L with R.S.D of 7.95%, 8.73%, and 1.09%, respectively. Therefore, the results displayed that the proposed strategy has great potential in practical applications.


**Table 2.** Recovery experiments of ALP in human serum samples. **Table 2.** Recovery experiments of ALP in human serum samples.

#### **4. Conclusions**

**4. Conclusions**  The proposed turn-off strategy shows high sensitivity, high selectivity with facile procedures in a short time in the quantification of ALP. Therefore, it has great potential to be utilized in the biological studies, early diagnosis and prognosis of some diseases related to the activity of ALP, such as diabetes, breast cancer, and prostatic cancer clinically [9– 11]. However, there are still some challenges to overcome in practical applications. For example, this method requires a different reaction buffer which is a challenge in practical The proposed turn-off strategy shows high sensitivity, high selectivity with facile procedures in a short time in the quantification of ALP. Therefore, it has great potential to be utilized in the biological studies, early diagnosis and prognosis of some diseases related to the activity of ALP, such as diabetes, breast cancer, and prostatic cancer clinically [9–11]. However, there are still some challenges to overcome in practical applications. For example, this method requires a different reaction buffer which is a challenge in practical applications.

applications. In conclusion, based on the poly T-DNA-templated formation of fluorescent CuNPs, we have proposed a facile but sensitive, selective, low-cost, and time-saving ALP assay. In conclusion, based on the poly T-DNA-templated formation of fluorescent CuNPs, we have proposed a facile but sensitive, selective, low-cost, and time-saving ALP assay.

Besides, the assay depends on Exo I, which can only split off the one-stranded DNA with

short time (40 min). The LOD value of 0.0098 U/L for the proposed assay demonstrates its high sensitivity. Compared with interfering enzymes, including UDG, T4 DNA Ligase, and Nb. BtsI without obvious variation of fluorescent signals, this method showed high selectivity to ALP. Moreover, when applied to test ALP levels in diluted human serum

Besides, the assay depends on Exo I, which can only split off the one-stranded DNA with the 3 0 -hydroxyl end hydrolyzed by ALP. The proposed strategy showed superiority in a short time (40 min). The LOD value of 0.0098 U/L for the proposed assay demonstrates its high sensitivity. Compared with interfering enzymes, including UDG, T4 DNA Ligase, and Nb. BtsI without obvious variation of fluorescent signals, this method showed high selectivity to ALP. Moreover, when applied to test ALP levels in diluted human serum samples, with high recovery rates and low R.S.D, the proposed strategy showed its potentially practical value with satisfactory results. Given the simplicity, wonderful sensitivity, and high selectivity of the proposed method, we can envisage that it may find a wide application in clinical diagnosis and prognosis.

**Author Contributions:** Conceptualization, C.M.; investigation, Y.W.; writing—original draft preparation, Y.W. and Y.Y.; writing—review and editing, X.L. and C.M.; supervision, C.M.; funding acquisition, X.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (No. 21205142) and the Research Innovation Program for Graduates of Central South University (2018zzts384, 2019zzts453).

**Institutional Review Board Statement:** The study was approved by the Ethics Committee of Central South University (protocol code 11 January 2020; date of approval 2 April 2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding authors.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

