**1. Introduction**

Alkaline phosphatase (ALP), a ubiquitous enzyme found in human tissues such as the liver, intestine, bone, kidney, and placenta, is a homodimeric enzyme with necessary cofactors, including one magnesium atom and two zinc atoms [1,2]. Studies have shown that ALP is able to catalyze alkaline hydrolysis of nucleic acids, proteins, and some small molecules, which are phosphate [1,3–5]. Owing to the indispensable role in many physiological processes such as cell cycle, growth, apoptosis, and signal transduction, ALP is closely connected to multiple human diseases, especially bone and hepatic diseases [5–7]. The concentration of ALP in healthy people's serum is 46–190 U/L [8]. Hence, any abnormal level of ALP in the serum may be an essential indicator of some diseases related to ALP function, such as diabetes, breast cancer, prostatic cancer, bone diseases, such as osteosarcoma, and hepatic diseases, e.g., Wilson's disease. So ALP levels in the serum may be an effective biomarker in medical diagnosis [9–11]. Furthermore, Prakash et al. have recently found that the level of ALP in saliva (readily accessible, safe, and noninvasive body fluid) may be able to serve as an early biomarker for diabetes mellitus and some potentially malignant tumors [12]. In addition, ALP is capable of generating signals for the analytes by conjugating to streptavidin or the second antibody in the biological analysis, such as enzyme-linked immunosorbent assay (ELISA), histochemical staining, and aptamer-based assay [13,14]. As a result, the detection limit of analytes is highly dependent on the detection limit of ALP in these biological analyses. Besides, due to its ability to monitor phosphorus-related water eutrophication, ALP is a good indicator of algal boom [15]. Therefore, it is of great importance to developing a facile and sensitive method to detect ALP.

**Citation:** Wang, Y.; Yan, Y.; Liu, X.; Ma, C. An Exonuclease I-Aided Turn-Off Fluorescent Strategy for Alkaline Phosphatase Assay Based on Terminal Protection and Copper Nanoparticles. *Biosensors* **2021**, *11*, 139. https://doi.org/10.3390/ bios11050139

Received: 9 April 2021 Accepted: 27 April 2021 Published: 29 April 2021

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A number of diverse methods and techniques, including colorimetric assay, electrochemistry, chromatography, photometric assay, photoelectrochemical assay and surfaceenhanced Raman scattering methods, have been developed to date to detect the concentration of ALP that catalyzes the dephosphorylation process of different substrates [16–23]. However, these traditional methods inevitably suffer from one or more limitations, such as time-consuming procedures, poor sensitivity, exorbitant material requirement, and use of complex devices. Recently, fluorometric methods have caught people's eyes for their advantages such as simplicity, convenience, rapid response, and high sensitivity [24,25].

Due to the simplicity, high sensitivity, low cost, and rapidness, nanomaterial-based probes have attracted considerable attention lately [26]. On account of the fluorescence of some nanoparticles, including copper nanoparticles (CuNPs), silver nanoparticles (AgNPs), and gold nanoparticles (AuNPs), the signals can be monitored by spectrometers [27,28]. In recent years, some efforts have been made towards the detection of ALP by measuring the fluorescence intensity of nanoparticles. Chen and his coworkers successfully screened the concentration of ALP with a LOD of 0.125 U/L by constructing a sequentially triggered nanoswitch depending on CuNPs using single-stranded poly-(thymine) (poly T) DNA as a template [29]. Chen et al. reported that AgNPs formed by Ag+ and CdTe quantum dots could detect ALP with LOD of 0.25 U/L [30]. Lin et al. proposed a rapid method to detect ALP, based on redox-modulated silver deposition on AuNPs, with LOD of 0.52 U/L [31]. Among these assays, poly T DNA-templated CuNPs is remarkable owing to their simple procedure, less necessity of DNA templates, and rapid formation with the support of ascorbate to reduce Cu2+ within just a few minutes [32]. Moreover, poly T DNAtemplated CuNPs are a prominent fluorescence probe, which exhibits a maximum λem at 615 nm with large MegaStrokes shifting with the ability to remove strong background signal from complex biological systems [33]. Considering that poly T DNA, an ideal template of fluorescent CuNPs, can be modified by the removal of a phosphate group from the 30 -end catalyzed by ALP, we have proposed a method for the measurement of ALP concentration with LOD of 0.0098 U/L requiring only 50 min based on terminal protection and fluorescent CuNPs. As far as we know, this is the first exonuclease I-aided turn-off fluorescent strategy for ALP assay based on terminal protection and CuNPs with high sensitivity in a short time.

#### **2. Materials and Methods**

## *2.1. Materials and Reagents*

The alkaline phosphatase and exonuclease I were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Tris (hydroxymethyl) methyl aminomethane hydrochloric acid (Tris-HCl), magnesium chloride (MgCl2), sodium chloride (NaCl), 30 -(Nmorpholino) propanesulfonic acid (MOPS), copper sulfate (CuSO4), and sodium ascorbate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Na3VO<sup>4</sup> was bought from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The DNA probe is T30: 50 -TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-p-30 , it was synthesized by Shanghai Sangon Biotech Co. Ltd. (Shanghai, China). All the other reagents were of analytical grade.

#### *2.2. Apparatus*

The fluorescence emission spectra were recorded on Hitachi F-2700 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan) at the excitation wavelength of 340 nm that was obtained from 550 nm to 650 nm at room temperature. The resulting error was obtained from three repeated measurements, and statistical methods were used to collate and analyze the data during the experiment.

#### *2.3. The Quantitative Detection of ALP*

To measure the activity of ALP quantitatively, T30 (2 µM) and ALP solutions of different concentrations were added into Tris-HCl buffer, and the resultant solution was incubated at 37 ◦C for 10 min and 80 ◦C for 20 min. After that, Exo I (40 U/mL) was added to the above tometer.

**3. Results** 

CuNPs.

solution, and the resulting solution was incubated at 37 ◦C for 10 min. Subsequently, 73 µL of MOPS buffer, 0.36 µL of CuSO<sup>4</sup> (28 mM), 1 µL of sodium ascorbate (500 mM) were added into the solution to get a final volume of 100 µL, and the resulting solution was incubated at room temperature for 10 min to form CuNPs. Finally, the fluorescence spectra of all the samples were recorded by the F-2700 fluorescence spectrophotometer. sequently, 73 µL of MOPS buffer, 0.36 µL of CuSO4 (28 mM), 1 µL of sodium ascorbate (500 mM) were added into the solution to get a final volume of 100 µL, and the resulting solution was incubated at room temperature for 10 min to form CuNPs. Finally, the fluorescence spectra of all the samples were recorded by the F-2700 fluorescence spectropho-

To measure the activity of ALP quantitatively, T30 (2 µM) and ALP solutions of dif-

cubated at 37 °C for 10 min and 80 °C for 20 min. After that, Exo I (40 U/mL) was added to the above solution, and the resulting solution was incubated at 37 °C for 10 min. Sub-

#### *2.4. Gel Electrophoresis Analysis 2.4. Gel Electrophoresis Analysis*

*Biosensors* **2021**, *11*, x FOR PEER REVIEW 3 of 10

The 20% denaturing urea polyacrylamide gel electrophoresis (Urea-PAGE) in 1xTBE (89 mM Tris-boric acid, 2 mM EDTA, pH 8.3) was used to analyze the feasibility of the proposed method at 150 V for 105 min. Afterwards, silver staining was employed as the staining method to show the different products formed under different conditions. The 20% denaturing urea polyacrylamide gel electrophoresis (Urea-PAGE) in 1xTBE (89 mM Tris-boric acid, 2 mM EDTA, pH 8.3) was used to analyze the feasibility of the proposed method at 150 V for 105 min. Afterwards, silver staining was employed as the staining method to show the different products formed under different conditions.

#### **3. Results**

*2.3. The Quantitative Detection of ALP* 

#### *3.1. Sensing Strategy of ALP Detection 3.1. Sensing Strategy of ALP Detection*

The method we proposed to measure the activity of ALP quantitatively is schematically illustrated in Scheme 1. A poly T-DNA with a phosphate modification at 30 -end is designated as the substrate of ALP. In the presence of ALP, the phosphate group is removed to liberate the phosphate group from the 30 -end so that there is a free 30 -OH which can be discerned by 30 single-stranded-specific exonuclease I (Exo I) [34]. Afterwards, the poly T-DNA can be split off into small fragments losing its ability to act as the template of CuNPs despite the existence of Cu2+ and sodium ascorbate. On the contrary, in the absence of ALP, the poly T-DNA with a 30 -phosphoryl can't be recognized by Exo I, so that integrated poly T-DNA becomes an ideal template for the formation of fluorescent CuNPs in the presence of Cu2+ and sodium ascorbate in the solution. Ultimately, the activity of ALP can be quantified by screening the fluorescent intensity changes. The method we proposed to measure the activity of ALP quantitatively is schematically illustrated in Scheme 1. A poly T-DNA with a phosphate modification at 3′-end is designated as the substrate of ALP. In the presence of ALP, the phosphate group is removed to liberate the phosphate group from the 3′-end so that there is a free 3′-OH which can be discerned by 3′ single-stranded-specific exonuclease I (Exo I) [34]. Afterwards, the poly T-DNA can be split off into small fragments losing its ability to act as the template of CuNPs despite the existence of Cu2+ and sodium ascorbate. On the contrary, in the absence of ALP, the poly T-DNA with a 3′-phosphoryl can't be recognized by Exo I, so that integrated poly T-DNA becomes an ideal template for the formation of fluorescent CuNPs in the presence of Cu2+ and sodium ascorbate in the solution. Ultimately, the activity of ALP can be quantified by screening the fluorescent intensity changes.

**Scheme 1.** Schematic illustration of fluorescent ALP activity analysis based on DNA-templated **Scheme 1.** Schematic illustration of fluorescent ALP activity analysis based on DNA-templated CuNPs.
