Construction of Aggregation-Induced Emission Molecule–MnO2 Composite Nanoprobe and Its Application in Alkaline Phosphatase Detection

Alkaline phosphatase (ALP) is among the most studied enzymes by far, playing an important role in the metabolism of organisms and the regulation of protein activity. Herein, a label-free composite nanoprobe is constructed by combining inorganic nanomaterials and aggregation-induced emission (AIE) molecule to achieve highly sensitive and selective detection of ALP. Negatively charged 9,10-bis [2-(6-sulfonatopropoxyl) naphthylethenyl] anthracene (BSNVA) molecule is synthesized, which has the AIE performance and can be assembled on the surface of amino–SiO2 nanoparticles through electrostatic interaction for fluorescence enhancement. MnO2 nanosheets are rich in negative charges, enabling them to be wrapped on the surface of the amino–SiO2 nanosphere to shield the positive charge on its surface, making it impossible for BSNVA to accumulate on the surface and then weakening the bio-fluorescence of the system. Furthermore, with catalyzed substrates induced by ALP, generating ascorbic acid and the redox reaction between ascorbic acid and MnO2, the nanoprobe helps in realizing the high-sensitivity detection of ALP with a detection limit of 0.38 mU/mL. The proposed strategy requires no complex cleaning and modification processes and can overcome the quenching effect caused by the aggregation of traditional organic dyes, proving to be a simple, low-cost and “turn-on” fluorescent detection method for ALP.


Introduction
Alkaline phosphatase (ALP) is one of the most commonly assayed biological enzymes, which can catalyze the hydrolysis process of a variety of substrates, such as proteins, nucleic acids and monophosphates, and plays a pivotal role in the metabolism of organisms and the regulation of protein activity [1]. In addition, ALP is an important biomarker closely related to the diagnosis of many diseases. Normally, the serum ALP concentration of normal adults is 40-190 U/L, while those of children and pregnant women can reach 500 U/L [2]. A variety of diseases have been found to be associated with abnormal ALP activity, such as bone cancer, prostate cancer, liver disease, breast cancer, nasopharyngeal carcinoma, colorectal cancer and pancreatic cancer [3][4][5][6][7][8][9][10]. Therefore, the detection of ALP activities is of great significance in clinical diagnosis and disease treatment. In recent years, a large number of ALP detection sensor systems have been reported, including colorimetric sensors, electrochemical sensors, surface-enhanced Raman scattering sensors and fluorescence sensors [11][12][13][14][15]. Although these methods can be applied in the detection of ALP, they all have some inherent defects. For instance, colorimetric sensors are developed based on gold nanoparticles (hence, they are susceptible to salts and other materials in the environment); electrochemical sensors require complex cleaning and modifications of electrodes; and surface-enhanced Raman scattering sensors require specialized and sophisticated instruments. In contrast, fluorescence-sensing systems have drawn extensive

Instrumentation
UV-vis spectra were characterized by a UV-2450 spectrometer (Shimadzu, Kyoto, Japan). Fluorescence spectra were measured using an RF-6000 (Shimadzu, Japan). The excitation wavelength was 410 nm and both the excitation and emission slits had a width of 5 nm. Transmission electron microscopy (TEM) images were measured on a JEOL 2010 transmission electron microscope operated at an accelerating voltage of 200 kV. The sample used for TEM characterization was prepared by dropping a suspension on a carbon-coated copper grid and drying at room temperature. Scanning electron microscope (SEM) images were measured on an S-8010 (Hitachi, Kyoto, Japan). The 1 H NMR spectra were measured on a JEOL 400 MHz spectrometer with DMSO (dimethyl sulfoxide) as the solvent. The zeta potential measurements were recorded with a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd., Malvern, UK).

Preparation of MnO 2 -SiO 2 NPs
In a typical experiment, the preparation of amino-functionalized SiO 2 nanospheres (SiO 2 -NH 2 NSs) was carried out through the Stöber process [35]. First, under the condition of stirring, NH 3 ·H 2 O (0.1 M, 200 µL) was added to CTAB (0.08 M, 50 mL) and the mixture was evenly mixed. Then, 0.08 mL of 20% TEOS methanol solution was rapidly added to the mixture and stirred at room temperature for 20 h. The mixture was centrifuged, washed and dried to obtain white SiO 2 nanospheres. Next, 120 mg of the SiO 2 nanospheres was added to the ethanol and stirred to fully dissolve it. Then, 30 µL of APTES was added to the solution and vigorously stirred for 10 h. The above solution was centrifuged and washed with ethanol three times to remove excess APTES, and the precipitate was dried to obtain SiO 2 -NH 2 NSs. MnO 2 nanosheets were synthesized following the method described in the literature [36]. MnCl 2 ·4H 2 O (0.5945 g) was dissolved in water (10 mL) and then 20 mL of the mixture of tetramethylammonium hydroxide solution (12.35 mL) and H 2 O 2 (2 mL, 30 wt. %) was added. The mixture solution immediately turned brown and was stirred at room temperature for 12 h. The precipitate was collected by centrifugation, then washed with methanol and water and dried to obtain massive MnO 2 . The bulk MnO 2 solid (0.0100 g) was dispersed in 20 mL distilled water and the MnO 2 solution was centrifuged at 2000 rpm/min for 10 min after 12 h ultrasonic treatment. The MnO 2 nanosheets were removed from the supernatant and stored at 4 • C for future use.
Then, 0.0050 g of SiO 2 -NH 2 NSs and 0.0050 g of MnO 2 nanosheets were dissolved in 10 mL of distilled water and dispersed by ultrasound. The dispersed SiO 2 -NH 2 NSs solution (40 µL) and nanosheets solution (20 µL) were mixed and incubated at room temperature for 30 min to form the complex. The mixed solution was centrifuged and washed Nanomaterials 2023, 13, 2138 4 of 12 three times to remove the unbound and unstable MnO 2 nanosheets. The precipitates were dispersed into the same volume of water to prepare MnO 2 -SiO 2 NPs solution.
2.4. Synthesis of 9,10-Bis [2-(6-sulfonatopropoxyl)naphthylethenyl]anthracene BSNVA was synthesized following the methods of our previous work [37]. The synthesis route is shown in Figure S1. The reaction took place in a nitrogen atmosphere, and 9,10-bis [2-(6-hydroxy)naphthyl-ethenyl]anthracene (0.515 g, 1 mmol) was taken as the reactant and dissolved in anhydrous ethanol (20 mL). Then, the anhydrous ethanol solution of sodium ethanolate (20 mL, 2.3 mmol) was added to the mixture dropwise. The solution was stirred until it turned orange-red, then 1,3-propanesultone (200 µL) was added. The mixed solution was stirred until the product was separated out. The filtered precipitate was then washed with ethanol and acetone to form 9,10-bis [2-

Detection of ALP in Human Serum Sample
The serum samples were diluted 100-fold with PBS buffer (10 mM, pH = 7.4) and incubated with NEM (0.2 mM) for 20 min at 37 • C. Then, different concentrations of ALP were added to the above solution for recovery tests. The measurement method and reaction conditions were the same as those for ALP detection above.

Results and Discussion
The construction of the AIE-MnO 2 composite nanosystem is illustrated in Scheme 1. The prepared BSNVA molecules were water-soluble and hardly emitted any light when dispersed in water. Due to the presence of a large number of amino groups on the surface of the SiO 2 -NH 2 NSs, they are positively charged and can bind negatively charged BSNVA through electrostatic interaction, so that BSNVA can accumulate on the surface of the SiO 2 -NH 2 NSs and emit strong green fluorescence. The MnO 2 nanosheet is an inorganic two-dimensional nanomaterial with excellent biocompatibility that can be reduced to Mn 2+ by AA and then decomposed; hence, it is widely used in the construction of biosensors. In addition, ALP catalyzes the process of dephosphorylation by removing the phosphate groups from the substrate to form hydroxyl groups. Based on the above principles, a MnO 2 nanosheet is introduced into the sensing system. A MnO 2 nanosheet has a negative charge and can be wrapped on the surface of SiO 2 -NH 2 NSs through electrostatic adsorption, so as to shield the positive charge on the surface and avoid binding with BSNVA, resulting in no luminescence in the system. With AAP and ALP coexisting, ALP can catalyze the former to generate AA, thereby inducing the decomposition of the MnO 2 nanosheet. Meanwhile, SiO 2 -NH 2 NSs are released, BSNVA accumulates on the surface of SiO 2 -NH 2 NSs and fluorescence is enhanced. The high sensitivity and specificity of ALP can be achieved following the dynamics of fluorescence intensity of the system.

Preparation and Characterization of Nanoparticles
SiO2-NH2 NSs and MnO2 nanosheets were synthesized and their sizes and morphology were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in Figure 1A, the synthesized SiO2-NH2 NSs were spherical and had good dispersion in aqueous solution, with a mean diameter of 57.9 nm. The morphology of the MnO2 nanosheet is shown in Figure 1B: the nanosheet was a two-dimensional lamellar structure with some folds. SiO2-NH2 NSs can be adsorbed on the surface of a MnO2 nanosheet through electrostatic interaction to form a MnO2-SiO2 complex; zeta potential measurements revealed that the potential changed ( Figure S2). TEM images showed that the MnO2 nanosheet had been successfully coated on the surface of SiO2-NH2 NSs ( Figure 1C). In addition, it can be clearly seen from Figure 1D that the MnO2 nanosheet was decomposed in the presence of AA, which is consistent with our expected results. Scheme 1. Schematic illustration of the AIE-MnO 2 composite nanosystem for alkaline phosphatase detection.

Preparation and Characterization of Nanoparticles
SiO 2 -NH 2 NSs and MnO 2 nanosheets were synthesized and their sizes and morphology were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in Figure 1A, the synthesized SiO 2 -NH 2 NSs were spherical and had good dispersion in aqueous solution, with a mean diameter of 57.9 nm. The morphology of the MnO 2 nanosheet is shown in Figure 1B: the nanosheet was a twodimensional lamellar structure with some folds. SiO 2 -NH 2 NSs can be adsorbed on the surface of a MnO 2 nanosheet through electrostatic interaction to form a MnO 2 -SiO 2 complex; zeta potential measurements revealed that the potential changed ( Figure S2). TEM images showed that the MnO 2 nanosheet had been successfully coated on the surface of SiO 2 -NH 2 NSs ( Figure 1C). In addition, it can be clearly seen from Figure 1D that the MnO 2 nanosheet was decomposed in the presence of AA, which is consistent with our expected results.

Preparation and Characterization of Nanoparticles
SiO2-NH2 NSs and MnO2 nanosheets were synthesized and their sizes and morphology were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in Figure 1A, the synthesized SiO2-NH2 NSs were spherical and had good dispersion in aqueous solution, with a mean diameter of 57.9 nm. The morphology of the MnO2 nanosheet is shown in Figure 1B: the nanosheet was a two-dimensional lamellar structure with some folds. SiO2-NH2 NSs can be adsorbed on the surface of a MnO2 nanosheet through electrostatic interaction to form a MnO2-SiO2 complex; zeta potential measurements revealed that the potential changed ( Figure S2). TEM images showed that the MnO2 nanosheet had been successfully coated on the surface of SiO2-NH2 NSs ( Figure 1C). In addition, it can be clearly seen from Figure 1D that the MnO2 nanosheet was decomposed in the presence of AA, which is consistent with our expected results.

Synthesis and Characterization of Anthracene Derivatives
The water-soluble molecule BSNVA was synthesized, with a large conjugated structure cored by 9,10-divinylanthracene, and its hydrogen on vinyl replaced by naphthalene ( Figure S1). Its structure was confirmed by 1 H NMR ( Figure S3). In order to explain its luminescence mechanism, the structure of BSNVA was optimized using the Gaussian 09 procedure and B3LYP method. As shown in Figure S4, the optimal structure of BSNVA presents a distorted conformation. When BSNVA is in the dissolved state, the vibration of 9,10-divinylanthracene and the rotation of the peripheral naphthalene can dissipate the energy of the excited state. In addition, the highly distorted conformation makes it easier for the peripheral aromatic ring of BSNVA to rotate in the aqueous solution, thus reducing the fluorescence yield. However, the formation of the aggregation state leads to blocked movement within the molecule, non-radiation attenuation channels are blocked and radiation attenuation is activated. At the same time, the highly distorted conformation hinders the π-π accumulation among molecules. Therefore, the luminous efficiency is improved and the compound emits strong fluorescence.
As shown in Figure 2A, the maximum absorption wavelength of BSNVA in aqueous solution was 410 nm. Its fluorescence was weak and its emission wavelength was 545 nm. Water was used as the good solvent and THF as the poor solvent to investigate the AIE performance of BSNVA. When the volume fraction (f T ) of THF was 20%, BSNVA began to accumulate. With the increase in f T , the fluorescence gradually became stronger and reached the maximum when the f T was 80%, which was 13 times higher than the fluorescence intensity in aqueous solution ( Figure 2C,D). The corresponding solution photo is shown in Figure 2B. All the solutions were light yellow and transparent under sunlight. Under the ultraviolet light, the green fluorescence first increased and then decreased, which is consistent with the fluorescence spectra results. of AA.

Synthesis and Characterization of Anthracene Derivatives
The water-soluble molecule BSNVA was synthesized, with a large conjugated structure cored by 9,10-divinylanthracene, and its hydrogen on vinyl replaced by naphthalene ( Figure S1). Its structure was confirmed by 1 H NMR ( Figure S3). In order to explain its luminescence mechanism, the structure of BSNVA was optimized using the Gaussian 09 procedure and B3LYP method. As shown in Figure S4, the optimal structure of BSNVA presents a distorted conformation. When BSNVA is in the dissolved state, the vibration of 9,10-divinylanthracene and the rotation of the peripheral naphthalene can dissipate the energy of the excited state. In addition, the highly distorted conformation makes it easier for the peripheral aromatic ring of BSNVA to rotate in the aqueous solution, thus reducing the fluorescence yield. However, the formation of the aggregation state leads to blocked movement within the molecule, non-radiation attenuation channels are blocked and radiation attenuation is activated. At the same time, the highly distorted conformation hinders the π-π accumulation among molecules. Therefore, the luminous efficiency is improved and the compound emits strong fluorescence.
As shown in Figure 2A, the maximum absorption wavelength of BSNVA in aqueous solution was 410 nm. Its fluorescence was weak and its emission wavelength was 545 nm. Water was used as the good solvent and THF as the poor solvent to investigate the AIE performance of BSNVA. When the volume fraction (fT) of THF was 20%, BSNVA began to accumulate. With the increase in fT, the fluorescence gradually became stronger and reached the maximum when the fT was 80%, which was 13 times higher than the fluorescence intensity in aqueous solution ( Figure 2C,D). The corresponding solution photo is shown in Figure 2B. All the solutions were light yellow and transparent under sunlight. Under the ultraviolet light, the green fluorescence first increased and then decreased, which is consistent with the fluorescence spectra results.

Construction of MnO 2 -SiO 2 -AIE Nanocomposite System
By synthesizing the above nanoparticles and BSNVA molecules, the MnO 2 -SiO 2 -AIE nanocomposite system was constructed. As shown in Figure 3A, the prepared SiO 2 -NH 2 NSs had no obvious UV-vis absorption in the range of 240-800 nm. The MnO 2 nanosheet showed obvious optical properties and had an obvious UV-vis absorption at 370 nm. The MnO 2 -SiO 2 complex also produced a UV-vis absorption at 370 nm, indicating that the MnO 2 nanosheet had successfully adsorbed on the surface of SiO 2 -NH 2 NSs. BSNVA has a negative charge and can be bounded to the surface of SiO 2 -NH 2 NSs through electrostatic interaction. The SiO 2 -BSNVA complex produced an obvious UV-vis absorption peak at 410 nm, which is consistent with the UV-vis absorption of BSNVA, indicating that BSNVA had been successfully concentrated on the surface of SiO 2 -NH 2 NSs. There was a blueshift in the UV-vis absorption of MnO 2 -SiO 2 -BSNVA complex at 410 nm, caused by the combination with MnO 2 nanosheet. The results showed that the MnO 2 -SiO 2 complex and SiO 2 -AIE complex had been successfully prepared.

Construction of MnO2-SiO2-AIE Nanocomposite System
By synthesizing the above nanoparticles and BSNVA molecules, the MnO2-SiO2-AIE nanocomposite system was constructed. As shown in Figure 3A, the prepared SiO2-NH2 NSs had no obvious UV-vis absorption in the range of 240-800 nm. The MnO2 nanosheet showed obvious optical properties and had an obvious UV-vis absorption at 370 nm. The MnO2-SiO2 complex also produced a UV-vis absorption at 370 nm, indicating that the MnO2 nanosheet had successfully adsorbed on the surface of SiO2-NH2 NSs. BSNVA has a negative charge and can be bounded to the surface of SiO2-NH2 NSs through electrostatic interaction. The SiO2-BSNVA complex produced an obvious UV-vis absorption peak at 410 nm, which is consistent with the UV-vis absorption of BSNVA, indicating that BSNVA had been successfully concentrated on the surface of SiO2-NH2 NSs. There was a blueshift in the UV-vis absorption of MnO2-SiO2-BSNVA complex at 410 nm, caused by the combination with MnO2 nanosheet. The results showed that the MnO2-SiO2 complex and SiO2-AIE complex had been successfully prepared. In addition, the formation of nanocomposites was further confirmed via fluorescence measurements. The MnO2-SiO2 complex had no emission in aqueous solution ( Figure 3B). BSNVA was soluble in water and hardly gave off light in aqueous solution. When BSNVA was combined with SiO2-NH2 NSs, the system emitted strong fluorescence. This was due to BSNVA molecules aggregation on the surface of SiO2-NH2 NSs, which limited their intramolecular motion, and, thus, turned on the fluorescence emission of BSNVA. It is worth noting that, when BSNVA was mixed with the MnO2-SiO2-NH2 nanosystem, its fluorescence remained almost unchanged. This was because the MnO2 nanosheet could wrap SiO2-NH2 NSs and shield the positive charge on the SiO2-NH2 NSs, resulting in the non-polymerization of BSNVA.

Concentration Optimization of MnO2 Nanosheet
The coating of the MnO2 nanosheet can shield the positive charge on the surface of SiO2-NH2 NSs, thus reducing the aggregation of BSNVA. In addition, it is also the target of reducing species and the key to the construction of an ALP sensor. Therefore, the concentration of MnO2 nanosheet was the main factor affecting the fluorescence intensity of the composite nanosystem. The fluorescence intensity of the system gradually decreased as the content of MnO2 nanosheet in the system gradually increased (Figure 4A,B). When the concentration of MnO2 nanosheet reached 100 µg/mL, the fluorescence intensity of the system hit its bottom, indicating that the SiO2-NH2 NSs were completely coated by MnO2 nanosheets. After that, the MnO2 nanosheets adsorbed on the surface of SiO2-NH2 NSs In addition, the formation of nanocomposites was further confirmed via fluorescence measurements. The MnO 2 -SiO 2 complex had no emission in aqueous solution ( Figure 3B). BSNVA was soluble in water and hardly gave off light in aqueous solution. When BSNVA was combined with SiO 2 -NH 2 NSs, the system emitted strong fluorescence. This was due to BSNVA molecules' aggregation on the surface of SiO 2 -NH 2 NSs, which limited their intramolecular motion, and, thus, turned on the fluorescence emission of BSNVA. It is worth noting that, when BSNVA was mixed with the MnO 2 -SiO 2 -NH 2 nanosystem, its fluorescence remained almost unchanged. This was because the MnO 2 nanosheet could wrap SiO 2 -NH 2 NSs and shield the positive charge on the SiO 2 -NH 2 NSs, resulting in the non-polymerization of BSNVA.

Concentration Optimization of MnO 2 Nanosheet
The coating of the MnO 2 nanosheet can shield the positive charge on the surface of SiO 2 -NH 2 NSs, thus reducing the aggregation of BSNVA. In addition, it is also the target of reducing species and the key to the construction of an ALP sensor. Therefore, the concentration of MnO 2 nanosheet was the main factor affecting the fluorescence intensity of the composite nanosystem. The fluorescence intensity of the system gradually decreased as the content of MnO 2 nanosheet in the system gradually increased (Figure 4A,B). When the concentration of MnO 2 nanosheet reached 100 µg/mL, the fluorescence intensity of the system hit its bottom, indicating that the SiO 2 -NH 2 NSs were completely coated by MnO 2 nanosheets. After that, the MnO 2 nanosheets adsorbed on the surface of SiO 2 -NH 2 NSs reached saturation and the fluorescence intensity of the system tended to be stable. As a result, 100 µg/mL was selected as the optimal concentration of MnO 2 nanosheet for further experiments.
reached saturation and the fluorescence intensity of the system tended to be stable. As a result, 100 µg/mL was selected as the optimal concentration of MnO2 nanosheet for further experiments.

Detection of ALP by MnO2-SiO2-BSNVA Composite System
ALP can catalyze the dephosphorylation of AA2P to produce AA [38]. Based on the above principles, the applicability of the constructed composite nanosystem in ALP detection was further explored. As shown in Figure S5A, the specific absorbing peak of MnO2 nanosheet disappeared after adding AA, indicating that the MnO2 nanosheet can be decomposed by AA, which was consistent with TEM results. Fluorescence detection results showed that adding AA to the composite system could enhance the fluorescence of the composite nanosystem. The fluorescence spectra in Figure S5B shows that the fluorescence of the composite nanosystem was enhanced with AA being added to the MnO2-SiO2-BSNVA composite system. The reason is that AA induced the MnO2 nanosheet to decompose into Mn 2+ , which destroyed the composite structure and released SiO2 nanospheres. The positive charge on its surface was exposed, which facilitated the formation of the SiO2-BSNVA complex by combining with BSNVA in the system, resulting in BSNVA fluorescence enhancement. This result further proves the successful construction of nanocomplexes and also proves that the label-free and "turn-on" strategy proposed by us is feasible for ALP detection.
On the basis of the above studies, AA2P and ALP of different concentrations were added to the MnO2-SiO2-BSNVA composite system, so as to detect the fluorescence of the composite system under optimal experimental conditions. As shown in Figure 5A, the fluorescence intensity of the composite system was positively correlated with the concentration of ALP in the system. With the increase in ALP concentration, the fluorescence of the system recovered rapidly, almost reaching full recovery at 500 mU/mL. As shown in Figure 5B, the probe showed a good linear response to ALP in the range of 1-160 mU/mL; the linear equation was y = 297.2 + 20.41x (R 2 = 0.998) and the detection limit was 0.38 mU/mL. The detection limit of this method is lower than those of other methods reported recently (Table 1).

Detection of ALP by MnO 2 -SiO 2 -BSNVA Composite System
ALP can catalyze the dephosphorylation of AA2P to produce AA [38]. Based on the above principles, the applicability of the constructed composite nanosystem in ALP detection was further explored. As shown in Figure S5A, the specific absorbing peak of MnO 2 nanosheet disappeared after adding AA, indicating that the MnO 2 nanosheet can be decomposed by AA, which was consistent with TEM results. Fluorescence detection results showed that adding AA to the composite system could enhance the fluorescence of the composite nanosystem. The fluorescence spectra in Figure S5B shows that the fluorescence of the composite nanosystem was enhanced with AA being added to the MnO 2 -SiO 2 -BSNVA composite system. The reason is that AA induced the MnO 2 nanosheet to decompose into Mn 2+ , which destroyed the composite structure and released SiO 2 nanospheres. The positive charge on its surface was exposed, which facilitated the formation of the SiO 2 -BSNVA complex by combining with BSNVA in the system, resulting in BSNVA fluorescence enhancement. This result further proves the successful construction of nanocomplexes and also proves that the label-free and "turn-on" strategy proposed by us is feasible for ALP detection.
On the basis of the above studies, AA2P and ALP of different concentrations were added to the MnO 2 -SiO 2 -BSNVA composite system, so as to detect the fluorescence of the composite system under optimal experimental conditions. As shown in Figure 5A, the fluorescence intensity of the composite system was positively correlated with the concentration of ALP in the system. With the increase in ALP concentration, the fluorescence of the system recovered rapidly, almost reaching full recovery at 500 mU/mL. As shown in Figure 5B, the probe showed a good linear response to ALP in the range of 1-160 mU/mL; the linear equation was y = 297.2 + 20.41x (R 2 = 0.998) and the detection limit was 0.38 mU/mL. The detection limit of this method is lower than those of other methods reported recently (Table 1).    In addition, the selectivity of the MnO 2 -SiO 2 -BSNVA composite-sensing system for ALP detection was explored by comparing the fluorescence signals in the presence of other interfering substances ( Figure 6). As demonstrated, the fluorescence of the sensing system remained unchanged or slightly changed upon addition of control proteins/enzymes (300 mU/mL), including insulin, lysozyme, human serum albumin, pepsin and trypsin. However, the fluorescence of the system could be enhanced when both the ALP (160 mU/mL) and the interfering substance were present, indicating that the system constructed has excellent selectivity for ALP.  In addition, the selectivity of the MnO2-SiO2-BSNVA composite-sensing system for ALP detection was explored by comparing the fluorescence signals in the presence of other interfering substances ( Figure 6). As demonstrated, the fluorescence of the sensing system remained unchanged or slightly changed upon addition of control proteins/enzymes (300 mU/mL), including insulin, lysozyme, human serum albumin, pepsin and trypsin. However, the fluorescence of the system could be enhanced when both the ALP (160 mU/mL) and the interfering substance were present, indicating that the system constructed has excellent selectivity for ALP.

Detection of ALP in Human Serum Sample
In order to evaluate the feasibility of the composite nanoprobe for practical application, the assay for detecting ALP in diluted human serum samples was carried out. To eliminate the interference of glutathione and cysteine, reduced N-ethylmaleimide was added to the serum samples [48]. As shown in Table 2, the average recoveries of the three real samples were 110.9, 108.2 and 107.0. The relative standard deviation (RSD) was between 2.24 and 2.81%, indicating that this composite nanoprobe could be used for detecting ALP in biological samples.

Conclusions
In conclusion, a "turn-on" composite nanosystem with high specificity and sensitivity based on AIE molecules was successfully constructed and used in the detection of ALP. Water-soluble BSNVA molecules, with good optical properties and negative charges in aqueous solution, were regarded as signal units. A MnO 2 nanosheet could not only be used as a positive protectant to shield the positive charge on the surface of SiO 2 -NH 2 NSs, but also as a response unit of the ALP-induced substrate decomposition reaction. It was decomposed to release SiO 2 -NH 2 NSs, so that BSNVA molecules could accumulate on the surface of SiO 2 -NH 2 NSs for fluorescence enhancement. The nanosensor did not require a complex modification process and overcame the disadvantages of traditional organic probes, such as poor water solubility and easy quenching at high concentration. In addition, the fluorescent probe was based on the "turn-on" strategy, which has the advantages of simple construction, fast response, low cost and being label-free. Furthermore, the combination of AIE molecules and inorganic nanomaterials provides a new perspective for the detection of enzyme activity, which has the potential to be applied in the diagnosis of diseases and biomedical research.