Enzyme cascade reaction-based ratiometric fluorescence probe for visual monitoring the activity of alkaline phosphatase

Journal Pre-proof Enzyme Cascade Reaction-Based Ratiometric Fluorescence Probe for Visual Monitoring the Activity of Alkaline Phosphatase Xia Cheng, Yuying Chai, Jian Xu, Lin Wang, Fangdi Wei, Guanhong Xu, Yong Sun, Qin Hu, Yao Cen

PII:

S0925-4005(20)30112-X

DOI:

https://doi.org/10.1016/j.snb.2020.127765

Reference:

SNB 127765

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

7 December 2019

Revised Date:

17 January 2020

Accepted Date:

21 January 2020

Please cite this article as: Cheng X, Chai Y, Xu J, Wang L, Wei F, Xu G, Sun Y, Hu Q, Cen Y, Enzyme Cascade Reaction-Based Ratiometric Fluorescence Probe for Visual Monitoring the Activity of Alkaline Phosphatase, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127765

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Enzyme Cascade Reaction-Based Ratiometric Fluorescence Probe for Visual Monitoring the Activity of Alkaline Phosphatase

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Xia Cheng†, Yuying Chai†, Jian Xu§, Lin Wang§, Fangdi Wei†,‡, Guanhong Xu†,‡, Yong Sun∥ ,

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Qin Hu†,‡,*, Yao Cen†,‡,* †

‡

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School of pharmacy, Nanjing medical university, Nanjing, Jiangsu 211166, PR China Key Laboratory of Cardiovascular & Cerebrovascular Medicine, School of pharmacy, Nanjing

Department of laboratory medicine, the first affiliated hospital of Nanjing medical university,

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§

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medical university, Nanjing, Jiangsu 211166, PR China

Nanjing, Jiangsu 211166, PR China ∥

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Key laboratory of toxicology, Ningde normal university, Ningde, Fujian 352000, PR China

*

Corresponding author. Tel/Fax: +86-25-86868468.

E-mail: [email protected] (Qin Hu); [email protected] (Yao Cen)

Highlights 

A ratiometric fluorescence probe based on CdTe/CdS/ZnS/SiO2 QDs and an enzyme cascade reaction for ALP sensing. 1



Sensitive and selective ALP activities sensing and inhibitors screening.



Simultaneous and highly sensitive detection of ALP in human serum.



4. Great potential for further clinical diagnosis and drug screening.

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Abstract Inspired by the intrinsic catalytic properties of alkaline phosphatase (ALP) and tyrosinase (TYR)

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as well as the fluorogenic reaction between levodopa and resorcinol, a ratiometric fluorescent

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probe based on the CdTe/CdS/ZnS/SiO2 QDs for visual monitoring the activity of ALP was developed. Phosphotyrosine was catalyzed into tyrosine by ALP, and then tyrosine was

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catalyzed into levodopa by TYR. Carboxyazamonardine, the reactive product of levodopa and

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resorcinol, gave rise to a strong blue fluorescence at 470 nm and quenched the red fluorescence of CdTe/CdS/ZnS/SiO2 QDs at 620 nm due to their inner filter effect. Thus, the resulting

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ratiometric fluorescence acted as the signal output for quantitative ALP detection, accompanying with the fluorescence color changing from red to purple and finally blue for semiquantitative ALP detection. A good linear relationship was obtained ranging from 0.08-500 U L-1 with the detection limit of 0.02 U L-1. Moreover, the probe was successfully applied to monitoring ALP activity in human serum samples without any pretreatment, and also used to screen ALP inhibitors. These results suggested that the developed method offered a highly sensitive and promising analytical platform for ALP in clinical diagnostics and paved a new way for monitoring enzymes activity visually for point-of-care diagnosis.

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Keywords: Visualization, Ratiometric fluorometry, Alkaline phosphatase, Inner filter effect, Inhibitors screening

1. Introduction As a nonspecific hydrolase in biological system, alkaline phosphatase (ALP) is able to

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catalyze hydrolysis of the phosphate group of various substrates including nucleic acids,

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proteins and small molecules and plays critical roles in cell growth, apoptosis and signal transmission [1-3]. To be specific, underexpressed ALP in serum is related to some metabolic

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disorders including hematological or Wilson's diseases such as chronic myelogenous leukemia

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and aplastic anemia [4]. While overexpressed ALP in serum is usually associated with liver dysfunction, bone disease (osteoblastic bone tumors, osteomalacia), prostatic cancer and

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diabetes [5-9]. In this regard, developing a reliable, sensitive, selective and simple method for clinical monitoring ALP activity in biological system is of great significance. date,

many analytical

methods

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To

for

ALP have

been reported,

including

chemiluminescence [10-11], electrochemistry [12-14], surface enhancement Raman scattering [15], colorimetry [16-18] and fluorescence method [19-20]. Among various methods, electrochemical assays and surface enhancement Raman scattering are well-established but limited by the shortcomings of requiring sophisticated steps and ponderous instruments, making the whole analysis process complicated. Colorimetry is facile and highly efficient, but has poor sensitivity [21-23]. Recently, the exploration of fluorescent method for ALP detection has made huge progress owing to the advantages of high sensitivity, low cost, simplicity and easy readout 3

[24-26]. Among them, the single-emission fluorescence probe is susceptible to different kinds of environmental factors including measurement conditions, matrix and fluorescence probe concentrations [27-28]. However, ratiometric fluorescent probes, i.e. dual-emission fluorescence probe, can provide a built-in correction to alleviate the analyte-independent interfering signals

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[29-30]. In addition, a ratiometric fluorescent probe is able to exhibit distinct color change with different concentrations of the analyte, which is conductive to on-site detection and diagnosis

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monitoring the activity of ALP still remains unexplored.

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[31-32]. To the best of our knowledge, developing a ratiometric fluorescent probe for visually

In this work, we presented a ratiometric fluorescence probe for visual monitoring ALP

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activity by making use of enzyme cascade reactions and CdTe/CdS/ZnS/SiO2 QDs.

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Phosphotyrosine was catalyzed into tyrosine by ALP, and then tyrosine was catalyzed into levodopa by tyrosinase (TYR). Carboxyazamonardine (CAZMON) which is the reactive product

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between levodopa and resorcinol yielded a novel blue fluorescence at 470 nm. CAZMON could quench the red fluorescence of CdTe/CdS/ZnS/SiO2 QDs at 620 nm. The resulting ratiometric fluorescence not only served as the signal output for quantitative ALP detection but also gave rise to the ratiometric fluorescence color changed from red to purple and finally blue for semiquantitative ALP detection. Importantly, the probe could detect ALP in human serum and screen inhibitors directly without any pretreatment. On the basis of these, the developed method holds great potential for sensitive, facile ALP activity detection in clinical diagnosis and opens a promising horizon for the on-site detection of enzymes conveniently.

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2. Experimental section 2.1 Materials and characterizations CdCl2,

3-mercaptopropionic

acid

(MPA),

tellurium

powder,

NaBH4,

ZnCl2,

tetraethoxysilane (TEOS), phosphotyrosine, levodopa and resorcinol were purchased from

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Aladdin Chemistry (Shanghai, China). Alkaline phosphatase (ALP) and tyrosinase (TYR) were purchased from Sigma-Aldrich Corporation (St. Louis, Mo, U.S.A.). The characterizations

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details have been provided in the Supporting Information.

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2.2 Detection assays for levodopa

The determination of levodopa was performed as follows: 50 μL of various concentrations

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of levodopa were mixed thoroughly with 50 μL of resorcinol (0.9 mM), followed by adjusting

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the volume to 290 μL with Britton-Robison buffer (40 mM, pH 11.5). After incubated at 37 ℃ for 40 min, 10 μL of CdTe/CdS/ZnS/SiO2 QDs synthesized by the method in supporting

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information (12 mg mL-1) was introduced. Then the fluorescence emission spectra were measured for quantitative analysis of levodopa (Ex=380 nm). 2.3 Detection assays for ALP activity

The determination of ALP was performed as follows: 25 μL of phosphotyrosine (550 μM,

pH 8.0) and 50 μL of TYR (3.5 U mL-1) were mixed with different concentrations of ALP. After incubated at 37 ℃ for 100 min, 50 μL of resorcinol (0.6 mM) was added and followed by adjusting the volume to 290 μL with BR buffer (40 mM, pH 11.5). After incubated at 37 ℃ for 40 min, 10 μL of CdTe/CdS/ZnS/SiO2 QDs (0.8 mg mL-1) was introduced. Then the fluorescence emission spectra were measured for quantitative analysis of levodopa (Ex=380 5

nm). 2.4 ALP inhibitor investigation 25 μL of different concentrations of Na3VO4 were incubated with 500.0 U L-1 ALP at 37 ℃ for 10 min. Then, 25 μL of phosphotyrosine (pH 8.0, 550 μM) and 50 μL of TYR (3.5 U mL-1) were introduced. After incubated at 37 ℃ for 100 min, 50 μL of resorcinol (0.6 mM) was added

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and followed by adjusting the volume to 290 μL with BR buffer (pH 11.5, 40 mM). After

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incubated at 37 ℃ for 40 min, 10 μL of CdTe/CdS/ZnS/SiO2 QDs (0.8 mg mL-1) was introduced.

2.5 Determination of ALP in human serum samples

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Then the fluorescence emission spectra were measured (Ex=380 nm).

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The human serum samples were provided by the first affiliated hospital-Nanjing medical

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university (Jiangsu, China). Prior to determination, the serum samples from five human bodies were diluted 15-fold with PBS buffer (20 mM, pH 8.0). Afterwards, the experimental process

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was performed as described above. 3. Results and discussion

3.1 Characterization of CdTe/CdS/ZnS/SiO2 QDs CdTe/CdS/ZnS/SiO2 QDs were prepared via a facile one-pot capping method [33]. The

TEM image (Figure 1A) presented that CdTe/CdS/ZnS/SiO2 QDs were spherical in shape with the diameter of approximately 4 nm. The fluorescence spectrum (Figure 1B) of CdTe/CdS/ZnS/SiO2 QDs revealed invariable emission wavelengths around 620 nm under the excitation wavelength ranging from 340 to 450 nm, indicating the excitation-independent

6

features [34]. CdTe/CdS/ZnS/SiO2 QDs presented strong red fluorescence under UV light (inset in Figure 1B) and the fluorescence significantly decreased in the acid conditions (Figure S1A). Furthermore, the fluorescence intensity showed no apparent change within 1 h (Figure S1B), implying that CdTe/CdS/ZnS/SiO2 QDs were stable enough to be applied for sensing. The FT-IR spectrum (Figure 1C) demonstrated that CdTe/CdS/ZnS/SiO2 QDs exhibited apparent absorption

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bands at 3471 cm-1 (O-H and N-H stretching vibration), 1419 and 1562 cm-1 (the symmetric and

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asymmetric stretching of the -COOH), which were responsible for the high aqueous solubility

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[35]. On the other hand, in order to analyze the chemical composition of CdTe/CdS/ZnS/SiO 2 QDs, XPS were recorded. XPS images (Figure 1D) confirmed the existence of Cd (404.97 eV),

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Te (572.00 eV), C (284.80 eV), Zn (1021.67 eV), Si (103.13 eV) and O (532.23 eV). As shown

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in Figure S2, the high-resolution XPS spectrum of Cd 3d revealed that the characteristic peaks were located at 404.93 eV (Cd 3d5/2) and 411.73 eV (Cd 3d3/2). There were two different states

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of C: C-O (288.48 eV) and C-N (284.88 eV). Furthermore, Te 3d were observed at 572.00 eV (Te 3d5/2), which was attributed to Cd-Te bonds. These results demonstrated the successful synthesis of the double shell CdTe/CdS/ZnS/SiO2 QDs [36].

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Figure 1. Characterizations of CdTe/CdS/ZnS/SiO 2 QDs: (A) TEM image. (B) The fluorescence

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emission spectra of CdTe/CdS/ZnS/SiO2 QDs at different excitation wavelength; Inset: the photographs under visible light (left) and UV light (right). (C) FT-IR spectrum. (D) The wide scan XPS spectrum.

3.2 Construction of the ratiometric fluorescence probe As illustrated in Scheme 1, phosphotyrosine can be dephosphorylated to produce tyrosine

by ALP, and then tyrosine was catalyzed into levodopa by TYR. Levodopa can react with resorcinol to generate CAZMON, which exhibited a strong blue fluorescence at 470 nm (Figure S3A) and the fluorescence intensity was also stable enough for detection (Figure S3B). The blue 8

fluorescence of CAZMON was in direct proportion to the concentration of levodopa as well as ALP. After the addition of CdTe/CdS/ZnS/SiO2 QDs to the above system, the red fluorescence of CdTe/CdS/ZnS/SiO2 QDs was quenched by CAZMON. So with the increase of ALP concentration, the blue fluorescence of CAZMON gradually increased whereas the red

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fluorescence of CdTe/CdS/ZnS/SiO2 QDs decreased, accompanying with the fluorescence color

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of the system changed from red to purple and finally blue.

Scheme 1. Schematic illustration of enzyme cascade reaction-based ratiometric fluorescence probe for visual monitoring ALP activity. Compared to levodopa and resorcinol respectively, CAZMON exhibited a characteristic 9

absorption band around 420 nm (Figure 2A), which was consistent with the results of fluorescence. Levodopa or resorcinol displayed no apparent fluorescence, and resorcinol could not react with phosphotyrosine or tyrosine (Figure S4A). However, levodopa can be oxidized to semiquinone and subsequently to quinone under alkaline conditions, which reacts with

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resorcinol to generate CAZMON with strong blue fluorescence around 470 nm (Figure 2B) [37]. Phosphotyrosine, tyrosine, levodopa or resorcinol has no effect on the fluorescence of QDs

(Figure

S4B).

However,

after

mixed

CAZMON

with

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CdTe/CdS/ZnS/SiO2

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CdTe/CdS/ZnS/SiO2 QDs, the red fluorescence of QDs was quenched. The absorption spectrum of CAZMON overlapped with the excitation spectrum of CdTe/CdS/ZnS/SiO2 QDs (Figure 2C).

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On the other hand, there was no evident variation in fluorescence lifetime of the

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CdTe/CdS/ZnS/SiO2 QDs in the presence and absence of CAZMON, manifesting that the

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quench effect resulted from inner filter effect (Figure 2D).

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Figure 2. (A) The UV-vis absorption spectra of CAZMON, resorcinol and levodopa. (B) The fluorescence spectra of resorcinol, levodopa, CAZMON, CdTe/CdS/ZnS/SiO 2 QDs, the mixture

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of CAZMON and CdTe/CdS/ZnS/SiO2 QDs. Inset: the photographs under visible light (upper) and UV light (down), and from left to right were resorcinol, levodopa, CAZMON, QDs and the mixture of CAZMON and QDs, respectively. (C) The UV-vis absorption spectrum of CAZMON and the fluorescence excitation spectrum of CdTe/CdS/ZnS/SiO2 QDs. (D) The fluorescence decay curve of CdTe/CdS/ZnS/SiO2 QDs in the absence and presence of CAZMON. 3.3 Quantitative determination of levodopa To achieve excellent analytical performance for levodopa sensing, the system pH, the concentration of resorcinol, the incubation time, the incubation temperature and the 11

concentration of CdTe/CdS/ZnS/SiO2 QDs were optimized to be pH 11.5, 0.9 mM, 40 min, 37 ℃ and 12 mg mL-1, respectively (Figure S5 and Figure S6). Upon the addition of levodopa ranging from 0.25-85 μM, the fluorescence intensity at 470 nm increased gradually accompanying with the generation of blue fluorescence (Figure 3A). An excellent linear relationship was achieved

F=102.47clevodopa+91.371

(R2=0.9909)

(Figure

3C).

However,

of

ranging from 0.25 to 25 μM and the linear regression equation was expressed as in

the

presence

of

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CdTe/CdS/ZnS/SiO2 QDs, the fluorescence of CAZMON at 470 nm increased while the

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fluorescence of CdTe/CdS/ZnS/SiO2 QDs at 620 nm decreased gradually with the increase of levodopa concentrations (Figure 3B). The levodopa could be determined in the range of

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0.085-500 μM with a linear range from 0.085-300 μM (Figure 3D). The linear relationship

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equation was expressed as F=0.2930clevodopa+0.2838 (R2=0.9989) with the detection limits (LOD) of 0.01 μM according to 3σ rule. Compared with the single-color probe, dual-color probe

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possessed a wider detection range and a higher sensitivity. More importantly, the photographs exhibited a distinct fluorescence color change from red to purple and finally blue under UV light, which showed more profuse color variation compared with single-color probe. The improved sensing performance was attributed to the advantages of the ratiometric fluorescence sensor constructed.

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Figure 3. The fluorescence spectra of levodopa assay with different concentrations of levodopa

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without (A) and with (B) CdTe/CdS/ZnS/SiO2 QDs. Insets: corresponding photographs under UV light. The curve of the fluorescence intensity without CdTe/CdS/ZnS/SiO2 QDs (C) and the curve of F470/F620 versus the concentrations of levodopa with CdTe/CdS/ZnS/SiO2 QDs (D). Inset: the linear relationship (λex = 380 nm). Error bars represent the standard deviations of three repetitive experiments.

3.4 Quantitative determination of ALP

The optimal reactive time, the concentration of phosphotyrosine, TYR, resorcinol and CdTe/CdS/ZnS/SiO2 QDs were recorded to be 100 min, 550 μM, 3.5 U mL-1, 0.6 mM and 0.8 mg mL-1, respectively (Figure S7 and Figure S8). Under optimal conditions, the fluorescence at 13

470 nm was continuously increased with ALP from 4-1000 U L-1 and was linearly correlated to ALP ranging from 4-600 U L-1 (Figure 4A and C). The linear regression equation was F=1.0689cALP+24.915 (R2=0.9904). In the presence of CdTe/CdS/ZnS/SiO2 QDs, the blue fluorescence at 470 nm increased gradually whereas the fluorescence at 620 nm slightly

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decreased with the increasing concentration of ALP from 0.08-1000 U L-1 (Figure 4B and D). A good linear relationship between F470/F620 and the ALP concentration ranging from 0.08-500 U

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L−1 (F=0.0752cALP+0.4369, R2=0.9970) was achieved with LOD of 0.02 U L−1 according to 3σ

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rule, which possessed a higher sensitivity. On the other hand, with increasing ALP concentrations, single-color probe displayed color change only from colorless to blue (the inset

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of Figure 4A). However, an apparent ratiometric fluorescence color change from red to purple

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and finally blue under UV light was observed in dual-color probe (the inset of Figure 4B). Hence, semiquantitative detection of ALP could be realized with the naked eye according to the

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fluorescence color variation. According to the values above, the developed ratiometric fluorescent method was comparable to those reported probes already (Table S1).

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Figure 4. The fluorescence spectra of ALP assay with different concentrations of ALP without

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(A) and with (B) CdTe/CdS/ZnS/SiO2 QDs. Insets: corresponding photographs under UV light. The curve of the fluorescence intensity without CdTe/CdS/ZnS/SiO2 QDs (C) and the curve of F470/F620 versus the concentrations of ALP with CdTe/CdS/ZnS/SiO2 QDs (D). Inset: the linear relationship (λex = 380 nm). Error bars represent the standard deviations of three repetitive experiments.

3.5 Selectivity evaluation of the method built for sensing ALP In order to assess the selective recognition capability of ALP assay, common interfering substances and various enzymes were investigated carefully. As shown in Figure 5, common interfering substances and various enzymes had no obvious effect on ALP detection. In addition, 15

the fluorescent responses of the method to ALP were not influenced by the addition of interfering substances and various enzymes, indicating that the developed method possessed

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superior selectivity to ALP.

Figure 5. Selectivity of ALP assay toward various ions, small molecules (A) and enzymes (B).

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The concentration of ALP was 100 U L -1, other potential interferent such as Na+, Mg2+, Ca2+,

lP

Zn2+, Fe3+, bovine serum albumin (BSA), glucose (Glu) and glycine (Gly) were 1 mM, various enzymes including lysozyme (Lys), galactosidase (Gal), thrombin (Thr), glutamate

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dehydrogenase (GLDH), cholesterol oxidase (COD), glucose oxidase (GOD), trypsin (Try) and acetylcholinesterase (AchE) were 100 U mL-1. 3.6 Detection of ALP in human serum samples To verify the excellent sensitivity and selectivity of the dual-color probe, the method was

further investigated the capability of detecting the level of endogenous ALP in real samples. Since the ALP concentration existing in human serum is relatively high (>46 U L-1) [38] ,with the LOD of 0.02 U L-1 and the linear range of 0.08-500 U L-1, the method satisfied the requirements for detecting ALP in human serum samples. The relative standard deviations (RSDs) ranged from 0.4-2.0% (Table S2). Additionally, the ALP levels measured by our method 16

were in good agreement with the standard values measured by the Beckman Coulter continuous monitoring assay which possessed a more complex detection process and expensive materials. These results confirmed the feasibility of the method in real samples detection. 3.7 Inhibition evaluation of ALP

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Considering that the ALP inhibitors reduce ALP activity through binding to the enzyme [39-40], the method was utilized for screening ALP inhibitors. It is well-known that Na3VO4 is a

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common ALP inhibitor, which can inhibit the catalytic effect of ALP efficiently. As Na 3VO4 was

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introduced in the ratiometric fluorescence system, the enzymatic catalytic reaction was retarded along with reduced concentration of tyrosine as well as levodopa, exhibiting a decreased blue

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fluorescence and an increase of red fluorescence (Figure S9A). The inhibition ratio (%) (IR%)

inhibitor).

inhibitor)/((F470/F620)0-(F470/F620)without

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was expressed as: IR%=((F470/F620)inhibitor-(F470/F620)without

A good linear relationship between the IR% and the concentration of Na 3VO4 was

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obtained ranging from 25.0 to 150.0 μM (Figure S9B). The IC50 was 90.7 μM, suggesting that the method holded great potential for screening the inhibitors of ALP. 4. Conclusions

We developed an enzyme cascade reaction-based ratiometric fluorescence probe by making

use of CdTe/CdS/ZnS/SiO2 QDs for visual monitoring the activity of ALP. By taking advantage of the enzymic catalytic reactions of ALP and TYR and the fluorescence of CdTe/CdS/ZnS/SiO2 QDs, a simple, sensitive and selective method for enzymes was proposed. Moreover, the semiquantitative detection for ALP activity based on the variation of colors varied from red to

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purple and finally blue under UV light was successfully fabricated which showed the potential on-site application. The advantages of the method mainly contained the following three points: (1) by constructing ratiometric fluorescence probe, the LOD was improved significantly; (2) it is very simple to construct the ratiometric fluorescence probe, avoiding the complex prepared

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procedures and pretreatment of samples; (3) importantly, the method provided a profuse fluorescence color variation, which paved a way for visually enzyme detection. However, the

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present method cannot be applied to monitoring ALP activity in cell and we will further

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investigate it. We expect that by exploration, this method can be extended to study the metabolism process as well as the biological roles of ALP in cellular homeostasis and

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Conflicts of interest

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pathophysiological situations.

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The authors declare no competing financial interest. Supplementary Information

Supplementary information is provided with this paper. ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (No. 61775099, 81973283, 21705080) and Natural Science Foundation of Jiangsu Province (No. BK20171487, BK20171043) and R&D fund for Smart Health Technology Innovation of Nanjing Medical University and Jiangsu Salt Group (No. NMU-SY201801).

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References [1] L. Li, J. Ge, H. Wu, Q.H. Xu, S.Q. Yao, Organelle-specific detection of phosphatase activities with two-photon fluorogenic probes in cells and tissues, J. Am. Chem. Soc. 134 (2012) 12157-12167. [2] C. Tang, Z. Qian, Y. Huang, J. Xu, H. Ao, M. Zhao, J. Zhou, J. Chen, H. Feng, A

of

fluorometric assay for alkaline phosphatase activity based on β-cyclodextrin-modified carbon

ro

quantum dots through host-guest recognition, Biosens. Bioelectron. 83 (2016) 274-280.

-p

[3] J.E. Coleman, Structure and mechanism of alkaline phosphatase, Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 441-483.

re

[4] Z. Tang, H. Chen, H. He, C. Ma, Assays for alkaline phosphatase activity: progress and

lP

prospects, TrAC Trends Anal. Chem. 113 (2019) 32-43.

[5] D.M. Goldberg, J.V. Martin, A.H. Knight, Elevation of serum alkaline phosphatase activity

Jo ur na

and related enzymes in diabetes-mellitus, Clin. Biochem. 10 (1977) 8-11. [6] A.J. Lorente, H. Valenzuela, J. Morote, A. Gelabert, Serum bone alkaline phosphatase levels enhance the clinical utility of prostate specifc antigen in the staging of newly diagnosed prostate cancer patients, Eur. J. Nucl. Med. 26 (1999) 625-632. [7] F. Abedini, H. Hosseinkhani, M. Ismail, A.J. Domb, A.R. Omar, P.P. Chong, P.D. Hong, D.S. Yu, I.Y. Farber, Cationized dextran nanoparticle-encapsulated CXCR4-siRNA enhanced correlation between CXCR4 expression and serum alkaline phosphatase in a mouse model of colorectal cancer, Int. J. Nanomed. 7 (2012) 4159-4168.

19

[8] K. Ooi, K. Shiraki, Y. Morishita, T. Nobori, High-molecular intestinal alkaline phosphatase in chronic liver diseases, J. Clin. Lab. Anal. 21 (2007) 133-139. [9] M.F. Cai, C.P. Ding, F.F. Wang, M.Q. Ye, C.L. Zhang, Y.Z. Xian, A ratiometric fluorescent assay for the detection and bioimaging of alkaline phosphatase based on near infrared Ag2S

of

quantum dots and calcein, Biosens. Bioelectron. 137 (2019) 148-153. [10] Z. Hai, J. Li, J. Wu, J. Xu, G. Liang, Alkaline phosphatase-triggered simultaneous

ro

hydrogelation and chemiluminescence, J. Am. Chem. Soc. 139 (2017) 1041-1044.

-p

[11] A.N. D́ıaz, F.G. Sánchez, M.C. Ramos, M.C. Torijas, Horseradish peroxidase solgel

Actuators B: Chem. 82 (2002) 176-179.

re

immobilized for chemiluminescence measurements of alkaline-phosphatase activity, Sens.

lP

[12] S. Goggins, C. Naz, B.J. Marsh, C.G. Frost, Ratiometric electrochemical detection of alkaline phosphatase, Chem. Commun. 51 (2015) 561-564.

Jo ur na

[13] P. Miao, L. Ning, X. Li, Y. Shu, G. Li, An electrochemical alkaline phosphatase biosensor fabricated with two DNA probes coupled with λ exonuclease, Biosens. Bioelectron. 27 (2011) 178-182.

[14] K. Ino, Y. Kanno, T. Arai, K.Y. Inoue, Y. Takahashi, H. Shiku, T. Matsue, Novel electrochemical methodology for activity estimation of alkaline phosphatase based on solubility diff erence, Anal. Chem. 84 (2012) 7593-7598. [15] C. Ruan, W. Wang, B. Gu, Detection of alkaline phosphatase using surface-enhanced raman spectroscopy, Anal. Chem. 78 (2006) 3379-3384.

20

[16] Y. Choi, N.H. Ho, C.H. Tung, Sensing phosphatase activity by using gold nanoparticles, Angew. Chem. Int. Ed. 46 (2007) 707-709. [17] C.M. Li, S.J. Zhen, J. Wang, Y.F. Li, C.Z. Huang, A gold nanoparticles-based colorimetric assay for alkaline phosphatase detection with tunable dynamic range, Biosens. Bioelectron. 43

of

(2013) 366-371. [18] Q. Hu, M. He, Y. Mei, W. Feng, S. Jing, J. Kong, X. Zhang, Sensitive and selective

ro

colorimetric assay of alkaline phosphatase activity with Cu(II)-phenanthroline complex, Talanta

-p

163 (2017) 146-152.

[19] C. Chen, J. Zhao, Y. Lu, J. Sun, X. Yang, Fluorescence immunoassay based on the

re

phosphate-triggered fluorescence turn-on detection of alkaline phosphatase, Anal. Chem. 90

lP

(2018) 3505-3511.

[20] R. Freeman, T. Finder, R. Gill, I. Willner, Probing protein kinase (CK2) and alkaline

Jo ur na

phosphatase with CdSe/ZnS quantum dots, Nano Lett. 10 (2010) 2192-2196. [21] J.J. Yang, L. Zheng, Y. Wang, W. Li, J.L. Zhang, J.J. Gu, Y. Fu, Guanine-rich DNA-based peroxidase mimetics for colorimetric assays of alkaline phosphatase, Biosens. Bioelectron. 77 (2016) 549-556.

[22] H.P. Jiao, J. Chen, W.Y. Li, F.Y. Wang, H.P. Zhou, Y.X. Li, C. Yu, Nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation: a new strategy for colorimetric sensing of alkaline phosphatase activity and inhibitor screening, ACS Appl. Mater. Interfaces 6 (2014) 1979-1985.

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[23] Q. Liu, H.X. Li, R. Jin, N. Li, X. Yan, X.G Su, Ultrasensitive detection alkaline phosphatase activity using 3-aminophenylboronic acid functionalized gold nanoclusters, Sens. Actuators B: Chem. 281 (2019) 175-181. [24] Y. Chen, W.Y. Li, Y. Wang, X.D. Yang, J. Chen, Y.N. Jiang, C. Yu, Q. Lin, Cysteine-directed

of

fluorescent gold nanoclusters for the sensing of pyrophosphate and alkaline phosphatase, J. Mater. Chem. C 2 (2014) 4080-4085.

ro

[25] Z. Song, R.T.K. Kwok, E. Zhao, Z. He, Y. Hong, J.W.Y. Lam, B. Liu, B.Z. Tang, A

-p

ratiometric fluorescent probe based on ESIPT and AIE processes for alkaline phosphatase activity assay and visualization in living cells, ACS Appl. Mater. Interfaces 6 (2014)

re

17245-17254.

lP

[26] W.J. Kang, Y.Y. Ding, H. Zhou, Q.Y. Liao, X. Yang, Y.G. Yang, J.S. Jiang, M.H. Yang, Monitoring the activity and inhibition of alkaline phosphatase via quenching and restoration of

Jo ur na

the fluorescence of carbon dots, Microchim. Acta 182 (2015) 1161-1167. [27] B.M. Cao, C. Yuan, B.H. Liu, C.L. Jiang, G.J. Guan, M.Y. Han, Ratiometric fluorescence detection of mercuric ion based on the nanohybrid of fluorescence carbon dots and quantum dots, Analytica Chimica Acta 786 (2013) 146-152. [28] Y. Cen, Y.M. Wu, X.J. Kong, S. Wu, R.Q. Yu, X. Chu, Phospholipid-modified upconversion nanoprobe for ratiometric fluorescence detection and imaging of phospholipase D in cell lysate and in living cells, Anal. Chem. 86 (2014) 7119-7127.

22

[29] S.A. Díaz, L. Giordano, T.M. Jovin, E.A. Jares-Erijman, Modulation of a photoswitchable dual-color quantum dot containing a photochromic fret acceptor and an internal standard, Nano Lett.n12 (2012) 3537-3544. [30] X. Huang, J. Song, B.C. Yung, X. Huang, Y. Xiong, X. Chen, Ratiometric optical

of

nanoprobes enable accurate molecular detection and imaging, Chem. Soc. Rev. 47 (2018) 2873-2920.

ro

[31] J.Y. Chen, M.K. Wang, X.G. Su, Ratiometric fluorescent detection of azodicarbonamide

-p

based on silicon nanoparticles and quantum dots, Sens. Actuators B: Chem. 296 (2019) 126643. [32] Y. Cui, R.J. Liu, F.G. Ye, S.L. Zhao, Single-excitation, dual-emission biomass quantum dots:

lP

Nanoscale 11 (2019) 9270-9275.

re

preparation and application for ratiometric fluorescence imaging of coenzyme A in living cells,

[33] F.D. Wei, G.H. Xu, Y.Z. Wu, X. Wang, J. Yang, L.P. Liu, P. Zhou, Q. Hu, Molecularly

Jo ur na

imprinted polymers on dual-color quantum dots for simultaneous detection of norepinephrine and epinephrine, Sens. Actuators B: Chem. 229 (2016) 38-46. [34] M.X. Gao, C.F. Liu, Z.L. Wu, Q.L. Zeng, X.X. Yang, W.B. Wu, Y.F. Li, C.Z. Huang, A surfactant-assisted redox hydrothermal route to prepare highly photoluminescent carbon quantum dots with aggregation-induced emission enhancement properties, Chem. Commun. 49 (2013) 8015-8017.

[35] N. Murase, P. Yang, Anomalous photoluminescence in silica-coated semiconductor nanocrystals after heat treatment, small 5 (2009) 800-803.

23

[36] J. Wang, N. Li, F. Shao, H.Y. Han, Microwave-assisted synthesis of high-quality CdTe/CdS@ZnS-SiO2 near-infrared-emitting quantum dots and their applications in Hg2+ sensing and imaging, Sens. Actuators B: Chem. 207 (2015) 74-82. [37] A.U. Acuna, M. Alvarez-Perez, M. Liras, P.B. Coto, F. Amat-Guerri, Synthesis and

of

photophysics of novel biocompatible fluorescent oxocines and azocines in aqueous solution, Phy. Chem. Chem. Phys. 15 (2013) 16704-16712.

ro

[38] K. Mukaiyama, M. Kamimura, S. Uchiyama, S. Ikegami, Y. Nakamura, H. Kato, Elevation

turnover, Aging Clin Exp Res 27 (2015) 413-418.

-p

of serum alkaline phosphatase (ALP) level in postmenopausal women is caused by high bone

re

[39] H.J. Liu, M. Li, Y.N. Xia, X.Q. Ren, A turn-on fluorescent sensor for selective and sensitive

lP

detection of alkaline phosphatase activity with gold nanoclusters based on inner filter effect, ACS Appl. Mater. Interfaces 9 (2017) 120-126.

Jo ur na

[40] L.S.B. Upadhyay, N. Verma, Alkaline phosphatase inhibition based conductometric biosensor for phosphate estimation in biological fluids, Biosens. Bioelectron. 68 (2015) 611-616.

Biographies

Xia Cheng is currently a graduate student focusing on synthesis and application of fluorescent sensors for Ph.D. at Nanjing Medical University. She was awarded a B.S. degree in pharmacy from the same university in 2017. Yuying Chai is currently a graduate student focusing on synthesis and application of 24

metal-organic frameworks for M.S. at Nanjing Medical University. She was awarded a B.S. degree in pharmacy from the same university in 2017. Jian Xu is currently an associate professor at department of laboratory medicine in the first affiliated hospital of Nanjing Medical University.

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Lin Wang is currently a pharmacist at department of laboratory medicine in the first affiliated hospital of Nanjing Medical University.

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Fangdi Wei is currently an associate professor at school of pharmacy, Nanjing Medical

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University. She obtained her M.S. on Analytical Chemistry from Nanjing University in 2002 and obtained Ph.D. from Nanjing Medical University in 2016. Her main research interests are in

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analysis of trace components in lifesystem.

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two areas: (1) synthesis and application of surface molecular imprinting polymer and (2)

Guanhong Xu received his M.S. degree in pharmacology from Nanjing Medical University in

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2009. He joined the Nanjing Medical University as a lecturer and was promoted to associate professor in 2014. His research interests include constructing novel functional fluorescent nanomaterials for bioassay and bioimaging. Yong Sun is currently a teacher at key laboratory of toxicology, Ningde Normal University. Qin Hu is a professor at school of pharmacy, Nanjing Medical University. She obtained her Ph.D. on Analytical Chemistry from East China Normal University in 2001. She is currently conducting research in two areas: (1) synthesis of nano-silica molecular imprinted polymer and its application in analysis of environmentalendocrine disrupting chemicals and (2) molecular imprinted quantum dots appli-cation in bioanalysis. 25

Yao Cen received her Ph.D. in Analytical Chemistry from Hunan University in 2016 under the mentorship of Prof. Xia Chu. She then joined the Nanjing Medical University as a lecturer. Her research interests include constructing novel functional nanostructures for bioassay, bioimaging

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and drug delivery.

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