Sensitive fluorometric detection of alkaline phosphatase using a water-soluble conjugated polymer

Abstract

In this paper, we developed a new fluorescence method for highly sensitive and selective detection of the alkaline phosphatase (ALP) activity with phenyl phosphate (PP) as the enzyme substrate and water-soluble conjugated polymer PPESO₃ as a fluorescent probe. Phenyl phosphate could be hydrolyzed to phenol via the ALP-catalyzed reaction. In the presence of horse radish peroxidase (HRP) and H₂O₂, phenol was rapidly oxidized to quinone which could efficiently quench the fluorescence of the conjugated polymer PPESO₃. The fluorescence intensity change of PPESO₃ is proportional to the concentration of ALP. Thus, we established a fluorescence method for ALP activity detection based on a water-soluble fluorescent conjugated polymer–enzyme hybrid system. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I₀ (I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of ALP, respectively) and the concentration of ALP in the range of 0–30 U L⁻¹, and the detection limit for ALP was 0.5 U L⁻¹. The present method was applied to the determination of ALP in spiked human serum samples with satisfactory results.

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Introduction

Recently, water-soluble conjugated polyelectrolytes (CPs) have attracted a great deal of research attention, and some optical platforms for sensitive detection of chemical and biological molecules by virtue of their fascinating optical, electrical, non-toxic and water-soluble properties have been established.^1–3 These unique features also hold great promise for potential applications in diagnosis, imaging, and therapy.^4–6 Comparing to other fluorescent materials like quantum dots and other small molecule dyes, CPs own superior signal amplification and superquenching properties owing to the conjugated polymer backbone. Up to now, many studies have been performed to develop CPs-based fluorescent assays for the detection of chemical and biological molecules including metal ions,^7,8 enzymes,^9,10 proteins^11,12 and nucleic acids.^13,14

Alkaline phosphatase (ALP), a prototypical phosphomonoesterase used as important biomarkers for several human diseases, is capable of catalyzing the hydrolysis and transphosphorylation of a series of monophosphate esters.¹⁵ ALP is distributed at different levels in mammalian tissues, higher concentration in the liver and bones and lower concentration in placenta, intestines and kidneys.¹⁶ The level of ALP is an important index for several diseases included breast and prostatic cancer, adynamic bone disease or liver dysfunction.¹⁷ Thus, it is extremely necessary to develop a simple and convenient method for monitoring the level of ALP in complex biological samples. In recent years, different approaches have been used for the determination of ALP activity in the biological samples, such as colorimetry,^18,19 electrochemistry,^20–22 chemiluminescence^23,24 and surface enhanced resonance Raman scattering.^25,26 Even though some of these methods shown high sensitivity, most of them were time-consuming, more expensive and difficult to perform compared with the fluorescence method. Recently, fluorescence approaches for ALP detection, superior in terms of sensitivity, simplicity, reproducibility and cost-effectiveness, have been reported.^27,28 ALP is homologous to a large variety of other enzymes and displays broad substrate specificities, so using a suitable substrate for the fluorescence detection of ALP activity is a topic of current interest. Several fluorometric substrates have been used for ALP detection.^29,30 In these studies, the fluorescence intensity variations were affected by enzymatic cleavage of phosphate groups which altered the photoinduced electron transfer or charge transfer processes. These fluorometric assays have some drawbacks, such as low fluorescence quantum yields, laborious procedures for preparation and low solubility in aqueous solutions. For example, the quantum yields of the synthesized fluorogenic substrates are 0.002 (ref. 31) and 0.0035.³² Phenyl phosphate (PP) is a fluorescence-labeled free substrate and commercial available which could significantly reduce the cost of the enzyme detection. Thus, we chose PP as the substrate for the activity assay of ALP in this work.

Some detection strategies for phenol derivative triggered by ALP-catalyzed hydrolysis have been reported.^22,33 In this paper, we developed a new fluorometric method for the simple, sensitive, inexpensive ALP activity detection with PP as enzyme substrate and water-soluble conjugated polymer poly(2,5-bis(3-sulfonatopropoxy)-1,4-phenylethynylene-alt-1,4-poly(phenylene ethynylene)) (PPESO₃) as fluorescence probe. PP could be rapidly hydrolyzed to phenol via the ALP-catalyzed reaction. Then, with the addition of horse radish peroxidase (HRP) and H₂O₂, phenol was oxidized to quinone which could efficiently quench the fluorescence of conjugated polymer PPESO₃. Although the most traditional sensing systems will be more straightforward and less procedure involved, the fluorescent polymer–enzyme hybrid system can obtain the high sensitivity and excellent selectivity.³⁴ Thus, we established a fluorescence method for ALP activity detection based on water-soluble fluorescent conjugated polymer–enzyme hybrid system.

Experiment

Reagents and chemicals

All chemicals used were of analytical reagent grade and used without further purification. Phenyl phosphate (PP) was obtained from Tianjin Guangfu Institute of elaborate chemical industry. Threonine (Thr), arginine (Arg) and glycine (Gly) were purchased from Shanghai Hui shi biochemical reagents Co. Ltd Alkaline phosphatase (ALP), bovine serum albumin (BSA), HRP, trypsin, pepsin and glucose (Glu) were attained from Sino-American Biotechnology Co. Ltd The water used in all experiments had a resistivity higher than 18 MΩ cm⁻¹.

Instrumentation

Fluorescence measurements were performed on a Shimadzu RF-5301 PC spectrafluorophotometer with a 1 cm path-length quartz cuvette. The fluorescence spectra were recorded with the excitation wavelength of 400 nm. The slit widths of the excitation and emission were both 5 nm.

Experimental method

Water-soluble fluorescence conjugated polymer PPESO₃ was synthesized according to the previously described method.^{35 1}H NMR was applied to characterize the polymer structure.³⁶ As shown in ESI (Fig. S1†), ¹H NMR (DMSO-d₆, δ_ppm): 2.10 (4H, four protons of methylene from –CH₂–SO₃Na), 2.72 (4H, four protons of methylene from –O–CH₂–CH₂–CH₂–), 4.17 (4H, four protons of methylene from –O–CH₂–), 7.21 (2H, two aromatic protons from –C₆H₂–), 7.62 (4H, four aromatic protons from –C₆H₄–).

In our experiment, ALP and PP were dissolved in 50 mM Tris–HCl buffer solution (pH 10.0), and PPESO₃ stock solution was diluted to 2 μM with 50 mM Tris–HCl buffer (pH 7.4). Fluorescence measurement of ALP activity was performed by incubating 10 μL ALP with various concentrations, 38 μL Tris–HCl (pH 10.0) and 2 μL, 100 mM PP for 30 min at 37 °C. Then, 940 μL, 50 mM Tris–HCl buffer solution (pH 7.4) and 1.0 mL, 2 μM PPESO₃ was introduced into the above solution, followed by addition of 5 μL, 1.0 mg mL⁻¹ HRP, 5 μL, 0.8 M H₂O₂. Subsequently, the final mixture was thoroughly shaken and incubated at room temperature for 10 min before the fluorescence measurement. The fluorescence spectra were measured from 420 nm to 650 nm, and the fluorescence intensity of the maximum emission peak at 530 nm was used for quantitative analysis.

ALP detection in real samples: human blood samples were collected from healthy volunteer at the China Japan Union Hospital, Changchun. All the blood samples were obtained by venipuncture and centrifuged at 10000 rpm for 10 min after standing for 2 h at room temperature. The human serum was diluted 100 times with 50 mM Tris–HCl buffer solution (pH 10.0). Then the diluted human serum was added with different concentration of ALP to prepare the spiked samples. Human serum samples detection was conducted using the procedure described above.

Results and discussion

The design of fluorescence method for ALP detection

In this paper, we used water-soluble conjugated polymers PPESO₃ as a fluorescent probe due to its signal superquenching property, which exhibiting a strong emission at around 530 nm in aqueous solution. As reported in our previous studies, quinone as a product of the enzymatic oxidation reaction can efficiently quench the fluorescence of PPESO₃.^34,37 Scheme 1 illustrated the principle of our fluorescence sensing system for the sensitively detection of ALP activity. Firstly, PP was rapidly hydrolyzed to phenol by ALP-catalyzed reaction. Secondly, phenol was oxidized to quinone with the addition of HRP and H₂O₂. And the generated quinone rapidly quenched the fluorescence of conjugated polymer PPESO₃. Thus, the quantified determination of ALP could be achieved by measuring the quenching fluorescence of PPESO₃.

Scheme 1 Schematic illustration of the fluorescence sensing system for the detection of ALP activity.

To further confirm the feasibility of this fluorescence sensing system for ALP detection, we compared the quenching effect of PPESO₃ by different components of this sensing system. As shown in Fig. 1, when PPESO₃ solution mixed with PP, ALP, HRP or H₂O₂ respectively, even the mixtures of two or three components, no obvious PPESO₃ fluorescence intensity changes were observed, which indicated that neither H₂O₂ nor hydroxyl radicals produced from the HRP-catalyzed reaction could quench the fluorescence intensity of PPESO₃. However, fluorescence superquenching could be observed remarkably with the addition of ALP into the sensing system. These results indicated that the oxidation from phenol to quinone took place in the presence of HRP and H₂O_2. Because the fluorescence intensity changes of PPESO₃ indirectly reflected the concentration of ALP. Therefore, a fluorescence method for ALP activity detection was successfully developed.

Fig. 1 The effect of ALP sensing system components on the fluorescence intensity of PPESO₃. (a) PPESO₃; (b) PPESO₃ + PP; (c) PPESO₃ + HRP; (d) PPESO₃ + H₂O₂; (e) PPESO₃ + ALP; (f) PPESO₃ + PP + H₂O₂; (g) PPESO₃ + PP + HRP + H₂O₂; (h) PPESO₃ + PP + ALP + HRP + H₂O₂. Concentrations: 1.0 μM PPESO₃, 100 μM PP, 30 U L⁻¹ ALP, 5 μg mL⁻¹ HRP, 2 mM H₂O₂.

Optimization for ALP detection

In order to optimize the conditions for ALP detection, we studied the effect of incubation times, pH and temperature on the fluorescence of PPESO₃. ALP detection in our designed strategy involves two processes: the ALP activity toward substrate hydrolysis and the oxidation of the ALP-hydrolyzed products in the present of HRP and H₂O₂. Fig. 2a showed the fluorescence intensity changes of PPESO₃ as a function of the incubating time of ALP-catalyzed hydrolysis of PP. From Fig. 2a, it could be seen that the fluorescence intensity of PPESO₃ was rapidly decreased with the incubating time increased from 0 to 40 min and kept slowly changing after 40 min. In order to shorten the reaction time, we chose 30 min as the optimum incubating time for substrate hydrolysis. Then we study the incubation time of the second process. As shown in Fig. 2b, the fluorescence intensity of the sensing system decreased quickly with the increasing of the incubation time and reached a plateau within 10 min at room temperature. The results indicated that the oxidation process of phenol in the presence of HRP and H₂O₂ finished within 10 min. In the following experiments, the incubation time of 10 min at room temperature for the oxidation process was adopted.

Fig. 2 The relationship between fluorescence intensity of PPESO₃ and the incubation time of the first (a) and second (b) process. Conditions: 1.0 μM PPESO₃, 100 μM PP, 20 U L⁻¹ ALP, 5 μg mL⁻¹ HRP, 2 mM H₂O₂.

As reported in the literatures, the catalytic activity of ALP towards a series of monophosphate esters depended on the value of pH and temperature.^21,22 Therefore, we first studied the effect of pH on the ALP-catalyzed hydrolysis process. From Fig. 3, it can be observed that the fluorescence intensity of PPESO₃ was nearly unchanged in the pH range of 6.0–8.0, then the remarkable decreasing of fluorescence intensity was observed in the pH range of 8.0–10.0 and the largest quenching effect appeared at pH 10.0, which indicated that the activity of ALP for hydrolyzing PP substrate was much higher in alkaline medium than that in acidic or neutral medium. So the pH of 10.0 was adopted in the following experiments. And the effect of temperature was also examined and the results were shown in Fig. 4. It can be seen that the fluorescence intensity of PPESO₃ decreased from 20 °C to 37 °C and then increased above 37 °C. The fluorescence quenching reached maximum at 37 °C. Thus, 37 °C was selected as the optimum temperature, which is also accord with the circumstance in human body.

Fig. 3 The effect of pH on the fluorescence intensity of PPESO₃. Conditions: 1.0 μM PPESO₃, 100 μM PP, 20 U L⁻¹ ALP, 5 μg mL⁻¹ HRP, 2 mM H₂O₂.

Fig. 4 The effect of temperature on the fluorescence intensity of PPESO₃. Conditions: 1.0 μM PPESO₃, 100 μM PP, 20 U L⁻¹ ALP, 5 μg mL⁻¹ HRP, 2 mM H₂O₂.

The detection of ALP activity

The quenching effect of ALP versus the concentration of ALP was further investigated under the optimized conditions. As shown in Fig. 5, the fluorescence intensity of PPESO₃ decreased successively upon the increasing concentration of ALP. The inset of Fig. 5 showed a good linear relationship between the fluorescence intensity ratio I/I₀ (I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of ALP, respectively) and the concentrations of ALP ranging from 0 to 30.0 U L⁻¹ with a correlation coefficient R² = 0.994. The linear regression equation is I/I₀ = 0.957−0.031C_ALP (U L⁻¹) and the detection limit for ALP is 0.5 U L⁻¹ based on 3σ rule, the standard deviation for nine replicate measurements of 2 U L⁻¹ ALP was 2.6%. These results indicated that the fluorescence sensing system by utilizing PP as ALP substrate in combination with conjugated polymers PPESO₃ as fluorescence probe could be successfully applied for ALP detection with high sensitivity. Compared with the previous reports for ALP determination in linear range and detection limit (Table 1), our method obtained the similar or superior detection limit and linear range.

Fig. 5 Fluorescence emission spectra of the sensing system with different concentrations of ALP, (a–g) represented the concentrations of ALP of 0, 1, 2, 4, 10, 20, 30 U L⁻¹. The inset showed the calibration curve between the fluorescence intensity ratio I/I₀ and the concentration of ALP in the range of 0 to 30 U L⁻¹, I₀ and I are the fluorescence intensity of the sensing system in the absence and presence of ALP, respectively.

Table 1 Comparison of different methods for the detection of ALP

Methods	Linear range (U L^−1)	Detection limit (U L^−1)	Ref.
Colorimetry	100–600	10	18
Electrochemistry	5 × 10^3 to 640 × 10^3	20	20
	0–300	—	22
Electrochemiluminescence	2–25	2	24
Fluorometry	0 to 5 × 10^3	10	27
	18–100	18	29
	0–30	0.5	This work

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Interference study

For further evaluating the selectivity and specificity of the present fluorescence method, a series of experiments were conducted by using ALP (20 U L⁻¹) and various potentially interfering substances included Na⁺, K⁺, Mg²⁺, glucose (Glu), arginine (Arg), glycine (Gly), threonine (Thr), bovine serum albumin (BSA), pepsin and trypsin. Blank represented the fluorescence intensity of the sensing system without ALP. The concentration ratio of coexisting substances to ALP was 1000-fold for Na⁺, K⁺, Mg²⁺ and 250-fold for Glu, Arg, Gly, Thr, BSA, pepsin and trypsin. As shown in Fig. 6, the results indicted that no obvious influence was observed even at high concentration of the coexistence substances and the present fluorescence method for ALP assay is highly selective.

Fig. 6 The interference of potentially interfering substances on the determination of 20 U L⁻¹ALP, including Na⁺, K⁺, Mg²⁺, glucose (Glu), arginine (Arg), glycine (Gly), threonine (Thr), bovine serum albumin (BSA), pepsin and trypsin.

Detection of ALP in human serum samples

To investigate whether the present fluorescence sensing system could be further used in complex biological samples, the developed method had been applied to determine ALP in two human serum samples. The results obtained by standard addition method were shown in Table 2. The recoveries of the added ALP were in the range of 95–106%, and the relative standard deviation (RSD) was less than 5.0%. These results demonstrated the potential applicability of this method for the detection of ALP in complicated real samples.

Table 2 Determination of ALP spiked in human serum samples

Sample	Added(U L^−1)	Found(U L^−1)	Recovery(%)	RSD(%, n = 3)
Serum1	4.00	4.15	103.8	2.3
	20.00	20.75	103.8	1.7
Serum2	4.00	3.81	95.3	4.6
	20.00	21.16	105.8	2.4

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Conclusion

In summary, we have developed a fluorescence method for ALP detection by using PP as substrate and water-soluble conjugated polymer PPESO₃ as fluorescence probe. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I₀ (I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of ALP, respectively) and the concentration of ALP in the range of 0–30 U L⁻¹ with the detection limit of 0.5 U L⁻¹. In comparison with the previous methods for ALP detection, the present method has some advantages: the use of water-soluble conjugated polymers imparts the method high sensitivity; the PP substrate is commercial available and does not require fluorescent labels; this method offers a convenient approach by simple mixing and incubation for the rapid ALP activity assay.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 21075050, no. 21275063) and the science and technology development project of Jilin province, China (no. 20110334).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra05844e

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