Electrochemiluminescence resonance energy transfer immunoassay for alkaline phosphatase using p-nitrophenyl phosphate as substrate

Analytica Chimica Acta xxx (xxxx) xxx

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Electrochemiluminescence resonance energy transfer immunoassay for alkaline phosphatase using p-nitrophenyl phosphate as substrate Wenjing Qi*, Yuling Fu, Maoyu Zhao, Hongkun He, Xue Tian, Lianzhe Hu, Yan Zhang Chongqing Key Laboratory of Inorganic Functional Materials, College of Chemistry, Chongqing Normal University, Chongqing, 401331, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Electrochemiluminescence resonance energy transfer (ECRET) immunoassay for alkaline phosphatase from 5 to 50 U/L.
Spectral overlap between absorption spectrum of p-nitrophenol (PNP) and ECL spectrum of luminol-SiNPs.
ECRET occurs from luminol-SiNPs to PNP and makes electrochemiluminescence intensity be quenched.
ALP converts p-nitrophenyl phosphate (PNPP) to PNP and results the absorption peak to shift from 360 nm to 450 nm.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 August 2019 Received in revised form 11 October 2019 Accepted 31 October 2019 Available online xxx

A sensitive electrochemiluminescent immunoassay for alkaline phosphatase (ALP) using p-nitrophenyl phosphate (PNPP) as substrate based on the electrochemiluminescence resonance energy transfer (ECRET) is developed. Luminol-doped silica nanoparticles (luminol-SiNPs) are prepared by water/oil (W/ O) microemulsion method. PNPP convertes to p-nitrophenol (PNP) in the presence of ALP, which results in the absorption peak shifting from 360 nm to 450 nm. Herein the spectral overlap between absorption spectrum of PNP and electrochemiluminescence (ECL) spectrum of luminol-SiNPs (425 nm) makes energy transfer occur from luminol-SiNPs to PNP. In the optimized conditions, a linear relationship was obtained using this ECRET method at the concentration of ALP from 5 to 50 U/L (r ¼ 0.9905) and with the limit of detection (LOD) of 0.8 U/L. This ECRET method exhibits sufficient specificity for ALP over other enzymes such as horseradish peroxidase, trypsin and lysozyme. © 2019 Elsevier B.V. All rights reserved.

Keywords: Alkaline phosphatase (ALP) Electrochemiluminescence resonance energy transfer (ECRET) Luminol-doped silica nanoparticles (luminol-SiNPs) P-nitrophenyl phosphate (PNPP) P-nitrophenol (PNP)

1. Introduction Electrochemiluminescence

(ECL)

is

a

* Corresponding author. E-mail address: [email protected] (W. Qi).

chemiluminescence

reaction initiated by the combination of electrochemical methods and luminescence techniques, has been extensively used in analytical chemistry [1e4]. ECL detection system has many significant advantages merits such as better reproducibility, lower background signal, easier temporal and spatial control [5,6]. It has been widely used in scientific research of clinical diagnostics, food, environmental samples and biological warfare reagents [7e9].

https://doi.org/10.1016/j.aca.2019.10.073 0003-2670/© 2019 Elsevier B.V. All rights reserved.

Please cite this article as: W. Qi et al., Electrochemiluminescence resonance energy transfer immunoassay for alkaline phosphatase using pnitrophenyl phosphate as substrate, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.073

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With the development of ECL technique, electrochemiluminescence resonance energy transfer (ECRET) attracts increasing attention owing to its rapidity, simplicity and high sensitivity [10,11]. ECRET strategy involves a pair of suitable materials constituting a pair of energy donor and energy acceptor. ECL emission spectrum of the donor effectively overlaps with the absorption spectrum of the acceptor, which leads to the absorption of the emitted light from the donor in the detection system by the receptor [12]. In the past decades, many researchers focused on developing ECRET systems. Cao and his co-researchers utilized CdS nanowires RuSi@Ru(bpy)2þ composite as ECL donors and gold 3 nanorods as ECL acceptors in ECRET simultaneous detection of two acute myocardial infarction markers [13]. Ke and his co-researchers utilizedECRET from Ru(bpy)2þ to gold nanorods (GNRs) in ECL 3 aptasensor for b-amyloid [14]. Lu and his co-researchers report an ECRET system using Ru(bpy)2þ silica nanoparticles 3 -doped (RuSiNPs) as ECL donor and hollow gold nanocages as ECL acceptor for the detection of miRNA141 [15]. Zhu and co-researchers developed immunosensor for prostate specific antigen detection by coupling ECRET using Fe3O4@MnO2 composites [10]. Liu and his co-researchers used pollution-free BN quantum dots in ECRET biosensor for Escherichia coli detection [11]. Since semiconductor quantum dots always have the potential toxicity caused by heavy metal ion leakage, which limiting their ECL application in bioanalysis. Therefore the search for benign or nontoxic nanomaterials with excellent ECL property used in ECRET system is imperative and meaningful. Alkaline phosphatase (ALP), a crucial enzyme in phosphate metabolism that responsible for phosphate metabolism and catalyzed the hydrolysis of phosphoryl esters in alkaline media [16e22], can be found in a variety of tissues such as intestine, liver, bone, and placenta [17,23]. In clinical practice, ALP is one of the most regularly assayed enzymes and has already been utilized as an important indicator for several diseases including liver dysfunction, bone diseases, breast and prostatic cancer, and diabetes [18e22,24e26]. Developing highly sensitive method of detecting ALP is essential to clinical diagnoses. Up to now, various methods are developed for sensitive detection of ALP including chromatography [27], electrochemistry [28], fluorescence [29e32], colorimetry [33e35], chemiluminescence [36], surface enhanced resonance Raman scattering [37], Plasma resonance absorption [38] and ECL methods [39]. Besides fluorescent and colorimetric methods are mainly utilized in ALP analysis owing to high sensitivity of fluorescent methods and the convenience of colorimetric methods. Although ECRET also exhibits high sensitivity and simple analysis, few ECRET reports about ALP detection have been reported until now. Herein, a simple and sensitive method for the determination of ALP activity based on the ECRET is designed in the present work. Luminol-doped silica nanoparticles (luminol-SiNPs) are prepared by water/oil (W/O) microemulsion method. The addition of numerous luminol molecules to silica matrix makes luminol-SiNPs exhibit ultra-sensitive electrochemical performance and much higher ECL efficiency than luminol [40e42]. Besides luminol-SiNPs have non-toxicity/low toxicity, which is of great significance to ECL analysis. Luminol-SiNPs which emit ECL at 425 nm are used as the energy donor [43]. Using p-nitrophenyl phosphate (PNPP) as a substrate for alkaline phosphatase (ALP), PNPP converts to PNP in the presence of ALP and brings the absorption peak shifting from 360 nm to 430 nm [43e46]. Thus the absorption of PNP overlaps with ECL emission of luminol-SiNPs is used in ECRET immunoassay for ALP in the present work (Scheme 1). When ALP is added, ECRET occurs from luminol-SiNPs to PNP and accordingly ECL of luminolSiNPs is quenched by PNP.

Scheme 1. Illustration of the principle of ECRET immunoassay for ALP using luminoldoped silica nanoparticles (luminol-SiNPs).

2. Experimental 2.1. Materials and reagents Luminol and p-nitrophenyl phosphate (PNPP) were obtained from Aladdin Reagent (Shanghai, China). Alkaline phosphatase (bovine intestinal mucosa), tetraethyl orthosilicate (TEOS), 3aminopropyltriethoxysilane (APTES) and Triton X-100 were purchased from Sigma-Aldrich (Beijing, China). 1-Hexanol was obtained from Beijing Yili Chemical Reagent Factory (Beijing, China). Cyclohexane was purchased from Beijing Chemical Reagent Factory (Beijing, China). Ammonium hydroxide (25%) was purchased from Chengdu Kelon Reagent (Chengdu, China). Lysozyme (LZM), trypsin, uricase and thrombin were purchased from Shanghai yuanye Bio-Technology. Horseradish peroxidase (HRP) was purchased from Tokyo chemical industry. All these chemicals were analytical-reagent grade and used without any further purification. Since luminol is slightly soluble in water and easily soluble in sodium hydroxide (NaOH), luminol powder was firstly dissolved with several drops of 0.2 M NaOH and then diluted with plenty of water. After setting volume with volumetric bottle, 20 mM luminol solution was obtained. 0.05 M Tris-HCl buffer solutions (pH 10.0) were used during the experiments. Doubly distilled water was used throughout all experiments. 2.2. Instrumentation All electrochemiluminescent measurements were conducted with an MPI-A capillary electrophoretic electrochemiluminescence detector produced by Xi’an Remex Electronics (Xi’an China) and Changchun Institute of Applied Chemistry (Changchun, China). A conventional three-electrode cell were used with a glassy carbon electrode (GCE) (4 ¼ 3 mm) as working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode (saturated KCl). ECL intensities were monitored through the bottom of the three electrode cell which was purchased from Xi’an Remex Electronics (Xi’an China). The photomultiplier tube voltage was kept at 1000 V during all the ECL measurements. GCE working electrode was polished with 0.05 mm alumina and then cleaned by ultrapure water in an ultrasonic bath prior to each use. Ultravioletevisible (UVevis) absorption spectra were employed a UV2550 UVevis spectrophotometer (Shimadzu, Japan). Scanning electron microscopy (SEM) images were taken by a SU8020 scanning electron microscope (Hitachi, Japan). Transmission electron microscopy

Please cite this article as: W. Qi et al., Electrochemiluminescence resonance energy transfer immunoassay for alkaline phosphatase using pnitrophenyl phosphate as substrate, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.073

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(TEM) images were taken by a JEM-2100F transmission electron microscope (JEOL, Japan). 2.3. Preparation of luminol-doped silica nanoparticles (LuminolSiNPs) Luminol-SiNPs were prepared with W/O microemulsion method similarly like the previous reports described with a little modification [40,42,47]. Typically, 20 mM luminol (2 mL) aqueous solution was firstly added into the mixture including cyclohexane (15 mL), 1-hexanol (3.2 mL), Triton X-100 (3.44 mL), water (1.26 mL) and kept magnetically stirred for 15 min. Next, TEOS (200 mL) was injected into the solution. After 30 min, ammonium hydroxide (120 mL) was added and a polymerization reaction was initiated. The solution was kept stirred for 24 h. Then, APTES (100 mL) was added and kept stirred for another 24 h. Finally, the resultant nanoparticles were collected by centrifugation and washed three times with ethanol and water, and then resuspended with 5 mL water. In the following ECL measurements, 40 mL of the resuspended luminol-SiNPs was used in one sample solution. 2.4. Detection of alkaline phosphatase (ALP) In a 1.5 mL plastic vial, 200 mL of 0.05 M Tris-HCl buffer (pH 10.0), 50 mL 20 mM PNPP, different activities (from 5 to 200 U/L) of ALP, 50 mL of the resuspended luminol-SiNPs and appropriate water were added to keep the final volume of the whole solution at 1 mL. All the samples were put in the water at 37  C for 50 min. The whole solution needed to be thoroughly vortex mixed before each ECL spectra measurement. Fig. 1. SEM (A) and TEM (B) images of luminol-SiNPs.

3. Results and discussion 3.1. Construction of ECRET immunoassay for ALP detection using luminol-SiNPs The synthesized luminol-NPs display spherical structure with uniform sizes. After calculated 100 particle size using software “Nano Measurer 1.2” and SEM mages, it can be seen that the average diameter of luminol-NPs is nearly 35 nm (Fig. 1). LuminolNPs solution can be stable for more than 1 month when stored in 4  C refrigerator by detecting ECL intensity. Luminol-NPs have maximal ECL emission at 425 nm similarly with luminol [40e42,48,49]. PNP has absorption at 430 nm. ECL emission of luminol-SiNPs effectively overlaps with the absorption of PNP (Supporting information Fig. S1). It is possible to trigger ECRET between luminol-SiNPs as ECL donors and PNP as acceptor. To verify the principle of ECRET from luminol-SiNPs to PNP, ECL of luminol-SiNPs system is detected. As shown in Fig. 2 and 44% ECL of luminol-SiNPs is quenched when 50 U/L ALP is added to the mixed solution of luminol-SiNPs and PNPP. Since electrochemical behavior is not changed from 0.5 V to 0. 75 V (Fig. 2B), ECL “turnoff” results can be ascribed to the fact that PNPP reacts with ALP to generate PNP and accordingly results in ECRET from luminol-SiNPs to PNP. Therefore it suggests the feasibility of ECRET from luminolSiNPs to PNP.

quenching efficiency ((I0eI)/I0) is obtained at 37  C. It is wellknown that the activity of ALP is relevant to the temperature. Lower temperature inhibits the activity of ALP and much high temperature causes the inactivation of ALP. Normally physiological temperature (37  C) is the optimal temperature to realize its high activity of ALP. Therefore the optimal temperature is kept at 37  C for the following ECRET detection of ALP. Fig. 4 shows the effect of time on ECRET detection of ALP. After the addition of ALP, ECL intensity of luminol-SiNPs system decreases sharply. To reach stable ECL quenching efficiency, 50 min is chosen as the optimal condition for ECL “turn-off” detection of ALP. Fig. 5 shows the effect of pH on ECRET detection of ALP. To reach stable and highest ECL quenching efficiency ((I0eI)/I0), pH 10.0 is chosen as the optimized conditions for ECL “turn-off” detection of ALP, which is in accordance with the principle of ALP usually used in alkaline condition. Since PNPP can induce the ECL to be quenched, the amount of PNPP has effect on ECRET detection of ALP. From the results in Fig. 6, it can be seen that with 1 mM PNPP is the turning point and high concentration of PNPP is not beneficial to ECL quenching effect. High concentrations of PNPP exceed the activity ability of enzyme (ALP) and besides can affect ECL intensity. Therefore 1 mM PNPP is chosen as the optimized conditions for ECL “turn-off” detection of ALP.

3.2. Optimization of ECRET detection of ALP using luminol-SiNPs The effect of temperature on ECL “turn-off” detection of ALP via the proposed ECRET strategy is optimized in Fig. 3. ECL intensities are measured from room temperature 25  Ce50  C. I0 represents ECL intensity of luminol-SiNPs and PNPP mixtures; I represents ECL intensity after the addition of ALP; (I0eI)/I0 represents ECL quenching efficiency on the whole system. The highest value of ECL

3.3. Sensitivity and selectivity of ECRET detection of ALP using luminol-SiNPs Fig. 7A shows ECL intensity of the proposed ECRET system in the presence of different concentrations of ALP. ECL intensity of luminol-SiNPs system gets decreased after the addition of ALP from 5 to 200 U/L. The plot of ECL quenching efficiency ((I0eI)/I0) versus

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Fig. 4. The effect of time on ECRET detection of ALP. Time(min): immediately, 10, 20, 30, 40, 50, 60, 80. c(PNPP): 1 mM; c(ALP): 50 U/L. 0.05 M Tris-HCl buffer solutions: pH 10.0; Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V. Black line represents ECL intensity of luminol-SiNPs and PNPP mixtures; Red line represents ECL intensity after the addition of ALP. All the error bars represent the standard deviation of three measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. ECL intensity-potential profiles (A) and linear sweep voltammetry (B) at glassy carbon electrode (GCE) of luminol-SiNPs in the absence and presence of ALP, PNPP, or both ALP and PNPP via the proposed ECRET strategy. c(PNPP): 1 mM; c(ALP): 50 U/L; 0.05 M Tris-HCl buffer solutions: pH 10.0; Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V.

Fig. 5. The effect of pH on ECRET detection of ALP. I0 represents ECL intensity of luminol-SiNPs and PNPP mixtures; I represents ECL intensity after the addition of ALP; (I0eI)/I0 represents ECL quenching efficiency after the addition of ALP to luminol-SiNPs and PNPP mixtures. 0.05 M Tris-HCl buffer solutions, pH: 8.0, 8.5, 9.0, 9.5, 10.0, 10.5; c(PNPP): 1 mM; c(ALP): 50 U/L; Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V. All the error bars represent the standard deviation of three measurements.

Fig. 3. The effect of temperature on ECRET detection of ALP. I0 represents ECL intensity of luminol-SiNPs and PNPP mixtures; I represents ECL intensity after the addition of ALP; (I0eI)/I0 represents ECL quenching efficiency after the addition of ALP to luminolSiNPs and PNPP mixtures. Temperature (oC): 25 (room temperature), 37, 50. c(PNPP): 1 mM; c(ALP): 50 U/L; 0.05 M Tris-HCl buffer solutions: pH 10.0; Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V. All the error bars represent the standard deviation of three measurements.

the concentration of ALP is shown in Fig. 7B. A linear relationship is obtained at the concentrations of ALP from 5 to 50 U/L with correlation coefficient (r) of 0.9905 and with the linear equation of

(I0eI)/I0 ¼ 0.00379 þ 0.00771c (U/L). It is concluded that this ECRET system can be utilized in ECL “turn-off” detection of ALP from 5 to 50 U/L. According to the reference [22], subjects with moderate-to-severe white matter hyper-intensities and silent lacunar infarct are more likely to have ALP levels 106 U/L than 64 U/L. Therefore the linear range of the method in the present work can meet the need of clinical analysis. The limit of detection (LOD) is calculated to be 0.8 U/L (S/N ¼ 3, n ¼ 8), which is calculated based on the standard deviation of the response and the slope of the calibration curve. Compared with other ALP detection reports (Table 1), LOD of the proposed ECRET method using luminol-SiNPs is lower than most of other ALP methods. Therefore the present ECRET method using luminol-SiNPs for ALP detection is a sensitive method for ALP detection. The effect of other enzymes during clinical measurements such as lysozyme (LZM), trypsin, peroxidase (HRP), uricase and thrombin

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Table 1 Comparison of the present ECRET detection with other methods for ALP detection. Method

Linear range (U/L)

LOD (U/L)

Refs

Colorimetry Colorimetry Colorimetry Colorimetry Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Electrochemistry ECRET

104e106 10e120 20e220 1e34 0.5e100 1e50 2.5e40 30e240 2e200 1e1000 6e600 5e50

870 5.4 1.27 0.19 0.2 0.94 1 5 0.8 0.25 2 0.8

[50] [51] [52] [53] [54] [55] [56] [57] [29] [58] [28] present work

Fig. 6. The effect of different concentrations of PNPP on ECRET detection of ALP. Black columns represent ECL intensity of luminol-SiNPs and PNPP mixtures; Red columns represent ECL intensity after the addition of ALP. c(PNPP, mM): 0.5, 0.8, 1, 3, 5, 8. c(ALP): 50 U/L; 0.05 M Tris-HCl buffer solutions: pH 10.0; Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V. All the error bars represent the standard deviation of three measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8. The selectivity of ECRET detection of ALP. All the concentrations of enzymes such as ALP, LZM, trypsin, HRP, uricase and thrombin are 50 U/L. Other substrates such as bovine serum albumin (BSA), glucose (Glu) and metal ions are 100 mM c(PNPP): 1 mM; 0.05 M Tris-HCl buffer solutions: pH 10.0; I0 represents ECL intensity of luminolSiNPs and PNPP mixtures; I represents ECL intensity after the addition of ALP, other enzymes and substrates; (I0eI)/I0 represents ECL quenching efficiency after the addition of enzymes and substrates to luminol-SiNPs and PNPP mixtures. Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V. All the error bars represent the standard deviation of three measurements.

such as Ca2þ, Mg2þ, Zn2þ and Fe3þ are also studied. Both of other enzymes or substrates during clinical measurements show similar ECL “turn-off” effect as ALP does. Because other enzymes and substrates cannot react with PNPP to form PNP and achieve ECRET from luminol-SiNPs to PNP. Therefore it suggests that the proposed ECRET method exhibits high selectivity for ALP detection towards other enzymes and substrates. The prepared luminol-SiNPs is also a sensitive and selective promising ECL probe for ALP detection.

4. Conclusions

Fig. 7. ECL intensity-time profiles (A) and linear correlation of ECRET detection of ALP. c(ALP, U/L): 5, 8, 10, 30, 50, 80, 100, 150, 200; c(PNPP): 1 mM; 0.05 M Tris-HCl buffer solutions: pH 10.0; Scan range: from - 1 V to 1 V; Scan rate: 0.1 V/s; Photomultiplier tube voltage: 1000 V. All the error bars represent the standard deviation of three measurements.

on ECRET system for “turn-off” detection of ALP using synthesized luminol-SiNPs is studied (Fig. 8). Moreover other substrates containing bovine serum albumin (BSA), glucose (Glu) and metal ions

In summary, user-friendly luminol-SiNPs are used as the donor and PNP generated by the reaction between PNPP and ALP is used as the receptor in this ECRET sensing platform. The spectral overlap between the absorption of the acceptor and the ECL emission of the donor (luminol-SiNPs) results in ECRET from luminol-SiNPs to PNP and achieves for ECL “turn-off” immunoassay of ALP. This ECRET method using luminol-SiNPs exhibits high sensitivity for ALP detection with LOD of 0.8 U/L and sufficient specificity for ALP over other enzymes such as HRP, trypsin and lysozyme. This ECRET system can be employed for ECL detection of other general targets such as proteins and drugs by coupling with proper ECL donors and acceptors.

Please cite this article as: W. Qi et al., Electrochemiluminescence resonance energy transfer immunoassay for alkaline phosphatase using pnitrophenyl phosphate as substrate, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.073

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[19]

[20]

Acknowledgements [21]

This project was supported by the National Natural Science Foundation of China (No. 21505011), Chongqing Research Program of Basic Research and Frontier Technology (No. cstc2018jcyjAX0742), Scientific and Technological Research Program of Chongqing Education Committee (No. KJQN201900521) and Program for Top-Notch Young Innovative Talents of Chongqing Normal University (No. 02030307-00042). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.10.073.

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Please cite this article as: W. Qi et al., Electrochemiluminescence resonance energy transfer immunoassay for alkaline phosphatase using pnitrophenyl phosphate as substrate, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.073