Photoelectrochemical determination for acid phosphatase activity based on an electron inhibition strategy

Sensors & Actuators: B. Chemical 307 (2020) 127654

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Photoelectrochemical determination for acid phosphatase activity based on an electron inhibition strategy

T

Mingjuan Huang, Jiuying Tian*, Chunhong Zhou, Ping Bai, Jusheng Lu* Jiangsu Key Laboratory of Green Synthetic Chemistry for Functional Materials, School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou 221116, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Photo-excited electron inhibition 3,5-diamino-1,2;4-triazole Acid phosphatase TNA/g-C3N4/DAT Coordination of Fe(II)

In the present work, inspired by the excellent photoelectrochemical (PEC) properties of TiO2 and g-C3N4, a simple and sensitive TNA/g-C3N4/DAT sensor for determination of acid phosphatase (ACP) was constructed based on the electron inhibition performance of 3,5-diamino-1,2,4-triazole (DAT) and coordination of Fe(II) with DAT. DAT was oxidized by sodium nitrite to its diazonium salt and then was electrochemically grafted onto the surface of TNA/g-C3N4 to form the TNA/g-C3N4/DAT, which significantly hindered the electron transfer of TNA/ g-C3N4, and reduced the photocurrent response of TNA/g-C3N4 under visible light irradiation. After immersion the TNA/g-C3N4/DAT in the mixing solution containing Fe(III), AAP and ACP enzyme, the photocurrent of the TNA/g-C3N4/DAT sensor increased due to the dephosphorylation of AAP to AA catalyzed by ACP enzyme, the reduction of Fe(III) to Fe(II) by AA, and the coordination reaction of Fe(II) with DAT, the increase value of photocurrent was linear with the concentration of ACP under the optimal experimental conditions. Therefore, the constructed TNA/g-C3N4/DAT PEC sensor exhibited a satisfying linear range (0.05–100 U L−1), low limit of detection (0.016 U L−1) and good selectivity towards ACP determination, which has been successfully applied for the analysis of real human serum samples with good precision of RSD less than 4.2 % and good accuracy of the recoveries ranged from 97 % to 104 %.

1. Introduction Acid phosphatase (ACP), a common hydrolase in the mammalian tissues and fluids, can catalyze the hydrolysis of phosphate monoesters in a weakly acidic medium [1], which plays an important role in many physiological processes, especially in human movements. The abnormal expression of ACP in the body may indicate the occurrence of many diseases, such as prostate cancer [2], Gaucher’s disease [3], hyperparathyroidism [4], multiple myeloma [5], etc. It has been served as a biomarker for the early diagnosis of related diseases [6–8]. Therefore, the establishment of an accurate and sensitive method for the determination of ACP activity is particularly important in clinical diagnosis. In recent years, there have been many reports on the determination of ACP activity, including high performance liquid chromatography [9], electrochemical methods [10,11], fluorescence spectrophotometry [12–14], and so on. All of these assays have acceptable sensitivity and selectivity, but some assays are time-consuming, or require sophisticated instrumentation and expensive equipment. To our knowledge, there is no report on the determination of ACP

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activity by photoelectrochemical (PEC) technology so far. As a biological sensing method that has attracted the interest of many researchers, PEC has a relatively high sensitivity and lower background due to its total separation of excitation signal and detection signal [15–20]. In some previous reports on PEC techniques [21–23], the researchers proposed an enzymatic biocatalytic precipitation method that used enzyme-catalyzed reactions to in-situ produce an insoluble product on the electrode surface, which may generate an insulating effect to block the interfacial electron transmission between photoactive material and electron donor, amplified or reduced the PEC signal, thereby improving the detection sensitivity. TiO2 nanotube arrays (TNAs) can be applied in the PEC biosensors due to the large surface area, good photochemical stability and biocompatibility [24–27]. However, under visible light irradiation, the electrons could not be excited from the valence band (VB) of TiO2 to the conduction band (CB) due to its wide band gap [28,29], limiting its PEC sensing application. Graphitic carbon nitride (g-C3N4) is an excellent visible light active semiconductor with a band gap of about 2.7 eV [30], and has high chemical stability and electronic property [31]. In addition, the functional groups on the g-C3N4 surface make it easy to deposit

Corresponding authors at: School of Chemistry and Materials Science, Jiangsu Normal University, 101 Shanghai Road, Xuzhou, 221116, PR China. E-mail addresses: [email protected] (J. Tian), [email protected] (J. Lu).

https://doi.org/10.1016/j.snb.2020.127654 Received 26 September 2019; Received in revised form 17 December 2019; Accepted 1 January 2020 Available online 02 January 2020 0925-4005/ © 2020 Elsevier B.V. All rights reserved.

Sensors & Actuators: B. Chemical 307 (2020) 127654

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Scheme 1. Construction process of the TNA/g-C3N4/DAT sensor for determination of ACP activity.

2.2. PEC determination of ACP activity

on the surface of TiO2, which is beneficial for the construction of TNA/ g-C3N4 [32–34], and make TNAs have good photocurrent response under visible light irradiation. 3,5-diamino-1,2,4-triazole (DAT) is a good organic ligand that can coordinated with Cu(II), Zn(II), Co(II) and Fe(II) ions [35–37] and can be oxidized by NaNO2 to form a DAT diazonium salt under acidic conditions [38]. The related reaction equation of DAT oxidization was shown in Scheme 1. The prepared DAT diazonium salt can be electrochemically grafted onto the surface of various materials such as carbon, metal and semiconductor [38–43] to form a dense DAT organic film. In the present work, the DAT diazonium salt was electrochemically grafted onto the surface of the TNA/g-C3N4, and the PEC response of the constructed TNA/g-C3N4/DAT would decrease significantly due to the electron hindrance effect of DAT organic films. After the hydrolysis of ascorbic acid-2-phosphate (AAP) catalyzed by ACP [44–46], the produced AA as a reducing agent could reduce Fe(III) to Fe(II) which coordinates with DAT, making the electronic conductivity of the TNA/gC3N4/DAT significantly enhanced, and the photocurrent of the TNA/gC3N4/DAT would increase correspondingly. Meanwhile, the produced AA could also be used as an electron donor for the TNA/g-C3N4/DAT photoanode, and further amplify the signal of the TNA/g-C3N4/DAT. Therefore, a simple and sensitive PEC sensor for the determination of ACP activity would be successfully constructed. The related construction process of the TNA/g-C3N4/DAT sensor was shown in Scheme 1.

Prior to PEC determination of ACP activity, the photocurrent of the TNA/g-C3N4/DAT under visible light irradiation was measured, which was I0. Then, the TNA/g-C3N4/DAT was immersed in 0.5 mL of 10 mM Tris−HCl solution (pH 5.0) containing 0.5 μM of Fe(III), 1.0 μM of AAP and ACP with different concentration and equilibrated for 15 min, determined the photocurrent value under visible light irradiation, which was I. Therefore, the ACP activity could be determined by the photocurrent change value of the TNA/g-C3N4/DAT sensor (△I = I - I0). 2.3. Pretreatment of real human serum sample A healthy human serum sample was obtained from Xuzhou Central Hospital (P. R. China) and had been ethically approved by the hospital. The experiments were carried out in compliance with the relevant laws and institutional guidelines. In 1.00 mL of the human serum sample, 0.5 mL of 10 mM pH 5.0 Tris−HCl buffer solution containing 4 wt% trichloroacetic acid (TCA) was added, stirred for 5 min, and centrifuged at 8000 rpm for 10 min. The supernatant was diluted 20-fold with 10 mM pH 5.0 Tris−HCl buffer solution and measured according to Section 2.2. All other materials and reagents and apparatus details can be seen in the Supporting Information. 3. Results and discussion

2. Experimental 3.1. Construction and characterization of the TNA/g-C3N4/DAT sensor 2.1. Construction of the TNA/g-C3N4/DAT sensor For construction of the TNA/g-C3N4/DAT sensor, the g-C3N4 nanosheet was first prepared. The results showed that, the obtained gC3N4 nanosheet could be dispersed in aqueous solution with the diameter about 100 nm as seen from the TEM image (Fig. 1A), and had a certain absorption intensity from the ultraviolet to the visible part in the UV–vis absorption spectroscopy (curve a of Fig. 1B). When the DAT diazonium salt was electrochemically grafted onto g-C3N4, the absorption peak of DAT blue-shifted to 363 nm from 376 nm due to the electron absorption of the triazine ring in the g-C3N4 (curve b and c of Fig. 1B). After immersing the g-C3N4/DAT in Fe(III) solution, the absorption peak of g-C3N4/DAT had not significant change. However, when the g-C3N4/DAT was immersed in the Tris−HCl buffer solution (pH 5.0) containing Fe(III), AAP and ACP, the absorption peak of DAT further blue-shifted to 354 nm (curve d of Fig. 1B). It indicated that, AAP was catalytically hydrolyzed by ACP to produce AA, which could

For construction of the TNA/g-C3N4/DAT sensor, 3,5-diamino1,2,4-triazole diazonium salt and g-C3N4 was prepared and seen in the Supporting Information. TNAs on titanium foil were fabricated by an electrochemical anodization technique according to our previously reported method [47], and 50.0 μL g-C3N4 suspension was dropped onto the on the surface of TNA and dried at 60 °C for 2 h to form the TNA/gC3N4. Then, a conventional three-electrode system was used, where a TNA/g-C3N4 was used as the working electrode, a Pt wire as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode, respectively. The DAT diazonium salts were electrochemically grafted onto the TNA/g-C3N4 by cyclic voltammetry scanning from -1.0–1.0 V for 10 cycles to obtain the TNA/g-C3N4/DAT, rinsed with water and dried with a nitrogen gas stream.

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Fig. 1. (A) TEM image of g-C3N4 nanosheets. (B) UV–vis absorption spectrum of g-C3N4 (a), DAT diazonium salt (b), g-C3N4/DAT (c), and g-C3N4/DAT-Fe(II) (d). FESEM top-view image of TNA (C) and TNA/g-C3N4 (D). (E) Energy dispersive spectroscopy (EDS) of TNA/g-C3N4. XPS survey spectrum of TNA/g-C3N4 (F) and highresolution XPS spectra of Ti 2p (G), O 1s (H), N 1s (I) and C 1s (J). (K) FESEM top-view image of TNA/g-C3N4/DAT. (L) FT-IR spectrum of g-C3N4 (a) and g-C3N4/DAT (b).

and b of Fig. 2A), however, one distinct redox peak at -0.85/-0.56 V was observed when the TNA/g-C3N4/DAT immersed in the Tris−HCl buffer solution (pH 5.0) containing Fe(III), AAP and ACP for a certain time (curve c of Fig. 2A), which was attributed to the Fe(II)/Fe(III) redox couples [52], illustrating the reduction of Fe(III) and the successful formation of the TNA/g-C3N4/DAT-Fe(II). With the increase of scan rate from 0.02 to 0.5 V s−1, the anodic and cathodic peak currents of the TNA/g-C3N4/DAT-Fe(II) all increased (Fig. 2B), and the corresponding currents were proportional to the scan rate (Fig. 2C), indicating a typical of surface-controlled quasireversible process of the TNA/g-C3N4/ DAT-Fe(II) [53]. In addition, the anodic and cathodic peak potentials were linearly depended on the logarithm of scan rate (Fig. 2D). Based on Laviron’s method [54], the electron transfer coefficient (α) was calculated to be 0.68, and the heterogeneous electron transfer rate constant (ks) was calculated to be 5.29 s−1 at the scan rate of 0.2 V s−1, which indicated the TNA/g-C3N4/DAT-Fe(II) had an excellent electron transfer ability. On the other hand, in the electrochemical impedance spectroscopy (EIS), the TNA/g-C3N4 substrate had a relatively small resistance value (1080 Ω, curve a of Fig. 3A), suggesting the preferable conductivity property of TNA/g-C3N4. After electrochemical grafting of DAT diazonium on the TNA/g-C3N4, the impedance value of the TNA/g-C3N4/ DAT sharply increased to 6520 Ω due to the poor conductivity of DAT (curve b of Fig. 3A). While the resistance value decreased to 4350 Ω after immersing the TNA/g-C3N4/DAT sensor in 10 mM Tris−HCl buffer solution (pH 5.0) containing 0.5 μM of Fe(III), 1.0 μM of AAP and 5.0 U L−1 of ACP enzyme for 15 min (curve c in Fig. 3A). With the increase of ACP concentration, the electrochemical resistance further decreased (curve d–f in Fig. 3A). It was due that, ACP had a catalytic effect on the hydrolysis of AAP to generate AA, which led to the reduction of Fe(III) to Fe(II), and coordination of Fe(II) with DAT, enhancing the electron conductivity of the TNA/g-C3N4/DAT electrode, which was in good agreement with the results from CV measurements (Fig. 3B).

reduce Fe(III) to Fe(II), promoting the successful coordination of Fe(II) with DAT. Compared to the naked TNA with an average nanotube inner diameter of ∼100 nm, the dropped g-C3N4 on the TNA surface were clearly observed and evenly distributed (SEM images in Fig. 1 C and D). The energy dispersive spectroscopy (EDS) of the TNA/g-C3N4 showed the existence of C, N, Ti and O (Fig. 1E), and X-ray photoelectron spectroscopy (XPS) measurement could further explain the surface composition and chemical state of TNA/g-C3N4 (Fig. 1 FeJ). The characteristic binding energy values of 459.3 and 465.0 eV for Ti 2p 3/2 and Ti 2p 1/ 2 (Fig. 1G), respectively, indicated a Ti4+ oxidation state originating in TiO2 [48]. The peak at 530.6 eV in the O 1s spectrum could be ascribed to the signal of Ti-O bond (Fig. 1H) [49]. Four divided peaks located at 398.7, 399.9, 401.0, and 404.1 eV in the N 1s XPS spectrum (Fig. 1I) were ascribed to the sp2 -bonded nitrogen (CeN = C), nitrogen in tertiary N-C3 groups, amino groups (C-N-H), and the π excitation of the C]N conjugated structure, respectively [50]. And the two peaks located at 284.4 eV and 288.2 eV (Fig. 1J) were ascribed to the CeC coordination and the NeC = N, respectively [51]. All of these XPS results indicated that g-C3N4 was successfully deposited on the surface of TNAs. After the electrochemical grafting of DAT onto the TNA/g-C3N4 substrate, the SEM image of TNA/g-C3N4/DAT exhibited a denser net structure and a uniform distribution (Fig. 1K). FT-IR spectroscopy (Fig. 1L) showed that one another absorption peak at 1687 cm−1 appeared for the g-C3N4/DAT compared with the pure g-C3N4, which was attributed to the stretching peak of –C = N- in the DAT, indicating the successful combination of DAT and g-C3N4. 3.2. Electrochemical behavior of the TNA/g-C3N4/DAT sensor The electrochemical behavior of different modified electrodes could be reflected by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), which was employed to investigated the intrinsic conductivity of the constructed TNA/g-C3N4/DAT sensor (Fig. 2 A–D). As illustrated in Fig. 2A, no obvious redox peaks appeared in the CV of the TNA/g-C3N4 or TNA/g-C3N4/DAT electrode (curve a 3

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Fig. 2. (A) Cyclic voltammograms of TNA/gC3N4 (a), TNA/g-C3N4/DAT (b), and TNA/gC3N4/DAT-Fe(II) (c). (B) Cyclic voltammograms of TNA/g-C3N4/DAT-Fe(II) at different scan rates. (C) Plot of the peak current versus the scan rate (ν). (D) Plot of the peak potential versus log ν. Electrolyte solution, anaerobic 10 mM Tris−HCl buffer solution (pH 5.0). Range of scan rate, 0.02 to 0.5 V s−1.

was because that the grafting of DAT produced a dense organic layer on the surface of the TNA/g-C3N4, hindered the photo-excited electron transfer of TNA/g-C3N4. Immersion of the TNA/g-C3N4/DAT in Tris−HCl buffer solution containing Fe(III), AAP and ACP enzyme was beneficial to the production of Fe(II) and the coordination of Fe(II) and DAT, and facilitated the photo-excited electron transfer, resulting in a re-increased photocurrent of TNA/g-C3N4/DAT (curve c of Fig. 3C). With the increase of ACP concentration, the photocurrent increased gradually (curve d–f of Fig. 3C). In addition, to verify the effect of AA and Fe(II) produced by ACP

3.3. Photoelectrochemical behavior of the TNA/g-C3N4/DAT sensor The experimental results showed that, under visible light irradiation, no significant photocurrent was observed for TNAs (Fig. S1A), however, a typically anodic photocurrent for TNA/g-C3N4 could be observed (curve a of Fig. 3C), which was due to the relatively narrow optical band gap of g-C3N4 with 2.73 eV and the appropriate energy level matching between TNA and g-C3N4 (Fig. S1B). After electrochemical grafting of DAT diazonium on the TNA/g-C3N4, the photocurrent of TNA/g-C3N4 decreased significantly (curve b of Fig. 3C). It

Fig. 3. (A) Nyquist plots of electrochemical impedance spectroscopy of different systems in 0.1 M PBS solution (pH 7.4) containing 5.0 mM Fe(CN)64−/3− and 0.1 M KCl; Cyclic voltammograms (B) and photocurrent responses (C) of different systems, (a) TNA/g-C3N4, (b) TNA/gC3N4/DAT, (c–f) TNA/g-C3N4/DAT immersed in 10 mM Tris−HCl buffer solution (pH 5.0) containing 0.5 μM of Fe(III), 1.0 μM of AAP and ACP with 5 (c), 10 (d), 20 (e) or 50 U L-1 (f). (D) Control experiment: Photocurrent responses of TNA/g-C3N4/DAT immersed in 10 mM Tris−HCl buffer solution (pH 5.0) containing 0.2 μM of Fe(II) (g), or 0.2 μM of Fe (II) +1.0 μM of AAP (h).

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Fig. 4. (A) Photocurrent responses of the TNA/ g-C3N4/DAT sensor under visible light irradiation after immersing in 10 mM Tris−HCl solution (pH 5.0) containing 0.5 μM of Fe(III), 1.0 μM of AAP and ACP with 0 (a), 0.05 (b), 0.1 (c), 0.2 (d), 0.5 (e), 1.0 (f), 2.0 (g), 5.0 (h), 10.0 (i), 20.0 (j), 50.0 (k), 80.0 (l), 100.0 (m), 120.0 (n) and 150.0 U L−1 (o), respectively; Inset: Calibration curve of photocurrent change value of the TNA/g-C3N4/DAT sensor versus ACP concentration within the range of 0.05–100.0 U L−1. (B) Photocurrent response of the TNA/ g-C3N4/DAT sensor after immersing in 10 mM Tris−HCl solution (pH 5.0) containing 0.5 μM of Fe(III), 1.0 μM of AAP (a, blank) and 1000 U L−1 of GOD (b), HRP (c), Try (d), LZM (e), TYR (f), ALP (g), or 50 U L−1 of ACP (h).

photocurrent response increased at first with the increase of pH, the maximum photocurrent response was obtained at pH 5.0. Further increased pH, the photocurrent decreased instead when the pH was larger than 8.0 (Fig. S2C). It was because that, the catalytic activity of ACP enzyme would be inhibited and Fe(III) would be hydrolyzed in a too alkaline solution. Also too small pH would affect the coordination ability of Fe(II) and DAT. So, 10 mM Tris−HCl buffer solution with pH 5.0 was used in the experiments, which not only ensured the high catalytic activity of ACP enzyme, but also ensured the coordination between Fe(II) and DAT. On the other hand, the influence of the incubation time after immersion of the TNA/g-C3N4/DAT in the 10 mM Tris−HCl buffer solution (pH 5.0) was investigated (Fig. S2D). The photocurrent increased with the increase of incubation time and remained essentially constant after 15 min, indicating that the ACP enzymatic reaction and coordination reaction of Fe(II) and DAT have been completed. Thus, we selected the incubation time with 15 min for the PEC determination of ACP activity. Except the above experimental conditions, we also studied the effect of irradiation intensity and turbidity of solution on the photocurrent response. The results showed that, the photocurrent increased with the increase of irradiation intensity from 10−100 mW cm−2 and reached a stable value when the irradiation intensity was greater than 100 mW cm−2 (Fig. S3A). In addition, the photocurrent decreased continuously with the increase of the turbidity of simulated sample solution (Fig. S3B). Therefore, the irradiation intensity was selected as 100 mW cm−2 in the present work, and TCA was added into the real serum sample to make the turbid solution clear and reduce the non-specific adsorption of the protein on the surface of the TNA/g-C3N4/DAT sensor.

catalysis on the photocurrent of the TNA/g-C3N4/DAT sensor, some control experiments were carried out in the present work. When the TNA/g-C3N4/DAT was immersed in the Fe(II) solution, the photocurrent of the TNA/g-C3N4/DAT increased (curve g of Fig. 3D). If AA was further added in the Fe(II) solution, the photocurrent of the TNA/gC3N4/DAT further increased (curve g of Fig. 3D), indicating that Fe(II) could improve the photo-excited electron transfer of the TNA/g-C3N4/ DAT, and AA could serve as an electron donor to ensure the continuous generation of photo-excited electrons, thereby generating a larger photocurrent. All of these indicate ACP enzyme could catalyze the hydrolysis of AAP to produce AA, which could not only reduce Fe(III) to Fe(II), promoting the coordination of Fe(II) and DAT, but also make the photocurrent of the TNA/g-C3N4/DAT photoanode increase as an electron donor. Therefore, the determination of ACP activity could be achieved by the change of photocurrent of the TNA/g-C3N4/DAT sensor. The relative mechanism of PEC measurement was shown in Scheme 1. 3.4. Optimization of the experimental conditions on the TNA/g-C3N4/DAT sensor for determination of ACP activity In order to accurately determine ACP activity by PEC technique, some experiments to optimize the experimental conditions were carried out, such as cycle numbers of cyclic voltammetry for electrochemically grafting of DAT diazonium salt, pH of the solution medium, the added amount of Fe(III) or AAP, and incubation time. Herein, the DAT diazonium salt was electrochemical grafted on the surface of TNA/g-C3N4 by cyclic voltammetry (CV), the cycle numbers of CV had a significant effect on the photocurrent change (△I) of the TNA/g-C3N4/DAT. The results showed that, too little or too many CV cycles all resulted in the small photocurrent change of TNA/g-C3N4/ DAT (Fig. S2A). The reason may be that, too small CV cycles made the amount of DAT covered on the surface is small, and the photocurrent of the TNA/g-C3N4/DAT substrate would be relatively large. When the number of CV cycles was too large, the surface DAT layer would be too dense, and the generated Fe(II) and AA could not significantly increase the photocurrent of the TNA/g-C3N4/DAT. So, we selected 10 CV cycles for DAT electrochemical grafting in the present work. In addition, the added amount of Fe(III) had a significant effect on the electron transfer of the TNA/g-C3N4/DAT sensor. The results showed that the photocurrent of the TNA/g-C3N4/DAT increased gradually with the increase of Fe(III) amount, and reached the maximum value with the concentration of Fe(III) of 0.5 and 0.8 μM (Fig. S2B). Excessive concentration of Fe(III) would consume AA unnecessarily, which reduced the function of AA as an electron donor for PEC. So, 0.5 μM of Fe(III) was used in the subsequent experiments. Furthermore, the medium pH affected the enzyme activity, the state of Fe(III) in solution, and coordination property of Fe(II) and DAT. In the range of pH 3.0–10.0 in 10 mM Tris−HCl buffer solution, the

3.5. Analytical performances of the TNA/g-C3N4/DAT for the determination of ACP activity Under the optimized experimental conditions, the ACP activity was determined by PEC technique with the proposed TNA/g-C3N4/DAT sensor. As shown in Fig. 4A, the photocurrent of the sensor increased with the increase of ACP concentration, and the increase value of photocurrent was linear proportional to the concentration of ACP in the range from 0.05–100 U L−1 (Inset of Fig. 4A), the linear regression equation was △I = 0.767 C (U L−1) + 1.595 (R2 = 0.9977), and a limit of detection (LOD) of such sensor based on a signal-to-noise ratio of 3 was 0.016 U L−1, which indicated the TNA/g-C3N4/DAT sensor could be used as a potential sensor for determining ACP, and also meet the analysis requirements of real samples due to the normal level of ACP in human serum in the range of 35–123 U L−1 [11]. Compared to other works summarized in Table 1, the proposed assay for determination of ACP activity exhibited a satisfying linear range and limit of detection. To investigate the selectivity of the TNA/g-C3N4/DAT sensor for ACP determination, some other enzymes including alkaline 5

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of the recoveries ranged from 97 % to 104 %.

Table 1 Comparison of the proposed method for ACP determination with other previously reported works. Methods

Linear range (U L−1)

LOD (U L−1)

Reference

Electrochemistry Potentiometric Ratiometric fluorescence “Switch-On” fluorescence FIA electrochemistry Fluorescence Reversible fluorescence Near-infrared fluorescence Colorimetric TNA/g-C3N4/DAT PEC

0.12-5.7 0.05-3.0 1.0-50 5-40 0.4-27 1.0-50 18.2-1300 0-150 0.25-2.5 0.05-100

0.12 0.031.5 0.43 0.1 0.4 0.45 5.5 1.2 0.016 0.016

[10] [11] [12] [46] [55] [56] [57] [58] [59] This work

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. Acknowledgements The authors gratefully acknowledge the financial support from the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP), the Natural Science Foundation of Jiangsu Provincial Department of Education (19KJB150026), the Science and Technology Project of Xuzhou City (KH17027), the Undergraduate Students Project of Jiangsu Province (201810320032Z), and the Natural Science Foundation of Jiangsu Normal University (15XLR007).

phosphatase (ALP), glucose oxidase (GOD), horseradish peroxidase (HRP), trypsin (Try), lysozyme (LZM) and tyrosinase (TYR) were introduced as control in the proposed system. As shown in Fig. 4B, the addition of these enzymes in the 10 mM Tris−HCl buffer solution (pH 5.0) containing Fe(II) and AAP has little effect on the photocurrent response of the TNA/g-C3N4/DAT sensor, even if their concentration is more than 20 times that of ACP, indicating the specificity of ACP toward AAP and the high selectivity of the proposed sensor for ACP determination. In addition to study the reproducibility of the TNA/gC3N4/DAT sensor, 10 continuous measurements of a single sensor were performed with the relative standard deviation (RSD) of 5.2 %, and RSD of a parallel analysis for 6 sensors was calculated to be 7.1 %, indicating a relatively good reproducibility of the sensor. Furthermore, the constructed TNA/g-C3N4/DAT sensor could be regenerated simply for repeated use by immersing the TNA/g-C3N4/DAT/Fe(II) in 0.1 M NaAcHAc buffer solution (pH = 4.0) containing 10 mM o-phenanthroline at room temperature for 30 min. Due to the strong chelation between Fe (II) and o-phenanthroline [60], Fe(II) ions would leave the surface the TNA/g-C3N4/DAT, and the sensor would be regenerated for the next usage cycle. In addition, the regenerated sensor could be used and maintain 96 % of its original performance after 15 repetitions of the regeneration process (Fig. S4), showing an excellent reusability.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2020.127654. References [1] H. Bull, P.G. Murray, D. Thomas, A. Fraser, P.N. Nelson, Acid phosphatases, J. Clin. Pathol. -Mol. Pathol. 55 (2002) 65–72. [2] C. Huggins, C.V. Hodges, Studies on prostatic cancer I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate, J. Urol. 167 (2002) 948–951. [3] L.R. Tuchman, M. Swick, High acid phosphatase level indicating Gaucher's disease in patient with prostatism, J. Am. Med. Assoc. 164 (1957) 2034–2035. [4] L.T. Yam, Clinical significance of the human acid phosphatases: a review, Am. J. Med. 56 (1974) 604–616. [5] E. Terpos, J. de la Fuente, R. Szydlo, E. Hatjiharissi, N. Viniou, J. Meletis, X. Yataganas, J.M. Goldman, A. Rahemtulla, Tartrate-resistant acid phosphatase isoform 5b: a novel serum marker for monitoring bone disease in multiple myeloma, Int. J. Cancer 106 (2003) 455–457. [6] E.S. Leman, R.H. Getzenberg, Biomarkers for prostate Cancer, J. Cell. Biochem. 108 (2009) 3–9. [7] L. Li, J.Y. 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. [8] A. Kirschenbaum, X.H. Liu, S. Yao, A. Leiter, A.C. Levine, Prostatic acid phosphatase is expressed in human prostate cancer bone metastases and promotes osteoblast differentiation, Ann. N. Y. Acad. Sci. 1237 (2011) 64–70. [9] C. Burgdorf, A. Prey, G. Richardt, T. Kurz, A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium, Anal. Biochem. 374 (2008) 291–297. [10] Z. Fredj, M.B. Ali, M.N. Abbas, E. Dempsey, Determination of prostate cancer biomarker acid phosphatase at a copper phthalocyanine-modified screen printed gold transducer, Anal. Chim. Acta 1057 (2019) 98–105. [11] S.S.M. Hassana, H.E.M. Sayour, A.H. Kamel, A simple potentiometric method for determination of acid and alkaline phosphatase enzymes in biological fluids and dairy products using a nitrophenylphosphate plastic membrane sensor, Anal. Chim. Acta 640 (2009) 75–81. [12] Z.M. Zhu, X.Y. Lin, L.N. Wu, C.F. Zhao, S.G. Li, A.L. Liu, X.H. Lin, L.Q. Lin, Nitrogendoped carbon dots as a ratiometric fluorescent probe for determination of the activity of acid phosphatase, for inhibitor screening, and for intracellular imaging, Microchim. Acta 186 (2019) 558. [13] S. Pang, S.Y. Liu, Lysozyme-stabilized bimetallic gold/silver nanoclusters as a turnon fluorescent probe for determination of ascorbic acid and acid phosphatase, Anal. Methods 9 (2017) 6713–6718. [14] Z.Y. Qu, W.D. Na, X.T. Liu, H. Liu, X.G. Su, A novel fluorescence biosensor for sensitivity detection of tyrosinase and acid phosphatase based on nitrogen-doped graphene quantum dots, Anal. Chim. Acta 997 (2018) 52–59. [15] W.W. Zhao, J.J. Xu, H.Y. Chen, Photoelectrochemical DNA biosensors, Chem. Rev. 114 (2014) 7421–7441. [16] C.X. Li, H.Y. Wang, J. Shen, B. Tang, Cyclometalated iridium complex-based labelfree photoelectrochemical biosensor for DNA detection by hybridization chain reaction amplification, Anal. Chem. 87 (2015) 4283–4291. [17] Q.M. Shen, J.Y. Jiang, S.L. Liu, L. Han, X.H. Fan, M.X. Fan, Q.L. Fan, L.H. Wang, W. Huang, Facile synthesis of Au-SnO2 hybrid nanospheres with enhanced photoelectrochemical biosensing performance, Nanoscale 6 (2014) 6315–6321. [18] L.F. Fan, G.H. Zhao, H.J. Shi, M.C. Liu, Y.B. Wang, H.Y. Ke, A Femtomolar level and highly selective 17β-estradiol photoelectrochemical aptasensor applied in environmental water samples analysis, Environ. Sci. Technol. 48 (2014) 5754–5761. [19] Y. Li, J.Y. Tian, T. Yuan, P. Wang, J.S. Lu, A sensitive photoelectrochemical

3.6. Analysis of real samples To evaluate analytical reliability and application potential of the proposed TNA/g-C3N4/DAT sensor for real sample, the activity of ACP in the pretreated serum sample was determined and the spiked experiment was carried out simultaneously. The analytical results were shown in Table S1, the RSD values were less than 4.2 % and the recoveries for the spiked solutions ranged from 97 % to 104 %, implying the proposed TNA/g-C3N4/DAT sensor could be applied for practical analysis with a good accuracy and precision. 4. Conclusions In summary, g-C3N4 nanosheets immobilized on the TNAs ensured the photocurrent response of TNAs under visible light irradiation. Electrochemical grafting of DAT on the surface of TNA/g-C3N4 constructed the TNA/g-C3N4/DAT sensor while also significantly reduced the photocurrent of TNA/g-C3N4. Immersion of the TNA/g-C3N4/DAT sensor in Tris−HCl buffer solution (pH 5.0) containing Fe(III), AAP and ACP could effectively adjust the photocurrent of TNA/g-C3N4/DAT due to the dephosphorylation of AAP to AA catalyzed by ACP enzyme, the reduction of Fe(III) to Fe(II) by AA, and the coordination reaction of Fe (II) and DAT, indicating the feasibility for determination of ACP activity. Under the optimal experimental conditions, the constructed TNA/g-C3N4/DAT sensor could quantitatively determine the ACP activity with excellent selectivity, high precision, and good accuracy, which has been successfully applied for the real sample analysis of ACP activity with good precision of RSD less than 4.2 % and good accuracy 6

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Mingjuan Huang is now a graduate student in School of Chemistry and Materials Science, Jiangsu Normal University. Her research interests are in the areas of biosensing and electrochemical analysis. Jiuying Tian obtained her M.S. degree from Sichuan University, PR China, in 2000. Now she is an associate professor in Jiangsu Normal University. Her research interests are in the areas of preparation and applications of nanomaterials. Chunhong Zhou is an undergraduate from Jiangsu Normal University. In the group, she worked on the construction of biosensor. Ping Bai is now an undergraduate student in School of Chemistry and Materials Science, Jiangsu Normal University., PR China. In the group, she worked on the preparation of nanomaterials. Jusheng Lu obtained his Ph.D. degree from Southeast University, PR China, in 2015. Now he is an associate professor in Jiangsu Normal University. His research interests are mainly in the areas of preparation of nanomaterials, fabrication and development of biosensors and bioreactors.

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